This invention relates to plants with improved yield-related traits, including delayed leaf senescence, improved seedling survival, fruit ripening, grain size/starch content and/or stress tolerance. Such plants have plastids, e.g. chloroplasts, which are altered in their membrane protein composition so that transport of proteins in and out of the plastids is modified and controlled. Such modified plants may be made by mutagenesis and selection, genetic engineering or gene editing. The invention therefore also concerns isolated nucleic acids, expression vectors or gene editing constructs and associated host cells and methods of using any of these to produce modified plants and germplasm. Such modified plant material has potential application in the generation and breeding of new plants.

BACKGROUND

Plastids are a diverse family of plant organelles. The family includes chloroplasts - the organelles responsible for photosynthesis - as well as a range of non-photosynthetic variants such as starch-containing amyloplasts in seeds, tubers and roots, carotenoid-rich chromoplasts in flowers and fruits, and chloroplast-precursor organelles in dark-grown plants called etioplasts (see Jarvis, P. and Lopez-Juez, E. (2013) Biogenesis and homeostasis of chloroplasts and other plastids Nat. Rev. Mol. Cell Biol. 14: 787-802.

Most plastid proteins are encoded by the nuclear genome and synthesized in the cytosol as precursors with N-terminal targeting signals called transit peptides. Import of precursors into chloroplasts is mediated by the TOC and TIC (Translocon at

the Outer/Inner envelope membrane of Chloroplasts) complexes. (See Jarvis, P. (2008) Targeting of nucleus-encoded proteins to chloroplasts in plants (Tansley Review) New Phytol. 179: 257-285 for more detail).

From Ling, Q., Huang, W., Baldwin, A. and Jarvis, P. (2012) Chioropiast biogenesis is regulated by direct action of the ubiquitin-proteasome system Science 338: 655-659 and Ling, Q. and Jarvis, P. (2013) Dynamic regulation of endosymbiotic organelles by ubiquitination Trends Cell Biol. 23: 399-408, it is known that plastid biogenesis is directly regulated by the ubiquitin-proteasome system (UPS). A screen of extragenic suppressors of the Arabidopsis plastid protein import mutation ppi1 - which is a knockout mutant of a translocon at the outer membrane of chloroplasts (Toc33) - identified SUPPRESSOR OF PPI1 LOCUS1_ ( SP1 ). SP1 encodes a RING-type ubiquitin E3 ligase in the plastid outer membrane that selectively targets the TOC machinery for ubiquitination and degradation. By controlling the levels of different TOC receptor isoforms, SP1 regulates which proteins are imported, and this in turn controls the plastid’s proteome, functions and developmental fate (/.e., which type of plastid is formed and when and at what point in time of plant development).

WO2014/037735 A1 UNIVERSITY OF LEICESTER discloses transgenic plants with altered expression of the SP1 gene or cDNA encoding SP1 (AT1G59560.1). Also, transgenic plants in which the activity of SP1 protein is altered by expression of a nucleic acid encoding by a mutant SP1 gene or cDNA encoding a mutated SP1 protein. Plastid development can be accelerated by these changes in expression. For example, in a seedling the transition from etioplasts to chloroplasts is accelerated, or in a seed the transition to amyloplasts may be accelerated. Alternatively, inactivation of SP1 such as by downregulation of SP1 expression results in delaying of plastid development. So, for example, the transition from chloroplast to gerontoplast is delayed whereby a“stay-green” phenotype may be achieved. The RING domain of SP1 is identified as a particular site for mutation. Also disclosed are nucleic acid expression vectors and host cells containing such vectors. Further disclosed are methods of increasing plant yield by increasing, inactivating, repressing or down-regulating regulating the expression of a nucleic acid comprising the SP1 gene or cDNA encoding SPI, or a functional homologue or variant thereof; or introducing and expressing in a plant a nucleic acid comprising a gene or cDNA encoding a mutant SP1. Other identified phenotypes include improved seedling emergence, growth and survival; alteration of fruit ripening; increased tolerance to stress such as salinity, osmotic stress and/or oxidative stress.

Also revealed was the role of SP1 in plant responses to abiotic stress. SP1 is activated under stress to deplete the TOC apparatus, thereby reducing the import of new

photosynthetic machinery components, attenuating photosynthetic activity, and reducing the potential for overaccumulation of harmful reactive oxygen species (see Ling, Q., et al., (2012) supra. The vitally important nature of the functions of SP1 , during development and stress, suggest agricultural applications linked to crop improvement.

Knowing that SP1 is an E3 ligase of the chloroplast outer membrane and operates to degrade TOC complexes, thus enabling reconfiguration of the import machinery essential for organellar protein changes occurring during development, this does not explain fully how the SP1 pathway in chloroplasts might work. The inventors undertook basic research in order to try and find suspected, but as yet unknown, further components of the SP1 pathway. BRIEF SUMMARY OF THE DISCLOSURE

Accordingly, the present invention provides a modified plant cell, modified plant or part thereof, wherein the expression and/or activity of plastid SP2 protein comprising an amino acid sequence of SEQ ID NO: 3, or a variant or homologue thereof, is altered compared to the expression and/or activity of plastid SP2 in an unmodified control or wild-type (WT) plant cell, plant or part thereof.

The expression and/or activity of plastid SP2 protein or variant or homologue thereof may be increased compared to expression and/or activity of plastid SP2 in the unmodified control or WT.

Alternatively, the expression and/or activity of plastid SP2 protein or variant or homologue thereof may be decreased or eliminated compared to expression and/or activity of plastid SP2 in the unmodified control or WT.

A“variant or homologue” of SP2 may be defined as being a protein which comprises an amino acid sequence of at least 50% identity to reference sequence SEQ ID NO: 3;

optionally at least 55% identity therewith. Additionally, such definition of“variant or homologue” may include the biological function of SP2 as further described herein, so that functional variants are disclosed herein, including such functional variants as defined by percentage sequence identity to the reference SEQ ID NO: 3.

Modified plant cells, modified plants or parts thereof as defined herein are preferably genetically modified, which may mean genetically engineered, e.g. transgenic, or it may mean gene edited, for example whereby a naturally occurring plant is modified with, for example, Cas9 gene editing, to alter from as few as a single nucleotide base in a genomic sequence. Plants arising from mutagenesis and screening are also included amongst what is meant by“modified plants”. Further included in the invention are modified plant cells, plants or plant parts where the expression and/or activity levels of SP2 (and the optional SP1 described later) may be achieved solely by epigenetic changes, preferably heritable and stable epigenetic changes.

Modified plant cells, modified plants or parts thereof as defined herein may be stably transformed with additional genetic material. Such additional genetic material is preferably under the control of at least one regulatory sequence, but a multiplicity of control points may be built in, whether using native of modified regulatory sequences.

The regulatory sequence may be a promoter; optionally an inducible promoter, preferably then one which may be induced by an external stress condition. In the alternative, a constitutive promoter may be employed, e.g. cauliflower mosaic 35S. The regulatory sequence may optionally be tissue specific and/or developmental^ regulated.

In modified plant cells, modified plants or parts thereof as defined herein, when SP2 is overexpressed there is preferably at least one copy of the additional genetic material compared to the unmodified or WT.

Such additional genetic material as is described herein may comprise a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a sequence of at least 50% identity therewith, or a fragment thereof of at least 17 contiguous nucleotides thereof.

A polynucleotide sequence or fragment thereof may be operably linked to a promoter in a sense or an antisense orientation if it is desired to employ an RNA-based suppression or knockdown of expression, of which there are many types known in the art.

In some modified plant cells, modified plants or parts thereof in accordance with any aspect of the invention, at least some of the expressed SP2 has itself altered activity compared to unmodified control or WT. This alteration of activity by way of mutation or by gene editing may be combined with increasing or decreasing the level of expression of SP2 compared to unmodified or WT.

The activity of SP2 which may be altered is preferably that of the SP2 protein association with plastid protein SP1 or with Toe proteins; wherein SP1 may comprise an amino acid sequence of SEQ ID NO: 6 or a sequence of at least 55% identity therewith.

The SP2 protein of altered activity is preferably lacking at least one, and up to 27 of the N- terminal amino acids of the protein sequence, e.g. SEQ ID NO: 3 or variants or homologs thereof.

As well as modified plant cells, modified plants or parts thereof based on altered SP2 expression and/or activity as hereinbefore defined, the invention includes the combination of all these possible aforementioned aspects and variations, together with an altered expression and/or activity of plastid SP1 protein. The SP1 protein comprises an amino acid sequence of SEQ ID NO: 6, or a sequence of at least 55% identity therewith, and is altered in expression and/or activity compared to plastid SP1 protein in an unmodified control or WT plant.

In these combined SP1 and SP2 aspects of the invention, expression and/or activity of SP1 may be increased compared to the expression and/or activity of SP1 in an unmodified control or WT plant. Alternatively, the expression and/or activity of SP1 is decreased or eliminated compared to the expression and/or activity of SP1 in an unmodified control or WT plant.

Where there is a polynucleotide encoding SP1 involved, this may comprise the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 5, or a sequence of at least 50% identity therewith, or a fragment of at least 17 contiguous nucleotides thereof.

In some aspects, the SP1 polynucleotide sequence or fragment thereof may be operably linked to a promoter in a sense or an antisense orientation if it is desired to employ an RNA-based suppression or knockdown of expression, of which there are many types known in the art. Possible within the scope of the invention for example is for differing methods of expression control for each of SP1 and SP2. So SP1 may be altered in expression by one method, e.g. mutation in a regulatory element for SP1 , and SP2 may be altered in expression by an RNA-based suppression approach.

At least some of the expressed SP1 may have an altered SP1 activity compared to unmodified control or WT. This may be achieved by genetic modification, e.g. known transgenic approaches or by gene editing usually performed using Cas9.

In particularly preferred aspects, the activity of the SP1 protein which is altered may be (a) reduced or abolished interaction with plastid SP2 protein; and/or (b) reduced or abolished E3 ligase activity; and/or (c) reduced or no association with plastid Toe proteins.

In any of the aforementioned possibilities of the invention for modified plant cells, modified plants or parts thereof, plastids may be altered in at least some cells, when compared to an unmodified control or WT. In this context, embodiments of the invention are concerned with spatial and/or temporal alterations in plastids and so considerations are for tissue specific and development specific controls being involved in making the alterations compared to WT.

The alteration may for example be in the numbers and/or type(s) and/or functions of plastid in the cell(s). This can also serve to better adapt an existing plant by modifying it to tolerate better particular environmental conditions such as light quality and level, temperature fluctuations, minima and/or maxima, and/or water, saline or osmotic stresses.

In accordance with the invention, the SP2 including any variant or homologue as herein defined by way of the percentage identities of sequence, may be from a different species, genus, family or order of plant. Where SP1 is altered in expression and/or activity in combination with SP2 alterations, then similarly this SP1 can independently be selected from a different species, genus, family or order of plant. The SP2 and SP1 genes being altered can be taken from the same or different plant species The invention therefore provides a modified plant as herein defined, which has increased expression and/or activity of SP2 protein; optionally also increased expression and/or activity of SP1 protein, in at least a particular cell type, and preferably wherein there is (a) increased tolerance to a stress condition compared to an unmodified control or WT plant; more preferably wherein the stress condition is one or more of saline stress, osmotic stress or oxidative stress; and/or (b) accelerated fruit ripening; and/or (c) the cell type is at a seed setting stage and there is an increase in seed/grain size or starch content; and/or (d) the cell type is at seedling stage and there is increased seedling survival, increased seedling growth and/or emergence

A plant as mentioned above may have a decreased or no expression and/or decreased or no activity of SP2 protein; optionally decreased or no expression and/or no decreased or no activity of SP1 protein, in at least a particular cell type; preferably wherein (a) the cell type is green photosynthetic and there is a delay of senescence; or (b) the plant is at seed setting stage and there is an increase in seed/grain size or starch content; or (c) the cell type is at fruiting stage and there is a delaying of fruit ripening.

In particularly preferred aspects the modified plants of the invention described herein are crop plants, biofuel plants or horticultural plants.

In another aspect, the present invention provides an isolated polynucleotide construct comprising a promoter and:

a. a polynucleotide comprising a nucleotide sequence: (i) of SEQ ID NO: 1 or a sequence of at least 50% identity thereto; or (ii) of SEQ ID NO: 2 or a sequence of at least 50% identity thereto; or (iii) encoding a protein of amino acid sequence SEQ ID NO: 3 or a sequence of at least 50% identity thereto;

b. a polynucleotide comprising a fragment of at least 17 contiguous nucleotides of: (i) SEQ ID NO: 1 or a sequence of at least 50% identity thereto; or (ii) SEQ ID NO: 2 or a sequence of at least 50% identity thereto.

Such polynucleotide constructs will be of assistance to persons of average skill in the making of altered plant cells, plants and parts thereof, wherein the SP2 gene is altered in expression and/or activity. The constructs of the invention lend themselves to the full range of known gene modification and gene expression modulation methods, including gene editing using Cas9 or Cpf1 , for example

In modification of the above, there may also be provided an isolated polynucleotide construct which additionally comprises: a. a polynucleotide comprising a nucleotide sequence: (i) of SEQ ID NO: 4 or a sequence of at least 55% identity thereto; or (ii) of SEQ ID NO: 5 or a sequence of at least 55% identity thereto; or (iii) encoding a protein of amino acid sequence SEQ ID NO: 6 or a sequence of at least 55% identity thereto;

b. a polynucleotide comprising a fragment of at least 17 contiguous

nucleotides of: (i) SEQ ID NO: 4 or a sequence of at least 55% identity thereto; or (ii) SEQ ID NO: 5 or a sequence of at least 55% identity thereto.

The SP2 encoding polynucleotide in the above may be downstream of the promoter, and the SP1 encoding polynucleotide may be downstream of the SP2 encoding polynucleotide. Alternatively, these positions of SP2 and SP1 may be reversed.

Described herein is also an isolated polynucleotide construct which comprises a promoter and:

a. a polynucleotide comprising a nucleotide sequence: (i) of SEQ ID NO: 4 or a sequence of at least 55% identity thereto; or (ii) of SEQ ID NO: 5 or a sequence of at least 55% identity thereto; or (iii) encoding a protein of amino acid sequence SEQ ID NO: 6 or a sequence of at least 55% identity thereto;

b. a polynucleotide comprising a fragment of at least 17 contiguous nucleotides of: (i) SEQ ID NO: 4 or a sequence of at least 55% identity thereto; or (ii) SEQ ID NO: 5 or a sequence of at least 55% identity thereto.

The invention also includes the aforementioned isolated polynucleotides as separate polynucleotides, each with their own same or different promoters and optional regulatory and other features, forming a portion of a kit of parts for use in a binary rather than single construct approach to the alteration of SP2 and SP1 protein activity and/or expression in plant cells, plants or parts thereof.

Such polynucleotides as aforementioned include Ti plasmids of Agrobacterium

tumefaciens which are well known in the art.

Included in the invention are vectors comprising a polynucleotide as aforementioned. This includes Agrobacterium tumefaciens, tobacco mosaic virus (TMV), potato virus X and cowpea mosaic virus.

Included therefore in the invention as another aspect are host cells comprising a polynucleotide or a vector as hereinbefore described. Such cells may not necessarily be plant cells when cloning, in vitro expression or genetic manipulation procedures are being carried out as part of a series of experimental or developmental steps to yield altered plant material. So, such host cells may include bacteria, e.g. Agrobacterium or Escherichia coli, or yeast, e.g. Saccharomyces cerevisiae.

In further aspect, the invention provides a method of altering plastids in a plant cell, comprising:

a. increasing, decreasing or eliminating the expression in the cell of plastid protein SP2 comprising an amino acid sequence of SEQ ID NO: 3 or a sequence of at least 50% identity therewith when compared to a control or WT plant cell; and/or

b. increasing, decreasing or eliminating the activity in the cell of plastid protein SP2 comprising an amino acid sequence of SEQ ID NO: 3 or a sequence of at least 50% identity therewith when compared to a control or WT plant cell, wherein the activity of SP2 is that of (i) SP2 association with plastid SP1 protein of SEQ ID NO: 6 or a sequence of at least 55% identity therewith, and/or (ii) SP2 association with Toe proteins.

In such a method of altering plastids, this may further (additionally) comprise:

a. increasing, decreasing or eliminating the expression in the cell of plastid protein SP1 comprising an amino acid sequence of SEQ ID NO: 6 or a sequence of at least 55% identity therewith when compared to a control or WT plant cell; and/or

b. increasing, decreasing or eliminating the activity in the cell of plastid protein SP1 comprising an amino acid sequence of SEQ ID NO: 6 or a sequence of at least 55% therewith when compared to a control or WT plant cell, wherein the activity of SP1 is that of SP1 association with plastid SP2 protein of SEQ ID NO: 3 or a sequence of at least 50% identity therewith, and/or (ii) SP1 association with Toe proteins, and/or (iii) SP1 alteration of E3 ligase activity.

In these aforementioned methods of altering plastids in a plant cell, the expression of SP2; optionally also the expression of SP1 , may be increased by the editing of a nucleotide sequence of at least one native SP2 regulatory element in the cell; optionally also at least one SP1 native regulatory element in the cell, and/or by the insertion of a polynucleotide construct or a vector as hereinbefore defined, into the cell.

In some aforementioned methods of altering plastids in a plant cell, plastid development may be accelerated; optionally wherein the cell is (a) a seedling cell and the transition of etioplasts into chloroplasts is accelerated; or (b) a seed cell and the transition to amyloplasts is accelerated; or (c) a fruit cell and the transition (a) from chloroplasts, and/or (b) to chromoplasts, is accelerated.

Alternatively, in other methods of altering plastids in plant cells, the expression of SP2; optionally also the expression of SP1 , may be decreased or eliminated by the editing of a nucleotide sequence of at least one native SP2 regulatory element in the cell; optionally also at least one SP1 native regulatory element in the cell, and/or by the insertion of a polynucleotide construct or a vector as hereinbefore defined, into the cell.

In methods of altering plastids where plastid development is delayed; the cell is optionally: (a) a green photosynthetic cell and the transition of chloroplasts into another type of plastid, e.g. gerontoplast, is delayed; or (b) a fruit cell and the transition from chloroplasts is delayed.

In any aspect of the invention herein, not excluded is the possibility that SP2 expression and/or activity may be increased whilst SP1 expression and/or activity may be decreased, with respect to a control or WT. And vice versa, not excluded, SP2 expression and/or activity may be decreased whilst SP1 expression and/or activity may be increased, with respect to a control or WT.

