Specifically, the present invention relates to several polynucleotides encoding calcium-dependent calmodulin-binding proteins isolated from tobacco and other plants and to functional homologs thereof.

Further specifically, the present invention relates to the use of these polynucleotides, functional portions thereof, functionally similar homologs, and/or portions of the functionally similar homologs to confer useful properties on plants expressing them. In particular, these useful properties include tolerance to heavy metal and other metal cations, such as, but not limited to Ni2+, and increased uptake of heavy and other metal cations, such as, but not limited to, Pb2+. Tolerance to Ni cations is useful because it allows plants to grow in an environment which would normally be considered toxic to sustain plant growth. Increased uptake of other heavy metal cations, such as Pb2+, has utility as a means of phytoremediation.

Citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

Toxic metal pollution, mainly from combustion of fossil fuels, from manufactured waste and the indiscriminate use of chemicals, has accelerated dramatically in recent years. The direct emission of trace metals such as Ni into the air, water and soil is by now measured in millions of tons per annum (Nriagu et al., 1988). The levels of most toxic metals emitted into the atmosphere because of humans are several times higher than these released by natural sources. For example, the anthropogenic emission of Pb into the atmosphere is more than 300-fold that of its natural emission

(Ayres, 1992). Increased acidification of soils and fresh waters, which leads to enhanced solubility of toxic metals, further increases toxic metal levels (Rengel, 1996).

Plants have developed unique protective strategies to deal with this environmental danger (Braam et al., 1990; Bowler et al., 1994; Knight et al., 1998; Snedden et al., 1998). By understanding these strategies, improved plant tolerance can be engineered and implementation of biotechnologies aimed at cleaning the environment can be undertaken.

Genes encoding proteins that are involved in the transport of metal ions are crucial in this endeavor. Specifically, such genes form potential targets for improving plant tolerance to toxic metals or increasing the accumulation of metal ions in plants as a means of phytoremediation (Raskin, 1996).

There is thus a widely recognized need for, and it would be highly advantageous to have, a means of regulating uptake and storage of heavy and other metal ions by plants which would, in some cases facilitate cultivation of plants in an environment containing high concentrations of metal ions, and in other cases facilitate phytoremediation of an area for future use as a cultivation site. The present invention represents a significant step forward towards these goals.

SUMMARY OF THE INVENTION According to one aspect of the present invention there is provided an isolated nucleic acid comprising a polynucleotide at least 60 % identical with any of SEQ ID NOs: 1,3,5,7,9,11 or portions thereof as determined using the Bestfit procedure of the DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the university of Wisconsin (gap creation penalty-50, gap extension penalty-3), in particular, SEQ ID NOs: 1,3,5,7,9,11 or portions thereof.

According to another aspect of the present invention there is provided an isolated nucleic acid comprising a polynucleotide encoding a polypeptide being at least 60 % homologous (identical + similar) with any of SEQ ID NOs: 2,4,6,8,10,12 or portions thereof as determined using the Bestfit procedure of the DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the university of Wisconsin (gap creation penalty-50, gap extension penalty-3).

According to yet another aspect of the present invention there is provided an isolated nucleic acid comprising a plant derived polynucleotide

encoding a transmembrane polypeptide having a cation channel activity when assembled in a plasmatic membrane of a plant cell, the polypeptide having overlapping cyclic nucleotide-binding domain site and calmodulin- binding site.

According to still another aspect of the present invention there is provided an isolated nucleic acid comprising a polynucleotide hybridizable with any of SEQ ID NOs: l, 3,5,7,9,11 or portions thereof under hybridization conditions of hybridization solution containing 10 % dextrane sulfate, 1 M NaCI, 1 % SDS and 5 x 106 cpm 32p labeled probe, at 65 °C, <BR> <BR> with a final wash solution of 1 x SSC and 0.1 % SDS and final wash at 50 ° C.

According to an additional aspect of the present invention there is provided an isolated nucleic acid comprising a polynucleotide hybridizable with a polynucleotide encoding a polypeptide as set forth in any of SEQ ID NOs: 2,4,6,8,10,12 or portions thereof under hybridization conditions of hybridization solution containing 10 % dextrane sulfate, 1 M NaCI, 1 % SDS and 5 x 106 cpm 32p labeled probe, at 65 °C, with a final wash solution of 1 x SSC and 0.1 % SDS and final wash at 50 °C.

According to yet an additional aspect of the present invention there is provided a nucleic acid construct comprising a polynucleotide as set forth herein downstream of a plant promoter. The polynucleotide can be in either sense or antisense orientation, whereas the plant promoter can be a constitutive promoter, a tissue specific promoter, an inducible promoter or a chimeric promoter.

According to still an additional aspect of the present invention there is provided a recombinant protein comprising a polypeptide encoded by any of the polynucleotides described herein.

According to a further aspect of the present invention there is provided a recombinant protein comprising a polypeptide at least 60 % homologous with any of SEQ ID NOs: 2,4,6,8,10,12 or portions thereof as determined using the Bestfit procedure of the DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the university of Wisconsin (gap creation penalty-50, gap extension penalty- 3), SEQ ID NOs: 2,4,6,8,10,12 or portions thereof, in particular.

According to yet a further aspect of the present invention there is provided a genetically transformed or virus infected cell or plant comprising any of the isolated nucleic acids or constructs described herein and preferably expressing any of the recombinant proteins described herein.

According to still a further aspect of the present invention there is provided an antibody specifically recognizing any of the recombinant proteins as described herein and/or natural equivalents thereof. Such an antibody can be, for example, a polyclonal antibody produced by a non- human mammal or in an egg, or alternatively, such an antibody can be a monoclonal antibody produced by a cell such as a hybridoma.

According to yet a further aspect of the present invention there is provided a method of increasing a tolerance of a plant to a metal cation, the method comprising the step of overexpressing in the plant a recombinant protein which reduces uptake and concentration of the metal cation within the plant cells.

According to still a further aspect of the present invention there is provided a method for phytoremediation of an area polluted with a metal cation, the method comprising the steps of (a) providing a plant characterized in resistivity to elevated concentrations of the metal cation, the plant overexpressing a recombinant protein which facilitates uptake and concentration of the metal cation within the plant cells; (b) planting the plant in the area; (c) following a time period, in which at least a fraction of the metal cation in the area has been accumulated in the plant, harvesting the plant, thereby removing at least the fraction of the metal cation from the area; and optionally (d) repeating steps (b)- (c) until a sufficient amount of the metal cation has been removed from the area.

According to still further features in the described preferred embodiments the plant is genetically transformed or viral infected with a nucleic acid including a polynucleotide at least 60 % identical with any of SEQ ID NOs: 1,3,5,7,9,11,15,17 or portions thereof as determined using the Bestfit procedure of the DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the university of Wisconsin (gap creation penalty-50, gap extension penalty-3).

According to further features in preferred embodiments of the invention described below, the polynucleotide is as set forth in any of SEQ ID NOs: 1,3,5,7,9,11,15,17 or portions thereof.

According to still further features in the described preferred embodiments the plant is genetically transformed or viral infected with a nucleic acid including a polynucleotide encoding a polypeptide being at least 60 % homologous with any of SEQ ID NOs: 2,4,6,8,10,12,16,18 or portions thereof as determined using the Bestfit procedure of the DNA sequence analysis software package developed by the Genetic Computer

Group (GCG) at the university of Wisconsin (gap creation penalty-50, gap extension penalty-3).

According to still further features in the described preferred embodiments the plant is genetically transformed or viral infected with a nucleic acid including a plant derived polynucleotide encoding a transmembrane polypeptide having a cation channel activity when assembled in a plasmatic membrane of a plant cell, the polypeptide having overlapping cyclic nucleotide-binding domain site and calmodulin-binding site.

According to still further features in the described preferred embodiments the plant is genetically transformed or viral infected with a nucleic acid including a polynucleotide hybridizable with any of SEQ ID NOs: 1,3,5,7,9,11,15,17 or portions thereof under hybridization conditions of hybridization solution containing 10 % dextrane sulfate, 1 M NaCI, 1 % SDS and 5 x 106 cpm 32p labeled probe, at 65 °C, with a final wash solution of 1 x SSC and 0.1 % SDS and final wash at 50 °C.

According to still further features in the described preferred embodiments the plant is genetically transformed or viral infected with a nucleic acid including a polynucleotide hybridizable with a polynucleotide encoding a polypeptide as set forth in any of SEQ ID NOs: 2,4,6,8,10,12, 16,18 or portions thereof under hybridization conditions of hybridization solution containing 10 % dextrane sulfate, 1 M NaCI, 1 % SDS and 5 x 106 cpm 32p labeled probe, at 65 °C, with a final wash solution of 1 x SSC and 0.1 % SDS and final wash at 50 °C.

According to still further features in the described preferred embodiments the plant is genetically transformed or viral infected with a nucleic acid construct comprising a polynucleotide as set forth herein downstream of a plant promoter in a sense orientation.

According to still further features in the described preferred embodiments the recombinant protein includes a polypeptide at least 60 % homologous with any of SEQ ID NOs: 16,18 or portions thereof as determined using the Bestfit procedure of the DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the university of Wisconsin (gap creation penalty-50, gap extension penalty-3).

According to still further features in the described preferred embodiments the polypeptide is as set fourth in any of SEQ ID NOs: 2,4,6, 8,10,12,16,18 or portions thereof.

According to still further features in the described preferred embodiments the recombinant protein includes a calmodulin binding domain.

According to still further features in the described preferred embodiments the recombinant protein further includes a cyclic nucleotide binding domain.

According to still further features in the described preferred embodiments the calmodulin binding domain and the cyclic nucleotide binding domain overlap.

According to still further features in the described preferred embodiments the metal cation is Ni, Na, Ba, Cd, Co, Cu, La, Mn, Zn and/or Pb cation.

According to still further features in the described preferred embodiments the area is selected from the group comprising of a terrestrial area and an aquatic area.

The present invention successfully addresses the shortcomings of the presently known configurations by providing and characterizing a new gene family which can be used for generating plants resistant to heavy metals and plants which accumulate heavy and other metals and which can be used for phytoremediation of soils and water reservoirs.

BRIEF DESCRIPTION OF THE DRAWINGS The invention is herein described, by way of example only, with reference to the accompanying drawings, wherein: FIG. 1A illustrates an amino acid sequence homology of NtCBP4 and NtCBP7 (SEQ ID NOs: 2 and 4). Numbers correspond to the deduced amino acids from the first ATG codon. Identical amino acids are boxed.

The six hydrophobic domains (S1-S6) are indicated by a solid underline, the predicted pore region (P) by a dashed underline, and the cyclic nucleotide- binding site is underlined with asterisks.

FIG. 1B illustrates hydropathic profiles of NtCBP4 and NtCBP7.

Mean hydropathy (ordinate) was plotted against amino acid residue number (abscissa) by using a moving window of 11 amino acids. Hydrophobic and hydrophilic regions are shown above and below the zero line, respectively.

