SUN, Sai Ming, Samuel (10A Staff Residence, Block 16The Chinese University of Hong Kong,Shatin, Hong Kong, Kong, CN)
SHAO, Guihua (Rm 1607, Block Gao-3The Chinese Academy of Agricultural Sciences12 Zhong Guan Cun Nan Da JieHai Dian District, Beijing, Beijing, CN)
LAM, Hon Ming (Rm 702, Daisy Li HallNew Asia College,The Chinese University of Hong Kong,Shatin, Hong Kong, Kong, CN)
SUN, Sai Ming, Samuel (10A Staff Residence, Block 16The Chinese University of Hong Kong,Shatin, Hong Kong, Kong, CN)
SHAO, Guihua (Rm 1607, Block Gao-3The Chinese Academy of Agricultural Sciences12 Zhong Guan Cun Nan Da JieHai Dian District, Beijing, Beijing, CN)
CLAIMS
1. A method of protecting a plant or plant cell from abiotic stress comprising modifying said plant or plant cell to produce an enzyme having a purple acid phosphatase (PAP) activity targeted to the mitochondria.
2. The method of claim 1 wherein the abiotic stress is one or more conditions selected from the group consisting of enhanced salinity, osmotic, and oxidative stress.
3. The method of claim 1 wherein said plant or plant cell has been modified to contain a heterologous nucleic acid which comprises a nucleotide sequence or a nucleotide sequence substantially identical thereto encoding an enzyme having a PAP activity fused to a mitochondrion-targeting transit peptide (MTTP).
4. The method of claim 3 wherein said enzyme having a PAP activity is soybean GmP AP3.
5. The method of claim 3 wherein the portion of said nucleotide sequence encoding the MTTP is heterologous to the portion of said nucleotide sequence encoding the enzyme having a PAP activity.
6. A transgenic plant cell which contains a heterologous nucleic acid which comprises a nucleotide sequence or a nucleotide sequence substantially identical thereto encoding an enzyme having a PAP activity fused to a mitochondrion-targeting transit peptide.
7. A recombinant nucleic acid construct which comprises a nucleotide sequence or a nucleotide sequence substantially identical thereto encoding an enzyme having a PAP activity fused to an MTTP operably linked to control sequences operable in plant cells.
8. The nucleic acid construct of claim 7 wherein the portion of the nucleotide sequence encoding the enzyme having a PAP activity is heterologous to the portion of the nucleotide sequence encoding the MTTP.
9. The nucleic acid construct of claim 7 or 8 wherein the control sequences are heterologous to the nucleotide sequence encoding an enzyme having a PAP activity and a fused MTTP.
10. The nucleic acid construct of claim 9 wherein the portion of said nucleotide sequence encoding the enzyme having a PAP activity is soybean GmPAP3.
11. The nucleic acid construct of claim 7 further including a second nucleotide sequence encoding a desired protein operatively linked to control sequences operable in plants.
12. A method of selecting for transformed cells or plants wherein the method comprises applying abiotic stress to cells or plants subjected to transformation with a recombinant construct comprising a nucleotide sequence or a nucleotide sequence substantially identical thereto encoding an enzyme having a PAP activity fused to MTTP as a selectable marker, whereby cells or plants that are resistant to said abiotic stress are selected.
13. The method of claim 12 wherein the abiotic stress is high salinity, osmotic and/or oxidation stress.
14. The method of any of claims 1-5 wherein said protecting results in maintenance of intact mitochondria, and/or in diminishing ROS, and/or maintaining viability.
15. A transgenic plant cell which contains a nucleic acid construct of any of claims 7-11.
16. The transgenic plant cell of claim 6 wherein the enzyme having a PAP activity is soybean GmP AP3.
17. A method for preparing a plant or a plant cell resistant to abiotic stress comprising modifying a plant or a plant cell to produce an enzyme having a purple acid phosphatase (PAP) activity targeted to the mitochondria.
18. The method of claim 17 wherein the abiotic stress is one or more conditions selected from the group consisting of enhanced salinity, osmotic, and oxidative stress.
19. The method of claim 17 wherein said plant or plant cell has been modified to contain a heterologous nucleic acid which comprises a nucleotide sequence or a nucleotide sequence substantially identical thereto encoding an enzyme having a PAP activity fused to a mitochondrion-targeting transit peptide (MTTP).
20. The method of claim 19 wherein said enzyme having a PAP activity is soybean GmP AP3.
21. The method of claim 19 wherein the portion of said nucleotide sequence encoding the MTTP is heterologous to the portion of said nucleotide sequence encoding the enzyme having a PAP activity. |
METHOD TO ALLEVIATE ABIOTIC STRESS IN PLANTS
Technical Field
[0001] The invention is in the field of plant genetic engineering. More specifically, it concerns the use of a purple acid phosphatase (PAP) protein targeted to the mitochondrion to alleviate stresses caused by salinity and drought and by oxidative stress.
Background Art
[0002] Plants may be subjected to a wide variety of stress conditions that are induced by non-living agents or circumstances such as drought, enhanced salinity, temperature stress, and the like. Deviations from the norm in either direction will constitute an abiotic stress. It is understood that hyper salinity and drought lead to oxidative stress through the accumulation of reactive oxygen species (ROS) and metabolic changes in the plant will generally arise as a result of these stress conditions. A general discussion of genes that are regulated in response to stress is set forth in U.S. Patent Publications 2002-0160378 and 2004-0009476, which appear to be based on the same application.
[0003] The present inventors, in an article appearing in 2003 (Liao, H, et al., GENE (2003) 318:103-111) cloned and sequenced a novel purple acid phosphatase-like gene in soybean, designated GmPAPi that was induced by enhanced salinity, but not by phosphorus deficiency as were many PAP genes. The authors further noted that the nucleotide sequence encoding this protein included a putative mitochondrion targeting transit peptide (MTTP) and speculated that the protein would reside mainly in the mitochondrion.