What is also possible in the present invention is that the ratio of SP2:SP1 expression or activity may be altered compared to a control or WT. This ratio may increase or decrease compared to control of WT. Ratios may be a ratio in the range of possibilities between 10:1 to 1 :10.

Also included as part of the invention are uses of the SP2 gene of SEQ ID NO: 1 or cDNA of SEQ ID NO: 2, or a polynucleotide sequence of at least 50% identity therewith, or an at least 17 nucleotide fragment thereof, for altering plastid protein composition in plant cells. Such uses therefore provide for alteration of plastid type, number, size, protein content, ultrastructural features, e.g. grana or inter-granal spaces. The alterations are referenced to unmodified control or WT. The alterations in plastids may be quantitative and/or qualitative, temporal and/or spatial in a plant, so as to realise advantageous physiological and phenotypic changes, e.g. stress tolerance and/or yield increase. Also, to achieve particular desired changes in plant growth and development, e.g. seedling emergence rates, de-etiolation rates, fruit ripening and starch accumulation. Where a method of the invention is disclosed this may be interpreted as being equivalent to“use” of the SP2 material for the state purpose(s).

The invention in various aspects opens up a practical approach to increasing plant yields by extending the duration of active photosynthesis. “Stay-green” is a term that is used to describe mutant and transgenic plants or cultivars with the trait of maintaining their leaves for a longer period of time than the wild-type or crosses from which they are derived. The sp2 mutants of the present invention and SP2 overexpressing plants may provide the necessary regulation of chloroplast longevity and thereby regulation of leaf senescence which allows the extension of duration of active photosynthesis in plants; ideally crop plants.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention are further described hereinafter with reference to the examples and accompanying drawings, in which:Figure 1A is a photograph showing phenotypes of 30-day-old sp2 suppressor and control plants grown on soil.

Figure 1 B is a chart showing Chlorophyll contents of 10-day-old sp2 suppressor and control seedlings grown in vitro.

Figure 1C shows micrographs of cotyledon chloroplasts in 10-day-old sp2 suppressor and control plants grown in vitro. Scale bar, 2 pm.

Figure 1 D is estimated chloroplast cross-sectional area from Figure 1C.

Figure 1 E is thylakoid development estimated from Figure 1C.

Figure 1 F is 2 shows protein import analysis using chloroplasts isolated from sp2 suppressor and control plants, and corresponding quantification of the maturation (mat) of 35S-labelled Rubisco small subunit precursor protein (pre).

Figure 1G is a domain map of the SP2 protein. Grey box, b-barrel domain; black boxes, predicted transmembrane spans. The sites of amino acid substitutions in two sp2 mutant alleles are indicated with grey triangles.

Figure 1 H is an immunoblot analyses of total leaf protein extracts from the indicated genotypes, including sp2 ppi1 suppressors.

Figure 11 shows protein abundances of immunoblot of Figure 1 H.

Figure 1J is an immunoblot analyses of total leaf protein extracts from the indicated genotypes of sp2 and toc75-lll-3 single and double mutants.

Figure 1 K shows protein abundances of immunoblot of Figure 1J.

Figure 1 L is an immunoblot analyses of total leaf protein extracts from SP2 overexpressors (OX).

Figure 1 M shows protein abundances of immunoblot of Figure 1 L. Figure 1 N is a photograph showing leaf senescence analysis of the indicated genotypes employing mature rosette leaves induced to senesce by covering with aluminium foil. Typical control (uncovered) and senescent (covered) leaves are shown (left).

Photochemical efficiency of photosystem II ( FJF _m ) was measured to estimate the extent of senescence (right).

Figure 10 shows the abiotic stress tolerance analysis of the indicated genotypes employing 14-day-old plants grown in vitro on NaCI medium. Typical plants (left) and chlorophyll contents (right) are shown. All values are means ± SEM (n ³ 3 experiments or samples).

Figure 2A is a photograph of WT and indicated genotype plants grown on agar.

Figure 2B is a chart showing chlorophyll content of the plants of Figure 2A.

Figure 3A is a shows genetic mapping of the sp2 locus.

Figure 3B shows a 40bp alignment of of sp2-1 ppi1 reads to the ppi1 reference genome.

Figure 3C shows a schematic representation of the SP2 (At3g44160) gene, annotated with the positions of the sp2 mutations.

Figure 3D shows analysis of SP2 mRNA expression in each of the sp2 mutant alleles by RT-PCR.

Figure 4A shows a Bayesian inference phylogenetic analysis of SP2 and OEP80.

Figure 4B shows structural models for the Arabidopsis SP2 and OEP80 proteins.

Figure 5 is an immunoblot showing P2 chloroplast localization and topology.

Figure 6 is an immunoblot showing enrichment of SP2 in isolated chloroplasts, and analysis of its interaction with TOC proteins by co-immunoprecipitation.

Figure 7 A is a photograph of soil grown plants of the identified genotypes used to demonstrate the effect of the sp2 mutation on other TOC mutants.

Figure 7B is a chart showing the chlorophyll content of the plants in Figure 7A.

Figure 7C is a photograph of agar grown plants of the identified genotypes used to demonstrate the effect of the sp2 mutation on other TOC mutants.

Figure 7D is a chart showing the chlorophyll content of the plants in Figure 7C.

Figure 8A is a photograph of agar grown plants of the identified genotypes used to demonstrate specificity of suppression mediated by the sp2 mutation.

Figure 8B is a chart showing the chlorophyll content of the plants in Figure 8A. Figure 9 is a chart of transcript levels for identified genotypes to show how SP2 does not affect TOC component transcript levels.

Figure 9B is a chart of transcript levels for SP2 overexpressor plants to show how SP2 does not affect TOC component transcript levels.

Figure 10A is a photograph of agar grown plants of identified genotype used to

demonstrate phenotypes of sp2 single-mutant and SP2 overexpressor plants.

Figure 10B is a chart showing the chlorophyll content of the plants of Figure 10A.

Figure 10C is a gel showing semi-quantitative RT-PCR analysis of SP2 expression in wild- type and SP2-OX plants.

Figure 10D is a chart quantitating the result of Figure 10C.

Figure 11A is a photograph of agar grown plants of identified genotype, including triple mutants.

Figure 11 B is a chart showing the chlorophyll content of the plants in Figure 11 A.

Figure 11C is an immunoblot showing abundance of Toc75 protein in the plants of Figure 1 1A.

Figure 11 D is chart quantitating the protein abundance found in the immunoblot of Figure 11C.

Figure 11 E is a photograph of agar grown SP2 overexpressing plants.

Figure 11 F is an immunoblot of extracts of plants of Figures 11 E.

Figure 11G is a chart quantitating the protein abundance found in the immunoblot of Figure 11 F.

Figure 11G is an immunoblot analysing TOC protein depletion in WT, sp2 mutant and SP2 overexpressing plants.

Figure 11G is a chart quantitating the protein abundance found in the immunoblot of Figure 11G.

Figure 13A is two-dimensional (2D)-blue native (BN)/SDS-PAGE analysis of SP1 and SP2 which shows how SP1 and SP2 proteins associate to form a complex.

Figure 13B is an in vitro pull-down analysis of the association between SP1 and SP2.

Figure 14A is a photograph of agar grown plants where the sp2 mutation is introduced into the SP1-OX ppi1 background.

Figure 14B is a chart showing the chlorophyll content of the plants of Figure 14A. Figure 15A is a table of sequence alignments using SP1 (At1g63900.2) as the reference sequence and whereby sequences were retrieved from EnsembIPIants by BLAST

(TBLASTN), and they were aligned (without any editing) using ClustalV in DNAstar Lasergene.

Figure 15B is a table of sequence alignments using SP2 (At3g44160) as the reference sequence and whereby sequences were retrieved from EnsembIPIants by BLAST

(TBLASTN), and they were aligned (without any editing) using ClustalW in DNAstar Lasergene.

Figure 16 is a model diagram for SP1-SP2 action in the chloroplast membrane.

DETAILED DESCRIPTION

In the following passages, different aspects of the invention are explained in more detail. Each aspect explained or defined may be combined with any other aspect or aspects, unless explicitly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

Conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology, gene editing and bioinformatics for use in employing the present invention are all readily known and available to a person of average skill in the art. Specific techniques are explained more fully in the referenced literature.

Described herein is a modified plant cell, modified plant or part thereof, wherein the expression and/or activity of plastid SP2 protein comprising an amino acid sequence of SEQ ID NO: 3, or a variant or homologue thereof, is altered compared to the expression and/or activity of plastid SP2 in an unmodified control or wild-type (WT) plant cell, plant or part thereof.

The terms "peptide", "polypeptide" and "protein" are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds.

The term“plastid” refers to any plant plastid, including etioplasts, chloroplasts,

amyloplasts, elaioplasts, chromoplasts or gerontoplasts. Preferably, the plastid is a chloroplast. Preferably, the development is the transition from one type of plastid to another. Preferably, plastid development is chloroplast development and more preferably refers to the transition of an etioplast into a chloroplast or the transition of a chloroplast into a gerontoplast. In another embodiment, plastid development is the transition from a chloroplast into a chromoplast.

The expression and/or activity of plastid SP2 protein or variant or homologue thereof may be increased compared to expression and/or activity of plastid SP2 in the unmodified control or WT.

Alternatively, the expression and/or activity of plastid SP2 protein or variant or homologue thereof may be decreased or eliminated compared to expression and/or activity of plastid SP2 in the unmodified control or WT.

The terms“altered”,“changed” and“modified” may be used interchangeably herein. A control plant as used herein is a plant which has not been modified. Accordingly, the control plant has not been genetically modified to alter either expression of a nucleic acid encoding SP2 or activity of a SP2 peptide as described herein. The control plant may be a wild type (WT) plant. Even if a plant were transgenic, but not in respect of SP2 (or additionally SP1) then it could function as a control plant. The WT or control need not be too specific, so long as it may provide a reliable reference against which SP2 expression and/or activity can be measured in a modified plant material. The control plant may be a transgenic plant that does not have altered expression of SP2 or altered activity of a SP2 peptide, but expresses a transgene that does not comprise a SP2 nucleic acid.

The terms "increase", "improve" or "enhance" are used interchangeably herein. Yield of SP2 expression levels for example are increased by at least a 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 35%, 40% or 50% or more in comparison to a control plant. SP2 expression levels can be measured by routine methods in the art and compared to control plants.

The terms "reduce" or "decrease" are also used interchangeably herein. A decrease, for example in SP2 expression may be 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 35%, 40% or 50% or more in comparison to a control plant.

Alternatively, a control plant may carry an expression vector only or carries a mutant SP2 gene expressing a non-functional SP2 peptide. The control plant is typically of the same plant species, preferably having the same genetic background as the modified plant.

The term "functional variant" as used herein refers to a variant gene or peptide sequence or part of the gene or peptide sequence which retains the biological function of the full non variant SP2 sequence, for example confers altered plastid development when expressed in a plant. A functional variant also comprises a variant of the gene of interest encoding a peptide which has sequence alterations that do not affect function of the resulting protein, for example in non-conserved residues. Also encompassed is a variant that is

substantially identical, i.e. has only some sequence variations, for example in non- conserved residues, to the wild type sequences as shown herein and is biologically active, for example complements the A. thaliana sp2 mutant.

A“variant or homologue” of SP2 may be defined as being a protein which comprises an amino acid sequence of at least 50% identity to reference sequence SEQ ID NO: 3;

optionally at least 55% identity therewith. Additionally, such definition of“variant or homologue” may include the biological function of SP2 as further described herein, so that functional variants are disclosed herein, including such functional variants as defined by percentage sequence identity to the reference SEQ ID NO: 3.

The term“homologue” as used herein also designates an SP2 orthologue from other plant species. A homologue (or variant) of AtSP2 polypeptide has, in increasing order of preference, at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %,

52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%,

67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,

82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%,

97%, 98%, or at least 99% overall sequence identity to the amino acid represented by SEQ ID NO: 3. (The“at least” prefixes each and every percentage identity listed above.)

Preferably, overall sequence identity is more than 49%, and in increasing order of preference more than 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,

61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68% or more than 69%. (The“more than” prefixes each and every percentage identity listed here.)

Preferably, overall sequence identity for SP2 homologues or variants is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, most preferably 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%.

The overall sequence identity may be determined using a global alignment algorithm known in the art, such as the Needleman Wunsch algorithm in the program GAP (GCG Wisconsin Package, Accelrys).

Suitable homologues can be identified by sequence comparisons and identifications of conserved domains. There are predictors in the art that can be used to identify such sequences. The function of the homologue can be identified as described herein and a skilled person would thus be able to confirm the function when expressed in a plant. Thus, one of skill in the art will recognize that analogous amino acid substitutions listed above with reference to SEQ ID NO: 3 can be made in SP2 from other plants by aligning the SP2 polypeptide sequence to be mutated with the AtSP2 polypeptide sequence as set forth in SEQ ID NO: 3.

Modified plant cells, modified plants or parts thereof as defined herein may be genetically modified, which may mean genetically engineered, e.g. transformed so as to be

transgenic, or it may mean gene edited, for example whereby a naturally occurring plant is modified with, for example, Cas9 gene editing, to alter from a few as a single nucleotide base in a genomic sequence. Plant arising from mutagenesis and screening are also included amongst what is meant by“modified plants”. Further included in the invention are modified plant cells, plants or plant parts where the expression and/or activity levels of SP2 (and the optional SP1 described later) may be achieved solely by epigenetic changes, preferably heritable and stable epigenetic changes.

The invention includes a method of producing a mutant plant expressing a SP2 variant and which is characterised by one of the phenotypes described herein, wherein said method uses mutagenesis and Targeting Induced Local Lesions in Genomes (TILLING) to target the gene expressing a SP2 polypeptide. The method comprises mutagenising a plant population and selecting a plant with altered plastid development and identifying the SP2 variant. For example, mutagenesis is carried out using TILLING where traditional chemical mutagenesis is flowed by high-throughput screening for point mutations. The plants are screened for one of the phenotypes described herein, for example a plant that shows delayed/accelerated plastid development or improved yield. A SP2 locus is then analysed to identify a specific SP2 mutation responsible for the phenotype observed. Plants can be bred to obtain stable lines with the desired phenotype and carrying a mutation in a SP2 locus. In one embodiment, germplasm is screened.

Thus, plants with different genotypes, induced through artificial means, or alternatively having originated through natural sequence divergence, may be screened for the expression of the endogenous SP2 gene to identify germplasm or plants with particular plastid development or yield characteristics.

In one embodiment, the method used to create and analyse mutations is targeting induced local lesions in genomes. In this method, seeds are mutagenised with a chemical mutagen. The mutagen may be fast neutron irradiation or a chemical mutagen, for example selected from the following non-limiting list: ethyl methanesulfonate (EMS), methylmethane sulfonate (MMS), N-ethyl-N-nitrosurea (ENU), triethylmelamine (1 ^' EM), N- methyl-N-nitrosourea (MNU), procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine,

dimethylnitosamine, N-methyl-N'-nitro-nitrosoguanidine (MNNG), nitrosoguanidine, 2- aminopurine, 7,12 dimethyl-benz(a)anthracene (DM BA), ethylene oxide, hexamethylphosphoramide, bisulfan, diepoxyalkanes (diepoxyoctane (DEO),

diepoxybutane (BEB), and the like), 2-methoxy-6-chloro-9 [3-(ethyl-2- chloroethyl)aminopropylamino]acridine dihydrochloride (ICR-170) or formaldehyde.

The resulting M1 plants are self-fertilised and the M2 generation of individuals is used to prepare DNA samples for mutational screening. DNA samples are pooled and arrayed on microtiter plates and subjected to gene specific PCR. The PCR amplification products may be screened for mutations in the SP2 target gene using any method that identifies heteroduplexes between wild-type and mutant genes. For example, but not limited to, denaturing high pressure liquid chromatography (dHPLC), constant denaturant capillary electrophoresis (CDCE), temperature gradient capillary electrophoresis (TGCE), or by fragmentation using chemical cleavage. Preferably the PCR amplification products are incubated with an endonuclease that preferentially cleaves mismatches in heteroduplexes between wild-type and mutant sequences. Cleavage products are electrophoresed using an automated sequencing gel apparatus, and gel images are analyzed with the aid of a standard commercial image-processing program. Any primer specific to the SP2 gene may be utilized to amplify the SP2 genes within the pooled DNA sample. Preferably, the primer is designed to amplify the regions of the SP2 gene where useful mutations are most likely to arise, specifically in the areas of the SP2 gene that are highly conserved and/or confer activity. To facilitate detection of PCR products on a gel, the PCR primer may be labelled using any conventional labelling method.

Rapid high-throughput screening procedures thus allow the analysis of amplification products for identifying a mutation conferring the reduction or inactivation of the expression of the SP2 gene as compared to a corresponding non-mutagenised wild-type plant. Once a mutation is identified in a gene of interest, the seeds of the M2 plant carrying that mutation are grown into adult M3 plants and screened for the phenotypic characteristics associated with the SP2 gene. Loss of and reduced function mutants with increased yield or increased/delayed plastid development compared to a control plant can thus be identified.

For example, a“plant part” may be green tissue, for example a leaf. In using green tissue the transition from chloroplast to gerontoplast might be desired to be delayed. A plant part may be a fruit and the transition from chloroplast to chromoplast might be desired to be delayed.

When employed in the invention herein, "transgenic", "transgene" or "recombinant" means with regard to, for example, a nucleic acid sequence, an expression cassette, gene construct or a vector comprising the nucleic acid sequence or an organism transformed with the nucleic acid sequences, expression cassettes or vectors according to the invention, all those constructions brought about by recombinant methods in which either

(a) the nucleic acid sequences encoding proteins useful in the methods of the invention, or

(b) genetic control sequence(s) which is operably linked with the nucleic acid sequence according to the invention, for example a promoter, or (c) a) and b) are not located in their natural genetic environment or have been modified by recombinant methods, it being possible for the modification to take the form of, for example, a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. The natural genetic environment is understood as meaning the natural genomic or chromosomal locus in the original plant or the presence in a genomic library. In the case of a genomic library, the natural genetic environment of the nucleic acid sequence is preferably retained, at least in part. The environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, especially preferably at least 1000 bp, most preferably at least 5000 bp. A naturally occurring expression cassette - for example the naturally occurring combination of the natural promoter of the nucleic acid sequences with the corresponding nucleic acid sequence encoding a polypeptide useful in the methods of the present invention, as defined above - becomes a transgenic expression cassette when this expression cassette is modified by non-natural, synthetic ("artificial") methods such as, for example, mutagenic treatment. Suitable methods are described, for example, in US 5,565,350 or WO 00/15815 both incorporated by reference.