The presumptive membrane-spanning and pore-lining regions are shown as S 1-S6 and P, respectively. Putative cyclic nucleotide-binding domain (cNBD) and calmodulin-binding domain (CaM) site are indicated by a horizontal bar.

FIG. 2 illustrates a comparison of NtCBP4 and NtCBP7 sequences with sequences of family members from other species. Amino-acid sequence alignment of tobacco (Nicotiana tabacum, NtCBP4), thale cress (Arabidopsis thaliana, AtCNGCl) and barley (Hordeum vulgare, HvCBTI) transporters (SEQ IDS NOs: 2,4,18 and 16, respectively). Amino acids identical to NtCBP4 are marked by dashes (-) and gaps by dots. The six hydrophobic domains (S 1-S6), predicted pore region (P), and putative cyclic nucleotide monophosphate binding domain are colored in red, blue and green, respectively. The CaM binding domain is underlined.

FIG. 3A illustrates a comparison of NtCBP4 and NtCBP7 sequences with that of other ion channels. Amino acid sequence alignment of the S4 regions of NtCBP4 and NtCBP7 (SEQ ID NOs: 2 and 4) with Arabidopsis AKT1 (SEQ ID NO: 35) and KAT1 (SEQ ID NO: 36), Drosophila Shaker (SEQ ID NO: 37) and EAG (SEQ ID NO: 38), and the rat CNG2 olfactory channels (SEQ ID NO: 34) (GenBank Accession Numbers X62907, X93022 and Swiss-Prot # P08510, Q02280, Q00195, respectively). Positively charged amino acids are highlighted by gray background.

FIG. 3B illustrates a comparison of NtCBP4 and NtCBP7 sequences with that of other ion channels. Amino acid sequence comparison of the putative pore region of NtCBP4 and NtCBP7 with the pore region of the ion channels of Figure 3A is shown. Residues identical or similar to the corresponding positions in NtCBP4 or NtCBP7 are highlighted by a black or gray background, respectively. The GYGD (SEQ ID NO: 43) motif of K+-selective channels is underlined by asterisks.

FIG. 3C illustrates a comparison of NtCBP4 sequence with that of other ion channels. Amino acid sequence comparison of the putative pore region and S6 region of NtCBP4 (SEQ ID NO: 2) with similar regions of KcsA, Dmshaker, AtAKTI, OCNG2 (SEQ ID NOs: 39,37,35,34).

Residues of the ion-selectivity filter, lining of the cavity and inner pore, and conserved amino acid residues, are colored in yellow, orange and gray, respectively.

FIG. 4 illustrates amino acid sequence alignment of tobacco channel- related proteins. Amino acid sequences spanning the S5 through S6 domains of four tobacco channel-related proteins (NtCBP4, NtCBP7, NtCBP10 and NtCBP63; SEQ ID NOs: 2,4,6 and 8, respectively) were aligned using the CLUSTALW multiple sequence alignment program (Devereux et al., 1984).

Positions with identical amino acids in at least three proteins are boxed.

Gaps in the alignment are indicated by dots. The S5, P, and S6 domains are underlined.

FIGs. 5A-B illustrate the purification of full-length NtCBP4 by affinity chromatography. Lysophosphatidylcholine-solubilized membrane proteins from Sf9-NtCBP4 cells were bound to the CaM-agarose column.

Equal volumes of total protein loaded to the column (T), the effluent fraction (FT), and the first to fifth EGTA-eluted fractions (1-5, respectively) were analyzed by electrophoresis and were either stained with Coomassie Blue (5A) or transferred to nitrocellulose membrane and probed with anti- NtCBP4 polyclonal antibodies (5B). The positions of molecular-mass standards (kDa) are indicated on the left.

FIG. 6 illustrates interaction between calmodulin and NtCBP4 C- terminal in yeast. p-galactosidase activity in liquid cultures (graph) and X- gal plates (streaks) of yeast expressing NtCBP4 C-terminal as bait and CaM as prey (NtCBP4C), or a negative control of yeast expressing non-specific bait and CaM as prey (Control) are shown. LacZ-specific activity units were calculated as described in materials and experimental methods in the Examples section that below.

FIG. 7A illustrates CaM binding by a GST-NtCPB4 fusion protein.

GST-NtCBP4 fusion proteins (see Figure 7B for details) or GST alone transferred to membranes after SDS-PAGE, were probed with either 35S- CaM in the presence of 1 mM Ca2+ or anti-GST antibodies.

FIG. 7B is a schematic map of the CaM-binding domain of NtCBP4.

Schematic presentation of the GST-NtCBP4 fusion proteins tested for CaM binding in Figure 7A. The numbers of the different fusion proteins correspond to the numbers of the samples loaded on the SDS-PAGE in Figure 7A.

FIG. 8A illustrates the a-helical portion of NtCBP4. The predicted a-helical wheel formed by NtCBP4 (SEQ ID NO: 2) amino acid residues 595-611 (using the GCG protein analysis program) is shown. Hydrophobic amino acids are boxed and basic amino acids are marked by +.

FIG. 8B illustrates that an NtCBP4 synthetic peptide forms a stable complex with CaM. The complex formation between CaM and a synthetic peptide corresponding to NtCBP4 amino acids 595-617 was assayed in the presence of 0.1 mM CaCl2. Increasing amounts of the peptide (peptide/CaM molar ratios indicated) were incubated with 100 pmole of bovine CaM, and then samples were separated by non-denaturing PAGE.

Arrows indicate the positions of free CaM and the peptide/CaM complex.

FIG 9A demonstrates the interaction of NtCBP4 CaM-binding peptide with dansyl CaM by fluorescence excitation. Fluorescence emission spectra of dansyl CaM and its complex with NtCBP4 CaM- binding peptide. Fluorescence emission spectra of 300 nM dansyl CaM without (empty circles) and with (fille circles) 300 nM peptide was measured at 23 OC in 50 mM Tris-HCI buffer (pH 7.5) containing 150 mM NaCI and 0.5 mM Ca2+, using an excitation wavelength of 340 nm with a band pass of 8 nm.

FIG. 9B illustrates the binding kinetics of the interaction of NtCBP4 CaM-binding peptide with dansyl CaM. Titration of the dansyl CaM with NtCBP4 CaM-binding peptide monitored by fluorescence enhancement.

The concentration of the dansyl CaM was 200 nM. Emitted fluorescence was measured at 480 nm. Data was fitted as described in the Examples section that follows.

FIG. 10 illustrates the alignment of the NtCBP4, NtCBP7 and olfactory channel cyclic nucleotide binding domains. Amino acid sequences of NtCBP4 (SEQ ID NO: 2; residues 487-616), NtCBP7 (SEQ ID NO: 4; residues 484-614), and rat olfactory channel (residues 464-586) are pictured in alignment. Regions corresponding to E. coli CAP secondary structure (Shabb et al., 1992) are underlined. Identical (boxed), conserved (gray shaded), and invariant (highlighted in black) amino acid residues are indicated. Residues that fit to the PROSITE cAMP/cGMP binding domain signature motif (PROSITE Accession Number PS50042) are overlined with asterisks. The NtCBP4 region that binds CaM is presented in bold letters.

FIG. 1 lA illustrates immunodetection of NtCBP4 expressed in insect cells. Non soluble fractions of SF9 cells (50 pLg total protein) were extracted from Sf9 cells infected with recombinant NtCBP4 (NtCBP4) or WT (Con) baculoviruses 1-3 days post infection. Proteins were separated by SDS-PAGE, stained by Coomassie Blue or transferred to a nitrocellulose membrane. The membrane was probed with [S] calmodulin and then with anti-NtCBP4 polyclonal antibodies. Immunoreactive proteins were detected by chemiluminescence. The positions of molecular-mass standards (kDa) are indicated.

FIG. 11B illustrates immunodetection of NtCBP4 expressed in transgenic tobacco. Tobacco proteins (25 g/lane) from soluble and membrane (microsome) fractions, and a sample of the full-length recombinant NtCBP4 protein (Rec) expressed in Sf9 insect cells were separated by SDS-PAGE and blotted to a nitrocellulose membrane. The

membrane was probed with anti-NtCBP4 polyclonal antibodies.

Immunoreactive proteins were detected by chemiluminescence. The positions of molecular-mass standards (kDa) are indicated.

FIG. 12A illustrates detection of NtCBP4 in tobacco plasma membranes with antibodies. Total microsomes from WT and transgenic tobacco seedlings were fractionated on a non-continuous sucrose gradient consisting of 20%, 30%, 34%, 38%, and 45% (w/w) sucrose. Membrane fractions were collected from interfaces between different sucrose concentrations. Proteins from each interface were electrophoresed, blotted and immiiiiodetected with anti-NtCBP4 antibodies, the 60-kDa subunit of vacuolar H-ATPase from oat roots (V-ATPas), endoplasmic reticulum BIP (ER-BIP), plasma membrane H-ATPase (P-ATPase), mitochondrial prohibitin (M-Prohibitin) and endoplasmic reticulum NADPH cytochrome- p450 reductase (ER-Cyt-p450).

FIG. 12B illustrated detection of NtCBP4 in tobacco plasma membranes with antibodies. Microsomal membranes (Mic) from roots of WT and transgenic plants (line 49-79) were fractionated by the aqueous two-phase partitioning method into a lower phase (L) enriched with intracellular membranes and an upper phase (U) enriched with plasma membranes. Proteins from each fraction (251lg/lane) were immunoblotted or stained with Coomassie Blue. Blots were probed with antibodies as described for Figure 12A.

FIG. 13A illustrates relative expression levels of NtCBP4 in transgenic tobacco lines. Immunodetection of NtCBP4 in microsomes of WT and transgenic plants with NtCBP4 cDNA in either antisense (AS), or sense (transgenic lines 15-89,13-93,49-79,10-2) orientation.

Immunodetection of vacuolar membrane H+-ATPase served as an internal standard. The relative levels of NtCBP4 in transgenic compared to WT (set as 1.00) plants were determined by densitometric scanning of the blot and are given below the immunoblot. 2+ FIG. 13B demonstrates tolerance of transgenic seedlings to Ni NiCl2-tolerance test of representative transgenic tobacco lines. WT and transgenic seedlings (line numbers indicated) were germinated in half- strength Hoagland's liquid medium in the absence or presence of NiC12 (concentration indicated). Photographs were taken after 12 days of exposure to NiCl2.

2+ FIG. 13C demonstrates tolerance of transgenic seedlings to Ni Photo enlargements of WT and transgenic seedlings (line 49-79) as in Figure 13B in the presence of 0.2 mM NiCl2.