[0004] In the present application, it has been confirmed that the expressed GmP AP3 protein is located in mitochondria and can confer tolerance of plants or plant cells with respect to abiotic stress conditions.
Disclosure of the Invention
[0005] Applicants have demonstrated that the purple acid phosphatase of soybean is mainly present in the mitochondria of plant cells and is able to counteract the accumulation of reactive oxygen species (ROS). Thus, mitochondrial-directed PAP protein is able to counteract abiotic stress by increasing the percentage of cells that retain intact mitochondria, reducing the percentage of dead cells, and reducing the accumulation of ROS. This has as well the effect on inducing better root growth and less lipid peroxidation in response to herbicide application.
[0006] Accordingly, in one aspect, the invention is directed to a method of protecting a plant or a plant cell from abiotic stress in which the method comprises modifying said plant or plant cell to produce an enzyme having a purple acid phosphatase (PAP) activity targeted to the mitochondria.
[0007] hi another aspect, the invention is directed to recombinant expression systems comprising a construct useful for modifying a plant or plant cell to conduct the method of the invention, hi an embodiment of the invention, the construct is used to express an enzyme having a PAP activity targeted to the mitochondria.
[0008] hi another aspect, the invention is directed to use of the recombinant expression systems as selectable markers.
[0009] hi another aspect, the invention is directed to a transgenic plant or a plant cell which contains a nucleotide sequence encoding an enzyme having a PAP activity or a construct of the invention.
[0010] In another aspect, the invention is directed to use of an enzyme having a PAP activity targeted to the mitochondria in preparation of a construct, or a plant or plant cell containing the enzyme.
[0011] In yet another aspect, the invention is directed to use of an enzyme having a PAP activity targeted to the mitochondria in manufacture of an agent or a kit useful for preparing a construct or a plant or plant cell containing the enzyme.
[0012] hi yet another aspect, the invention is directed to a method for preparing or producing a plant or a plant cell resistant to abiotic stress comprising modifying a plant or a plant cell to produce an enzyme having a PAP activity targeted to the mitochondria. Therefore, a plant or a plant cell resistant to abiotic stress is obtained according to the methods of the present invention.
Brief Description of the Drawings
[0013] Figures 1A-1H show a comparison of wild type tobacco BY-2 cells (Figures IA, 1C, IE and IG) and transgenic tobacco BY-2 cells (Figures IB, ID, IF and IH) that have been modified to contain an expression vector for GmPAP3-T7 fusion protein.
[0014] Figures 2A-2B show immunodetection of GmP AP3. Figure 2A shows a Western blot indicating the presence of soybean GmPAP3 protein in mitochondria-enriched protein fractions from GmPAP3-T7 transgenic BY-2 cell lines.
[0015] Figure 2B shows electron microscope detection using the GmP AP3 antibody employed in Figure 2A in two independent GmPAP3-T7 transgenic cell lines (1535-1, 1535-2). Arrowheads indicate the location of gold particles that were mostly found in mitochondria. Scale bar = 200 nm.
[0016] Figures 3A-3L show the effect of GmP AP 3 on the ability of transgenic BY-2 cells to survive salinity and osmotic stress.
[0017] Figures 4A-4L show the effect of GmPAPS on the ability of BY-2 cell lines to remain viable in the presence of salinity or osmotic stress.
[0018] Figures 5A-5L are pictorial representations of the effect of expression of mitochondrion-targeted GmPAP3 on the ROS accumulation in BY-2 cells.
[0019] Figure 6 is a graphical representation of the results in Figures 5A-5L, a representation of the effect of expression of mitochondrion-targeted GmPAP 3 on the ROS accumulation in BY-2 cells.
[0020] Figure 7 shows the effect of GmP AP3 on the root growth of transgenic Arabidopsis under salt stress.
[0021] Figure 8 shows the effect of GmP AP3 on transgenic Arabidopsis under osmotic stress as measured by root growth.
[0022] Figure 9 shows the effect of GmP AP3 on root growth of transgenic Arabidopsis under oxidative stress provided by the herbicide Paraquat.
[0023] Figure 10 is a graph showing the effect of GmP AP3 on transgenic Arabidopsis under oxidative treatment as measured by lipid peroxidation.
Detailed Description of the Invention Definitions
[0024] The following definitions are set forth to facilitate explanation of the invention.
[0025] The term "purple acid phosphatases (PAPs)" as used herein represents purple acid phosphatases present in some bacteria, plants and animals, preferably, in plants. AU members of this family contain a characteristic set of seven amino-acid residues involved in metal ligation. PAPs of the present invention particularly refer to those with peroxidase activities or could be induced by stresses including high salinity, drought, and oxidative stress. Examples of such PAPs include, but are not limited to, soybean GmP AP3 (Liao et al, 2003 Gene 318:103-111), Arabidopsis thaliana AtACP5 (del Pozo et al, 1999
Plant J. 19 (5): 579-589), tomato IAP (Bozzo et al., 2004 Biochem J. 377: 419-428), tomato SAPl and SAP2 (Bozzo et al, 2002 Eur. J. Biochem 269: 6278-6286). Preferably, the PAP is soybean GmP AP3.