Where the invention may provide a transgenic plant, the nucleic acids used in the method of the invention are not at their natural locus in the genome of said plant, it being possible for the nucleic acids to be expressed homologously or heterologously. Thus, the plant expresses a transgene. However, as mentioned, in certain embodiments, transgenic may means that, while the nucleic acids according to the different embodiments of the invention are at their natural position in the genome of a plant, the sequence has been modified with regard to the natural sequence, and/or that the regulatory sequences of the natural sequences have been modified, for example by mutagenesis.

Transgenic is preferably understood as meaning the expression of the nucleic acids according to the invention at an unnatural locus in the genome, i.e. homologous or, preferably, heterologous expression of the nucleic acids takes place. According to the invention, the transgene is stably integrated into the plant and the plant is preferably homozygous for the transgene.

Modified plant cells, modified plants or parts thereof as defined herein may be stably transformed with additional genetic material. Such additional genetic material is preferably under the control of at least one regulatory sequence, but a multiplicity of control points may be built in, whether using native of modified regulatory sequences.

Transformation of plants is now a routine technique in many species. Advantageously, any of several transformation methods may be used to introduce the gene of interest into a suitable ancestor cell. The methods described for the transformation and regeneration of plants from plant tissues or plant cells may be utilized for transient or for stable

transformation. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts, electroporation of protoplasts, microinjection into plant material, DNA or RNA-coated particle bombardment, infection with (non-integrative) viruses and the like. Transgenic plants, including transgenic crop plants, are preferably produced via Agrobacterium tumefaciens mediated transformation.

Transformation methods are well known in the art. Thus, according to the various aspects of the invention, a nucleic acid comprising a SP2 nucleic acid, for example SEQ ID NO: 1 or 2, or a functional variant or homolog thereof, is introduced into a plant and expressed as a transgene. The nucleic acid sequence is introduced into said plant through a process called transformation. The term "introduction" or "transformation" as referred to herein encompasses the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention and a whole plant regenerated there from. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem). The polynucleotide may be transiently or stably introduced into a host cell and may be maintained non- integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome. The resulting transformed plant cell may then be used to regenerate a transformed plant in a manner well known in the art.

To select transformed plants, plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants. For example, seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying. A further possibility is growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants. Alternatively, the transformed plants are screened for the presence of a selectable marker such as the ones described above. Following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, both techniques being well known in the art.

The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or Ti) transformed plant may be selfed and homozygous second-generation (or T2)

transformants selected, and the T ₂ plants may then further be propagated through classical breeding techniques. The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).

The regulatory sequence may be a promoter; optionally an inducible promoter, preferably then one which may be induced by an external stress condition. In the alternative, a constitutive promoter may be employed, e.g. cauliflower mosaic 35S.

The regulatory sequence may optionally be tissue specific.

The term "regulatory element" as used herein may be considered interchangeably with "control sequence" and "promoter" and all terms are to be taken in a broad context to refer to regulatory nucleic acid sequences capable of effecting expression of the sequences to which they are ligated. The term "promoter" typically refers to a nucleic acid control sequence located upstream from the transcriptional start of a gene and which is involved binding of RNA polymerase and other proteins, thereby directing transcription of an operably linked nucleic acid. Encompassed by the aforementioned terms are

transcriptional regulatory sequences derived from a classical eukaryotic genomic gene (including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence) and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue- specific manner. Also included within the term is a transcriptional regulatory sequence of a classical prokaryotic gene, in which case it may include a -35 box sequence and/or -10 box transcriptional regulatory sequences.

The term "regulatory element" also encompasses a synthetic fusion molecule or derivative that confers, activates or enhances expression of a nucleic acid molecule in a cell, tissue or organ.

A "plant promoter" comprises regulatory elements, which mediate the expression of a coding sequence segment in plant cells. Accordingly, a plant promoter need not be of plant origin, but may originate from viruses or micro-organisms, for example from viruses which attack plant cells. The "plant promoter" can also originate from a plant cell, e.g. from the plant which is transformed with the nucleic acid sequence to be expressed. This also applies to other "plant" regulatory signals, such as "plant" terminators. The promoters upstream of the nucleotide sequences useful in the methods of the present invention can be modified by one or more nucleotide substitution(s), insertion(s) and/or deletion(s) without interfering with the functionality or activity of either the promoters, the open reading frame (ORF) or the 3'-regulatory region such as terminators or other 3' regulatory regions which are located away from the ORF. Also possible is that the activity of the promoters is increased by modification of their sequence, or that they are replaced completely by more active promoters, even promoters from heterologous organisms. For expression in plants, the nucleic acid molecule is, as described above, preferably linked operably to or comprises a suitable promoter which expresses the gene at the right point in time and with the required spatial expression pattern. For the identification of functionally equivalent promoters, the promoter strength and/or expression pattern of a candidate promoter may be analysed for example by operably linking the promoter to a reporter gene and assaying the expression level and pattern of the reporter gene in various tissues of the plant.

Suitable well-known reporter genes are known to the skilled person and include for example beta-glucuronidase or beta-galactosidase.

The term "operably linked" as used herein refers to a functional linkage between the promoter sequence and the gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest.

For example, the nucleic acid sequence may be expressed using a promoter that drives overexpression. Overexpression according to the invention means that the transgene is expressed at a level that is higher than expression of endogenous counterparts driven by their endogenous promoters. For example, overexpression may be carried out using a strong promoter, such as a constitutive promoter. A "constitutive promoter" refers to a promoter that is transcriptionally active during most, but not necessarily all, phases of growth and development and under most environmental conditions, in at least one cell, tissue or organ. Examples of constitutive promoters include the cauliflower mosaic virus promoter (CaMV35S or 19S), rice actin promoter, maize ubiquitin promoter, rubisco small subunit, maize or alfalfa H3 histone, OCS, SAD1 or 2, GOS2 or any promoter that gives enhanced expression. Alternatively, enhanced or increased expression can be achieved by using transcription or translation enhancers or activators and may incorporate enhancers into the gene to further increase expression. Furthermore, an inducible expression system may be used, where expression is driven by a promoter induced by environmental stress conditions (for example the pepper pathogen-induced membrane protein gene CaPIMPI or promoters that comprise the dehydration-responsive element (DRE), the promoter of the sunflower HD-Zip protein genes Hahbl or Hahb4, which is inducible by water stress, high salt concentrations and ABA or a chemically inducible promoter (such as steroid- or ethanol-inducible promoter system). The promoter may also be tissue-specific. The types of promoters listed above are described in the art. Other suitable promoters and inducible systems are also known to a person of average skill.

A promoter specific for seed development (e.g. HaFAD2-1 from sunflower or a seed storage protein promoter, such as zein, glutenin or hordein) or seed maturation (e.g. soybean pm36) may be used, or one specific for seed germination (e.g. barley or wheat alpha-amylase or carboxypeptidase) or a seedling-specific promoter (such as the Pyk10 promoter) may be used. The patatin promoter may be used for tubers.

A green tissue-specific promoter may be used. For example, a green tissue-specific promoter may be selected from the maize orthophosphate kinase promoter, maize phosphoenol pyruvate carboxylase promoter, rice phosphoenol pyruvate carboxylase promoter, rice small subunit rubisco promoter, rice beta expansin EXB09 promoter, pigeonpea small subunit rubisco promoter or pea RBS3A promoter.

The promoter may be a constitutive or strong promoter. In a preferred embodiment, the regulatory sequence is an inducible promoter or a stress inducible promoter. The stress inducible promoter is selected from the following non limiting list: the HaHB1 promoter, RD29A (which drives drought inducible expression of DREB1A), the maize rabl7 drought- inducible promoter, P5CS1 (which drives drought inducible expression of the proline biosynthetic enzyme P5CS1), ABA- and drought-inducible promoters of Arabidopsis clade A PP2Cs (ABM , ABI2, HAB1 , PP2CA, HA11 , HAI2 and HAI3) or their corresponding crop orthologues.

In modified plant cells, modified plants or parts thereof as defined herein, when SP2 is overexpressed there is preferably at least one copy of the additional genetic material compared to the unmodified or WT. Such additional genetic material as is described herein may comprise a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a sequence of at least 50% identity therewith, or a fragment thereof of at least 17 contiguous nucleotides thereof.

In the various aspects of the invention, including the methods and uses, encompass not only a SP2 nucleic acid or protein, but also a fragment or part thereof. By "fragment" or "part" is intended a portion of the nucleotide sequence or a portion of the amino acid sequence and hence of the protein encoded thereby. Fragments of a nucleotide sequence may encode protein fragments that retain the biological activity of the native protein.

A polynucleotide sequence or fragment thereof may be operably linked to a promoter in a sense or an antisense orientation if it is desired to employ an RNA-based suppression or knockdown of expression, of which there are many types known in the art as will be described in more detail below.

Gene silencing may be used to achieve inactivation, repression or down-regulation of SP2 (and additionally SP1). For example, RNA-mediated gene suppression or RNA silencing. "Gene silencing" is generally used to refer to suppression of expression of a gene via sequence-specific interactions that are mediated by RNA molecules. The degree of reduction may be so as to totally abolish production of the encoded gene product, but more usually the abolition of expression is partial, with some degree of expression remaining. The term should not therefore be taken to require complete "silencing" of expression.

Transgenes may be used to suppress endogenous plant genes. This was discovered originally when chalcone synthase transgenes in petunia caused suppression of the endogenous chalcone synthase genes and indicated by easily visible pigmentation changes. Subsequently it has been described how many, if not all plant genes can be "silenced" by transgenes. Gene silencing requires sequence similarity between the transgene and the gene that becomes silenced. This sequence homology may involve promoter regions or coding regions of the silenced target gene. When coding regions are involved, the transgene able to cause gene silencing may have been constructed with a promoter that would transcribe either the sense or the antisense orientation of the coding sequence RNA. It is likely that the various examples of gene silencing involve different mechanisms that are not well understood. In different examples there may be

transcriptional or post-transcriptional gene silencing and both may be used according to the methods of the invention. The mechanisms of gene silencing and their application in genetic engineering, which were first discovered in plants in the early 1990s and then shown in Caenorhabditis elegans are extensively described in the literature.

RNA-mediated gene suppression or RNA silencing according to the methods of the invention includes co-suppression wherein over-expression of the SP2 (and optionally SP1) sense RNA or mRNA leads to a reduction in the level of expression of the genes concerned. RNAs of the transgene and homologous endogenous gene are co-ordinately suppressed. Other techniques used in the methods of the invention include antisense RNA to reduce transcript levels of the endogenous SP2 gene (optionally additionally SP1 gene) in a plant. In this method, RNA silencing does not affect the transcription of a gene locus, but only causes sequence-specific degradation of target mRNAs. An "antisense" nucleic acid sequence comprises a nucleotide sequence that is complementary to a "sense" nucleic acid sequence encoding a SP2 protein, or a part of a SP2 protein, i.e. complementary to the coding strand of a double-stranded cDNA molecule or

complementary to an mRNA transcript sequence. The antisense nucleic acid sequence is preferably complementary to the endogenous SP2 gene to be silenced. The

complementarity may be located in the "coding region" and/or in the "non-coding region" of a gene. The term "coding region" refers to a region of the nucleotide sequence comprising codons that are translated into amino acid residues. The term "non-coding region" refers to 5' and 3' sequences that flank the coding region that are transcribed but not translated into amino acids (also referred to as 5' and 3' untranslated regions).

Antisense nucleic acid sequences can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid sequence may be complementary to the entire SP2 nucleic acid sequence, but may also be an oligonucleotide that is antisense to only a part of the nucleic acid sequence (including the mRNA 5' and 3' UTR). For example, the antisense oligonucleotide sequence may be complementary to the region surrounding the translation start site of an mRNA transcript encoding a polypeptide. The length of a suitable antisense oligonucleotide sequence is known in the art and may start from about 50, 45, 40, 35, 30, 25, 20, 15 or 10 nucleotides in length or less. An antisense nucleic acid sequence according to the invention may be constructed using chemical synthesis and enzymatic ligation reactions using methods known in the art. For example, an antisense nucleic acid sequence (e.g., an antisense oligonucleotide sequence) may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acid sequences, e.g., phosphorothioate derivatives and acridine-substituted nucleotides may be used. Examples of modified nucleotides that may be used to generate the antisense nucleic acid sequences are well known in the art. The antisense nucleic acid sequence can be produced biologically using an expression vector into which a nucleic acid sequence has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest). Preferably, production of antisense nucleic acid sequences in plants occurs by means of a stably integrated nucleic acid construct comprising a promoter, an operably linked antisense oligonucleotide, and a terminator.

Nucleic acid molecules used in a silencing method of the invention hybridize with or bind to mRNA transcripts and/or insert into genomic DNA encoding a polypeptide to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid sequence which binds to DNA duplexes, through specific interactions in the major groove of the double helix. Antisense nucleic acid sequences may be introduced into a plant by transformation or direct injection at a specific tissue site. Alternatively, antisense nucleic acid sequences can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense nucleic acid sequences can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid sequence to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid sequences can also be delivered to cells using vectors.

RNA interference (RNAi) is another post-transcriptional gene-silencing phenomenon which may be used according to the methods of the invention. This is induced by double- stranded RNA in which mRNA that is homologous to the dsRNA is specifically degraded.

It refers to the process of sequence-specific post-transcriptional gene silencing mediated by short interfering RNAs (siRNA). The process of RNAi begins when the enzyme,

DICER, encounters dsRNA and chops it into pieces called small-interfering RNAs (siRNA). This enzyme belongs to the RNase III nuclease family. A complex of proteins gathers up these RNA remains and uses their code as a guide to search out and destroy any RNAs in the cell with a matching sequence, such as target mRNA.

Artificial and/or natural microRNAs (miRNAs) may be used to knock out gene expression and/or mRNA translation. MicroRNAs (miRNAs) miRNAs are typically single stranded small RNAs typically 19-24 nucleotides long. Most plant miRNAs have perfect or near perfect complementarity with their target sequences. However, there are natural targets with up to five mismatches. They are processed from longer non-coding RNAs with characteristic fold-back structures by double-strand specific RNases of the Dicer family. Upon processing, they are incorporated in the RNA-induced silencing complex (RISC) by binding to its main component, an Argonaute protein. miRNAs serve as the specificity components of RISC, since they base-pair to target nucleic acids, mostly mRNAs, in the cytoplasm. Subsequent regulatory events include target mRNA cleavage and destruction and/or translational inhibition. Effects of miRNA overexpression are thus often reflected in decreased mRNA levels of target genes. Artificial microRNA (amiRNA) technology has been applied in Arabidopsis thaliana and other plants to efficiently silence target genes of interest. The design principles for amiRNAs have been generalized and integrated into a Web-based tool (http://wmd.weigelworld.org).

A plant may be transformed to introduce a RNAi, snRNA, dsRNA, siRNA, miRNA, ta- siRNA, amiRNA or cosuppression molecule that has been designed to target the expression of an SP1 gene and selectively decreases or inhibits the expression of the gene or stability of its transcript. Preferably, the RNAi, snRNA, dsRNA, siRNA, miRNA, amiRNA, ta-siRNA or cosuppression molecule used according to the various aspects of the invention comprises a fragment of at least 17 nt, preferably 22 to 26 nt and can be designed on the basis of the information shown in SEQ I D No: 1. Guidelines for designing effective siRNAs are known to the skilled person. Briefly, a short fragment of the target gene sequence (e.g., 19-40 nucleotides in length) is chosen as the target sequence of the siRNA of the invention. The short fragment of target gene sequence is a fragment of the target gene mRNA. In preferred embodiments, the criteria for choosing a sequence fragment from the target gene mRNA to be a candidate siRNA molecule include 1) a sequence from the target gene mRNA that is at least 50-100 nucleotides from the 5' or 3' end of the native mRNA molecule, 2) a sequence from the target gene mRNA that has a G/C content of between 30% and 70%, most preferably around 50%, 3) a sequence from the target gene mRNA that does not contain repetitive sequences (e.g., AAA, CCC, GGG, TTT, AAAA, CCCC, GGGG, TTTT), 4) a sequence from the target gene mRNA that is accessible in the mRNA, 5) a sequence from the target gene mRNA that is unique to the target gene, 6) avoids regions within 75 bases of a start codon. The sequence fragment from the target gene mRNA may meet one or more of the criteria identified above. The selected gene is introduced as a nucleotide sequence in a prediction program that takes into account all the variables described above for the design of optimal oligonucleotides. This program scans any mRNA nucleotide sequence for regions susceptible to be targeted by siRNAs. The output of this analysis is a score of possible siRNA oligonucleotides. The highest scores are used to design double stranded RNA oligonucleotides that are typically made by chemical synthesis. In addition to siRNA which is complementary to the mRNA target region, degenerate siRNA sequences may be used to target homologous regions. siRNAs according to the invention can be synthesized by any method known in the art. RNAs are preferably chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA RNA synthesizer. Additionally, siRNAs can be obtained from commercial RNA oligonucleotide synthesis suppliers.

siRNA molecules may be double stranded. A double stranded siRNA molecule may comprise blunt ends. Or, a double stranded siRNA molecule may comprise overhanging nucleotides (e.g., 1 -5 nucleotide overhangs, preferably 2 nucleotide overhangs). In some examples, the siRNA may be a short hairpin RNA (shRNA); and the two strands of the siRNA molecule may be connected by a linker region (e.g., a nucleotide linker or a non nucleotide linker). siRNAs described herein may contain one or more modified nucleotides and/or non-phosphodiester linkages. Chemical modifications well known in the art are capable of increasing stability, availability, and/or cell uptake of the siRNA. A person of average skill will be well aware of other types of chemical modification which may be incorporated into RNA molecules.

In some modified plant cells, modified plants or parts thereof in accordance with any aspect of the invention, at least some of the expressed SP2 has itself altered activity compared to unmodified control or WT. This alteration of activity by way of mutation or by gene editing may be combined with increasing or decreasing the level of expression of SP2 compared to unmodified or WT.