FIG. 14A is a graphic representation of the effects of Ni on transgenic chlorophyll accumulation in transgenic seedlings. Relative chlorophyll content (%) (y axis) for WT and transgenic seedlings (line numbers on x axis). Data are given as the mean SD of three independent experiments. The values determined in seedlings grown without NiC12 were set as 100%. Chlorophyll accumulation corresponds to NtCBP4 expression level. 2+ FIG. 14B is a graphic representation of the effects of Ni on root growth of transgenic seedlings. Root length of seedlings of WT and transgenic line 49-79 grown for 12 days in the absence or presence of 0.1 mM NiC12. Root length was calculated as the mean SD of 25 seedlings for each measurement. The values determined in seedlings grown without NiC12 were set as 100%.. Root growth corresponds to NtCBP4 expression level.

FIG. 14C is a graphic representation of the effects of Ni2+ on transgenic seedling growth. Relative fresh weight of WT or transgenic (lines 49-79,10-2) seedlings grown in the absence or presence of different concentrations of NiCl2. Data are given as the mean SD of three independent experiments (150 seedlings were used for each point). In (a), (b), and (c), The values determined in seedlings grown without NiC12 were set as 100%. Resistance to NI2+ increases as NtCBP4 expression level increases.

FIG. 15A demonstrates tolerance of developed transgenic plants to Ni2+. WT and transgenic plants were grown for four weeks in half-strength Hoagland's liquid medium and then transferred to the same medium supplemented with 0.2 mM NiCl2. Photographs were taken after two weeks of exposure to NiCl2. Immunodetection of the vacuolar membrane H+- ATPase served as an internal standard.

FIG. 15B illustrates tolerance of developed transgenic calli to Ni2+.

Calli were regenerated from leaves of WT and transgenic line 10-2 on B5+0.8% agar medium. Tolerance of calli to Ni2+ was studied as described in the experimental procedures. Photographs were taken two weeks after recovery of calli on Ni2+-free medium (bar = 10 mm). Immunodetection of the vacuolar membrane H+-ATPase served as an internal standard.

FIG. 15C illustrates tolerance of developed transgenic calli to Ni2+.

Immunodetection of NtCBP4 in microsomes from calli of WT and transgenic line 10-2 is demonstrated. Immunodetection of the vacuolar membrane H+-ATPase served as an internal standard.

FIG. 16 illustrates Ni2+ accumulation in WT and transgenic plants.

Plants were grown for four weeks before exposing them to different concentrations of NiC12 (0-0.2 mM) for 24 hours. Ni content was determined by ICP-AES as described in the Experimental procedures. Data are the mean SD of three independent experiments.

FIG. 17A illustrates Pb2+ hypersensitivity and accumulation in transgenic plants. WT and transgenic seedlings grown with or without 1 mM Pb (N03) 2 for 12 days. Immunodetection of the vacuolar membrane H+-ATPase served as an internal standard.

FIG. 17B illustrates Pb2+ hypersensitivity and accumulation in transgenic plants. Relative chlorophyll content (%) in WT and transgenic seedlings (line numbers indicated). Chlorophyll content without Pb (N03) 2 was set as 100 % for each line. Data are the mean A SD of three independent experiments. Pb2+ accumulation correlates to NtCBP4 expression levels.

FIG. 17C illustrates Pb2+ hypersensitivity and accumulation in transgenic plants. Pb accumulation in shoots of WT and transgenic line 49- 79. The plants were grown for four weeks before exposing them to 0.1 mM Pb (N03) 2 for 24 hours. Pb content was determined as described in the Examples section that follows. Data are the mean SD of three independent experiments. Pb2+ accumulation correlates to NtCBP4 expression levels.

FIGs 18A-B show schematic presentations of DNA constructs for expression of the full-length and C-terminal truncated NtCBP4 in transgenic tobacco. Figure 18A shows the full-length NtCBP4 cDNA in a binary Ti plant transformation vector, as described by Arazi et al. (1999). Regions with the 35S CaMV promoter and the transcription termination sequence are shown in hatched boxes. The NtCBP4 six-transmembrane core, putative cyclic-nucleotide monophosphate-binding domain (cNBD, in grey; Arazi et al., 1999) and the overlapping calmodulin-binding domain (CaMBD, cross hatched; Arazi et al., 2000) are indicated. The amino acid sequence of part of the cNBD is shown on top. The CaMBD (bold letters) and the aB and aC predicted helices (underlined) of the cNBD are according to Arazi et al.

(2000). The downward arrow shows the site of the deletion used to create

the C-terminal truncated protein designated NtCBP4AC (amino acids Met,- Sers93). Numbers denote the terminal amino acid residues shown in the sequence, based on Arazi et al. (1999; 2000). Figure 18B shows the NtCBP4AC cDNA in a binary Ti-plant transformation vector. The NtCBP4AC cDNA, as described in Figure 18A, was prepared by cloning a corresponding PCR-amplified DNA fragment into the Xhol and EcoRI sites of the same vector used for the full-length NtCBP4.

FIGs 19A-B demonstrates Transgenic seedlings expressing NtCBP4AC are tolerant to Pb2+. Figure 19A-seedlings of WT and transgenictobacco expressing either the full-length NtCBP4 (NtCBP4FL; Arazi et al., 1999) or the NtCBP4AC mRNA (transgenic lines AC-22-11 and AC-42-11, AC-29) were germinated and grown in the presence of the indicated concentrations of Pb (NO3) 2 in modified Blaydes solution (pH 4.5) for 12 days and then photographed. Figure 19B-photo enlargements of seedlings of WT and representative transgenic lines in the presence of 0 and 0.75 mM Pb (N03) 2.

FIGs. 20A-B show expression analysis in transgenic plants. Figure 20A-twenty pg of total RNA samples from WT and the transgenic plants indicated were separated by gel electrophoresis, blotted, and hybridised with an NtCBP4-specific probe (upper panel), or with an actin-specific probe (lower panel). The gel positions of the 25S and 18S ribosomal RNA bands are indicated. Arrows point to the full-length NtCBP4 (FL) and the C- terminal truncated NtCBP4 (AC) mRNAs in the upper panel. Figure 20B- expression analysis of the full length NtCBP4 mRNA in WT, NtCBP4FL, and NtCBP4AC (line AC-42-11) plants by RT-PCR. Poly-A+ mRNA samples from the lines indicated were treated as described in Experimental procedures and a 404-bp DNA fragment corresponding to a region of the full-length NtCBP4 mRNA was amplified with the primers designated NtCBP4. Amplification of the corresponding region from the NtCBP4 cDNA clone with the same primers served as a positive control (cDNA control). The amounts of poly-A+ RNA were normalise using the expression of the tobacco ß-ATPase gene as a standard. Amplified DNA samples and DNA size markers (indicated in bp on the left) were fractionated by agarose gel electrophoresis, stained with ethidium bromide and photographed.

FIGs 21A-B show that Pb2+ tand accumulation in transgenic seedlings. Figure 21 A-relative fresh weight of WT and transgenic tobacco seedlings expressing the full-length NtCBP4 mRNA (NtCBP4FL;

Arazi et al., 1999), and two transgenic lines expressing the truncated NtCBP4 mRNA (AC-22-11 and AC-42-11) in the presence of different concentrations of Pb (N03) 2. Data are given as the mean fresh weight of 150 seedlings for each concentration SD of three independent experiments.

Fresh weight of seedlings of each line grown without Pb (N03) 2 was set as 100%. Figure 21B-lead accumulation in 12-day-old seedlings grown in the presence of 0.2 mM Pb (N03) 2. Seedlings were dried at 80°C for 3 days and lead content was determined by ICP-AES as described (Arazi et al., 1999). Data are the mean SD of three independent experiments.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is of (i) polynucleotides encoding calcium- dependent calmodulin-binding proteins from plants; (ii) polypeptides which are the translation products of these polynucleotides; (iii) expression and antisense vectors containing the polynucleotides or portions thereof ; (iv) cultured cells and transgenic or viral infected plants expressing the polynucleotides or portions thereof ; and methods of (v) using such plants to assist agriculture and/or (vi) phytoremediation, by providing plants characterized by heavy and other metal resistivity or plants capable of accumulating high concentration of a heavy or other toxic metal and thereby removing the heavy or other toxic metal from the environment.

As used herein the term"heavy metal"refers to a metal having a density above 5 g per cubic cm (see, Prasad M. N. V. and Hagemeyer J.

1999, Heavy Metal Stress in Plants, Springer-Verlag, Berlin). As used herein the term"toxic metal"refers to toxic concentrations of that metal.

The principles and operation of a according to the present invention may be better understood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

Isolation and analysis of tobacco cDNAs designated NtCBP4 and NtCBP7 and other family members are herein described. These cDNAs

encode Ca2+/Calmodulin (CaM)-binding proteins with sequences similar to K+-selective channels. NtCBP4 family members contain transmembrane domains, a pore motif, and a highly conserved cyclic nucleotide-binding domain in their C-terminus. Sequence analysis indicates that NtCBP4 and its family members (NtCBP7, NtCBP10, NtCBP63, StCBP18 and MsCBP15) are most similar to the olfactory cyclic nucleotide-gated channel (Dhallan et al., 1990). Differences between NtCBP4 family members and other dated ion channel proteins suggest a selectivity for cations other than potassium. Differences in amino acid sequences among family members, imply differences in ion permeability between members of this novel protein family.

The full-length NtCBP4 binds CaM in a Ca2+-dependent manner via a single CaM-binding domain ofapproximately 23 amino acid (Phe595- Trp, SEQ ID NO: 2). This domain is characterized by aromatic and long-chain aliphatic residues separated by positively charged amino acids.

Only NtCBP4 and its family members, and the olfactory channel, contain both CaM-and cyclic nucleotide-binding domains. In contrast to NtCBP4, in which the CaM-binding domain coincides with the cyclic nucleotide- binding domain, the CaM-binding domain is separate from the cyclic nucleotide-binding domain in the olfactory channel.

NtCBP4 resembles recently cloned ion transporters from Arabidopsis (Kohler et al., 1999) and Hordeum vulgare (Schuurink et al., 1998). Kohler et al. (1999) demonstrated that AtCNGC1 partially complements a yeast mutant deficient in K+ uptake and suggested that this protein is a non- selective cation channels. However, a major study of the function of this or other members of this plant protein family was not done (Leng, et al. 1999).

Transgenic techniques were used to investigate the functions of NtCBP4 in tobacco. Independently derived transgenic lines differing in the level of expressed NtCBP4 were assayed. The NtCBP4 protein was found in tobacco root and shoot microsomes, indicating its presence in the membranes of both organs. As predicted from its sequence, membrane solubilization studies revealed that NtCBP4 is an integral membrane protein. To determine the subcellular localization of NtCBP4, separation of membranes by sucrose gradients and by aqueous two-phase partitioning was performed. These experiments demonstrated that both WT and transgenic NtCBP4 co-fractionate with the plasma membrane. In conclusion, data submitted as part of the present invention support the postulated function of

NtCBP4 and its family members as a component of a plasma membrane ion channel.

. Analysis of the effect of the expression of NtCBP4 on metal uptake and sensitivity to toxic metals in transgenic plants was performed.