[0026] The term "substantially identical", as used herein to describe a degree of similarity between nucleotide sequences, refers to two or more sequences that have at least about 60%, preferably at least about 70%, more preferably at least about 80%, more preferably about 90% to about 99%, still more preferably about 95% to about 99%, and most preferably about 99% nucleotide identity, when compared and aligned for maximum correspondence. The substantial identity exists most preferably in nucleotide sequences comprising a full length coding sequence. The term "full length" as used herein to refer to a complete open reading frame encoding a functional PAP polypeptide or protein, or amino acid sequences of the polypeptide or protein. Preferably, the nucleotide sequence of the invention comprises a nucleotide sequence or a sequence substantially identical thereto encoding an enzyme selected from a group consisting soybean GmP AP3 (Liao, H, et al., GENE (2003) 318:103-111), Arabidopsis thaliana AtACP5 (Accession No.: AJ133747), and tomato IAP.
[0027] Another indication that two nucleotide sequences are substantially identical is that the two molecules specifically or substantially hybridize to each other under stringent conditions.
[0028] "Stringent hybridization conditions" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and Northern blot analysis are both sequence- and environment-dependent. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, part I chapter 2, Elsevier, New York, N. Y. Generally, highly stringent hybridization and wash conditions are selected
to be about 5 degree C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
[0029] The term "reactive oxygen species (ROS)" refer to partially reduced or activated derivatives of oxygen, such as singlet oxygen, superoxide anion, hydrogen peroxide and hydroxyl radical. At low levels, these species may function in cell signaling processes. At higher levels, these species are highly reactive and toxic, and can lead to the oxidative destruction of cells.
[0030] The term "abiotic stress" as used herein refers to nonliving environmental factors that exert a disadvantageous influence on plants or plant cells. In most cases, stress is measured in relation to plant survival, crop yield, growth (biomass accumulation), or the primary assimilation processes (CO 2 and mineral uptake), which are related to overall growth. Examples of abiotic stresses include, but are not limited to, high salinity, drought, oxidative stress, and extreme cold or heat. Particularly, "abiotic stresses" in the context of the invention refer to the stresses whose adverse effects are generated via ROS.
[0031] The terms "promoter" or "transcriptional control sequence" or "transcriptional regulatory sequence" as used herein refers to a regulator region a short distance from the 5' end of a gene that acts as the binding site for RNA polymerase. Such sequences have the functions of directing or regulating the transcription of nucleic acid sequences immediately downstream them. There are various types of promoters, including constitutive promoters, tissue-specific promoters, inducible promoters and those triggered by specific physiological conditions, such as high levels of reactive oxygen species. The examples of promoters could be used in the present invention include, but are not limited to, CaMV 35S, Ubi, SAG12 promoter, and the like. More suitable promoters can be found in the PlantProm database (Shahmuradov et al., Nucleic Acids Research, 2003, Vol. 31, No. 1 114-117).
[0032] The term "mitochondria targeting transit peptide (MTTP)" as used herein refers to a signal peptide which migrates into mitochondria. By its migration, a polypeptide fused to 3 ' end of it is also directed into mitochondria. The mitochondria targeting transit peptides can be found in the N-terminus of proteins synthesized in cytoplasm and then transported into mitochondria, such as that from the ATP synthase beta-subunit.
[0033] The term "construct", as used herein to describe an expression construct, refers to an expression vector further comprising a nucleotide sequence operatively inserted within the vector, such that the nucleotide sequence is expressed.
[0034] The term "vector" as used herein refers to a nucleic acid molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, or plant virus, into which a nucleic acid sequence may be inserted or cloned. A vector preferably contains one or more unique restriction sites and may be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible. Accordingly, the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into a cell, is integrated into the genome of the recipient cell and replicated together with the chromosome(s) into which it has been integrated. A vector system may comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The choice of the vector will typically depend on the compatibility of the vector with the cell into which the vector is to be introduced. The vector may also include a selection marker such as an antibiotic resistance gene that can be used for selection of
suitable transformants. Examples of such resistance genes are well known to those of skill in the art. In the present invention, the terms "construct" and "vector" can be alternately used.
[0035] The term "heterologous nucleic acids" as used herein are those that are not operatively linked or are not contiguous to each other in nature. Usually, heterologous nucleic acids refer to sequences that originate from different sources such as the tissues or DNA of different species. For example, a mitochondria targeting peptide from Arabidopsis is considered heterologous to a soybean coding domain sequence. Also, a promoter from a species other than soybean is considered heterologous to a soybean coding sequence.
[0036] The term "operably linked" as used herein refers to that transcriptional and translational regulatory nucleic acids are positioned relative to a polypeptide-encoding polynucleotide in such a manner that the polynucleotide is, under the control of the regulatory nucleic acids, transcribed and optionally the polypeptide is translated.
[0037] The phase "operable in plant cells" as used herein means that a promoter or control sequence is capable of performing its normal function of direct and/or regulate expression of coding sequences immediately downstream to it in a plant cell, wherein the plant cell can be a part of a living plant or iri cell culture.
[0038] The term "selectable marker" as used herein refers to a gene that imparts a distinct phenotype to cells expressing the marker gene and thus allows such transformed cells to be distinguished from cells that do not have the marker. A selectable marker gene confers a trait for which one can "select" based on resistance to a selective agent (e.g., a herbicide, antibiotic, radiation, heat, or other treatment damaging to untransformed cells, including abiotic stresses). A screenable marker gene (or reporter gene) confers a trait or a phenotype that one can identify through observation or testing, i.e., by "screening" (e.g. .beta. -glucuronidase, luciferase, or other enzyme activity not present in untransformed cells).
Embodiments of Carrying Out the Invention
[0039] In the present invention, it has been found that PAP activity targeted to the mitochondria is successful in conferring tolerance in plant cells and plants with respect to abiotic stress factors and in particular hypersalinity, osmotic, and oxidative stress, which oxidative stress may be exhibited by accumulation of reactive oxygen species (ROS) and may be a secondary response to primary stress factors. Plants and plant cells can exhibit this tolerance by virtue of transgenic modification to include expression systems or constructs of the present invention which result in the production of proteins that target PAP activity to the mitochondria. This is illustrated below in tobacco plant cells and in Arabidopsis plants, but is by no means limited to these examples. Any higher plant or cell of a higher plant is a suitable subject for the methods and materials of the present invention.