The activity of SP2 which may be altered is preferably that of the SP2 protein association with plastid protein SP1 ; wherein SP1 may comprise an amino acid sequence of SEQ ID NO: 6 or a sequence of at least 55% identity therewith.

The SP2 protein of altered activity is preferably lacking at least one, and up to 27 of the N- terminal amino acids of the protein sequence, e.g. SEQ ID NO: 3 or variants or homologs thereof.

As well as modified plant cells, modified plants or parts thereof based on altered SP2 expression and/or activity as hereinbefore defined, the invention includes the combination of all these possible aforementioned aspects and variations, together with an altered expression and/or activity of plastid SP1 protein. The SP1 protein comprises an amino acid sequence of SEQ ID NO: 6, or a sequence of at least 55% identity therewith, and is altered in expression and/or activity compared to plastid SP1 protein in an unmodified control or WT plant.

In these combined SP1 and SP2 aspects of the invention, expression and/or activity of SP1 may be increased compared to the expression and/or activity of SP1 in an unmodified control or WT plant. Alternatively, the expression and/or activity of SP1 is decreased or eliminated compared to the expression and/or activity of SP1 in an unmodified control or WT plant.

Where there is a polynucleotide encoding SP1 involved, this may comprise the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 5, or a sequence of at least 50% identity therewith, or a fragment of at least 17 contiguous nucleotides thereof.

In some aspects, the SP1 polynucleotide sequence or fragment thereof may be operably linked to a promoter in a sense or an antisense orientation if it is desired to employ a gene silencing approach, of which there are many types known in the art. Possible within the scope of the invention for example is for differing methods of expression control for each of SP1 and SP2. So SP1 may be altered in expression by one method, e.g. mutation in a regulatory element for SP1 , and SP2 may be altered in expression by an RNA-based suppression approach.

In all of the above where there may be gene silencing of SP2, the same applies where optionally expression and/or activity of SP1 being altered as well.

At least some of the expressed SP1 may have an altered SP1 activity compared to unmodified control or WT. This may be achieved by genetic modification, e.g. known transgenic approaches or by gene editing usually performed using Cas9.

In particularly preferred aspects, the activity of the SP1 protein which is altered may be (a) reduced or no interaction with plastid SP2 protein; and/or (b) reduced or no E3 ligase activity; and/or (c) reduced or no association with plastid Toe proteins.

In any of the aforementioned possibilities of the invention for modified plant cells, modified plants or parts thereof, plastids may be altered in at least some cells, when compared to an unmodified control or WT. In this context, embodiments of the invention are concerned with spatial and/or temporal alterations in plastids and so considerations are for tissue specific and development specific controls being involved in making the alterations compared to WT.

Altered plants in accordance with the invention advantageously may provide better yield characteristics. These may be designed into the alterations being made. Yield

characteristics, also known as yield traits may comprise one or more of the following non- limitative list of features: early flowering time, yield, biomass, seed yield, seed viability and germination efficiency, seed/grain size, starch content of grain, early vigour, greenness index, increased growth rate, delayed senescence of green tissue. The term "yield" in general means a measurable produce of economic value, typically related to a specified crop, to an area, and to a period of time. Individual plant parts directly contribute to yield based on their number, size and/or weight, or the actual yield is the yield per square meter for a crop and year, which is determined by dividing total production (includes both harvested and appraised production) by planted square metres. The term "yield" of a plant may relate to vegetative biomass (root and/or shoot biomass), to reproductive organs, and/or to propagules (such as seeds) of that plant. Thus, according to the invention, yield comprises one or more of and can be measured by assessing one or more of: increased seed yield per plant, increased seed filling rate, increased number of filled seeds, increased harvest index, increased viability/germination efficiency, increased number or size of seeds/capsules/pods, increased growth or increased branching, for example inflorescences with more branches, increased biomass or grain fill. Preferably, increased yield comprises an increased number of grains/seeds/capsules/pods, increased biomass, increased growth, increased number of floral organs and/or increased floral branching. Yield is usually measured relative to a control plant.

The alteration in plants of the invention may for example be in the numbers and/or type(s) of plastid in the cell(s). This can also serve to better adapt an existing plant by modifying it to tolerate better particular environmental conditions such as light quality and level, temperature fluctuations, minima and/or maxima, and/or water, saline or osmotic stresses. (This may also tie in with better yield traits, as described above).

In accordance with the invention, the SP2 including any variant or homologue as herein defined by way of the percentage identities of sequence, may be from different species, genus, family or order of plant. Where SP1 is altered in expression and/or activity in combination with SP2 alterations, then similarly this SP1 can independently be selected from a different species, genus, family or order of plant. The SP2 and SP1 genes being altered can be taken from the same or different plant species

The invention therefore provides a modified plant as herein defined, which has increased expression and/or activity of SP2 protein; optionally also increased expression and/or activity of SP1 protein, and which has (a) increased tolerance to a stress condition compared to an unmodified control or WT plant; preferably wherein the stress condition is one or more of saline stress, osmotic stress or oxidative stress; and/or (b) accelerated fruit ripening.

Where the plant stress condition is concerned, this may be one or more of salinity, osmotic stress and/or oxidative stress. The tolerance of plants to different types of abiotic stresses is not necessarily conferred through related mechanisms and indeed occurs via different signal transduction pathways. Thus, it cannot be expected that a gene that confers, when expressed, one type of stress, could also confer a different type of stress.

Stress can thus refer to moderate or severe salt stress and is present when the soil is saline. Soils are generally classified as saline when the ECe is 4 dS/m or more, which is equivalent to approximately 40 mM NaCI and generates an osmotic pressure of approximately 0.2 MPa. Most plants can however tolerate and survive about 4 to 8 dS/m although this will impact on plant fitness and thus yield. For example, in rice, soil salinity beyond ECe ~ 4 dS/m is considered moderate salinity while more than 8 dS/m becomes high. Similarly, pH 8.8 - 9.2 is considered as non-stress while 9.3 - 9.7 as moderate stress and equal or greater than 9.8 as severe stress. Thus, salt stress as used herein refers to an ECe of 4 dS/m or more, for example about 4 to about 8 dS/m or about 40 mM NaCI or more, for example about 40 mM NaCI to about 100 mM NaCI or about 40 mM NaCI to 200 mM NaCI. Exposure to high levels of NaCI not only affects plant water relations but also creates ionic stress in the form of cellular accumulation of Cl and, in particular, Na ⁺ ions. Salt stress also changes the homeostasis of other ions such as Ca ²⁺ , K ⁺ , and NO ₃ levels (water deficit, ion toxicity, nutrient imbalance, and oxidative stress), and at least two main responses can be expected: a rapid protective response together with a long-term adaptation response. During initial exposure to salinity, plants experience water stress, which in turn reduces leaf expansion. During long-term exposure to salinity, plants experience ionic stress, which can lead to premature senescence of adult leaves, and thus a reduction in the photosynthetic area available to support continued growth. Thus, by increasing tolerance to salt stress, plant yield is increased.

When plant cells are under environmental stress, several chemically distinct reactive oxygen species (ROS) are generated by partial reduction of molecular oxygen and these can cause oxidative stress damage or act as signals. Oxidative stress can be induced by various environmental and biological factors such as hyperoxia, light, drought, high salinity, cold, metal ions, pollutants, xenobiotics, toxins, reoxygenation after anoxia, experimental manipulations, pathogen infection and aging of plant organs. Auto-oxidation of

components of the photosynthetic electron transport chain leads to the formation of superoxide radicals and their derivatives, hydrogen peroxide and hydroxyl radicals. These compounds react with a wide variety of biomolecules including DNA, causing cell stasis and death. Thus, by increasing tolerance to oxidative stress, plant yield is increased.

The invention therefore permits increasing or enhancing plant response to oxidative stress, caused for example by extreme temperatures, drought UV light, irradiation, high salinity, cold, metal ions, pollutants, toxins, or pathogen infection by bacteria, viruses or fungi or a combination thereof.

Osmotic adjustment plays a fundamental role in water stress responses and growth in plants. Drought, salinity and freeze-induced dehydration constitute direct osmotic stresses; chilling and hypoxia can indirectly cause osmotic stress via effects on water uptake and loss. By increasing tolerance to osmotic stress, plant yield is increased. Osmotic stress in accordance with the invention refers to osmotic stress caused by salinity, freezing or chilling and drought.

According to the invention, plant stress responses may be increased, enhanced or improved. This is understood to mean an increase compared to the level as found in a control, for example a wild-type plant. A skilled person will appreciate that such stress responses can be measured and the increase can be 2- to 10-fold.

The stress may be severe or preferably moderate stress. In Arabidopsis research, stress is often assessed under severe conditions that are lethal to wild-type plants. For example, drought tolerance is assessed predominantly under quite severe conditions in which plant survival is scored after a prolonged period of soil drying. However, in temperate climates, limited water availability rarely causes plant death, but restricts biomass and seed yield. Moderate water stress, that is suboptimal availability of water for growth can occur during intermittent intervals of days or weeks between irrigation events and may limit leaf growth, light interception, photosynthesis and hence yield potential. Leaf growth inhibition by water stress is particularly undesirable during early establishment. There is a need for methods for making plants with increased yield under moderate stress conditions. In other words, whilst plant research in making stress tolerant plants is often directed at identifying plants that show increased stress tolerance under severe conditions that will lead to death of a wild-type plant, these plants do not perform well under moderate stress conditions and often show growth reduction which leads to unnecessary yield loss.

So, in pursuing certain aspect of the invention, plant yield may be improved under moderate stress conditions. The terms moderate or mild stress/stress conditions are used interchangeably and refer to non-severe stress. In other words, moderate stress, unlike severe stress, does not lead to plant death. Under moderate, that is non-lethal, stress conditions, wild-type plants are able to survive, but show a decrease in growth and seed production and prolonged moderate stress can also result in developmental arrest. The decrease can be at least 5%-50% or more. Tolerance to severe stress is measured as a percentage of survival, whereas moderate stress does not affect survival, but growth rates. The precise conditions that define moderate stress vary from plant to plant and also between climate zones, but ultimately, these moderate conditions do not cause the plant to die. With regard to high salinity for example, most plants can tolerate and survive about 4 to 8 dS/m. Specifically, in rice, soil salinity beyond ECe ~ 4 dS/m is considered moderate salinity while more than 8 dS/m becomes high. Similarly, pH 8.8 - 9.2 is considered as non-stress while 9.3 - 9.7 as moderate stress and equal or greater than 9.8 as higher stress. So, in accordance with the invention are methods relating to increasing resistance to moderate (non-lethal) stress or severe stress. Modified plants according to the invention show increased resistance to stress and therefore, the plant yield is not or less affected by the stress compared to wild type yields which are reduced upon exposure to stress. In other words, an improve in yield under moderate stress conditions can be observed.

Preferred homologues of AtSP2 peptides are SP2 peptides from crop plants, for example cereal crops. In one embodiment, preferred homologues include SP2 in maize, rice, wheat, sorghum, sugar cane, oilseed rape (canola), soybean, cotton, potato, tomato, tobacco, grape, barley, pea, bean, field bean or other legumes, lettuce, sunflower, alfalfa, sugar beet, broccoli or other vegetable brassicas or poplar.

Preferably, the plant is a crop plant. By crop plant is meant any plant which is grown on a commercial scale for human or animal consumption or use. In a preferred embodiment, the plant is a cereal or legume.

A plant according to the various aspects of the invention, including the transgenic plants, methods and uses described herein may be a monocot or a dicot plant.

The term "plant" as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, fruit, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs, wherein each of the aforementioned comprise the gene/nucleic acid of interest. The term "plant" also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the gene/nucleic acid of interest.

A monocot plant may, for example, be selected from the families Arecaceae,

Amaryllidaceae or Poaceae. For example, the plant may be a cereal crop, such as wheat, rice, barley, maize, oat, sorghum, rye, millet, buckwheat, turf grass, Italian rye grass, sugarcane or Festuca species, or a crop such as onion, leek, yam or banana.

A dicot plant may be selected from the families including, but not limited to Asteraceae, Brassicaceae (e.g. Brassica napus ), Chenopodiaceae, Cucurbitaceae, Leguminosae ( Caesalpiniaceae , Aesalpiniaceae Mimosaceae, Papilionaceae or Fabaceae ), Malvaceae, Rosaceae or Solanaceae. For example, the plant may be selected from lettuce, sunflower, Arabidopsis, broccoli, spinach, water melon, squash, cabbage, tomato, potato, yam, capsicum, tobacco, cotton, okra, apple, rose, strawberry, alfalfa, bean, soybean, field (fava) bean, pea, lentil, peanut, chickpea, apricots, pears, peach, grape vine, bell pepper, chilli or citrus species. In one embodiment, the plant is oilseed rape. Also included are biofuel and bioenergy crops such as rape/canola, sugar cane, sweet sorghum, Panicum virgatum (switchgrass), linseed, lupin and willow, poplar, poplar hybrids, Miscanthus or gymnosperms, such as loblolly pine. Also included are crops for silage (maize), grazing or fodder (grasses, clover, sanfoin, alfalfa), fibres (e.g. cotton, flax), building materials (e.g. pine, oak), pulping (e.g. poplar), feeder stocks for the chemical industry (e.g. high erucic acid oil seed rape, linseed) and for amenity purposes (e.g. turf grasses for golf courses), ornamentals for public and private gardens (e.g. snapdragon, petunia, roses, geranium, Nicotiana sp.) and plants and cut flowers for the home (African violets, Begonias, chrysanthemums, geraniums, Coleus spider plants, Dracaena, rubber plant).

Most preferred plants are maize, rice, wheat, oilseed rape/canola, sorghum, soybean, sunflower, alfalfa, potato, tomato, tobacco, grape, barley, pea, bean, field bean, lettuce, cotton, sugar cane, sugar beet, broccoli or other vegetable brassicas or poplar.

A plant as mentioned above may have a decreased or no expression and/or decreased or no activity of SP2 protein; optionally decreased or no expression and/or no decreased or no activity of SP1 protein, in at least a particular cell type; optionally wherein (a) the cell type is at seedling stage and there is increased seedling survival, increased seedling growth and/or emergence; or (b) the cell type is green photosynthetic and there is a delay of senescence; or (c) the cell type is at seed setting stage and there is an increase in seed/grain size or starch content; or (d) the cell type is at fruiting stage and there is a delaying of fruit ripening.

In particularly preferred aspects the modified plants of the invention described herein are crop plants, biofuel plants or horticultural plants.

In another aspect, the present invention provides an isolated polynucleotide construct comprising a promoter and:

a. a polynucleotide comprising a nucleotide sequence of: (i) SEQ ID NO: 1 or a sequence of at least 50% identity thereto; or (ii) SEQ ID NO: 2 or a sequence of at least 50% identity thereto; or

b. a polynucleotide comprising a fragment of at least 17 contiguous nucleotides of: (i) SEQ ID NO: 1 or a sequence of at least 50% identity thereto; or (ii) SEQ ID NO: 2 or a sequence of at least 50% identity thereto.

As used herein, the terms "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), naturally occurring, mutated, synthetic DNA or RNA molecules, and analogues of the DNA or RNA generated using nucleotide analogues. They can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, anti- sense sequences, and non-coding regulatory sequences that do not encode mRNAs or protein products. These terms also encompass a gene.

As to a "gene" or "gene sequence", these broadly to refer to a DNA nucleic acid associated with a biological function. Thus, genes may include introns and exons as in the genomic sequence, or may comprise only a coding sequence as in cDNAs, and/or may include cDNAs in combination with regulatory sequences.

If orthologues of nucleotide sequences encoding SP2 are included in the aforementioned, then they may have, in increasing order of preference, an identity at least 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%,

43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,

58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,

73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,

88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity to the nucleic acid represented by SEQ ID NO: 1 or 2. (The“at least” prefixes each and every percentage identity listed above.)

Preferably though, in the context of polynucleotides of the invention, overall sequence identity may be in order of increasing preference, more than 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68% or more than 69%. (The“more than” prefixes each and every percentage identity listed here.)

Preferably though, overall sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, in increasing order of preference is at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%,

58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,

73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,

88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity to the nucleic acid represented by SEQ ID NO: 1 or 2. (The“at least” prefixes each and every percentage identity listed above.)

As may be estimated from Figure 15A, a preferred limit of percentage identity which defines SP1 may be at least 65% compared to SEQ ID NO: 6. A similar preferred limit of variation for full length genomic and cDNA may also apply, so at least 65% identity to SEQ ID NO: 4 or SEQ ID NO: 5. Each and every individual percentage limit as recited above from at least 56% to at least 99% identity with the reference sequences is contemplated. As may be estimated from Figure 15B, the preferred limit of percentage identity which defines SP2 is at least 78% compared to SEQ ID NO: 3. A similar preferred limit of variation for full length genomic and cDNA may also apply, so at least 78% identity to SEQ ID NO: 1 or SEQ ID NO: 2. Optionally the percentage identity for these reference sequences may be at least 80% or at least 85%; preferably at least 90%. However, each and every individual percentage limit as recited above from at least 78% to at least 99% identity with the reference sequences is contemplated.

The degree of sequence identity of polynucleotides of the invention may, instead of being expressed as a percentage identity to reference sequence, may instead be defined in terms of hybridization to a polynucleotide of reference sequence SEQ ID NO: 1 or SEQ ID NO: 2. Hybridization of such sequences may be carried out under stringent conditions. By "stringent conditions" or "stringent hybridization conditions" is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the salt concentration is less than about 1 .5 M Na ion, typically about 0.01 to 1 .0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Duration of hybridization is generally less than about 24 hours, usually about 4 to 12. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.

Thus, a nucleotide sequence as described herein can be used to isolate corresponding sequences from other organisms, particularly other plants, for example crop plants. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences described herein.

Topology of the sequences, e.g. for SP2 the characteristic 16 x TMD pattern and N- terminal 27 amino acids of SP2 can also be considered when identifying and isolating SP2 homologues for example. Sequences may be isolated based on their sequence identity to the entire sequence or to fragments thereof. In hybridization techniques, all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e. , genomic or cDNA libraries) from a chosen plant. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labelled with a detectable group, or any other detectable marker. Thus, for example, probes for hybridization can be made by labelling synthetic oligonucleotides based on the ABA-associated sequences of the invention. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook, et al., (1989) Molecular Cloning: A Library Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

Such polynucleotide constructs will be of assistance to persons of average skill in the making of altered plant cells, plants and parts thereof, wherein the SP2 gene is altered in expression and/or activity. The constructs of the invention lend themselves to the full range of known gene modification and gene expression modulation methods, including gene editing using Cas9 or Cpf1 , for example.