Transgenic plants were tested with different toxic metal ions. Transgenic plants with higher levels of NtCBP4 are relatively tolerant to Ni2+ but hypersensitive to Pb2+. Because of the dual role of Ni2+ as an essential microelement on the one hand and as a toxic environmental factor on the other, complete exclusion is not possible. Therefore, genetically engineering plants to have a reduced metal uptake can contribute to improving their tolerance to Ni2+. Exclusion of toxic metal ions by the plasma membrane is conceptually the best mechanism for preventing damage to cellular functions under metal stress conditions.

The tolerance of transgenic lines expressing relatively high levels of NtCBP4 to Ni2+ was apparent in very young seedlings and in plants grown for four weeks and then transferred to a Ni2+-containing medium for two weeks, as well as in transgenic calli. Thus, resistance to Ni2+ is both cell autonomous and independent of developmental stage. Determination of the concentration-dependent Ni accumulation in shoots of wild type and transgenic plants revealed that overexpression of NtCBP4 restricts the accumulation of Ni.

Because Pb is a non-essential element for plants and is extremely toxic, plant cells are not likely to possess specific Pb transporters.

However, certain metal uptake transporters in plants are relatively non- selective, such that both metal nutrients and non-essential toxic metals are taken up (Rubio et al., 1995; Huang et al., 1994). Consequently, the uptake of heavy metal ions, including Pb, by crop plants is a major cause for the accumulation of these toxic ions in the human body (Foy et al., 1978). On the other hand, plants that hyperaccumulate heavy and other metals should be useful for phytoremediation. Use of plants to extract Pb from contaminated soils requires a better understanding of the mechanisms of Pb2+ uptake, translocation and accumulation by plants. As part of the present invention, enhancement of the accumulation of Pb in plants by NtCBP4 is demonstrated. In animals, voltage-gated Ca2+ channels are permeable to Pb2+ (Simons and Pocock, 1987; Tomsing and Suszokiw, 1991). However, to date, plant transporters mediating Pb2+ uptake into plants have not been characterized at the molecular level. Therefore, NtCBP4 is the first example of a plant protein that can modulate Pb

tolerance and accumulation in plants. As such, it has unique value for improving phytoremediation strategies.

In spite of the fact that the physiological roles of NtCBP4 and the related genes from barley, Arabidopsis, and other plants, remains to be resolved, the remarkable effects of NtCBP4 on heavy and other metal tolerance and accumulation in transgenic plants can not be ignored. The transgenic plants prepared and characterized as part of the present invention, as well as other transgenic plants expressing modified NtCBP4 proteins with changes in their ion selectivity and regulatory properties will undoubtedly find myriad uses in sustaining commercial agriculture in an environment increasingly prone to contamination with toxic metal cations.

In summary, a channel-like protein family in plants with a new structural motif in which a phylogenetically conserved helix in the cyclic nucleotide-binds CaM with high affinity. This makes members of the new gene family communication points for cross-talk between Ca2+ and cyclic nucleotide signal transduction pathways in plants. Transgenic plants overexpressing NtCBP4 exhibit increased Ni2+ tolerance and hypersensitivity to Pb2+. Different family members are expected to influence tolerance or sensitivity to other metal cations. These qualities make the new gene family useful for cultivating plants in soil contaminated with toxic metal cations on the one hand, and for phytoremediation on the other hand.

By expressing a modified version of NtCBP4, lacking presumed regulatory domains, the phenotype, with respect to Pb2+ tolerance and accumulation, is the opposite of that exhibited by plants expressing the full- length protein. The contrasting phenotypes obtained by expressing two variants of the same gene strongly supports the likelihood that NtCBP4 is a component of an ion transport system that is responsible for Pb2+ entry into plant cells.

Because Pb2+ is a non-essential toxic metal, the presumed NtCBP4- associated ion transport mechanism is likely to have other yet unknown physiological roles. Of particular interest is the possible involvement of NtCBP4 and related proteins in Ca2+ signal transduction, either by the regulation of NtCBP4 by Ca2+ signals through calmodulin, by being permeable to Ca2+, or by both, like the mammalian cyclic nucleotide-gated non-selective cation channels. This would be consistent with earlier reports identifying Ca2+-permeable channels as a pathway for Pb2+ entry into animal cells (Simons and Pocock, 1987; Tomsig and Suszkiw, 1991) and plants

(Huang and Cunningham, 1996), and with the recent demonstration of cyclic nucleotide-dependent Ca2+ entry into human embryonic kidney cells transfected with the Arabidopsis AtCNGC2 gene (Leng et al., 1999).

The mammalian cyclic nucleotide-gated channels function as tetrameric complexes (Liu et al., 1996). Therefore, the inhibitory effect of NtCBP4AC on Pb2+ accumulation, and the concomitant improved tolerance to Pb2+ may have resulted from the formation of non functional NtCBP4/NtCBP4AC heteromeric complexes, since the endogenous native NtCBP4 gene was expressed in the background of the NtCBP4AC transgene. Regardless of the mechanism, an inhibitory effect of NtCBP4AC on the activity of NtCBP4-associated channels might influence certain physiological and developmental processes. One interesting difference between the plants expressing the full-length NtCBP4 and the plants expressing the truncated protein is the apparent male sterility in the latter.

Although the reason for this phenotype is at present unknown, further analysis of the two types of transgenic plants described here should be useful for elucidating the physiological role of NtCBP4. Based on the phenotypes that was characterised regarding Pb2+ uptake into plant cells, it is expect that the physiological responses that are mediated by NtCBP4- associated channels will be enhanced in transgenic lines overexpressing the full-length channel but attenuated in plants expressing the truncated one.

Thus, according to one aspect of the present invention there is provided an isolated nucleic acid comprising a polynucleotide at least 60 %, preferably at least 65 %, more preferably at least 70 %, still preferably at least 75 %, still preferably at least 80 %, yet preferably at least 90-100 % identical with any of SEQ ID NOs: 1,3,5,7,9,11 or portions thereof as determined using the Bestfit procedure of the DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the university of Wisconsin (gap creation penalty-50, gap extension penalty- 3), SEQ ID NOs: 1,3,5,7,9,11 or portions thereof, in particular.

According to another aspect of the present invention there is provided an isolated nucleic acid comprising a polynucleotide encoding a polypeptide being at least 60 %, preferably at least 65 %, more preferably at least 70 %, still preferably at least 75 %, still preferably at least 80 %, yet preferably at least 90-100 % homologous (similar + identical acids) with any of SEQ ID NOs: 2,4,6,8,10,12 or portions thereof as determined using the Bestfit procedure of the DNA sequence analysis software package

developed by the Genetic Computer Group (GCG) at the university of Wisconsin (gap creation penalty-50, gap extension penalty-3).

According to yet another aspect of the present invention there is provided an isolated nucleic acid comprising a plant derived polynucleotide encoding a transmembrane polypeptide having a cation channel activity when assembled in a plasmatic membrane of a plant cell, the polypeptide having overlapping (coinciding) cyclic nucleotide-binding domain site and calmodulin-binding site. The polypeptide, when incorporated in a plasmatic membrane functions as a metal channel, i. e., in some embodiments it transports metal ions into the cell cytoplasm, wherein in other embodiments it transports metal ions out of the cell cytoplasm.

According to still another aspect of the present invention there is provided an isolated nucleic acid comprising a polynucleotide hybridizable with any of SEQ ID NOs: l, 3,5,7,9,11 or portions thereof.

According to an additional aspect of the present invention there is provided an isolated nucleic acid comprising a polynucleotide hybridizable with a polynucleotide encoding a polypeptide as set forth in any of SEQ ID NOs: 2,4,6,8,10,12 or portions thereof.

Hybridization for long nucleic acids (e. g., above 200 bp in length) is effected according to preferred embodiments of the present invention by stringent or moderate hybridization, wherein stringent hybridization is effected by a hybridization solution containing 10 % dextrane sulfate, 1 M NaCI, 1 % SDS and 5 x 106 cpm 32p labeled probe, at 65 °C, with a final wash solution of 0.2 x SSC and 0.1 % SDS and final wash at 65°C; whereas moderate hybridization is effected by a hybridization solution containing 10 % dextrane sulfate, 1 M NaCI, 1 % SDS and 5 x 106 cpm 32p labeled probe, at 65 °C, with a final wash solution of 1 x SSC and 0.1 % SDS and final wash at 50 °C.

It will be appreciated that by using the above hybridization conditions and/or other molecular techniques, such as those further exemplified in the Examples section that follows, one of ordinary skills, without undue experimentation, would readily isolate additional members of the new gene family. The scope of the present invention also encompasses such sequences which are the result of man induced variation, such as site directed or non-specific mutagenesis. Methods of man induced variation of nucleic acids are well known in the art and are further described in detail Sambrook et al., molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. (1989), which is

incorporated herein by reference. The scope of the present invention also encompasses such sequences which are the result of naturally occurring variations. Such variations can be characterized and such sequence alterations isolated by one ordinarily skilled in the art using the assays and procedures described hereinunder in the Examples section that follows. In addition, the scope of the present invention also encompasses both cDNA and genomic sequences. cDNA sequences have been isolated while reducing the present invention to practice. By employing the stringent hybridization conditions described herein one of skills in the art would be able, without undue experimentation, to readily isolate genomic sequences corresponding to the isolated or isolatable cDNA sequences.

According to yet an additional aspect of the present invention there is provided a nucleic acid construct comprising a polynucleotide as set forth herein downstream of a plant promoter.

As used herein in the specification and in the claims section that follows the phrase"plant promoter"includes a promoter which can direct gene expression in plant cells (including DNA containing organelles). Such a promoter can be derived from a plant, bacterial, viral, fungal or animal origin. Such a promoter can be constitutive, i. e., capable of directing high level of gene expression in a plurality of plant tissues, tissue specific, i. e., capable of directing gene expression in a particular plant tissue or tissues, inducible, i. e., capable of directing gene expression under a stimulus, or chimeric, i. e., formed of portions of at least two different promoters.

Thus, the plant promoter employed can be a constitutive promoter, a tissue specific promoter, an inducible promoter or a chimeric promoter.

Examples of constitutive plant promoters include, without being limited to, CaMV35S and CaMV19S promoters, FMV34S promoter, sugarcane bacilliform badnavirus promoter, CsVMV promoter, Arabidopsis ACT2/ACT8 actin promoter, Arabidopsis ubiquitin UBQ1 promoter, barley leaf thionin BTH6 promoter, and rice actin promoter.

Examples of tissue specific promoters include, without being limited to, bean phaseolin storage protein promoter, DLEC promoter, PHSp promoter, zein storage protein promoter, conglutin gamma promoter from soybean, AT2S1 gene promoter, ACT11 actin promoter from Arabidopsis, napA promoter from Brassica napus and potato patatin gene promoter.