[0040] The method according to the invention can be applied to any plant, preferably higher plants belonging to the classes of Angiospermae and Gymnosperniae. Plants of the subclasses of the Dicotylodenae and the Monocotyledonae are particularly suitable. Dicotyledonous plants belong to the orders of the Magniolales, Illiciales, Laurales, Piperales Aristochiales, Nymphaeales, Ranunculales, Papeverales, Sarraceniaceae, Trochodendrales, Hamamelidales, Eucomiales, Leitneriales, Myricales, Fagales, Casuarinales, Caryophyllales, Batales, Polygonales, Plumbaginales, Dilleniales, Theales, Malvales, Urticales, Lecythidales, Violales, Salicales, Capparales, Ericales, Diapensales, Ebenales, Primulales, Rosales, Fabales, Podostemales, Haloragales, Myrtales, Cornales, Proteales, Santales, Rafflesiales, Celastrales, Euphorbiales, Rhamnales, Sapindales, Juglandales, Geraniales, Polygalales, Umbellales, Gentianales, Polemoniales, Lamiales, Plantaginales, Scrophulariales, Campanulales, Rubiales, Dipsacales, and Asterales. Monocotyledonous plants belong to the orders of the Alismatales, Arales, Arecales, Bromeliales, Commelinales, Cyclanthales, Cyperales, Eriocaulales, Hydrocharitales, Juncales, Najadales, Restionales, Poales, Triuridales, Typhales, Zingiberales, Pandanales,
Lilliales, and Orchidales. Plants belonging to the class of the Gymnospermae are Cycadales, Pinales, Ginkgoales, and Gnetales.
[0041] The method of the invention is preferably used with plants that are important or interesting for agriculture, horticulture, energy industry, biomass for bioconversion and/or forestry. Examples are tobacco, oilseed rape, sugar beet, potatoes, tomatoes, cucumbers, peppers, beans, peas, citrus fruits, avocados, peaches, apples, pears, berries, plumbs, melons, eggplants, cotton, soybean, sunflowers, roses, poinsettia, petunia, guayule, cabbages, spinach, alfalfa, artichokes, corn, wheat, rice, rye, barley, grasses such as switch grass or turf grass, millet, hemp, bananas, poplars, eucalyptus trees and conifers.
[0042] In order to provide the requisite targeted PAP protein, plant cells are modified to contain nucleotide sequences encoding the relevant protein such as PAP or MTTP-PAP, optionally operably linked to control sequences operable in plants, or integrated into the genome so as to be expressed under the control of endogenous control sequences.
[0043] hi one embodiment of the invention, the plant cells or plants are transfected or transformed with nucleic acid constructs or vectors of the invention. Preferably, the nucleic acid constructs may contain control sequences operable in plants operably linked to the MTTP-PAP encoding sequence, which control sequences can be selected to result in constitutive, tissue-specific or non tissue-specific, or inducible expression. A wide variety of such control sequences is available in the art, and appropriate vectors for genetic modification are also well known and, indeed, commercially available.
[0044] hi one embodiment of the invention, the binary systems derived from the Ti plasmid of Agrobacteria are particular suitable for the present invention. A binary system typically includes two vectors of different sizes. The larger vector is a helper vector containing the necessary genes for integration of the T-DNA carried on the smaller vector into the genome of a plant cell. The smaller vector, usually called as a binary vector, carries the gene to be inserted or cloned. Typically, the helper vector has been transformed
to a suitable Agrobacterium strain in advance. Many of such Agrobacterium strains are well known in the art, such as LBA4404, GV3101, EHAlOl, EHA105, ABI, and the like (Plant Molecular Biology Manual. Ed. S. B. Gelvin and R. A. Schilperoort, 2 nd Edition. Springer, 1994). Similarly, there are many binary vectors available for practicing the invention, such as pBR322, pUC series, pBI series, pMON series, pCambia series, pGreen series, and the like. The choice of the binary vectors will depend on the plant species to be transformed. For example, LBA4404 is suitable for transforming a dicot, while GV3101 should be chosen for a monocot. Knowledge about choosing suitable vectors is within the ability of one of ordinary skill in the art and can be found, for example, in Plant Molecular Biology Manual. Ed. S. B. Gelvin and R. A. Schilperoort, 2 nd Edition. Springer, 1994.
[0045] Similarly, techniques for effecting genetic modification of plant cells and reconstituting intact plants are now well known in the art. A useful summary of the state of the art in this respect, including a reasonably comprehensive list of the types of plants and plant cells that can form the subjects of the present invention is found in U.S. Patent Publication 2004-0009476, published on 14 January 2004, noted above, and incorporated herein by reference with respect to its disclosure of appropriate techniques for genetic manipulation of plants and the range of plants and plant cells to which these techniques may be applied.
[0046] Further, because the modified cells and plants of the invention are resistant to stress caused by osmotic, high salinity and/or oxidative stress, an expression system or a construct comprising a nucleotide sequence encoding the MTTP-PAP fusion operably linked to control sequences operable in plants can be used as a selectable marker for successful transformation of cells. Successful transformants are more highly resistant and survive an applied stress for which the marker confers tolerance. Hence, successful transformants can be identified by virtue of their ability to survive such stress conditions.