Recombinant DNA constructs may be made and used as described in US 6635805, incorporated herein by reference.

A silencing RNA molecule may be introduced into a plant using conventional methods, for example a vector and Agrobacterium-medi atedi transformation. Stably transformed plants are generated and expression of the SP2 gene compared to a wild type control plant is analysed.

Silencing of the SP2 gene may also be achieved using virus-induced gene silencing.

A modified plant cell, plant or part thereof of the invention may express a nucleic acid construct comprising a RNAi, snRNA, dsRNA, siRNA, miRNA, ta-siRNA, amiRNA or co suppression molecule that targets the SP2 gene as described herein and reduces expression of the endogenous SP2 gene. A gene is targeted when, for example, the RNAi, snRNA, dsRNA, siRNA, miRNA, ta-siRNA, amiRNA or cosuppression molecule selectively decreases or inhibits the expression of the gene compared to a control plant.

Alternatively, a RNAi, snRNA, dsRNA, siRNA, miRNA, ta-siRNA, amiRNA or

cosuppression molecule targets SP2 when the RNAi, snRNA, dsRNA, siRNA, miRNA, ta- siRNA, amiRNA or cosuppression molecule hybridises under stringent conditions to the gene transcript. Within the context of the invention, preferably, to specifically target SP2, the RNA must comprise at least the same seed sequence. Thus, any RNA that targets SP2 is preferably identical in positions 2-8 of the antisense strand. Gene silencing may also occur if there is a mutation on an endogenous gene and/or a mutation on an isolated gene/nucleic acid subsequently introduced into a plant. The reduction or substantial elimination may be caused by a non-functional polypeptide. For example, the polypeptide may bind to various interacting proteins; one or more mutation(s) and/or truncation(s) may therefore provide for a polypeptide that is still able to bind interacting proteins (such as receptor proteins) but that cannot exhibit its normal function (such as signalling ligand).

A further approach to gene silencing is by targeting nucleic acid sequences

complementary to the regulatory region of the gene (e.g., the promoter and/or enhancers) to form triple helical structures that prevent transcription of the gene in target cells. Other methods, such as the use of antibodies directed to an endogenous polypeptide for inhibiting its function in planta, or interference in the signalling pathway in which a polypeptide is involved are well known. In particular, manmade molecules may be useful for inhibiting the biological function of a target polypeptide, or for interfering with the signalling pathway in which the target polypeptide is involved.

In modification of the above, there may also be provided an isolated polynucleotide construct which additionally comprises:

a. a polynucleotide comprising a nucleotide sequence of: (i) SEQ ID NO: 4 or a sequence of at least 55% identity thereto; or (ii) SEQ ID NO: 5 or a sequence of at least 55% identity thereto; or

b. a polynucleotide comprising a fragment of at least 17 contiguous nucleotides of: (i) SEQ ID NO: 4 or a sequence of at least 55% identity thereto; or (ii) SEQ ID NO: 5 or a sequence of at least 55% identity thereto.

This is for achieving a dual alteration of expression and/or activity of SP2 and SP1 in a plant, plant cell or plant part. There are various possibilities whereby single or binary constructs can be used. In a single construct, the SP2 encoding polynucleotide in the above may be downstream of the promoter, and the SP1 encoding polynucleotide may be downstream of the SP2 encoding polynucleotide. Alternatively, these positions of SP2 and SP1 may be reversed.

Described herein is also an isolated polynucleotide construct which comprises a promoter and:

a. a polynucleotide comprising a nucleotide sequence of: (i) SEQ ID NO: 4 or a sequence of at least 55% identity thereto; or (ii) SEQ ID NO: 5 or a sequence of at least 55% identity thereto; or b. a polynucleotide comprising a fragment of at least 17 contiguous nucleotides of: (i) SEQ ID NO: 4 or a sequence of at least 55% identity thereto; or (ii) SEQ ID NO: 5 or a sequence of at least 55% identity thereto.

The invention also includes the aforementioned isolated polynucleotides as separate polynucleotides, each with their own same or different promoters and optional regulatory and other features, forming a portion of a kit of parts for use in a binary rather than single construct approach to the alteration of SP2 and SP1 protein activity and/or expression in plant cells, plants or parts thereof.

Such polynucleotides as aforementioned include Ti plasmids of Agrobacterium

tumefasciens which are well known in the art.

In further aspect is a single polynucleotide construct comprising a regulatory sequence, e.g. promoter, and comprising Included in the invention are vectors comprising a polynucleotide as aforementioned. This includes Agrobacterium tumefasciens, tobacco mosaic virus (TMV), potato virus X and cowpea mosaic virus. Preferably, the vector further comprises a regulatory sequence which directs the desired expression of the nucleic acid, spatially or temporally, or possibly in reaction to an inducer or stress condition.

Included therefore in the invention as another aspect are host cells comprising a polynucleotide or a vector as hereinbefore described. Such cells may not necessarily be plant cells when cloning, in vitro expression or genetic manipulation procedures are being carried out as part of a series of experimental or developmental steps to yield altered plant material. So, such host cells may include isolated plant cells or protoplasts, bacteria, e.g. Agrobacterium or Escherichia coli, or yeast, e.g. Saccharomyces cerevisiae.

Also included in the invention is a culture medium or kit comprising a culture medium and an isolated host cell as described herein.

In further aspect, the invention provides a method of altering plastids in a plant cell, comprising:

a. increasing, decreasing or eliminating the expression in the cell of plastid protein SP2 comprising an amino acid sequence of SEQ ID NO: 3 or a sequence of at least 50% identity therewith when compared to a control or WT plant cell; and/or

b. increasing, decreasing or eliminating the activity in the cell of plastid protein SP2 comprising an amino acid sequence of SEQ ID NO: 3 or a sequence of at least 50% identity therewith when compared to a control or WT plant cell, wherein the activity of SP2 is that of SP2 association with plastid SP1 protein of SEQ ID NO: 6 or a sequence of at least 55% identity therewith.

In such a method of altering plastids, this may further (additionally) comprise:

a. increasing, decreasing or eliminating the expression in the cell of plastid protein SP1 comprising an amino acid sequence of SEQ ID NO: 6 or a sequence of at least 55% identity therewith when compared to a control or WT plant cell; and/or

b. increasing, decreasing or eliminating the activity in the cell of plastid protein SP1 comprising an amino acid sequence of SEQ ID NO: 6 or a sequence of at least 55% therewith when compared to a control or WT plant cell, wherein the activity of SP1 is that of SP1 association with plastid SP2 protein of SEQ ID NO: 3 or a sequence of at least 50% identity therewith.

In these aforementioned methods of altering plastids in a plant cell, the expression of SP2; optionally also the expression of SP1 , may be increased by the editing of a nucleotide sequence of at least one native SP2 regulatory element in the cell; optionally also at least one SP1 native regulatory element in the cell, and/or by the insertion of a polynucleotide construct or a vector as hereinbefore defined, into the cell.

In some aforementioned methods of altering plastids in a plant cell, plastid development may be accelerated; optionally wherein the cell is (a) a seedling cell and the transition of etioplasts into chloroplasts is accelerated; or (b) a seed cell and the transition to amyloplasts is accelerated; or (c) a fruit cell and the transition from chloroplasts is accelerated.

Alternatively, in other methods of altering plastids in plant cells, the expression of SP2; optionally also the expression of SP1 , may be decreased or eliminated by the editing of a nucleotide sequence of at least one native SP2 regulatory element in the cell; optionally also at least one SP1 native regulatory element in the cell, and/or by the insertion of a polynucleotide construct or a vector as hereinbefore defined, into the cell.

In methods of altering plastids where plastid development is delayed; the cell is optionally: (a) a green photosynthetic cell and the transition of chloroplasts into another type of plastid, e.g. gerontoplast, is delayed; or (b) a fruit cell and the transition from chloroplasts is delayed.

In any aspect of the invention herein, not excluded is the possibility that SP2 expression and/or activity may be increased whilst SP1 expression and/or activity may be decreased, with respect to a control or WT. And vice versa, not excluded, SP2 expression and/or activity may be decreased whilst SP1 expression and/or activity may be increased, with respect to a control or WT.

What is also possible in the present invention is that the ratio of SP2:SP1 expression or activity may be altered compared to a control or WT. This ratio may increase or decrease compared to control of WT. Ratios may a ratio in the range of possibilities between 10:1 to 1 : 10.

Some aspects of the invention involve recombinant DNA technology and in preferred embodiments exclude embodiments that are solely based on generating plants by traditional breeding methods.

The various aspects of the invention described herein clearly extend to any plant cell or any plant produced, obtained or obtainable by any of the methods described herein, and to all plant parts and propagules thereof unless otherwise specified. The present invention extends further to encompass the progeny of a primary transformed or transfected cell, tissue, organ or whole plant that has been produced by any of the aforementioned methods, the only requirement being that progeny exhibit the same genotypic and/or phenotypic characteristic(s) as those produced by the parent in the methods according to the invention.

The invention also extends to harvestable parts of a plant of the invention as described above such as, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs. The invention furthermore relates to products derived, preferably directly derived, from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch or proteins. The invention also relates to food products and food supplements comprising the plant of the invention or parts thereof.

In another aspect, the invention relates to a plant obtainable or obtained by a method as described herein.

In all embodiments of methods where SP2 expression and/or activity is modified, then these apply equally to situations where SP1 expression and/or activity is in addition modified

EXAMPLES

Hereinafter, reference numbers in parenthesis correspond to the references listed at the end of the description.

Chloroplasts are plant organelles responsible for the bulk of photosynthetic primary production, and they evolved via endosymbiosis from a cyanobacterial organism more than 1 Bya (7). The modern chloroplast proteome comprises -3000 proteins, most of which are nucleus-encoded and imported post-translationally by translocases in the chloroplast envelope membranes (2-5). Turnover of internal chloroplast proteins is governed by several prokaryotic-type proteases inherited from the endosymbiont (6). In contrast, chloroplast outer envelope membrane (OEM) proteins are degraded by the cytosolic ubiquitin-proteasome system (UPS) via poorly understood mechanisms ( 1 ).

The RING-type ubiquitin E3 ligase SP1 is located in the chloroplast OEM where it mediates the ubiquitination of OEM components of the chloroplast protein import machinery (so-called TOC proteins; these act in conjunction with TIC translocases in the inner membrane (2-5)), thereby promoting their degradation by the cytosolic 26S proteasome (7). The TOC components affected by SP1 include the receptors Tod 59 and Toc33, and the channel protein Toc75. Such SP1- mediated regulation of the TOC apparatus changes the organellar proteome, which in turn influences the developmental fate and functions of the organelle (e.g., enabling plant adaptation to abiotic stress) (7, 8). While the role of SP1 in marking proteins for degradation is clear, other aspects of this chloroplast protein degradation system have remained obscure. Because TOC proteins are integral membrane components, additional factors are most likely required to overcome the energetic barrier to their extraction from the membrane, prior to degradation in the cytosol, as is the case in other membrane-associated proteolytic systems (9-11).

The inventors have discovered mutants with lesions at a locus unlinked to sp1. These are termed suppressor ofppH locus2 ( sp2 ). Double-mutant sp2 ppi1 plants were found by the inventors to be larger and greener than the ppi1 progenitor and exhibited substantial improvements in chloroplast development and protein import capacity.

Example: Identification of SP2 and its role in plastid development

Genetic Resources

Seeds of Arabidopsis thaliana ecotype Columbia-0 (Col-0), SALK_137135 were obtained on 9 ^th April 2014 from Nottingham Arabidopsis Stock Centre (NASC), School of

Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, United Kingdom.

Seeds of Arabidopsis thaliana ecotype Landsberg erecta (Ler) have been in possession of the inventors since earlier than 12 ^th October 2014 from the laboratory of Joanne Chory at the Salk Institute 10010 N Torrey Pines Rd., La Jolla, CA 92037, California, USA.

The sp2-4 (SALK_137135) mutant was obtained from the Salk Institute Genomic Analysis Laboratory. Materials and methods:

Plant material and growth conditions

All Arabidopsis thaliana plants were of the Columbia-0 (Col-0) ecotype, except the ppi1 line used for the genetic mapping of sp2 which was introgressed into Landsberg erecta (Ler) through seven outcrosses. The sp1-1, sp1-3, ppi1, tic40-4, hsp93-V-1, ppi2-3 (fts1), toc75-\\\-3 (marl) mutants, and the 35S promoter-driven SP1 overexpressor (SP1-OX) transgenic line, have all been described previously (7, 12, 16, 19, 30, 31). The sp2-4 (SALK_137135) mutant was obtained from the Salk Institute Genomic Analysis Laboratory, and confirmed by PCR and RT-PCR analysis, as described previously (32); this mutant was phenotypically similar to the three chemically-induced sp2 alleles identified in this study, and unlike a previously-described T-DNA mutant (33). For in vitro growth, seeds were surface sterilized, sown on Murashige-Skoog (MS) agar medium in petri plates, cold- treated at 4°C, and thereafter kept in a growth chamber, as described previously (34). All plants were grown under a long-day cycle (16 h light, 8 h dark).

Physiological studies

Chlorophyll measurements were performed by using a Konica-Minolta SPAD-502 meter (35), or by photometric quantification following extraction in N,N’-dimethylformamide (DMF) as described previously (36).

Dark treatments for the induction of senescence were conducted as previously described (7, 37). Developmentally-equivalent leaves of 28-day-old plants were wrapped in aluminium foil whilst still attached to the plant, and then left under standard growth conditions for 5 days. Photochemical efficiency of photosystem II (Fv/Fm) was determined by measuring chlorophyll fluorescence using a CF Imager (Technologica, UK) as described previously (38). Three experiments were performed, and approximately five leaves (each one from a different plant) were analysed per genotype in each experiment.

Salt stress experiments were conducted as described previously with minor modifications (8). All seeds of the different genotypes used in this work were harvested at the same time. Seeds were germinated directly on MS agar medium (supplemented with 1% sucrose) containing 150-170 mM NaCI. Stress tolerance was assessed by measuring chlorophyll accumulation after 14 days. Three experiments were performed, and ~25 seedlings per genotype were analysed in each experiment. Detection of hydrogen peroxide was performed by staining with 3,3’-diaminobenzidine (DAB) (Sigma) as previously described (39). The plants were left to grow for further 2 days before initiating DAB staining. Each experiment used approximately 5 seedlings per genotype. Three experiments were performed with the same result, and typical images are presented. The area of staining was quantified using ImageJ as described previously (8).

Identification of the sp2 mutants and genetic mapping

The original sp2 mutants ( sp2-1 [ sp2-310 ], sp2-2 [sp2-416\ and sp2-3 [sp2-555\) were identified by screening the M2 progeny of 7,000 M1 ppi1 seeds that had been treated with 100 mM ethyl methanesulfonate for 3 h using a published procedure (7, 40). Initial mapping of sp2 was conducted by analysing the greenest plants in F2 populations from crosses between sp2-1 ppi1 (Col-0) and ppi1 introgressed into the Ler ecotype, using PCR markers that detect Col-0/Ler polymorphisms. In a mapping population of 190 such F2 plants, six were heterozygous for the marker F21A17 at position 12285000 on the upper arm of chromosome 3, but homozygous for Col-0 downstream of that, suggesting that the suppressor mutation was in the downstream Col-0 region; F3 seedlings from these six plants were grown and verified visually to be non-segregating, as expected for a homozygous sp2-1 ppi1 double mutant. In a second mapping population of 192 plants, the sp2 mutation was further mapped to the south of a more southerly marker, MJI6-2 at position 12597802 on the upper arm of chromosome 3. However, it was not possible to determine the position of the sp2 locus precisely owing to the persistence of an“island” of Col-0 DNA in the Ler-introgressed ppi1 line, near the sp2 locus (around the chromosome 3 centromere). Thus, final identification of the gene was achieved by whole-genome sequencing.

Whole-genome sequencing and assembly

Approximately 100 mg of plant inflorescence tissue from each of the original sp2 alleles (sp2-1 ppi1, sp2-2 ppi1, and sp2-3 ppi1), and from ppi1-1, was harvested and flash-frozen in liquid nitrogen. Total genomic DNA was then extracted using an E.Z.N.A. Plant DNA Kit (Omega Bio-tek) following the manufacturer’s guidelines. The DNA samples were quantified by comparison with standards.

Library preparation and sequencing were conducted at the Earlham Institute (Norwich,

UK). Approximately 1-5 pg genomic DNA per sample at a minimum concentration of 20 ng/mI was used in sequencing library preparation. Individual barcoded lllumina TruSeq DNA libraries were generated for each genotype. The four samples were then sequenced on one lane of lllumina HiSeq 2000, which generated between 32.2-42.3 million 100 bp paired-end reads for each sample. The first five bases of the 5' ends of the reads were removed using fastx trimmer v.0.0.14 (http://hannoniab.cshi.edu/fastx_toolkit/), and any bases with a Phred quality score below 15 were removed from the 3' end using cutadapt (v.1.3) {41). I llumina TruSeq adaptors where also removed using cutadapt, where a minimum overlap of 10 bases with the adaptors, a maximum error rate of 0.1 , and a minimum final read length of 50 bases were set.

Finally, fastq_q ual ity_fi Iter v0.0.14 (htp://hannoniab.cshi.edu/fastx_tooikit/) was used to remove sequences with a Phred score below 20 in more than 5% of the bases. Read pairs were identified using pairSeq.py (https://github.com/topei-research-group/pairSeq). The reads from the ppi1 single mutant were mapped to the TAIR10 Arabidopsis thaliana reference genome (ftp://ftp.jgi- psf.org/pub/compgen/phytozome/v9.0/Athaliana/assembly/Athali ana_167. fa. gz) of the Phythozome v.9.0 release (http://www.phytozome.net/) using clc_mapper v. 4.0.13.86165 (https://www.qiagenbioinfonYiatics.com/). A consensus sequence in FASTA format was then generated using clc_find_variations v. 4.0.13.86165. Also, the transcript sequences from the TAIR10 release were aligned to the reference genome in order to facilitate manual examination of identified mutations and visualization of whether a particular mutation occurs in an exon, intron, etc.

The three datasets from the individual sp2 double mutants were independently aligned to the ppi1 reference genome using clc_mapper v. 4.0.13.86165. The ppi1 reads were also aligned to the same reference in order to identify any variable sites resulting from allelic variation in the ppi1 line.