The inducible promoter is a promoter induced by a specific stimuli such as stress conditions comprising, for example, light, temperature, chemicals, drought, high salinity, osmotic shock, oxidant conditions or in

case of pathogenicity and include, without being limited to, the light- inducible promoter derived from the pea rbcS gene, the promoter from the alfalfa rbcS gene, the promoters DRE, MYC and MYB active in drought; the promoters INT, INPS, prxEa, Ha hspl7.7G4 and RD21 active in high salinity and osmotic stress, and the promoters hsr203J and str246C active in pathogenic stress.

According to still an additional aspect of the present invention there is provided a recombinant protein comprising a polypeptide encoded by any of the polynucleotides described herein.

According to a further aspect of the present invention there is provided a recombinant protein comprising a polypeptide at least 60 %, preferably at least 65 %, more preferably at least 70 %, still preferably at least 75 %, still preferably at least 80 %, yet preferably at least 90-100 % homologous with any of SEQ ID NOs: 2,4,6,8,10,12 or portions thereof as determined using the Bestfit procedure of the DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the university of Wisconsin (gap creation penalty-50, gap extension penalty- 3), SEQ ID NOs: 2,4,6,8,10,12 or portions thereof, in particular.

As used herein in the specification and in the claims section that follows the term"polypeptide"refers also to a protein, in particular a transmembrane protein, which may include a transit peptide, and further to a post translationally modified protein, such as, but not limited to, a phosphorylated protein, glycosylated protein, ubiquitinylated protein, acetylated protein, methylated protein, etc.

According to a preferred embodiment of the present invention, the polypeptide includes an N terminal transit peptide fused thereto which serves for directing the polypeptide to a specific membrane. Such a membrane can be, for example, the cell membrane, wherein the polypeptide will serve to transport metal ions from the apoplast into the cytoplasm or vice versa, or, such a membrane can be the outer and preferably the inner chloroplast membrane, wherein the polypeptide will serve to transport metal ions from the cytoplasm to the intermembranal space and the stroma, respectively, or vice versa. Transit peptides which function as herein described are well known in the art. Further description of such transit peptides is found in, for example, Johnson et al. The Plant Cell (1990) 2: 525-532; Sauer et al. EMBO J. (1990) 9: 3045-3050; Mueckler et al.

Science (1985) 229: 941-945; Von Heijne, Eur. J. Biochem. (1983) 133: 17- 21; Yon Heijne, J. Mol. Biol. (1986) 189: 239-242; Iturriaga et al. The

Plant Cell (1989) 1: 381-390; McKnight et al., Nucl. Acid Res. (1990) 18: 4939-4943; Matsuoka and Nakamura, Proc. Natl. Acad. Sci. USA (1991) 88: 834-838. A recent text book entitled"Recombinant proteins from plants", Eds. C. Cunningham and A. J. R. Porter, 1998 Humana Press Totowa, N. J. describe methods for the production of recombinant proteins in plants and methods for targeting the proteins to different compartments in the plant cell. The book by Cunningham and Porter is incorporated herein by reference. It will however be appreciated by one of skills in the art that a large number of membrane integrated proteins fail to poses a removable transit peptide. It is accepted that in such cases a certain amino acid sequence in said proteins serves not only as a structural portion of the protein, but also as a transit peptide.

According to yet a further aspect of the present invention there is provided a genetically transformed or virus infected cell or plant comprising any of the isolated nucleic acids or constructs described herein and preferably expressing any of the recombinant proteins described herein.

As used herein in the specification and in the claims section that follows the term"plant"includes organisms, both unicellular or multicellular, both prokaryotes or eukaryotes, both soil grown or aquatic, capable of producing complex organic materials, especially carbohydrates, from carbon dioxide using light as the source of energy and with the aid of chlorophyll and optionally associated pigments.

As used herein in the specification and in the claims section that follows the term"transformed"and its conjugations such as transformation, transforming and transform, all relate to the process of introducing heterologous nucleic acid sequences into a cell or an organism, which nucleic acid are propagatable to the offspring. The term thus reads on, for example,"genetically modified","transgenic"and"transfected", which may be used herein to further described the present invention. The term relates both to introduction of a heterologous nucleic acid sequence into the genome of an organism and/or into the genome of a nucleic acid containing organelle thereof, such as into a genome of chloroplast or a mitochondrion.

As used herein in the specification and in the claims section that follows the phrase"viral infected"includes infection by a virus carrying a heterologous nucleic acid sequence. Such infection typically results in transient expression of the nucleic acid sequence, which nucleic acid sequence is typically not integrated into a genome and therefore not

propagatable to offspring, unless further infection of such offspring is experienced.

There are various methods of introducing foreign genes into both monocotyledonous and dicotyledenous plants (Potrykus, I., Annu. Rev.

Plant. Physiol., Plant. Mol. Biol. (1991) 42: 205-225; Shimamoto et al., Nature (1989) 338: 274-276). The principle methods of causing stable integration of exogenous DNA into plant genomic DNA include two main approaches: (i) Agrobacterium-mediated gene transfer: Klee et al. (1987) Annu. Rev. Plant Physiol. 38: 467-486; Klee and Rogers in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 2-25; Gatenby, in Plant Biotechnology, eds. Kung, S. and Arntzen, C. J., Butterworth Publishers, Boston, Mass. (1989) p.

93-112.

(ii) direct DNA uptake: Paszkowski et al., in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 52-68; including methods for direct uptake of DNA into protoplasts, Toriyama, K. et al. (1988) Bio/Technology 6: 1072-1074.

DNA uptake induced by brief electric shock of plant cells: Zhang et al.

Plant Cell Rep. (1988) 7: 379-384. Fromm et al. Nature (1986) 319: 791- 793. DNA injection into plant cells or tissues by particle bombardment, Klein et al. Bio/Technology (1988) 6: 559-563; McCabe et al.

Bio/Technology (1988) 6: 923-926; Sanford, Physiol. Plant. (1990) 79: 206- 209; by the use of micropipette systems: Neuhaus et al., Theor. Appl.

Genet. (1987) 75: 30-36; Neuhaus and Spangenberg, Physiol. Plant. (1990) 79: 213-217; or by the direct incubation of DNA with germinating pollen, DeWet et al. in Experimental Manipulation of Ovule Tissue, eds.

Chapman, G. P. and Mantell, S. H. and Daniels, W. Longman, London, (1985) p. 197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83: 715- 719.

The Agrobacterium system includes the use of plasmid vectors that contain defined DNA segments that integrate into the plant genomic DNA.

Methods of inoculation of the plant tissue vary depending upon the plant species and the Agrobacterium delivery system. A widely used approach is the leaf disc procedure which can be performed with any tissue explant that provides a good source for initiation of whole plant differentiation. Horsch

et al. in Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A supplementary approach employs the Agrobacterium delivery system in combination with vacuum infiltration.

The Agrobacterium system is especially viable in the creation of transgenic dicotyledenous plants.

There are various methods of direct DNA transfer into plant cells. In electroporation, the protoplasts are briefly exposed to a strong electric field.

In microinjection, the DNA is mechanically injected directly into the cells using very small micropipettes. In microparticle bombardment, the DNA is adsorbed on microprojectiles such as magnesium sulfate crystals or tungsten particles, and the microprojectiles are physically accelerated into cells or plant tissues.

Following transformation plant propagation is exercised. The most common method of plant propagation is by seed. Regeneration by seed propagation, however, has the deficiency that due to heterozygosity there is a lack of uniformity in the crop, since seeds are produced by plants according to the genetic variances governed by Mendelian rules. Basically, each seed is genetically different and each will grow with its own specific traits. Therefore, it is preferred that the transformed plant be produced such that the regenerated plant has the identical traits and characteristics of the parent transgenic plant. Therefore, it is preferred that the transformed plant be regenerated by micropropagation which provides a rapid, consistent reproduction of the transformed plants.

Micropropagation is a process of growing new generation plants from a single piece of tissue that has been excised from a selected parent plant or cultivar. This process permits the mass reproduction of plants having the preferred tissue expressing the protein. The new generation plants which are produced are genetically identical to, and have all of the characteristics of, the original plant. Micropropagation allows mass production of quality plant material in a short period of time and offers a rapid multiplication of selected cultivars in the preservation of the characteristics of the original transgenic or transformed plant. The advantages of cloning plants are the speed of plant multiplication and the quality and uniformity of plants produced.

Micropropagation is a multi-stage procedure that requires alteration of culture medium or growth conditions between stages. Thus, the micropropagation process involves four basic stages: Stage one, initial tissue culturing; stage two, tissue culture multiplication; stage three,

differentiation and plant formation; and stage four, greenhouse culturing and hardening. During stage one, initial tissue culturing, the tissue culture is established and certified contaminant-free. During stage two, the initial tissue culture is multiplied until a sufficient number of tissue samples are produced to meet production goals. During stage three, the tissue samples grown in stage two are divided and grown into individual plantlets. At stage four, the transformed plantlets are transferred to a greenhouse for hardening where the plants'tolerance to light is gradually increased so that it can be grown in the natural environment.

The basic bacterial/plant vector construct according to the present invention will preferably provide a broad host range prokaryote replication origin; a prokaryote selectable marker; and, for Agrobacterium transformations, T DNA sequences for Agrobacterium-mediated transfer to plant chromosomes. Where the heterologous sequence is not readily amenable to detection, the construct will preferably also have a selectable marker gene suitable for determining if a plant cell has been transformed. A general review of suitable markers for the members of the grass family is found in Wilmink and Dons, Plant Mol. Biol. Reptr. (1993) 11: 165-185.

Sequences suitable for permitting integration of the heterologous sequence into the plant genome are also recommended. These might include transposon sequences and the like for homologous recombination as well as Ti sequences which permit random insertion of a heterologous expression cassette into a plant genome.

Suitable prokaryote selectable markers include resistance toward antibiotics such as ampicillin or tetracycline. Other DNA sequences encoding additional functions may also be present in the vector, as is known in the art.

The constructs of the subject invention will include an expression cassette for expression of the protein of interest. Usually, there will be only one expression cassette, although two or more are feasible. The recombinant expression cassette will contain in addition to the heterologous sequence one or more of the following sequence elements, a promoter region, plant 5'untranslated sequences, initiation codon depending upon whether or not the structural gene comes equipped with one, and a transcription and translation termination sequence. Unique restriction enzyme sites at the 5'and 3'ends of the cassette allow for easy insertion into a pre-existing vector.

Viruses are a unique class of infectious agents whose distinctive features are their simple organization and their mechanism of replication.

In fact, a complete viral particle, or virion, may be regarded mainly as a block of genetic material (either DNA or RNA) capable of autonomous replication, surrounded by a protein coat and sometimes by an additional membranous envelope such as in the case of alpha viruses. The coat protects the virus from the environment and serves as a vehicle for transmission from one host cell to another.