[0047] The MTTP-PAP fusion proteins of the invention comprise an amino acid sequence that confers PAP activity and a sequence that affects the transit of the fused
protein to the mitochondria, positioned in such a way that the MTTP is operable to transport the fusion protein. One such fusion protein is found natively, as described above, in soybeans and is designated GmPAP3 by Liao, H., et al. (supra). However, nucleotide sequences or nucleotide sequences substantially identical thereto that encode proteins with PAP activity from other sources or indeed other PAP encoding sequences from soybeans, for example, Arabidopsis thaliana AtACP5, and tomato IAP, can also be used in the invention by preparing a construct which includes the MTTP-encoding sequence in operable linkage thereto. Accordingly, the invention includes such constructs.
[0048] The ability of the MTTP-PAP fusion to confer tolerance to hypersalinity, osmotic and/or oxidative stress such as conferred by the herbicide Paraquat, can be exhibited in numerous ways, including amelioration of ROS, retention of intact mitochondria, maintenance of viability, enhanced growth of various plant parts, and general improvement in cellular health.
[0049] The following examples and results confirm and illustrate the success of the methods and constructs of the invention.
[0050] The following protocols resulted in the data set forth in Examples 1-6 below.
Establishment of Transgenic Tobacco BY-2 Cell Lines and Transgenic Arabidopsis thaliana [0051] Recombinant constructs containing GmPAPS or GmPAP3-T7 under the control of the constitutive Cauliflower Mosaic Virus 35S promoter were cloned into a binary vector (Brears et al, Plant Physiol. (1993) 103: 1285-1290) and introduced into Agrobacterium. The constructs were transformed into BY-2 cells (GmPAP3 and GmPAP3-T7) or A. thaliana (GmPAP3) respectively using a co-cultivation method (An, G., Plant Physiol. (1985) 79:568-570) or a vacuum infiltration protocol (Bechtold and Pelletier, J. Martinez-Zapater, J. Salinas, eds, Arabidopsis Protocols. Humana Press Inc., Totowa(1993) pp 259-266). After selecting the transformants on antibiotic-containing
media, PCR screening using gene specific primers was performed to verify the successful integration of the transgene into the genomes; and Northern blot analysis was performed to confirm the expression of the transgenes in the transgenic cell and plant lines. For transgenic A. thaliana, seeds of T 3 homozygous lines with single insert were obtained and used in subsequent physiological studies.
Gene Expression Study
[0052] To study the gene expression patterns of GmPAPS under NaCl and PEG treatment, surface-sterilized soybean {Glycine max L. Merr. cv. Union) seeds were first germinated in filter papers containing modified Hoagland's solution (4.5 mM KNO 3 , 3.6 niM Ca(NO 3 ) 2 , 1.2 mM NH 4 NO 3 , 3.0 mM MgSO 4 , 1.2 mM (NH 4 ) 2 SO 4 , 0.25 mM KH 2 PO 4 , 4.5 μM MnSO 4 , 4.5 μM ZnSO 4 , 1.5 μM CuSO 4 , 0.4 μM (NH 4 ) 6 Mo 7 0 24 , 0.09 mM Fe-EDTA, and 1.5 μM H 3 BO 3 ). After germination, one-week-old seedlings of uniform growth stage were transferred to a hydroponic system containing the same culture medium. After opening of the first trifoliate, seedlings were treated with modified Hoagland's solution supplemented with 125 mM NaCl and 5 % PEG, respectively. The youngest fully expanded trifoliate of treated plants were collected for total RNA extraction after 48 h. To study the gene expression pattern of GmPAPS under PQ treatment, surface sterilized seeds were germinated in silicon sand containing half Hoagland's solution. After germination, 10-day-old seedlings of uniform growth stage were transferred to a hydroponic system containing the same culture medium. After equilibration of seedlings for 24 d, 10 mM PQ solution was sprayed on both surfaces of trifoliate leaves and leaf samples were collected after 4 h.
[0053] Northern blot analysis was performed and Antisense single-stranded DNA probes labeled with digoxygenin (DIG) (Roche, Mannheim, Germany) used as a probe. Since GmPAPS was cloned into the pBluescript II KS (+) vector, the T3-(5 '-AATTAACCCTCACTAAAGGG-S') and
T7-(5 '-GTAATACGACTCACTATAGGGC-S') promoter primers were used for synthesizing the PCR probes.
Analysis of Mitochondria Integrity
[0054] Cells were treated with 200 mM NaCl for 1 h or 2 % PEG for 1 h before staining with lOμg/ml rhodamine-123 (Rhl23) (R302, Molecular Probes) for 1 h. The signal of Rhl23 was excited by green HeNe laser at 543nm. The filter set HQ590/70 was used and confocal images were collected by the Bio-Rad Radiance 2100 system. 10-25 cells were counted for each sample for statistical analysis.
Cell Viability Assay
[0055] Cells were treated with 200 mM NaCl or 2 % PEG for 24 h before staining with 0.4 % trypan blue (T8154, Sigma). Stained cells were observed under light microscope. Around 200 cells were counted for each sample.
Detection of ROS
[0056] The chemical probe H 2 DCFDA has been used extensively as a non-invasive, in vivo measure of intracellular ROS (Allan, et al., Plant Cell (1997) 9: 1559-1572); D.P. Maxwell et al, Proc. Natl. Acad. ScL USA (1999) 96:8271-8276). Cells were pre-stained with dichlorodihydrofluorescein diacetate (H 2 DCFDA) for 30 min before treatment with 200 mM NaCl for 1 h or 2 % PEG for 1 h. The signals Of H 2 DCFDA were excited by 488nm Argon laser and the HQ 515/30 filter set was used. The same level of laser excitation, iris and gain were used for each cell analyzed. The fluorescence intensity of H 2 DCFDA was estimated by using the program described in the National Institute of Health website on the World Wide Web located at http://rsb.info.nih.gov/ij/. Quantitative analysis was done by tracing the entire cell (by using the selection tools) and the total fluorescence intensity was measured. This fluorescence intensity measurement (in pixels)
was then divided by the area of the cell to obtain average pixel fluorescence intensity. In addition, background fluorescence intensity was measured in the same field and was subtracted. 10-20 cells were analyzed for each sample to perform statistical analysis.