In silico identification of mutations

The mutagen ethyl methanesulfonate (EMS) used to generate the three sp2 mutants reacts with guanine in the DNA molecule and is likely to (1) cause point mutations that change guanine to adenosine (or cytosine to thymidine on the reverse strand). The respective mutations affecting the three sp2 mutants were furthermore expected to (2) occur in the same gene (or corresponding promotor region) in (3) all three mutants but (4) not necessarily in the exact same position. These four search criteria were implemented in the program“find_sp2.py” (https://github.com/topel-research-group/sp2) which takes as input a gff3 file with gene coordinates and the SNP variant output from clc_mapper, and outputs a list of names of mutated genes from each dataset. Genes found to be mutated in all the sp2 datasets were then manually examined by visualizing the alignment data using the genome viewer IGV (v.2.3) (42).

This analysis showed that each sp2 allele contains a G-to-A point mutation within the At3g44160 gene, just to the south of the chromosome 3 centromere. In sp2-1, a mutation was detected at the splice junction preceding the final exon; this was later shown to cause mis-splicing, frame-shifts, and premature termination, implying that sp2-1 is a knockout allele. In sp2-2 and sp2-3, the detected mutations were both predicted to cause an amino- acid substitution. The transmembrane beta-strands and the three-dimensional structure of the SP2 protein were predicted using Phyre2, with a model based on structures for bacterial TamA and BamA proteins (c4c00a, c5ekqA, c4k3bA, c4n75A, c4k3cA) (43).

Phylogenetic analysis

Sequences were obtained by BLAST searches of the Phytozome 12 database (44) (table S1). Sequences were aligned by multiple ajignment using fast Fourier transform (MAFFT) (45), and manual alignment adjustments were made using Mesquite 1.12 (Tangient). Phylogeny was inferred using MrBayes 3.2 software (46). Two runs were performed in parallel, with each using 8 MCMC chains for 8 million generations and the temperature set to 0.2. The standard deviation of split frequencies (StdDev) was 0.001228 at the end of the analysis and therefore assumed to have converged. Trees were sampled every 1000 generations, reaching a total of 8000 trees. Burn-in was set to 25%, and so the first 2000 trees were discarded. The resulting phylogeny was a minimum 50% consensus of the remaining 6000 sampled trees. Parameters not mentioned were retained at the default setting.

Gene identifiers

The following gene sequences from Arabidopsis thaliana were employed experimentally in this study: SP2 (At3g44160); Toc33 (At1g02280); CDKA1 (At3g48750); Tod 59

(At4g02510); OEP7 (At3g52420); OEP80 (At5g19620); SSU (At1g67090).

Plasmid constructs

All primers used are listed in the tables below:

(A) Primers used to generate various constructs and mutations.

(B) Primers used in RT-PCR experiments.

(C) Primers used to genotype mutant plants by dCAPS (derived Cleaved Amplified Polymorphic Sequence) analysis. The PCR products amplified using dCAPS primers were digested with restriction enzyme, and thereafter resolved on 3% agarose gels. Genotypes were determined by comparing the sizes of the bands with controls corresponding to wild type and the homozygous mutant.

(D) Primers used to genotype T-DNA insertion mutant plants.

The SP1-HA, YFP-HA, SP1-YFP, GST-SPWex and YFP-Toc33 constructs have all been described previously (7, 47). All other Arabidopsis CDSs were PCR-amplified from Col-0 cDNA. The Gateway cloning system (Invitrogen) was used to make most of the constructs, and all entry clones were verified by DNA sequencing. To generate C-terminal 6*Myc tag fusion proteins, the SP1 and SP2 CDSs were cloned into the pE3c vector (49), and then subcloned into the p2GW7 35S-driven expression vector (50) for protoplast transfection (generating the SP1-Myc and SP2-Myca constructs). The SP2 CDS, with and without the Myc tag, was cloned into the pB2GW7 binary 35S-driven overexpression vector (50) for stable plant transformation (generating the SP2-OX and SP2-Myc constructs). To generate N-terminally TAP-tagged Toc33, the corresponding CDS was cloned into the NTAPi binary vector (52) (generating the TAP-Toc33 construct). Transient assays and stable plant transformation

Protoplast isolation and transient assays were carried out as described previously (7, 54). When required, MG132 (Sigma), epoxomicin (Merck), bortezomib (Selleckchem) (all three chemicals prepared as a 10 mM stock solution in DMSO), or E-64 (Melford) (prepared as a 10 mM stock solution in water) was added to the protoplast culture medium at 15 h following transfection, to a final concentration of 1-30 mM, 1-10 pM, 5 pM, or 1-10 pM, respectively; subsequently, the culture was incubated for a further 2-3 h before analysis. For XFP fluorescence and immunoprecipitation assays, 0.1 ml (105) or 1 ml (106) aliquots of protoplasts were transfected with 5 pg or 100 pg of DNA, respectively, and the fluorescence signals were analysed after 15-18 h.

Transgenic lines carrying the SP2-OX and SP2-Myc constructs were generated by Agrobacterium-medi atedi transformation (16, 32). Transformants were selected using MS medium containing phosphinothricin for these SP2 constructs. At least 12 T2 lines for each transformation were analysed, and at least two lines with a single T-DNA insertion (which showed a 3:1 segregation on selective MS medium in the T2 generation) were chosen for further analysis.

Microscopy

Transmission electron microscopy was performed as described previously (16).

Measurements were recorded using at least 30 different plastids per genotype, and were representative of three individuals per genotype. Chloroplast cross-sectional area was estimated as described previously (16, 30), using the equation: p ^c 0.25 ^c length ^c width. Numbers of thylakoid lamellae per granal stack, and of interconnections between granal stacks, were counted as previously described (7, 16) in at least 96 resolvable grana across three individuals per genotype.

All fluorescence microscopy and BiFC experiments were conducted at least twice with the same results, and typical images are presented. For the imaging of CFP, YFP and chlorophyll fluorescence signals, in most cases protoplasts were examined using a Zeiss LSM 510 META laser- scanning confocal microscope (Carl Zeiss Ltd.), as described previously (8). To visualize signals associated with chloroplasts without interference from cytosolic signals, protoplasts were ruptured by gently tapping the cover glass; this enabled the release of the cytosol and of intact chloroplasts. Fluorescence images were captured using a Nikon Eclipse TE-2000E inverted microscope as described previously (32).

In vitro translation and in vitro pull-down analysis The SP2 and OEP80 CDSs were cloned into pBlueScript II SK- using a single Smal restriction site and verified by DNA sequencing. The preSSU construct was described previously, as was the in vitro transcription/translation procedure (16, 55).

The GST-SP1flex and GST proteins were purified from bacteria as described previously, as was the procedure employed for in vitro pull-down analysis (7).

Chloroplast isolation, protein import, and topology analysis

Chloroplasts were isolated from 14-day-old in vitro grown plants (or, when stated, from protoplasts). Isolations, protein import, and protease treatments were performed as described previously (16, 34, 56-59). The presented chloroplast protein import data are representative of three independent experiments.

Immunoblotting, immunoprecipitation and blue native (BN) PAGE

Immunoblotting was performed as previously described (30, 60) with minor modifications. Primary antibodies were as follows. To identify TOC proteins or components of the translocon at the inner envelope membrane of chloroplasts (TIC), we employed: anti- atToc75-lll antibody (32); anti-atToc159 antibody (67); anti-atToc33 (G-domain) antibody (32); anti-atTid 10 antibody (62, 63); and anti-atTic40 antibody (32). To identify non-TOC outer envelope membrane proteins, we employed: anti-OEP80 antibody (64); and anti- SFR2 antibody (65). To identify chloroplast stromal proteins, we employed: anti-cpHsc70 (AgriSera, AS08 348) (66); anti-Hsp93 (heat shock protein, 93 kD) antibody (16, 67); and anti- PRPL35 antibody (7). To identify proteins of other cellular compartments, we employed: anti-Slp1 (mitochondria) (68); anti-calreticulin (ER) (69, 70); and anti-H3 histone (Abeam; nucleus) (32). Other primary antibodies we employed were: anti-HA tag (Sigma); anti-c-Myc tag (Sigma); anti-GFP (detects both GFP and YFP; Sigma); and anti-FLAG tag (Sigma).

Secondary antibodies were anti-rabbit IgG conjugated with horseradish peroxidase (Santa Cruz Biotechnology), or, in the case of anti-c-Myc and anti-FLAG, anti-mouse IgG conjugated with horseradish peroxidase (GE Healthcare). Chemiluminescence was detected using ECL Plus Western Blotting Detection Reagents (GE Healthcare) and an LAS-4000 imager (Fujifilm). Band intensities were quantified using Aida software

(Raytest). Guantification data were based on results from at least three experiments all showing a similar trend. Typical images are shown in all figures. For the immunoprecipitation of HA-tagged proteins, total protein (-500 mg) was extracted from protoplasts in IP buffer (25 mM Tris-HCI, pH 7.5, 150 mM NaCI, 1 mM EDTA, 1% Triton X-100) containing 0.5% plant protease inhibitor cocktail (PPIC, Sigma), and centrifuged at 20,000g for 10 min at 4°C. The clear lysate was then incubated with 50 pi EZview Red Anti-HA Affinity Gel (Sigma) for 2 h to overnight at 4°C with slow rotation. After six washes with 500 mI IP-washing buffer (25 mM Tris-HCI, pH7.5, 150 mM NaCI, 1 mM EDTA, 0.5% Triton X-100), bound proteins were eluted by boiling in 2* SDS- PAGE loading buffer (50 mM Tris-HCI, pH 6.8, 20% glycerol, 1% sodium dodecyl sulphate [SDS], and 0.1 M DTT) for 5 min, and analysed by SDS-PAGE and immunoblotting. A similar procedure was adopted for the immunoprecipitation of Myc-tagged proteins, except that 50 mI EZview Red Anti-c-Myc Affinity Gel (Sigma) was used instead of the anti-HA gel. When detecting ubiquitinated proteins, the IP buffer also contained 10 mM N-ethylmaleimide (NEM; Sigma).

Two-dimensional BN-PAGE was performed using a procedure described previously (71).

Tandem affinity purification (TAP) and mass spectrometry

Chloroplasts were isolated from a complemented ppi1 mutant line carrying the TAP:Toc33 construct, and then used as starting material for TAP. The TAP procedure was performed as described previously (72), omitting the secondary affinity purification step which was not essential for our analysis. The Tobacco Etch Virus (TEV) nuclear-inclusion-a

endopeptidase eluates were concentrated 1 :10 by using Vivaspin 500 ultrafiltration spin columns (Sartorius Stedim Biotech), boiled with 1 volume 2* SDS- PAGE loading buffer, and loaded on SDS-PAGE gels for analysis. Silver staining was used to visualize proteins and estimate their sizes and migration positions. For identification of CDC48, the 75-100 kD region of a Coomassie Brilliant Blue-stained SDS-PAGE gel slice was subjected to in gel trypsin digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Scaffold (Proteome Software) and Mascot database searches were used to interpret the results.

In vivo retrotranslocation assays

The method used was adapted and modified from similar approaches commonly applied in ERAD studies (21). First, SP1-HA and ubiquitin were transiently overexpressed in 10 ⁶ protoplasts for each genotype to increase detection sensitivity for higher molecular weight (ubiquitinated) forms of SP1 substrate (73). The transformed protoplasts were incubated for 15 h, and then bortezomib was applied to a final concentration of 5 mM before an additional 3 h incubation. Subsequent fractionation steps to produce separate chloroplast and cytosol samples were all carried out on ice or at 4°C, and used previously described procedures with modifications (56, 74). Protoplasts were pelleted by centrifugation at 100g for 2 min, and gently resuspended with protoplast-washing buffer (500 mM mannitol, 4 mM 4-morpholineethanesulfonic acid [MES]-KOH, pH 5.6). Then, the protoplasts were pelleted again and resuspended by gentle agitation in 500 pi HS buffer (50 mM 4-(2- hydroxyethyl)piperazine-1-ethanesulfonic acid [HEPES]-NaOH, pH 8.0, 0.3 M sorbitol) containing 0.5% PPIC and 5 mM bortezomib, and gently forced twice through 10 pm nylon mesh to release chloroplasts. The collected flow-through was centrifuged at 1 ,000g for 5 min to produce a chloroplast-containing pellet and a cytosol-containing supernatant (S1). The pellet was gently resuspended in 500 mI HS buffer, and the chloroplasts were purified by a two-step Percoll (Fisher Scientific) gradient (34). Intact chloroplasts were washed with 500 mI HS buffer, and then pelleted by centrifugation at 1 ,000g for 5 min. The S1 sample was centrifuged at 10,000g for 15 min. The resulting supernatant (S10) was recovered and ultracentrifuged at 100,000g for 1 h, producing a further supernatant (S100) that was concentrated to 50 mI by using Vivaspin 500 ultrafiltration spin columns; this was the cytosolic fraction. The pelleted chloroplasts were lysed in 100 mI denaturing buffer (25 mM Tris-HCI, pH 7.5, 150 mM NaCI, 5 mM EDTA, 10 mM NEM, 1% SDS, 2% Sarcosyl, 5 mM dithiothreitol [DTT]) containing PPIC, while the cytosolic fraction was mixed with 50 mI

2*denaturing buffer containing PPIC. Finally, the SP1-HA protein was purified by immunoprecipitation using a previously described procedure to improve sensitivity of detection of the ubiquitinated protein (7). Experiments were repeated three times, and similar results were obtained.

Statistical analysis

Statistical calculations (mean, standard error of the mean, t-test) were performed using Microsoft Excel software. Statistical significance of differences between two experimental groups was assessed by using a two-tailed Student’s t-test. Differences between two datasets were considered significant at p <0.05.

Results

As shown in Figures 1A - F, and Figures 2A and B, double-mutant sp2 ppH plants were larger and greener than the ppi1 progenitor and exhibited substantial improvements in chloroplast development and protein import capacity. Three independent mutant alleles of sp2 suppress the ppi1 mutation, in similar fashion to sp2-1. The sp2-2 ppi1 and sp2-3 ppi1 mutants were identified using the same EMS mutagenesis screen as sp2-1 ppH. Allelism tests confirmed that all of these mutations are allelic. All three sp2 ppi1 mutants were backcrossed to ppi1 three times before analysis. Subsequently, the sp2-4 mutant (SALK_137135) was obtained from SIGnAL via the Nottingham Arabidopsis Stock Centre. It was crossed with ppi1, and the sp2-4 ppi1 double mutant was selected and verified by phenotype analysis and using PCR-based genotyping. All sp2 ppi1 and control plants were grown under standard conditions on MS agar medium for 10 days before

photography (Figure 2A). Chlorophyll was measured photometrically following DMF extraction using plants similar to those shown in Figure 2A. About 10 seedlings per genotype were measured in each experiment, and the values shown are means ± SEM derived from four experiments per genotype. In Figure 2B the data indicate nmol total chlorophyll per mg tissue fresh weight. The chlorophyll value for sp2-2 was highly statistically significantly different from that for sp2-1 (Student's t-test, p <0.003), suggesting that sp2-2 is a weak allele.

The SP2 locus was identified using a combination of genetic mapping and whole-genome sequencing. Figure 3A shows genetic mapping of the sp2 locus. Initial analysis of an F ₂ population of 190 individuals from crosses between sp2-1 ppi1 (Col-0 ecotype) and ppi1 (introgressed into the Ler ecotype) placed the sp2 locus to the south of marker F21A17 at position 12285000 on the upper arm of chromosome 3. A second mapping population of 192 plants placed the sp2 mutation to the south of a more southerly marker, MJI6-2 at position 12597802 on the upper arm of chromosome 3. However, the genetic mapping failed to define a southern boundary concerning the location of sp2, owing to the existence of an“island” of Col-0 DNA in the Ler-introgressed ppi1 line, around the chromosome 3 centromere. Numbers of recombinants at key markers are shown.

Figure 3B shows Identification of the sp2 mutations by whole-genome sequencing. The figure shows 40 bp of the alignment of sp2-1 ppi1 reads to the ppi1 reference genome.

The vertical grey lines represent the read coverage at each base (this is 34 at the mutated site), and the horizontal grey lines indicate individual sequence reads. Positions in different shading in the centres of the reads show the mutated site (C-to-T on the strand shown; the brightness of the red colour indicates the mapping quality) that is responsible for the sp2 phenotype. The SP2 (At3g44160) gene is coded on the reverse strand relative to the reference genome, and so the complement of the SP2 gene, in the ppi1 reference sequence, is shown at the bottom. Genomic position in the ppi1 reference genome is indicated at the top of the figure. Similar results were obtained for the sp2-2 and sp2-3 alleles, although the mutation sites within the gene differed. Figure 3C is a schematic representation of the SP2 (At3g44160) gene, annotated with the positions of the sp2 mutations. Information on the nature and consequence of each mutation is shown. The positions of PCR primers used in D are indicated with arrows. Black boxes and interconnecting black lines, exons and introns, respectively; white boxes, untranslated regions; LB, left border sequence of the SALK T-DNA insertion.

Figure 3D is an analysis of SP2 mRNA expression in each of the sp2 mutant alleles by RT- PCR. The primer pairs used in each case are indicated at left, and their positions are shown in Figure 3C. Amplicon sizes are indicated at right. The sp2-1 allele carries a splice site mutation and exhibited slicing defects. Two different sp2-1 amplicons were detected, and sequenced: the larger, minor amplicon retains a portion of intron 10, and the corresponding transcript is predicted to encode a truncated protein; the smaller, more abundant amplicon carries a single guanine nucleotide deletion (at position 924 of the CDS, in exon 10), causing a frameshift such that the corresponding transcript is also predicted to encode a truncated protein. This indicated that sp2-1, like sp2-4, is most likely a null allele. In contrast, sp2-2 and sp2-3 may be weak alleles, which is in line with the chlorophyll data shown in Figures 2A - F. The encoded protein (At3g44160; Figure 1G) is a member of the Omp85 superfamily of beta-barrels involved in protein transport, which are widely distributed in the outer membranes of bacteria, mitochondria and chloroplasts (13, 14).