Viruses that have been shown to be useful for the transformation of plant hosts include CaV, TMV and BV. Transformation of plants using plant viruses is described in U. S. Pat. No. 4,855,237 (BGV), EP-A 67,553 (TMV), Japanese Published Application No. 63-14693 (TMV), EPA 194,809 (BV), EPA 278,667 (BV); and Gluzman, Y. et al., Communications in Molecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, New York, pp. 172-189 (1988). Pseudovirus particles for use in expressing foreign DNA in many hosts, including plants, is described in WO 87/06261.

Construction of plant RNA viruses for the introduction and expression of non-viral foreign genes in plants is demonstrated by the above references as well as by Dawson, W. O. et al., Virology (1989) 172: 285- 292; Takamatsu et al. EMBO J. (1987) 6: 307-311; French et al. Science (1986) 231: 1294-1297; and Takamatsu et al. FEBS Letters (1990) 269: 73- 76.

When the virus is a DNA virus, the constructions can be made to the virus itself. Alternatively, the virus can first be cloned into a bacterial plasmid for ease of constructing the desired viral vector with the foreign DNA. The virus can then be excised from the plasmid. If the virus is a DNA virus, a bacterial origin of replication can be attached to the viral DNA, which is then replicated by the bacteria. Transcription and translation of this DNA will produce the coat protein which will encapsidate the viral DNA. If the virus is an RNA virus, the virus is generally cloned as a cDNA and inserted into a plasmid. The plasmid is then used to make all of the constructions. The RNA virus is then produced by transcribing the viral sequence of the plasmid and translation of the viral genes to produce the coat protein (s) which encapsidate the viral RNA.

Construction of plant RNA viruses for the introduction and expression of non-viral foreign genes in plants is demonstrated by the above references as well as in U. S. Pat. No. 5,316,931

In one embodiment, a plant viral nucleic acid is provided in which the native coat protein coding sequence has been deleted from a viral nucleic acid, a non-native plant viral coat protein coding sequence and a non-native promoter, preferably the subgenomic promoter of the non-native coat protein coding sequence, capable of expression in the plant host, packaging of the recombinant plant viral nucleic acid, and ensuring a systemic infection of the host by the recombinant plant viral nucleic acid, has been inserted. Alternatively, the coat protein gene may be inactivated by insertion of the non-native nucleic acid sequence within it, such that a protein is produced. The recombinant plant viral nucleic acid may contain one or more additional non-native subgenomic promoters. Each non-native subgenomic promoter is capable of transcribing or expressing adjacent genes or nucleic acid sequences in the plant host and incapable of recombination with each other and with native subgenomic promoters.

Non-native (foreign) nucleic acid sequences may be inserted adjacent the native plant viral subgenomic promoter or the native and a non-native plant viral subgenomic promoters if more than one nucleic acid sequence is included. The non-native nucleic acid sequences are transcribed or expressed in the host plant under control of the subgenomic promoter to produce the desired products.

In a second embodiment, a recombinant plant viral nucleic acid is provided as in the first embodiment except that the native coat protein coding sequence is placed adjacent one of the non-native coat protein subgenomic promoters instead of a non-native coat protein coding sequence.

In a third embodiment, a recombinant plant viral nucleic acid is provided in which the native coat protein gene is adjacent its subgenomic promoter and one or more non-native subgenomic promoters have been inserted into the viral nucleic acid. The inserted non-native subgenomic promoters are capable of transcribing or expressing adjacent genes in a plant host and are incapable of recombination with each other and with native subgenomic promoters. Non-native nucleic acid sequences may be inserted adjacent the non-native subgenomic plant viral promoters such that said sequences are transcribed or expressed in the host plant under control of the subgenomic promoters to produce the desired product.

In a fourth embodiment, a recombinant plant viral nucleic acid is provided as in the third embodiment except that the native coat protein coding sequence is replaced by a non-native coat protein coding sequence.

The viral vectors are encapsidated by the coat proteins encoded by the recombinant plant viral nucleic acid to produce a recombinant plant virus. The recombinant plant viral nucleic acid or recombinant plant virus is used to infect appropriate host plants. The recombinant plant viral nucleic acid is capable of replication in the host, systemic spread in the host, and transcription or expression of foreign gene (s) in the host to produce the desired protein.

Thus, according to a preferred embodiment of the present invention the polynucleotide or nucleic acid molecule of the present invention further includes one or more sequence elements, such as, but not limited to, a nucleic acid sequence encoding a transit peptide, an origin of replication for propagation in bacterial cells, at least one sequence element for integration into a plant's genome, a polyadenylation recognition sequence, a transcription termination signal, a sequence encoding a translation start site, a sequence encoding a translation stop site, plant RNA virus derived sequences, plant DNA virus derived sequences, tumor inducing (Ti) plasmid derived sequences and a transposable element derived sequence.

Thus, according to preferred embodiments of the present invention, the step of transforming the cell or plant with a polynucleotide encoding polypeptide according to the present invention is effected by a method such as genetic transformation and transient transformation (viral infection).

Genetic transformation can be effected by, for example, Agrobaterium mediated transformation, whereas transient transformation can be effected by, for example, viral infection. Both transient and genetic transformation can be effected by electroporation, particle bombardment or any of the other methods listed and further described hereinabove.

A technique for introducing heterologous nucleic acid sequences to the genome of the chloroplasts is known. This technique involves the following procedures. First, plant cells are chemically treated so as to reduce the number of chloroplasts per cell to about one. Then, the heterologous nucleic acid is introduced via particle bombardment into the cells with the aim of introducing at least one heterologous nucleic acid molecule into the chloroplasts. The heterologous nucleic acid is selected such that it is integratable into the chloroplast's genome via homologous recombination which is readily effected by enzymes inherent to the chloroplast. To this end, the heterologous nucleic acid includes, in addition to a gene of interest, at least one nucleic acid stretch which is derived from the chloroplast's genome. In addition, the heterologous nucleic acid

includes a selectable marker, which serves by sequential selection procedures to ascertain that all or substantially all of the copies of the chloroplast genomes following such selection will include the heterologous nucleic acid. Further details relating to this technique are found in U. S. Pat.

Nos. 4,945,050; and 5,693,507 which are incorporated herein by reference.

A polypeptide can thus be produced by the protein expression system of the chloroplast and become integrated into the chloroplast's inner membrane.

According to still a further aspect of the present invention there is provided an antibody specifically recognizing any of the recombinant proteins as described herein and/or natural equivalents thereof. Such an antibody can be, for example, a polyclonal antibody produced by a non- human mammal or in an egg, or alternatively, such an antibody can be a monoclonal antibody produced by a cell such as a hybridoma.

Thus, the present invention can utilize serum immunoglobulins, polyclonal antibodies or fragments thereof, (i. e., immunoreactive derivative of an antibody), or monoclonal antibodies or fragments thereof.

Monoclonal antibodies or purified fragments of the monoclonal antibodies having at least a portion of an antigen binding region, including such as Fv, F (abl) 2, Fab fragments (Harlow and Lane, 1988 Antibody, Cold Spring Harbor), single chain antibodies (U. S. Patent 4,946,778), chimeric or humanized antibodies and complementarily determining regions (CDR) may be prepared by conventional procedures. Purification of these serum immunoglobulins antibodies or fragments can be accomplished by a variety of methods known to those of skill including, precipitation by ammonium sulfate or sodium sulfate followed by dialysis against saline, ion exchange chromatography, affinity or immunoaffinity chromatography as well as gel filtration, zone electrophoresis, etc. (see Goding in, Monoclonal Antibodies: Principles and Practice, 2nd ed., pp. 104-126,1986, Orlando, Fla., Academic Press). Under normal physiological conditions antibodies are found in plasma and other body fluids and in the membrane of certain cells and are produced by lymphocytes of the type denoted B cells or their functional equivalent. Antibodies of the IgG class are made up of four polypeptide chains linked together by disulfide bonds. The four chains of intact IgG molecules are two identical heavy chains referred to as H-chains and two identical light chains referred to as L-chains. Additional classes includes IgD, IgE, IgA, IgM and related proteins.

Methods for the generation and selection of monoclonal antibodies are well known in the art, as summarized for example in reviews such as

Tramontano and Schloeder, Methods in Enzymology 178,551-568,1989.

A recombinant protein of the present invention may be used to generate antibodies in vitro. More preferably, the recombinant protein of the present invention is used to elicit antibodies in vivo. In general, a suitable host animal or egg is immunized with the recombinant protein of the present invention. Advantageously, the animal host used is a mouse of an inbred strain. Animals or eggs are typically immunized with a mixture comprising a solution of the recombinant protein of the present invention in a physiologically acceptable vehicle, and any suitable adjuvant, which achieves an enhanced immune response to the immunogen. By way of example, the primary immunization conveniently may be accomplished with a mixture of a solution of the recombinant protein of the present invention and Freund's complete adjuvant, said mixture being prepared in the form of a water in oil emulsion. Typically the immunization may be administered to the animals intramuscularly, intradermally, subcutaneously, intraperitoneally, into the footpads, or by any appropriate route of administration. The immunization schedule of the immunogen may be adapted as required, but customarily involves several subsequent or secondary immunizations using a milder adjuvant such as Freund's incomplete adjuvant. Antibody titers and specificity of binding to the protein can be determined during the immunization schedule by any convenient method including by way of example radioimmunoassay, or enzyme linked immunosorbant assay, which is known as the ELISA assay.

When suitable antibody titers are achieved, antibody producing lymphocytes from the immunized animals are obtained, and these are cultured, selected and cloned, as is known in the art. Typically, lymphocytes may be obtained in large numbers from the spleens of immunized animals, but they may also be retrieved from the circulation, the lymph nodes or other lymphoid organs.

Lymphocytes are then fused with any suitable myeloma cell line, to yield hybridomas, as is well known in the art. Alternatively, lymphocytes may also be stimulated to grow in culture, and may be immortalized by methods known in the art including the exposure of these lymphocytes to a virus, a chemical or a nucleic acid such as an oncogene, according to established protocols. After fusion, the hybridomas are cultured under suitable culture conditions, for example in multiwell plates, and the culture supernatants are screened to identify cultures containing antibodies that recognize the hapten of choice. Hybridomas that secrete antibodies that recognize the recombinant protein of the present invention are cloned by limiting dilution

and expanded, under appropriate culture conditions. Monoclonal antibodies are purified and characterized in terms of immunoglobulin type and binding affinity.

According to yet a further aspect of the present invention there is provided a method of increasing a tolerance of a plant to a metal cation.

The method according to this aspect of the present invention comprising the step of overexpressing in the plant a recombinant protein which reduces uptake and concentration of the metal cation within the plant cells.

According to still a further aspect of the present invention there is provided a method for phytoremediation of an area polluted with a metal cation. The method according to this aspect of the present invention is effected by implementing the following method steps, in which, in a first step, a plant is provided characterized in resistivity to elevated concentrations of the metal cation, the plant overexpressing a recombinant protein which facilitates uptake and concentration of the metal cation within the plant cells. In a second step of the method according to this aspect of the present invention the plant in planted in the polluted area. Then, following a time period, in which at least a fraction of the metal cation in the polluted area has been accumulated in the plant, the plant is harvested, thereby at least the fraction of the metal cation is removed from the area.