Subcellular Localization of GmPAP3
[0057] The subcellular localization of GmP AP3 was studied by using a GmPAP3-T7 fusion protein. The location of the T7 tag was visualized by immunolabeling with FITC-conjugated secondary antibody. BY-2 cell fixation and confocal immunofluorescence were carried out according to (Jiang and Rogers, J.Cell Biol. (1998) 143:1183-1199) with minor modifications. Cells were prestained with the mitochondria marker MitoTracker Orange™ (M7510, Molecular Probes) before the fixation and immunolabeling process. The signal of MitoTracker Orange was excited by 543nm Green HeNe laser and the HQ 590/70 filter set was used. The signal of FITC was excited by 488nm Argon laser and the HQ 515/30 filer set was used. All confocal images were collected by Bio-Rad Radiance 2100 system.
[0058] For quantification of the colocalization of FITC and MitoTracker Orange™ signals, superimposition of green (FITC) and magenta (MitoTracker Orange ) images resulted in yellow where the green and magenta signals overlapped. By using the program ImageJ1.34n (Sukumvanich et al., supra), the pixel area occupied by yellow divided by the pixel area occupied by green was calculated. Images from at least 10 different cells from the double-labeling experiment were analyzed to calculate the colocalization of FITC and MitoTracker Orange™ signals.
Extraction of Mitochondrial Protein
[0059] Mitochondria protein was extracted by means of differential centrifugation as described in Douce, R., et al. Methods Enzymol. (1987) 148:403-415
with modifications. Plant material was gently homogenized in 2 volumes of ice-cold extraction medium (0.25M Sucrose, 5 mM EDTA, 1 mM EGTA, ImM dithioerythritol, 0.1% BSA, 0.6% PVPP in 1OmM HEPES-TRIS pH 7.4). The homogenate was filtered and squeezed through Miracloth™ and the mitochondria were immediately separated from the cytoplasmic fraction by centrifugation at 10,000 g, 10 min. The resulting crude mitochondrial pellet was resuspended in medium I (0.25M Sucrose, 5mM EDTA, ImM EGTA, 0.1% BSA in 1OmM HEPES-TRIS pH 7.4) and centrifuged at 60Og, 5 min to remove nuclei and heavy cell debris. This washing procedure was repeated two times. Washed mitochondria were resuspended in medium II (Sucrose 0.25 M, EGTA 30 μM in HEPES-TRIS 10 mM pH 7.4) and stored on ice.
Root Growth Assay of Transgenic Arabidopsis Under Salt Treatment
[0060] Seeds of transgenic lines (GmPAP 3 and the empty vector) and their untransformed parent Columbia-0 (Col-0) were sown on vertical MS plates containing 3 % sucrose and 0.9 % (w/v) agar. Seedlings (7 d after germination) were transferred onto either the control MS agar plates or MS agar plates supplemented with 150 mM NaCl. The root length of each individual seedlings before and 7 d after treatment was recorded and percentage root growth was calculated.
Root Growth Assay of Transgenic Arabidopsis Under PEG Treatment
[0061] Seeds of transgenic lines (GmPAP3 and the empty vector) and their untransformed parent Columbia-0 (Col-0) were sown on MS plates containing 3 % sucrose and 0.9 % (w/v) agar. Seedlings (7 d after germination) were transferred onto either the control MS agar plates or MS agar plates supplemented with 15% Polyethylene-glycol 6000 (PEG). The root length of each individual seedlings before and 7 d after treatment was recorded and percentage root growth was calculated.
[0062] Since PEG cannot be dissolved in the agar before pouring plates (PEG will prevent agar from polymerizing), PEG treatment was brought about by using PEG-infused agar plates. The PEG-infused agar plates were prepared as described by Verslues, P. E., et al, The Plant Journal (2006) 47:776-787 with slight modifications. Appropriate amount of MS were prepared for both MS agar media and PEG overlay for the number of plates needed and adjusted to pH 5.7. To the solution used to prepare the agar plates, 9 g per liter agar was added. To the solution used for the PEG overlay, no agar is added, but instead autoclaved directly. After autoclaving, 20ml of MS media were poured into 100mm square plates. For the MS medium (without agar), 15% PEG was added. After the MS agar plates solidified, 30ml of PEG overlay was added on each plate. The plate were infused in PEG overlay and allowed to equilibrate for at least 16 hr. The PEG overlay was poured off just before use and the PEG-infused plates were used immediately.
Root Growth Assay of Transgenic Arabidopsis Under Paraquat Treatment
[0063] Seeds of transgenic lines (GmPAP3 and the empty vector) and their untransformed parent CoI-O were sown on vertical MS plates containing 3 % sucrose and 0.9 % (w/v) agar. Seedlings (7 d after germination) were transferred onto either the control MS agar plates or MS agar plates supplemented with 1 μM PQ. The root length of each individual seedlings before and 7 d after treatment was recorded and percentage root growth was calculated.
Lipid Peroxides Detection
[0064] FOX assay was used to determine lipid peroxides as described (Sattler et al., Plant Cell (2004) 16: 1419-1432). Twelve seedlings were extracted with 400 μL of methanol:dichloromethane (1:1 [v/v]) containing 0.05 % butylated hydroxytoluene and 50 μL of 150 mM acetic acid. Lipids were partitioned into the organic phase by adding 300 μL of water, vortexing and centrifugation at 3,750 g. The lipid extracts were incubated at
room temperature with FOX solution (23280, Pierce) for 30 min. Immediately after incubation, the absorbance was measured at 595 nm by a microplate reader. A standard curve was constructed using hydrogen peroxide as suggested in the manufacturer's protocol. The reactivity of 18:2-derived lipid hydroperoxides (LOOHs) with the FOX reagent is nearly identical to hydrogen peroxide (DeLong et al., J, Agric. Food. Chem. (2002) 50:248-254).