The SP2 protein is of unknown function, but Figure 4A shows that it is broadly conserved in the angiosperms and closely related to the chloroplast outer membrane protein OEP80 (Toc75-V) (75); the function of OEP80 is also uncertain (15), although it has been proposed to mediate outer membrane protein biogenesis (16, 17) by analogy with well- characterized homologues in bacteria (BamA) and mitochondria (Sam50/Tob55) (13, 14). For Figure 4A, Bayesian inference phylogenetic analysis of SP2 and OEP80 was undertaken. Predicted amino acid sequences homologous to Arabidopsis SP2 and OEP80 from a variety of plants and algae were retrieved from the Phytozome 12 database (44). Bacterial Omp85 sequences were also obtained and were included in the analysis to act as an outgroup. Sequences were aligned using the MAFFT algorithm (45), and the phylogeny was inferred using MrBayes 3.2 software (46). The tree represents a 50% consensus of 6000 trees generated from two runs, each using 8 Markov chain Monte- Carlo (MCMC) chains for 8 million generations (the first 2000 trees were discarded as burn-in) where standard deviation of split frequencies was 0.001 at the end of the analysis. Posterior probability values are shown. Scale bar, 0.3 changes per site.

As shown in figures 5 and 6, SP2 is located in the chloroplast OEM, and it was previously shown to form a membrane channel (18). SP2 CDS was fused with sequence encoding a C-terminal 6*Myc tag and cloned downstream of the strong, constitutive CaMV 35S promoter in the pB2GW7 binary vector (50), and then the resultant SP2- Myc construct was used to stably transform sp2-4 ppi1 and sp2-4 plants. Approximately 12 T2 transformants for each genotype were analysed, and from these, representative single locus lines were selected for further investigation based on segregation of the resistance phenotype on petri plates containing phosphinothricin, SP2 mRNA expression, and phenotype analysis. The SP2-Myc construct could fully complement the phenotype of sp2- 4 ppi1, indicating that the Myc tag does not affect the function of SP2. Therefore, SP2- Myc sp2-4 plants selected in this way were analysed as shown in in Figure 5). Chloroplast localization and topology of SP2 were analysed by chloroplast isolation and subsequent protease treatment. Chloroplasts were prepared from 14-day-old wild-type (WT) and SP2- Myc sp2-4 plants. The isolated SP2-Myc sp2-4 chloroplasts were treated: with thermolysin or trypsin at different concentrations (100 or 500 pg/ml); with either protease plus 1%

Triton X-100 detergent (TX100); or, with buffer lacking protease (Mock). Then, the samples (together with a wild-type chloroplast control; Mock) were analysed by SDS- PAGE and immunoblotting using antibodies against the Myc tag and a number of chloroplast proteins. Immunoblotting analysis showed that SP2-Myc is localized in chloroplasts. Specificity of the Myc signal was verified by comparing the WT and SP2-Myc Mock chloroplasts. Partial resistance to thermolysin indicated that the C-terminal tag of SP2-Myc is oriented towards the intermembrane space, as this protease does not penetrate the outer membrane (so that only protein domains exposed at the cytosolic surface are fully sensitive to thermolysin) (58); assuming that SP2 has an even number of transmembrane beta-strands, one may infer that its N-terminus is also located in the intermembrane space. In this assay, SP2 behaved similarly to Toc75, which is as expected as our Toc75 antibody reacts with the N-terminal POTRA domain that is localized in the intermembrane space (77, 78). Tod 59 has a large cytosolic domain and was almost completely degraded by thermolysin, confirming efficacy of the treatments. Unlike thermolysin, trypsin can penetrate the outer membrane to the intermembrane space (58). Increasing trypsin concentrations progressively depleted SP2-Myc, like Toc75, which indicated that SP2 is an outer membrane protein with similar topology as Toc75. In contrast, TIC-associated proteins located largely or wholly in the stroma (Tic40 and Hsp93, respectively) were not sensitive to trypsin, indicating that the protease was behaving as expected. When co-applied with Triton X-100 to solubilize the envelope membranes, both proteases completely degraded SP2-Myc, confirming that the signals observed were due to protection by the membranes and not a result of intrinsic protease-resistance.

In more detail, for Figure 6, chloroplasts were isolated from 14-day-old wild-type and SP2- Myc sp2-4 plants, and subsequently lysed with hypotonic buffer (25 mM HEPES, pH 8.0, 4 mM MgCI2); the chloroplast pellet was broken up with a sterile plastic pestle and then the sample was rotated at 4°C for 1 h to ensure efficient lysis. The membranes were recovered by ultracentrifugation at 110,000g for 1 h at 4°C, and were then subjected to immunoprecipitation using anti-c-Myc antibody. Input (i.e. , before immunoprecipitation was initiated; Chloroplast lysate), flow-through, and immunoprecipitated (Elution) samples were then analysed by immunoblotting using antibodies against: the Myc tag, to verify the enrichment of SP2-Myc; the TOC components Tod 59, Toc75 and Toc33, to detect putative SP2-partner interactions; and, Tid 10 and Tic40, to assess whether the above interactions are specific. In addition, whole-plant protein extracts (Total protein) were prepared from wild-type and SP2-Myc sp2-4 plants (equivalent to those from which the chloroplasts were isolated), and analysed in parallel. Enrichment of SP2-Myc in the chloroplast lysates (relative to the total protein samples), in a similar way to other chloroplast proteins, further confirmed that SP2 is localized in chloroplasts. The SP2-Myc sp2-4 plants analysed here are as described in relation to Figure 5.

Unlike OEP80, SP2 lacks an N-terminal POTRA domain (such domains typically mediate protein interactions), as shown in Figure 1G, suggesting that the two proteins have functionally diverged. Figure 4B shows structural models for the Arabidopsis SP2 and OEP80 proteins. Three-dimensional models for SP2 and OEP80 were derived by homology modelling using the crystal structures of bacterial TamA and BamA proteins, using the Phyre2 server (43). Both protein models are oriented with the N-terminal domain facing downwards. The beta-barrel and polypeptide transport associated (POTRA; OEP80 only) domains are indicated. OEP80 and SP2 have opposing effects on TOC protein abundance, as discussed previously (OEP80 knockdown depletes TOC proteins (16)) and below.

Referring to Figures 7 and 8, in addition to ppi1, two other TOC mutations (hypomorphic alleles of the genes encoding Tod 59 and Toc75) (16, 19) were suppressed by sp2, whereas mutations that cause chlorosis for other reasons were not suppressed. This implies a close functional relationship between SP2 and the TOC apparatus, a notion that is supported by the restored accumulation of Toc75 in sp2 toe double mutants (see Figure 1 , H - K, and Figure 9). In all of these respects, the sp2 mutants were phenotypically very similar to sp1 mutants (7).

In more detail for Figure 7, panels A and C shows assessment of the effect of sp2 on the chlorotic phenotypes of two additional TOC mutants. The sp2-4 mutation was introduced into the toc75-lll-3 (mart, affecting Toc75) (16) and pp/2-3 (fts1\ affecting Toc159) (19) mutant backgrounds by crossing. Double mutants were identified as described in relation to Figure 2, and subsequently grown alongside wild-type and single-mutant controls under standard conditions on soil for 22 days (A), or on MS agar medium for 10 days (C), prior to photography; typical plants are shown. Both TOC mutations were suppressed by sp2, indicating that the effect of the latter is not specific to ppi1. In Figures 7B and D, chlorophyll contents in the genotypes shown in Figures 7 A and C were measured photometrically following DMF extraction. About ten 10-day-old seedlings per genotype were measured in each experiment, and the values shown are means ± SEM derived from four experiments per genotype. The data indicate nmol total chlorophyll per mg tissue fresh weight.

In more detail for Figure 8, panel A shows assessment of the effect of sp2 on the chlorotic phenotypes of two mutants with protein import defects linked to the TIC apparatus of the inner envelope membrane. The sp2-4 mutation was introduced into the hsp93-V-1 background (30) by crossing. The sp2-1 mutation was isolated from the ppi1 background by crossing to wild type followed by PCR genotyping, and then introduced into the tic40-4 background (30) by further crossing. Double mutants were obtained as described in relation to Figure 2, and subsequently grown alongside appropriate control genotypes under standard conditions on MS agar medium for 10 days prior to photography; typical plants are shown. Neither TIC-associated mutant was suppressed by sp2, indicating that the suppression effect of the latter is specific. Figure 8B shows chlorophyll contents in the genotypes shown in Figure 8A were measured photometrically following DMF extraction. About ten 10-day-old seedlings per genotype were measured in each experiment, and the values shown are means ± SEM derived from four experiments per genotype. For comparison purposes, the individual measured values (nmol per mg tissue fresh weight) were expressed as percentages of the corresponding wild-type value.

In more detail for Figure 9, this shows total RNA samples isolated from 10-day-old plants of the indicated genotypes analysed by quantitative real-time PCR using gene-specific primers for atTOC75-lll (A), or atTOC75-lll, atTOC159 and atTOC33 (B). Expression data for the TOC genes were normalized using equivalent data for two reference genes:

GAPDH and ACTIN2 (ACT2). For each genotype tested, four biological replicates were analysed with three technical replicates of each, and the values shown are means ± SEM (n = 4 biological replicates).

Overexpression of SP2 triggered the specific depletion of TOC proteins (see Figures 1 L and M), resembling closely the effect of SP1 overexpression (7). Like SP1 , SP2 interacted physically with TOC components (see Figure 6). Further similarities with SP1 were observed when sp2 mutant and SP2 overexpressor plants were analysed physiologically, in relation to leaf senescence (see Figure 1 N) and abiotic stress tolerance (see Figure 10 and Figure 10). Activity of SP1 promotes both of these processes (which it does by reconfiguring the chloroplast protein import machinery to produce the necessary organellar proteome changes) (7, 8), and a similar pattern was observed here for SP2 (see Figures 1 N and O).

In more detail for Figure 10A - B, these show phenotypic analysis of sp2 single-mutant and SP2 overexpressor (OX) plants. The three original sp2 mutants were isolated from the ppi1 background by crossing to wild type, followed by PCR genotyping. The SP2 CDS was cloned downstream of the 35S promoter in the pB2GW7 binary vector (50), and the resultant construct ( SP2-OX) was used to stably transform wild-type plants. Using procedures similar to those described in relation to Figure 5, representative single-locus lines were chosen for further analysis, based on the segregation of phosphinothricin resistance and the level of SP2 mRNA overexpression (see Figure 10C and D); note that the selected transformants are also shown in Figure 1 L (#12 and #15), and in Figures 11A - G and Figure 12G and H (#12 only). Images of typical plants (Figure 11A) and chlorophyll content measurements determined photometrically following extraction in DMF (Figure B) are shown. In each case 10- day-old seedlings grown under standard conditions on MS agar medium were analysed. For the chlorophyll assays, about 10 seedlings per genotype were measured in each experiment, and the values shown are means ± SEM derived from four experiments per genotype. The data indicate nmol total chlorophyll per mg tissue fresh weight.

In more detail for Figures 10C - D, these show semi-quantitative RT-PCR analysis of SP2 expression in wild-type and SP2-OX plants. Total RNA samples isolated from 10-day-old plants were analysed by RT-PCR using gene-specific primers for SP2 and the reference gene ACT2. Amplifications employed a limited number of cycles to avoid saturation, and products were analysed by agarose gel electrophoresis and staining. A representative gel image is shown (Figure 10C), along with quantification of four biological replicates (Figure 10D). The amplicon bands were quantified using Aida software, and the data obtained for SP2 were normalized relative to equivalent data for ACT2. Values shown are means ± SEM (n = 4).

In more detail for Figures 11A - D, these show analysis of an sp1 sp2 ppi1 triple mutant. Triple mutant plants were compared with both sp ppi1 single mutants in relation to the extent of suppression of ppi1. No phenotypic additivity in the triple mutants was apparent upon analysing visible phenotypes (Figure 11 A), chlorophyll contents (Figure 11 B), or the abundance of Toc75 protein by immunoblotting (Figures 11C and D). In Figures 11 E - G, there is analysis of plants simultaneously overexpressing (OX) both SP1 and SP2. Double OX plants were compared with both single SP-OX genotypes, revealing a synergistic interaction in relation to visible phenotype (Figure 11 E, right), chlorophyll content (Figure 11 E, left), and TOC protein depletion as analysed by immunoblotting (Figures 11 F and G).

In more detail for Figures 12G and H, these show analysis of the role of SP2 in

retrotranslocation of SP1-HA substrate. In vivo retrotranslocation assays were performed using protoplasts from wild-type, sp2 mutant, and SP2- OX plants. Typical immunoblotting results are shown (Figure 12G), along with quantification (Figure 12H). All values are means ± SEM (n = 3-4 experiments).

Figure 13 shows how SP1 and SP2 proteins associate to form a complex. Figure 13A shows a two-dimensional (2D)-blue native (BN)/SDS-PAGE analysis of SP1 and SP2. The SP2 CDS was fused with sequence encoding a C-terminal 6*Myc tag and cloned downstream of the 35S promoter in the p2GW7 vector. The resulting SP2-Myca construct, together with the SP1-HA construct (7), were used to co-transform Arabidopsis

protoplasts. Chloroplasts were isolated from the transformed cells as described previously (56), solubilized in BN-PAGE sample buffer containing 1% DDM, and subjected to 2D- BN/SDS-PAGE followed by immunoblotting using the indicated antibodies. Molecular weights (MW) of standards analysed in the first dimension are shown at the bottom in kD. The SP1-HA and SP2-Myc proteins co-migrated at two different positions: the first (>669 kD) overlapped with the TOC complex (80, 81) (and may indicate an intermediate step preceding TOC component degradation); the second (232-440 kD) may correspond to a resting or inactive core complex for CHLORAD (see discussion below).

Figure 13B shows an In vitro pull-down analysis of the association between SP1 and SP2. In vitro translated (IVT), 35S-radiolabelled SP2 or OEP80 (as a control OEM protein) were used as“prey” in pull-down assays with bacterially-expressed, purified GST-SP1flex or an excess of GST (negative control) as the“baits”. Eluted GST proteins, along with any associated partner proteins, were resolved by SDS-PAGE and then analysed by phosphorimaging (to detect the radiolabelled“prey” proteins) or Coomassie Brilliant Blue staining (to detect the GST“baits”). The result showed that SP1 specifically associates with SP2 in vitro.

An absence of phenotypic additivity in sp1 sp2 double mutants (in the ppi1 background), in relation to plant greening and Toc75 protein accumulation (Figure 12A - D), supports SP1 and SP2 functioning together. Indeed, SP2 is essential for SP1 action, as the sp2 mutation abrogated the effect of SP1 overexpression, as shown in Figure 14.

In more detail for Figure 14, the sp2 mutation was introduced into the SP1-OX ppi1 background (7) by crossing the latter with the sp2-4 ppi1 double mutant. A resultant F ₂ population was grown on phosphinothricin plates to select for the SP1-OX construct, and the phosphinothricin-resistant plants were PCR genotyped to identify those that were sp2- 4 homozygous. Individual F3 families were grown on phosphinothricin plates to select those that were homozygous for the SP1-OX construct. The SP1-OX sp2-4 ppi1 plants thus identified were then, together with corresponding control genotypes, grown on MS agar medium for 10 days before photography (Figure 14A) and photometric chlorophyll content analysis following DMF extraction (Figure 14B). About 10 seedlings per genotype were measured in each experiment, and the values shown are means ± SEM derived from four experiments per genotype and indicate nmol total chlorophyll per mg tissue fresh weight. As reported before (7), SP1-OX enhanced the chlorotic phenotype of ppi1 by making the plants even paler. This effect of SP1-OX on ppi1 was abolished by the sp2-4 mutation, indicating that SP1 action is dependent on SP2.

Moreover, whereas the overexpression of neither SP1 nor SP2 individually affected plant greening in the wild-type background under standard conditions (see Figure 10), the simultaneous overexpression of both genes caused strong chlorosis linked to severe depletion of TOC proteins (see Figure. 3E - G), indicating functional interdependency between SP1 and SP2. Such interactions are often observed where the components interact physically, and may arise through mutual stabilization (26, 27). Indeed, SP1 and SP2 co-migrated (with each other and with the TOC apparatus) on native gels and interacted specifically in vitro (see Figure 13).

Chloroplast-Associated Protein Degradation (CHLORAD).

Without wishing to be bound by any particular hypothesis, the inventors consider that SP1 and SP2 exist at the core of a system for chloroplast protein removal, designated

Chloroplast-Associated Protein Degradation (CHLORAD). This system targets chloroplast substrates for degradation, either as a homeostatic, quality-control process for the removal damaged proteins under stress, or as a regulatory mechanism to control plastid

development and functions. Figure 16 is a detailed diagram showing the proposed model of action of the SP1 and SP2 proteins that the inventors have.