These steps are optionally repeated until a sufficient amount of the metal cation has been removed from the polluted area. Methods well known in the art can be employed to monitor the phytoremediation process of a polluted area. Such methods include both chemical/physical methods which are used to directly determine the concentration of the metal cation in the polluted area, and biological methods in which a series of plants with variable sensitivity to the metal cation are planted and their growth or growth inhibition monitored to thereby provide an insight to the level of phytoremediation so far achieved. It will be appreciated that according to the present invention terrestrial or aquatic phytoremediation can be employed, provided that suitable terrestrial or aquatic plants are selected for such purposes. It will further be appreciated that removing plant harvest can be effected both from terrestrial and aquatic environments. In the latter case nets or filters can be employed to effect plant harvesting, depending on the size of the plants employed.

The following provides several preferred embodiments employed when implementing any one of the two methods according to the present invention described above.

Thus, according to a preferred embodiment of the present invention, and in order to implement the methods according to the present invention, the plant is genetically transformed or viral infected with a nucleic acid including a polynucleotide at least 60 %, preferably at least 70 %, more preferably at least 80 %, still preferably at least 90 %, yet preferably at least 100 % identical with any of SEQ ID NOs: I, 3,5,7,9,11,15,17 or portions thereof as determined using the Bestfit procedure of the DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the university of Wisconsin (gap creation penalty-50, gap extension penalty-3), SEQ ID NOs: l, 3,5,7,9,11,15,17 or portions thereof, in particular.

According to anther preferred embodiment of the present invention, and in order to implement the methods according to the present invention, the plant is genetically transformed or viral infected with a nucleic acid including a polynucleotide encoding a polypeptide being at least 60 %, preferably at least 70 %, more preferably at least 80 %, still preferably at least 90 %, yet preferably at least 100 % homologous (identical + similar) with any of SEQ ID NOs: 2,4,6,8,10,12,16,18 or portions thereof as determined using the Bestfit procedure of the DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the university of Wisconsin (gap creation penalty-50, gap extension penalty- 3).

According to still anther preferred embodiment of the present invention, and in order to implement the methods according to the present invention, the plant is genetically transformed or viral infected with a nucleic acid including a plant derived polynucleotide encoding a transmembrane polypeptide having a cation channel activity when assembled in a plasmatic membrane of a plant cell, the polypeptide having overlapping cyclic nucleotide-binding domain site and calmodulin-binding site.

According to still anther preferred embodiment of the present invention, and in order to implement the methods according to the present invention, the plant is genetically transformed or viral infected with a nucleic acid including a polynucleotide hybridizable with any of SEQ ID NOs: l, 3,5,7,9,11,15,17 or portions thereof under hybridization conditions of hybridization solution containing 10 % dextrane sulfate, 1 M NaCl, 1 % SDS and 5 x 106 cpm 32p labeled probe, at 65 °C, with a final wash solution of 1 x SSC and 0.1 % SDS and final wash at 50 °C.

According to still anther preferred embodiment of the present invention, and in order to implement the methods according to the present invention, the plant is genetically transformed or viral infected with a nucleic acid including a polynucleotide hybridizable with a polynucleotide encoding a polypeptide as set forth in any of SEQ ID NOs: 2,4,6,8,10,12, 16,18 or portions thereof under hybridization conditions of hybridization solution containing 10 % dextrane sulfate, 1 M NaCl, 1 % SDS and 5 x 106 cpm 32p labeled probe, at 65 °C, with a final wash solution of 1 x SSC and 0.1 % SDS and final wash at 50 °C.

According to a further preferred embodiment of the present invention, and in order to implement the methods according to the present invention, the plant is genetically transformed or viral infected with a nucleic acid construct comprising a polynucleotide as set forth herein downstream of a plant promoter in a sense orientation. Plant promoters and plant transformation/viral infection methods are further described hereinabove.

According to a further preferred embodiment of the present invention, and in order to implement the methods according to the present invention, the recombinant protein includes a polypeptide at least 60 %, preferably at least 70 %, more preferably at least 80 %, still preferably at least 90 %, yet preferably at least 100 % homologous with any of SEQ ID NOs: 2,4,6,8,10,12,16,18 or portions thereof as determined using the Bestfit procedure of the DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the university of Wisconsin (gap creation penalty-50, gap extension penalty-3).

According to a further preferred embodiment of the present invention, and in order to implement the methods according to the present invention, the recombinant protein includes a calmodulin binding domain.

According to a further preferred embodiment of the present invention, and in order to implement the methods according to the present invention, the recombinant protein further includes a cyclic nucleotide binding domain.

According to a further preferred embodiment of the present invention, and in order to implement the methods according to the present invention, the calmodulin binding domain and the cyclic nucleotide binding domain overlap (coincide).

According to still further features in the described preferred embodiments the metal cation is Ni, Na, Ba, Cd, Co, Cu, La, Mn, Zn and/or

Pb. Using the molecular, physical and physiological methods described in the Examples section that follows, one of ordinary skills would be able without undue experimentation to characterize additional sequences of the new gene family according to the present invention in order to find sequences associated with accumulation of or resistance to any of the above listed metal cations.

According to another aspect of the present invention there is provided a male sterile plant expressing a C termainl truncated form of a calcium-dependent calmodulin-binding protein as herein described.

Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

EXAMPLES Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures in recombinant DNA technology described below are these well known and commonly employed in the art. Standard techniques are used for cloning, DNA and RNA isolation, amplification and purification.

Generally enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like are performed according to the manufacturers'specifications. These techniques and various other techniques are generally performed according to Sambrook et al., molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. (1989). The procedures described therein and in other citations are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

MATERIALSAND EXPERIMENTAL METHODS The following experimental methods were employed in conjunction with the examples described hereinbelow.

Library Screenings and Sequence Analysis : Nicotiana tabacum (tobacco var. Samsun NN) leaf cDNA library was obtained from R. Fluhr (Weizmann Institute of Science). Library screening was performed using high specific activity (approximately 2 x 106 cpm Hg-l) 35S-labeled recombinant CaM as a probe (Fromm, et al., 1992). Full-length clones were obtained by screening 1 x 106 plaque forming units of the above cDNA library with 32P-labeled partial NtCBP4 DNA as a probe. Both strands of the NtCBP4 and NtCBP7 full-length cDNAs were sequenced at the Weizmann Institute Sequencing Unit (SEQ ID NOs: 1 and 3). Sequence analysis was performed using the GCG (Genetics Computer Group, Inc., Wisconsin, USA), version 9.0 (Devereux et al., 1984) and the PROSITE server (European Bioinformatics Institute) software. Hydropathy was calculated by published methods (Lockow et al., 1988).

Additional screenings of Potato guard cell cDNA library (given by Berned Muller-Rober, Max Planck Institute of Molecular Plant Physiology, Karl-Liebknecht-Strabetae 25, Haus 20, D-14476 Golm/Potsdam, Germany) and Alfalfa root cDNA library (given by Aviha Zilberstein, Department of Plant Sciences, Tel-Aviv university) produced clones StCBP18 (SEQ ID NO: 9) and MsCBP15 (SEQ ID NO: 11) respectively.

Isolation of cDNAs Encoding Channel-Like Proteins Using RT- PCR: Degenerate oligonucleotides corresponding to highly conserved sequences flanking the S5 and S6 transmembrane domains were used as sense and antisense primers as follows: Sense-5'-TGGAA (T/C) AA (A/G) AT (/C/A) TT (T/C) GT-3' (SEQ ID NO: 13) and antisense-5'-GA (A/G) ACIGCITGGG (C/G) IGG-3' (SEQ ID NO: 14, I = inosine). As a template served cDNA that was synthesized from tobacco leaf poly (A) + RNA using Superscript II reverse transcriptase (GIBCO- BRL). The amplified DNA fragments were subcloned into pGEM vector (Promega) and their nucleotide sequences were determined. PCR conditions were: 1 minute at 94 °C, followed by 40 cycles oh : 1 minute at 94 °C, 1 minute at 48 °C and 1 minute at 72 °C, in the presence an appropriate PCR buffer containing 2.5 mM Mg.

Bacterial expression of GST fusion proteins and antibody preparation: Extracts of Escherichia coli cells expressing a glutathione S- transferase (GST)-NtCBP4 (amino acids 394-708 of SEQ ID NO: 2) fusion protein were separated by SDS-PAGE. An acrylamide band containing the recombinant fusion protein was excised from the gel, crushed, and mixed (1: 1) with complete Freund's adjuvant. Three ml of the mixture containing

100 ug ofrecombinant protein were injected into two rabbits. Each rabbit was given two booster injections about two weeks apart and bled 10 days after each injection. The serum was depleted from antibodies recognizing GST by passage through a GST column (Pierce, USA).

In order to express NtCBP4 in insect cells, a transfer plasmid pFBNtCBP4 was constructed by inserting the NtCBP4 cDNA into a pFastBac vector, downstream of the baculovirus polyhedrin gene promoter.

The resultant plasmids were then transformed into DH10BAC E. coli cells (GibcoBRL, UK) for transposition into the bacamid. The screening and isolation of recombinant bacamid DNA were according to the manufacturer's instructions.

Sf9 insect cells were transfected with recombinant bacamid DNA using CelIFECTIN (GibcoBRL, United Kingdom). Recombinant baculoviruses were harvested 72 hours after the start of transfection.

Subsequently, the Sf9 cells were layered at a density of 5 x 106 cells per 90- mm plate and infected with high recombinant baculoviruses. After the indicated days of incubation at 27 oC, cells were harvested by centrifugation at 500 x g for 10 minutes, washed once with PBS and the total protein was extracted. Proteins were separated by SDS-PAGE, blotted to nitrocellulose membranes and subjected to either immunodetection or [35S] calmodulin- overlay by published methods (Arazi et al., 1995).

Expression of NtCBP4 in Spodoptera frugiperda Sf7 Insect Cells and its Affinity Purification : Cells were cultured as previously described (Lockow et al., 1988) in Graces medium supplemented with 10 % fetal calf serum and 10 pg/ml gentamycin. Recombinant proteins were expressed using the Bac-to-Bac system (Gibco-BRL). Transfer plasmid pFBNtCBP4 was constructed by inserting the NtCBP4 cDNA into a pFastBac vector.