Statistical Analysis
[0065] Data were analyzed using the SPSS (ver. 12.0) statistical package. Samples exhibiting significant differences (p<0.01) were indicated.
Example 1
Location of GmP AP3 In Mitochondria
[0066] The nucleotide sequence encoding soybean GmPAPi described in Liao, H. et al. (Gene (2003) 318:103-111, supra.) was cloned into an expression vector compatible with tobacco cells as a fusion protein with T7 as described above. This expression vector was used to transform tobacco BY-2 cells.
[0067] Subcellular localization of GmPAP3 was studied by confocal immunofluorescence localization. FITC-conjugated secondary antibody was used to label the GmPAP3-Tl tag fusion protein produced in BY-2 cells. MitoTracker-Orange™ fluorescence dye was used to specifically label mitochondria. Percent of colocalization for the signal of FITC and MitoTracker- Orange™ was expressed as amount of FITC colocalized with MitoTracker- Orange™ signal. Percent colocalization is expressed as the mean ± standard deviation (SD) for the number of cells analyzed, as shown below.
[0068] The results for wild type and 1535-2 are also shown in Figure 1.
Example 2
Alternative Measure of Colocalization of GmPAP3 in Mitochondria [0069] The transgenic BY-2 cells provided in Example 1 were cultured to effect production of GmP AP3. Mitochondrial protein was extracted and GmP AP3 specific antibody was used for immunodetection. The results are shown in Figure 2A. where WT indicates mitochondrial protein fraction from wild type BY-2 cell lines; 9111 and 9112 show mitochondrial protein extracted from GmPAP 3 transgenic cell lines; J23 shows mitochondrial protein extracted from the soybean cultivar J23. As seen GmP AP3 migrates to the mitochondrial fraction.
[0070] Figure 2B shows an Electron Microscope Observation of Localization of GmP AP3 in Mitochondria which is an alternative demonstration of the results in Figure 2 A. Embedding and electron microscopy were performed as described (Tse YC, et al., Dynamic response of prevacuolar compartments to Brefeldin A in Plant Cells. Plant Physiol. (2006)142: 1442-1459) with slight modifications. Samples were fixed in a primary fixative solution contain 0.25% (v/v) glutaraldehyde and 1.5% (v/w) paraformaldehyde in 50 niM phosphate buffer, pH 7.4, for 15 min at room temperature before incubating at 4 0 C for an additional 16 h. After washing with phosphate buffer at room temperature, cells were dehydrated in an ethanol series and then embedded in Lowicryl ® (HM20) resin. Ultrathin sections were than prepared from these blocks using Ultra Cut S (Leica, Wetzlar, Germany). The GmP AP3 specific antibody was used as the primary antibody followed by detection using the gold-conjugated anti-rabbit secondary
antibody. Immuno-labeled sections were then post-stained with 4% uranyl acetate and examined using a transmission electron microscope (JEM-1200EXII, JEOL, Tokyo, Japan).
Example 3
Effect of GmPAPS on Mitochondrial Membrane Integrity Under Salinity and
Drought Stresses
[0071] Transgenic BY-2 cell lines prepared as in Example 1 were cultured to express the GmP AP3 protein and subjected to treatment with sodium chloride or polyethylene glycol (PEG, osmotic stress) as explained above and in the description of the results as shown in Figures 3 A-3L. The results on mitochondrial integrity were compared with the effect of the presence of ascorbic acid. The results are shown in the table below which summarizes the data in Figure 3A-3L. The data are the results of counting 10-25 cells and the experiment repeated twice. The percentage was presented as the mean value of 3 experiments± SD, where ** denotes statistical difference (p<0.01) from the wild type BY-2 cells under the same treatment, based on one-way ANOVA followed by the Turkey test.
Cell lines % cells with intact mitochondria
Control NaCl PEG
Wild type BY-2 100 % 45.27±6.3 47.98+3.1
Wild type BY-2 + 100 % 86.20+3.3** 82.16±3.7**
10 mM ascorbic acid
GmPAPS transgenic cell line 20 100 % 81.36+3.9** 81.32+5.6**
GmPAPS transgenic cell line 29 100 % 80.40±4.7** 79.5±6.9**
[0072] Wild type (WT) BY-2 cells (Figures 3A-3C) and GmPAP3 transgenic BY-2 cell lines #20 and #29 (Figures 3G-3I & 3J-3L, respectively) without ascorbic acid supplements and wild type BY-2 cells with 10 mM ascorbic acid supplements (+Asc) (Figures 3D-3F) were pre-treated in a cell culture medium without stress (Figures 3A, 3D, 3G and 3J), with 200 mM NaCl for 1 h (Figures 3B, 3E, 3H and 3K), or with 2 % PEG for Ih (Figures 3C, 3F, 31, 3L) before staining with 10 μg/mL Rhl23 for another hour. The signal of RhI 23 was observed using a confocal laser scanning microscope (see materials and methods). 10-25 cells were counted for each line. Scale bar = 50 μm.