The full length genomic sequence of SP2 At3g44160 [SEQ ID NO:1] is:

1 AAAAATATCC AAAGCATCAA ATCCTTAACC TCTCTGCTAA TTCATTCACT

51 CCTGAAGAAG AAGAAAGAAG AAATAAGTAA ATAAAAATTC CTCCTTTTTC 101 TGGTCATTGC TTGTCTAATG CCAATTCCTA AATTGGGTTC TCTTCATGGT 151 TGATTCTTCT CTCATTCCAT CGCCATGGGA GCTCAGAAGA GTATCCACGC 201 TGGTAGAGGT CATCACTCTT ATCTTAGATT CTCGATTTTT CGAATTTTGT 251 GTTGTTGCTG GATCAGTAGG TAATGTGATA CTGTGCTGGA AATGGTACGA 301 TTCTGGAATT TGATTGTGGA TTTAACCTTA ACCAATGGAG GGCTTTGCTT 351 TGTTTCCATT TTTCTTCTAT TATCTTACAT TCATTTTCAA TGTATAAGAT 401 ATGATTCATT TCATTTGTAT AGTTATGATG ATGTTTATGT TTGTGGTAAT 451 TGATGAATTA GAATGTTTTA GGCATGAGTA CACATTGTTT TTTTTGGTAA 501 AAGAAACATT TTTCTAAGAC TTTTGTTACC AACCATTATG TGTGATACGT 551 CTTGCTTTTG GGTTTTGTGA AAGATTGGTT TTGATATGTG GATTTCCTAA 601 ATGTCTTTTT GGGGAAAAGT ATCGATTTTG ATCTGAATGT TGTAACTTTT 651 TCTGTATCTT TTTGTTACTA ACTCATCATG TCGAATATGT CTTTCTGTTG 701 AATATAGTGA AAGATTAAGA TCGGACTGGT TTGCAATTGC AATGTAGTTT 751 TCTAGCAGCG GCATTGATTT TGATATTCTT GTGTTGTTGA TTGCAGCCAA 801 GATTGATGTT AATGTGGACT TCACTCACAA GCTTTGTACT TCTTTAATGT 851 TTCCTGCTTT CAGGTTTGCT CTCATCATCC ATACTTTGAC GTCACTTACA 901 TTTTAGCTAA TACTCATGAA ACTGTACCGC CTTTAGCTTT CTCGACGTAT 951 TAAAGCTCAG TTTGTTACTT TTTGAACAGG GACACTAGCA GTCCTCTTTC 1001 TCTAGTGATT GGCAGGCAAG TTTCGAGACT TAATGCTACA GCTTACAGAA 1051 ATATTGTTGG TGTTCATATA ATCTGCAGAA CTAAATTTTC ATATCTTTCT 1101 TTTCGATTCA AGTGTTCATT TGGATCTGTT TGTTGCAGCC TCTGTATCAA 1151 ACATCCAAAT TTGTTTGGAG GAAGCGAGAA GCTTGATGTA TCATGGGATA 1201 AAGGATTGTA TGATTCCAAT GTACTTGTGG CTTTTAGGAG ACCTAGACCT 1251 GAATGGCGTC CACAACAGTG TTTCTTCATA CAGGTACTCA ATGCTTATTT 1301 GTTATTGTAC TGATCTGTGA AGCTATCTTA GAAAGTGAAT TTTAAAGCTA 1351 TGTTACATTT AAATTTGTTA ATCTGGCGCA GCATTCTCTC TCACCCGAGA 1401 TAGGGGTCCA CGGCACCCCA GTCGACAACT TTTCTCGGTC AGGAAGTGGA 1451 GGTGTAAATC TGTCTAAATT GGCTCTTGGT TTAGACTTGA GTGAGCCAGC 1501 GAGTTCAAAA TGGAGCAGCA CAACCAGCAT AAAGTTTGAG GTGCCCGCAC 1551 ATTACCTTCT TCAGACATTG TAGAACAATA TTTTTCTCTT TGCTGTTTTG 1601 CTTTGGTATA AAAGAGTAAT ATTTTCCATT GGTGCAGCAT GTGCGTCCGA 1651 TTAACGATGA TGGACGCGCG ATAACCAGAG ATCTGGACGG ATTTCCTATA 1701 ACATGCAGGT GATATTAGAT CTCGATTCCC TTAATTTGTT TCTTTAAGTA 1751 CTAGCATAAA ACTGATATTT ACATGTGTAG AATTCTCTGC TATGCAGTGG 1801 AAATACCCAT GACAGTATGG TAGTTTTAAA GCAAGAATCC CGGTTTGCAA 1851 AGGCTACCGA CCAAGGTCTT TCTCATGTAA GAGACTCTTC ATTTCTGTTT 1901 TATAGCCTAG GCAAACCACA CAGCCATTTT TGCAGTAACT TTGTCAACGT 1951 TTCTTTTCTT TCATACTGGT TTCTTGCTTT TTCAGTTTAG CATGCAAATA 2001 GAACAAGGTA TTCCGGTTGT GTCCAAGTGG CTTATCTTCA ACCGTTTCAA 2051 ATTTGTTGCA TCAAAAGGTG TCAGGTTTGG ACCGGCTTTT CTCTTAGCAA 2101 GGTACTGACA GAATCGTACA CACTTGATCT AGAAAACTAC ACGTAGAAGA 2151 TGCTTTGTAA AGATGTCCTA GTTTTGCCGT GGTTCTTACC TCTTCTGTGA 2201 CAGCTTGACA GGTGGTTCAA TTGTAGGAGA CATGGCACCT TATCAAGCAT 2251 TTGCCATTGG TGGGCTAGGC AGTGTTCGCG GATATGGTGA GGGTGCTGTT 2301 GGATCCGGTC GGTCATGCCT TGTTGCCAAT ACAGAATTGG CGTTACCTTT 2351 GGTACGTGGA GCTGTTGCAT GATTTGGTCA TCAGAACCAT TAAATGGTTT 2401 CCCCTGTTAA GCTTTCATTG AGCTTTGGTG TCTTTTTGTG TACAGAACAA 2451 GATGACAGAA GGGACCATCT TCTTGGATTG TGGAACAGAC CTAGGCTCAA 2501 GCCGCCTTGT CCCCGGTAAG TTCACTTTAC CTTCTCCGAA CAAGAATCAT 2551 AAAGAAGTTG AGACACAAAA ATGATAAAGC TGTTGCGGAA TTGGTTTCAG 2601 GAAACCCTTC AATGAGACAA GGGAAGCCAG GGTTTGGGTA TGGATTCGGA 2651 TATGGGCTAC GGTTCAAGTC TCCATTGGGT CACCTTCAGG TTGACTATGC 2701 CATAAATGCT TTCAACCAGA AGACTCTTTA CTTCGGTGTC ACCAATCTTG 2751 CTTCATCAAC ATAGTCAAAT AGAATAAAGC AATCAAGAAA AGCACATATT 2801 CAATGTCTTG ATTCAAAGAT ATATTTGTGT TTGCGTTGGA ACCAGAAACG 2851 CCACAAGATG AGGCAAACAC AGTCCAAGGA GAAGCTGTAT ATGACAGAGA 2901 TCTTGAGAAG ATAAATGTAG TGTTGTCATT AGAAATCATG TAATATTACA 2951 CGGGTATAAG TTTTCATTGT TTTGGTATAT ACAGCTACCA GAGTTTTGTC 3001 TGAAAAGCTG CAGGTTTCAT AGAGAAGAGA ACACACATTT GATTTGATGG 3051 TGTTGCACTG TTGCGAATTA GTATCTATTG CTTTTACATT TGTACGTATA 3101 TCTATGGCTC GACTCTCGAC TAACTCTGAA TACAAGTAGT GGTTGAACC

The full-length cDNA sequence of SP2 [SEQ ID NO: 2] is:

1 AAAAATATCC AAAGCATCAA ATCCTTAACC TCTCTGCTAA TTCATTCACT 51 CCTGAAGAAG AAGAAAGAAG AAATAAGTAA ATAAAAATTC CTCCTTTTTC 101 TGGTCATTGC TTGTCTAATG CCAATTCCTA AATTGGGTTC TCTTCATGGT 151 TGATTCTTCT CTCATTCCAT CGCCATGGGA GCTCAGAAGA GTATCCACGC 201 TGGTAGAGCC AAGATTGATG TTAATGTGGA CTTCACTCAC AAGCTTTGTA 251 CTTCTTTAAT GTTTCCTGCT TTCAGGGACA CTAGCAGTCC TCTTTCTCTA 301 GTGATTGGCA GCCTCTGTAT CAAACATCCA AATTTGTTTG GAGGAAGCGA 351 GAAGCTTGAT GTATCATGGG ATAAAGGATT GTATGATTCC AATGTACTTG 401 TGGCTTTTAG GAGACCTAGA CCTGAATGGC GTCCACAACA GTGTTTCTTC 451 ATACAGCATT CTCTCTCACC CGAGATAGGG GTCCACGGCA CCCCAGTCGA 501 CAACTTTTCT CGGTCAGGAA GTGGAGGTGT AAATCTGTCT AAATTGGCTC 551 TTGGTTTAGA CTTGAGTGAG CCAGCGAGTT CAAAATGGAG CAGCACAACC 601 AGCATAAAGT TTGAGCATGT GCGTCCGATT AACGATGATG GACGCGCGAT 651 AACCAGAGAT CTGGACGGAT TTCCTATAAC ATGCAGTGGA AATACCCATG 701 ACAGTATGGT AGTTTTAAAG CAAGAATCCC GGTTTGCAAA GGCTACCGAC 751 CAAGGTCTTT CTCATTTTAG CATGCAAATA GAACAAGGTA TTCCGGTTGT 801 GTCCAAGTGG CTTATCTTCA ACCGTTTCAA ATTTGTTGCA TCAAAAGGTG 851 TCAGGTTTGG ACCGGCTTTT CTCTTAGCAA GCTTGACAGG TGGTTCAATT 901 GTAGGAGACA TGGCACCTTA TCAAGCATTT GCCATTGGTG GGCTAGGCAG 951 TGTTCGCGGA TATGGTGAGG GTGCTGTTGG ATCCGGTCGG TCATGCCTTG 1001 TTGCCAATAC AGAATTGGCG TTACCTTTGA ACAAGATGAC AGAAGGGACC 1051 ATCTTCTTGG ATTGTGGAAC AGACCTAGGC TCAAGCCGCC TTGTCCCCGG 1 101 AAACCCTTCA ATGAGACAAG GGAAGCCAGG GTTTGGGTAT GGATTCGGAT 1 151 ATGGGCTACG GTTCAAGTCT CCATTGGGTC ACCTTCAGGT TGACTATGCC 1201 ATAAATGCTT TCAACCAGAA GACTCTTTAC TTCGGTGTCA CCAATCTTGC 1251 TTCATCAACA TAGTCAAATA GAATAAAGCA ATCAAGAAAA GCACATATTC 1301 AATGTCTTGA TTCAAAGATA TATTTGTGTT TGCGTTGGAA CCAGAAACGC 1351 CACAAGATGA GGCAAACACA GTCCAAGGAG AAGCTGTATA TGACAGAGAT 1401 CTTGAGAAGA TAAATGTAGT GTTGTCATTA GAAATCATGT AATATTACAC 1451 GGGTATAAGT TTTCATTGTT TTGGTATATA CAGCTACCAG AGTTTTGTCT 1501 GAAAAGCTGC AGGTTTCATA GAGAAGAGAA CACACATTTG ATTTGATGGT 1551 GTTGCACTGT TGCGAATTAG TATCTATTGC TTTTACATTT GTACGTATAT 1601 CTATGGCTCG ACTCTCGACT AACTCTGAAT ACAAGTAGTG GTTGAACC

The amino acid sequence of the SP2 protein [SEQ ID NO: 3] is:

1 MGAQKSIHAG RAKI DVNVDF THKLCTSLMF PAFRDTSSPL SLVIGSLCIK

51 HPNLFGGSEK LDVSWDKGLY DSNVLVAFRR PRPEWRPQQC FFIQHSLSPE 101 IGVHGTPVDN FSRSGSGGVN LSKLALGLDL SEPASSKWSS TTSIKFEHVR

151 PINDDGRAIT RDLDGFPITC SGNTHDSMW LKQESRFAKA TDQGLSHFSM

201 QIEQGIPVVS KWLIFNRFKF VASKGVRFGP AFLLASLTGG SIVGDMAPYQ

251 AFAIGGLGSV RGYGEGAVGS GRSCLVANTE LALPLNKMTE GTIFLDCGTD 301 LGSSRLVPGN PSMRQGKPGF GYGFGYGLRF KSPLGHLQVD YAINAFNQKT 351 LYFGVTNLAS ST

The full length genomic sequence of SP1 At1g63900 [SEQ ID NO: 4] exons are marked in upper case is:

ATGATTCCTTGGGGTGGAGTTACTTGCTGCCTCAGCGCCGCTGCTCTTTATCTTCT

CGGCCGGAGTAGTGGCAGgtttgtctgatctcttttatatttcatcttcccaaagag attatcaatcaatcaaatcc tttcttatccttttgagtgcagGGATGCTGAAGTACTCGAAACAGTCACTAGGGTTAATC AGCT

CAAGGAGTTAGgtaatcttcttctcccctgattgcttcatctactctcaggatgaag ttttgatcatgttttctgattgttctgt atgtgtagCT C AATT GCT AG AATT AG AT AGCAAG ATT CTGCCTTT CATT GTTGCGGTAT

CAGGAAGAGTCGGCTCT GAGACACCT ATCAAATGCGAGCAT AGTGGCAT ACGCG

GTGTTATTGTTGAAGAAACGgtatgttgtagactgatgattagcgcatggaacttag tttgttttctggtttaatcg attaggttttatgtgaaactctagtaattgcatgatcttttcagGCGGAACAACATTTCC TGAAACATAATG

AGACGGGTTCTTGGGT ACAAGAT AGT GCGTT GATGCT ATCAAT GAGCAAAGAGGT

TCCTTGGTTTCTGgtaagtctagtctagtagcatagtgtttgaaacaactgtgattg gatgttttcttaataccatttcc aaaactgtttgggatagGACGATGGGACAAGCCGTGTCCATGTAATGGGAGCTCGTGGTG

CG ACG G GTTTTG CCTTG ACTGTTG GTAGTG AAGTTTTTG AAG AGTCAG

GACGCTCGCTTGTACGAGGAACACTTGATTATCTTCAAGGACTTAAGgtttttactt ttctttccgg ttctttgtttgttggcttctctatttgcttaagcggccattgttttgtttcagATGCTTG GAGTTAAGCGCATTGAGCGT

GT TCTTCCAACTGGAATACCGCTAACAATTGTTGGTGAGgtatgtcgtattctcagtgtttt cgggtcct ctcttttgcttaagttgtaactgttgatagagatacatagcacactaactccttcatcag tctggtatttgcctcttgaaattttctc aaagttcctttaatagcaatatttgtaggaagtgggattgatctatgtatagaggcttac cgatgagtttaaatctaatttgtgtt gctgccatgtataacagGCTGTCAAGGACGATATTGGAGAATTCAGGATTCAAAAACCTG A

CAGGGGCCCTTTCTACGTCTCTTCTAAATCACTCGATCAGCTCATTTCTAATTTGG

GAAAATGGTCAAGgtcgtgtctctctcctctctcggttcttctcctatactcttgta gaaaaacggcaatgagccaaa ctgattgagaagagtataatttacagGTT GT ACAAAT AT GCCTCCATGGGTTTT ACT GTTCTTGG

TGT GTTCCT AATT ACG AAGCAT GT CATT G ACT CT GTT CT AGAG AG AAG ACGGCGG

AGACAGTTACAAAAAAGgtatgtcacagatttgtctgtctaaaagtgaataaccgtt ctcaagcatgagtactag atcggcttgtttctctcgaaactatgtacacacaaaattaagtagtcagctgtttttgca gAGTGCTTGACGCAGC

AGCAAAGAGAGCTGAGCTAGAGAGTGAAGgtatccattggtgaatctctttattcta catataggttgca ctggctctgactacaatctcttctgaccagGTTCAAACGGGACACGTGAGAGCATTTCAG ATTCTA

CCAAG AAAGAAGACGCT GTTCCT GAT CTCTGT GT GAT ATGCCT AGAGC AGG AGT A

CAACGCTGTGTTTGTCCCgtaagcattcttccgccatttttggttgattctgcattt gcaacttgctaaaatgcttgt ggttggtactcgcagGT GT GGTCAT AT GTGCTGCT GCACCGCAT GCTCCTCCCACTT GACCA

GCTGTCCACTTTGTCGGAGACGAATAGATCTGGCGGTTAAGACATATCGTCAC TGA

The full-length cDNA sequence of SP1 [SEQ ID NO: 5] is:

atgagaatattgagagagatcgaagcaaaggatcattcaattccaaccctctgaatc ttttaatttcccctttcgaaattctcc tcttctttcactgcttctagtttctaattcttcaaactcttcctcgattcatactcataa ctctcattagctaatttcgcatgatcttcttc catctctctgtgttctaaatccagattcgtttcactcccatctctatttcattcaattcg ctgcatccagattcaaaacctacctcta tctctctgctcatcaataacttcaaaggtattgttgttcttctgcaaacaagtaagagtg acttcagagtctgatgattccttggg gtggagttacttgctgcctcagcgccgctgctctttatcttctcggccggagtagtggca gggatgctgaagtactcgaaac agtcactagggttaatcagctcaaggagttagctcaattgctagaattagatagcaagat tctgcctttcattgttgcggtatc aggaagagtcggctctgagacacctatcaaatgcgagcatagtggcatacgcggtgttat tgttgaagaaacggcgga acaacatttcctgaaacataatgagacgggttcttgggtacaagatagtgcgttgatgct atcaatgagcaaagaggttcc ttggtttctggacgatgggacaagccgtgtccatgtaatgggagctcgtggtgcgacggg ttttgccttgactgttggtagtg aagtttttgaagagtcaggacgctcgcttgtacgaggaacacttgattatcttcaaggac ttaagatgcttggagttaagcg cattgagcgtgttcttccaactggaataccgctaacaattgttggtgaggctgtcaagga cgatattggagaattcaggattc aaaaacctgacaggggccctttctacgtctcttctaaatcactcgatcagctcatttcta atttgggaaaatggtcaaggttgt acaaatatgcctccatgggttttactgttcttggtgtgttcctaattacgaagcatgtca ttgactctgttctagagagaagacg gcggagacagttacaaaaaagagtgcttgacgcagcagcaaagagagctgagctagagag tgaaggttcaaacgg gacacgtgagagcatttcagattctaccaagaaagaagacgctgttcctgatctctgtgt gatatgcctagagcaggagta caacgctgtgtttgtcccgtgtggtcatatgtgctgctgcaccgcatgctcctcccactt gaccagctgtccactttgtcggag acgaatagatctggcggttaagacatatcgtcactgaacaacaactcaggcctcagaaac attctctacttgagtcttgtct gtaaataccgcaaaatcaaaacattacacagtttagcgttcgatattccctttggtttga tttcgacaacaaaacattttgaatt atatagaaacataaggtgtttactcgatttgcaaaacagtacattcgtgtttacttattc gtgttgttgccaatgccatgaggtg

The amino acid sequence of the SP1 protein [SEQ ID NO: 6] is

MIPWGGVTCCLSAAALYLLGRSSGRDAEVLETVTRVNQLKELAQLLELDSKILPFIVAV

SGRVGSETPIKCEHSGIRGVIVEETAEQHFLKHNETGSWVQDSALMLSMSKEVPWFL

DDGTSRVHVMGARGATGFALTVGSEVFEESGRSLVRGTLDYLQGLKMLGVKRIERVL

PTGIPLTIVGEAVKDDIGEFRIQKPDRGPFYVSSKSLDQLISNLGKWSRLYKYASMG FT

VLGVFLITKHVIDSVLERRRRRQLQKRVLDAAAKRAELESEGSNGTRESISDSTKKE DA

VPDLCVICLEQEYNAVFVPCGHMCCCTACSSHLTSCPLCRRRIDLAVKTYRH

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of them mean“including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

References

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