Example 4
Effect of Stress on Viability Protection by GmPAP3
[0073] The experiment of Example 3 was performed but using viability as an assessment rather than mitochondrial integrity. Viability was evaluated by staining with trypan blue. Cells were treated with 200 mM NaCl or 2 % PEG for 24 h before staining with trypan blue. The percentage was presented as the mean value of around 200 cells ± SD, and shown in the table below, where ** denotes statistical difference (p<0.01) from the wild type BY-2 cells under the same treatment, based on one-way ANOVA followed by the Turkey test. The exponents a and b indicate two separate sets of experiments. Cell lines % of viable cells
Untreated 200 mM NaCl 2% PEG
Wild type BY-2 a 98.70+2.5 48.30+8.8 66.81+8.2
Wild type BY-2 + 97.52+5.9 92.68+8.0** 94.87+9.2**
10 mM ascorbic acid a
Wild type BY-2 b 93.41+10.6 42.14+6.9 60.63+13.2
GmPAPZ transgenic cell line 20 b 94.61+6.0 79.70+16.7** 95.96+6.1**
GmPAPl) transgenic cell line 29 b 98.14+2.6 85.97±18.1** 91.76+12.6**
[0074] Similar results were obtained using histological evaluation as shown in Figures 4A-4L. Wild type (WT) BY-2 cells (Figures 4A-4C) and GmPAPS transgenic BY-2 cell lines #20 and #29 (G-I & J-L, respectively) without ascorbic acid supplements and wild type BY-2 cells with 10 mM ascorbic acid supplements (+Asc) (Figures 4D-4F) were pre-treated in a cell culture medium without stress (Figures 4A, 4D, 4G and 4J), with 200 mM NaCl (Figures 4B, 4E, 4H and 4K) for 24 h, or with 2 % PEG (Figures 4C, 4F, 41, 4L) for 24 h. Treated cells were stained with 0.4 % trypan blue. Non-viable cells were stained blue. Around 200 cells were counted for each line. Scale bar = 50 μm.
Example 5 Effect of GmPAPS on ROS
[0075] GmPAP 3 reduces ROS accumulation in BY-2 cells when subjected to salt and osmotic stresses, as shown in Figures 5A-5L. Wild type (WT) BY-2 cells (Figures 5A-5C) and GmPAPS transgenic BY-2 cell lines #20 and #29 (Figures 5G-5I & Figures 5J-5L, respectively) without ascorbic acid supplements and wild type BY-2 cells with 10 mM ascorbic acid supplements (+Asc) (Figures 5D-5F) were pre-stained with H 2 DCFDA for 30 min before placed in a cell culture medium without stress (Figures 5 A, 5D, 5 G, 5J), with 200 mM NaCl for 1 h (Figures 5B, 5E, 5H, 5K), or with 2 % PEG for Ih (Figures 5C, 5F, 51, 5L). The signals of H 2 DCFDA were observed using a confocal laser scanning microscope. 10-20 cells were counted for each line. Scale bar = 50 μm.
[0076] These results can also be shown graphically as shown in Figure 6.
[0077] Fluorescence intensity was calculated by ImageJ and data obtained were analyzed by one-way analysis of variance (ANOVA) test, in which case significant differences between individual lines were determined by the Turkey's test. ** indicated that the mean difference (compared to wild type) is significant at p<0.01 level.
Example 6
Effect of GmPAPS on Response to Stress in Arabidopsis
[0078] Arabidopsis lines F42 and C25 which have been modified to produce GmPAPS as described above were used in these experiments. First, the effects of salt stress on root growth of Arabidopsis were studied. Seeds from the wild type parent (CoIO), empty vector transgenic control (V7) and two independent GmPAPS transgenic lines (F42 and C25) were sown on MS agar plates. Young seedlings were transferred to either the control MS agar plates (CT) or MS agar plates supplemented with 150 mM NaCl. Percentage growth of root was estimated and shown in Figure 7. Error bar: standard error. N=48. Data obtained were analyzed by one-way analysis of variance (ANOVA) test, in which case significant differences between individual lines were determined by the Turkey's test. ** indicates that the mean difference (compared to wild type) is significant atpO.Ol level.
[0079] As shown in Figure 7, the presence of salt actually enhanced the growth of roots in the transformance, although it diminished the growth in wild type.
[0080] Next, the effects of PEG stress on root growth of Arabidopsis were studied. Seeds from the wild type parent (CoIO), empty vector transgenic control (V7) and two independent GmPAPS transgenic lines (F42 and C25) were sown on MS agar plates. Young seedlings were transferred to either the control MS agar plates (CT) or MS agar plates supplemented with 15% PEG. Percentage growth of root was estimated and shown in Figure 8. Error bar: standard error. N=48. Data obtained were analyzed by one-way analysis of variance (ANOVA) test, in which case significant differences between individual lines were determined by the Turkey's test. ** indicates that the mean difference (compared to wild type) is significant at p<0.01 level.
[0081] The effects of oxidative stress on root growth of Arabidopsis were studied. Seeds from the wild type parent (CoIO), empty vector transgenic control (V7) and two independent GmPAPS transgenic lines (F42 and C25) were sown on MS agar plates. Young seedlings were transferred to either the control MS agar plates (CT) or MS
agar plates supplemented with lμM Paraquat. Percentage growth of root is shown in Figure 9. Error bar: standard error. N=48. Data obtained were analyzed by one-way analysis of variance (ANOVA) test, in which case significant differences between individual lines were determined by the Turkey's test. ** indicates that the mean difference (compared to wild type) is significant at p<0.01 level.
[0082] Lipid peroxidation in GmPAPS transgenic Arabidopsis under oxidative treatment. Seedlings were grown and treated with Paraquat as described above. Lipid peroxidation was measured by the FOX assay and the results shown in Figure 10. The reactivity of 18:2-derived lipid hydroperoxides (LOOHs) levels were expressed in mole LOOHs per g fresh weight (FW). Error bar: standard error. N=4 (four sets of 12 seedlings for each data point). Data obtained were analyzed by one-way analysis of variance (ANOVA) test, in which case significant differences between individual lines were determined by the Turkey's test. ** indicates that the mean difference (compared to wild type) is significant at p<0.01 level. Open and close bars indicate untreated control and treated samples, respectively.
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