Plasmid pFBNtCBP4 was recombined in vivo with Bacmid DNA, resulting in a recombinant virus DNA termed vir-NtCBP4. Protein expression was carried out by infecting Sf9 cells with vir-NtCBP4. The cells were collected at 72 hours post-infection and microsomal fractions containing recombinant protein were prepared by breaking cells with a polytron in hypotonic buffer (5 mM Tris, 2 mM EDTA) at 4 oC. The homogenate was subjected to differential centrifugation (100 x g for 10 minutes and 100,000 x g for 1 hour), and the resultant microsomal fraction was collected. Recombinant NtCBP4 was solubilized by resuspending NtCBP4-containing microsomes with CaM-binding buffer (0.1 % lysophosphatidylcholine, 50 mM Hepes- NaOH (pH 7.4), 150 mM KC1 and 1 mM CaCl2), incubated overnight with

constant slow shaking, and centrifuged at 100,000 x g for 1 hour. The supernatant containing solubilized NtCBP4 was collected and loaded on a CaM-agarose (Sigma) column, which was pre-equilibrated with CaM- binding buffer. The column was washed with 10 volumes of CaM-binding buffer, and subsequently CaM-binding proteins were eluted with CaM- Binding buffer containing 2 mM EGTA instead of 1 mM CaCl2.

Construction of DNA Templates Coding for Truncated NtCBP4 Proteins: Templates for amino-and carboxy-termini-deleted forms of NtCBP4 were produced by PCR amplification of the corresponding NtCBP4 coding regions, with specific oligonucleotides as primers: AS 5'-GAAGGAATTCTAATTATCTTCAGCAGTAAAATC-3'SEQ ID NO: 19 S 5'-CTAGCGGATCCAGATAAAGCGTCATCTTTGTTTAG-3'SEQ ID NO: 20 S 5'-GTCTAGCGGATCCAAGCACTCTCAGAAGTTGAAG-3'SEQ ID NO : 2 l AS 5'-GAAGGAATTCTTATGCTTGGGCAGTTCTAGTTGAG-3'SEQ ID NO: 22 AS 5'-GAAGGAATTCTTAACGAAGAGACTCTTCTACATTC-3'SEQ ID NO: 23 S 5'-GTCTAGCGGATCCGTGATGAAGAAAACAGGTTGC-3'SEQ ID NO: 24 SEQIDNO:25S5-CCGCTCGAGCCCGGGATCAATCACCGCCAAGACGAG-3' AS 5'-CGGAATTCCCCGGGTTACCTGAGGTCTTTCGGAAGGTTG-3'SEQ ID NO: 26 S = Sense; AS = Antisense The sense primers used for PCR amplification contained a BamHI site and the antisense primers contained an EcoRI site. The PCR fragments were digested with EcoRl and BamHI and then cloned into the BamHI- EcoRI sites of a pGEX-3X vector (Pharmacia), creating an in-frame fusion of the coding sequence for GST and the deleted forms of NtCBP4. The nucleotide sequences of all cloned fragments derived by PCR amplification were confirmed by sequencing.

35S-CaM Overlay Assay : Total E. coli extracts containing recombinant proteins were separated by SDS-PAGE, electrotransferred to nitrocellulose and overlayed with 35S-recombinant CaM as previously described (Arazi et al., 1995). After autoradiography, specific proteins were identified by immunodetection assays. Nitrocellulose membranes were washed for 30 minutes in PBS-T (PBS containing 0.05 % Tween 20) and 1 % non-fat milk. All additional immunostaining steps were performed as previously described (Arazi et al., 1995).

CaM/Peptide Complex Detection on Non-Denaturing PAGE: Samples containing 100 pmole (1 pg) of bovine CaM (Sigma) and different quantities of a HPLC-purified synthetic peptides (kindly provided by Prof.

M. Fridkin, Department of Organic Chemistry, Weizmann Institute of Science, amino acids 595-617 and 1-13 of SEQ ID NO: 2) in 100 mM Tris- HC1 (pH 7.2), and 0.1 mM CaCl2, making a total volume of 30 ul, were incubated for 1 hour at room temperature. Samples were analyzed by non- denaturing gel electrophoresis as previously described (Arazi et al., 1995).

Fluorescence Measurements of the Interaction Between NtCBP4 CaM-Binding Peptide and Dansyl CaM. Dansylated bovine CaM (300 nM; Sigma) was incubated alone or with different concentrations of the C terminal derived synthetic peptide (amino acids 595-617 of SEQ ID NO: 2) or a single concentration of the N terminal derived synthetic peptide (amino acids 1-13 of SEQ ID NO: 2) in 50 mM Tris-HCl (pH 7.5), 150 mM NaCI, and 0.5 mM CaCl2. After each addition of peptide, the CaM/peptide solution was mixed and incubated for 5 minutes at 23 oC. Emission fluorescence at 480 nm was then measured using a SLM AMINCO 8000 fluorimeter (SLM instruments); excitation wavelength was at 340 nm. Each measurement was the average of 3 readings. The dissociation constant (Kd) was determined by directly fitting fluorescence measurements of dansyl CaM at different peptide concentrations as previously described (Faiman et al., 1998; equation-1) using Kaleidagraph (version 2.1; Synergy Software).

Detecting Protein-Protein Interactions with a Yeast Two-Hybrid System: The cDNA corresponding to the NtCBP4 C-terminal (amino acids 401-708, SEQ ID NO: 2) was amplified by PCR using specific primers: 5'- CGGAATTCCAATCCTCAACATTAAGATTAGAG-3' (SEQ ID NO: 27) and 5'-TACCGCTCGAGCTAATTATCTTCAGCAGTAAAATCT-3' (SEQ ID NO: 28) and cloned into pEG202 vector (bait) EcoRI-XhoI sites, to fuse with the DNA-binding domain of the bacterial repressor LexA. The cDNA of a full length plant CaM (Swiss-Prot No. P27162) was amplified by PCR using specific primers: 5'-CCGCTCGAGTCACTTGGCCA TCATGACCT-3' (SEQ ID NO: 29) and 5'-CGGAATTCATGGCGGA TCAGCTTACAGA-3' (SEQ ID NO: 30), and cloned into pJG4-5 vector (prey) EcoRI-XhoI sites to create in-frame fusions with the bacterial B42 transcriptional-activator domain. Interaction trap assay was performed as previously described (Golemis et al., 1997). Briefly, the yeast strain EGY48 was transformed with the LacZ reporter vector pSH18-34. The pRFHMl vector, which encodes LexA fused to the N-terminus of the Drosophila protein bicoid, served as a non specific bait. Protein-protein interactions were identified by the ability of the yeast transformed with bait

and prey plasmids to grow on galactose plates lacking leucine, and by the appearance of blue and white color on the galactose-X-gal and glucose-X- gal plates, respectively. ß-galactosidase activity in liquid cultures was determined as previously described (Lundblad, 1997).

Preparation of transgenic plants : DNA constructs with NtCBP4 in the sense or anti-sense orientation were prepared by cloning an EcoRI fragment of the entire NtCBP4 cDNA into the unique EcoRI site of a binary Ti plant transformation vector (Cuozzo et al., 1988) downstream of the Cauliflower Mosaic Virus 35S promoter (Guilley et al., 1982). The orientation of the cDNA was determined by PCR amplification using gene- specific primers corresponding to NtCBP4 and vector sequences (SEQ ID NOs: 31,32 and 33). Transgenic tobacco plants (Nicotiana tabacum var.

Samsun, NN) were prepared by the leaf-disk transformation procedure (Horsch et al., 1985) and selected on kanamycin (50 g per ml). Two- month-old primary transformants were initially screened for NtCBP4 mRNA by Northern analysis and later by immunodetection of NtCBP4.

NtCBP4 expression in each transgenic line was confirmed at the T2 generation stage.

Preparation of tobacco membranes, fractionation and immunodetection ofproteins : Tobacco leaves or roots were homogenized using a pestle and mortar in a buffer (1.5 ml g'fresh weight) containing 0.5 M sucrose, 50 mM HEPES-KOH, pH 7.5,5 mM ascorbic acid, 1 mM DTT, 0.6 % (w/v) polyvinylpyrrolidone, and the protease inhibitors phenylmethylsulfonyl fluoride (2 mM), aprotinin (1 g ml-1), leupeptin (1 pg ml-l), and pepstatin (1 pLg ml~l). The homogenate was filtered through one layer of Miracloth (Calbiochem, LaJolla, CA), and the resulting filtrate centrifuged at 10,000 x g for 15 minutes. Microsomal membranes were pelleted from the supernatant by centrifugation at 50,000 x g for 30 minutes.

Membrane proteins were separated by SDS-PAGE, blotted and immunodetected with polyclonal antibodies against NtCBP4 or plant vacuolar H+-ATPase (Ward et al., 1992). For fractionation by non- continuous sucrose gradients, microsomes (7.4 mg protein) were gently resuspended in 0.5 ml resuspension buffer (5 mM KPO4, pH 7.8,0.25 M sucrose) and layered onto a non-continuous gradient containing 20 %, 30 %, 34 %, 38 %, 45 % (W/W) sucrose in centrifugation buffer (10 mM Tris- MES pH 7.2,2.5 mM DTT, 1 mM MgSO4). The gradient was then centrifuged at 100,000 x g for 2 hours and the membranes were collected from the interface between different sucrose concentrations, frozen in liquid

nitrogen, and stored at-80 oC. For aqueous two-phase partitioning, microsomes from tobacco roots (57 grams) were gently resuspended in 0.33 M sucrose and 5 mM potassium phosphate, pH 7.8, and then fractionated by the aqueous two-phase partitioning method as described (Larsson, 1985).

Phase separations were carried out in a series of 5-g phase systems with a final composition of 6.2 % (w/w) dextran T500,6.2 % (w/w) polyethylene glycol 3350,0.33 M sucrose and 5 mM potassium phosphate, pH 7.8. Three successive rounds of partitioning yielded a colorless upper phase enriched in plasma membranes and a brown lower phase containing intracellular membranes. After washing, pelleting, and resuspending in homogenization buffer, the partitioned membranes were stored in liquid nitrogen for further analysis. Antibodies used for immunodetection on blots were against NtCBP4 (described above), the 60-kDa subunit of vacuolar H-ATPase from oat roots (V-ATPase, from H. Sze, University of Maryland; (Ward et al., 1992)), endoplasmic reticulum BIP (ER-BIP, from G. Galili, The Weizmann Institute), plasma membrane H-ATPase (P-ATPase, from Dr.

Orit Shaul, of the Weizmann Institue of Science, Israel), mitochondrial prohibitin (M-Prohibitin; Snedden and Fromm, 1997) and endoplasmic reticulum NADPH cytochrome-p450 reductase (ER-Cyt-p450, from I.

Benveniste, CNRS, Institute de Biologie moleculaire des plants, Strasbourg, France).

Plant culture and metal toxicity assays : Seeds were sterilized with 70 % ethanol for 10 seconds, NaOCI (10 % active chlorine) for 15 minutes, then rinsed with distilled water and placed in plates containing either half- strength Hoagland's nutrient solution (Hoagland et al., 1950), pH 5.7, for Ni treatment, or in modified Blaydes solution (Parrot et al., 1990), pH 4.5, for Pb treatment. The seedlings were grown in a controlled growth room for 12 to 13 days, with a day/night cycle of 16h/8h at 25 °C. Chlorophyll content was measured as described (Arnon, 1949). For the analysis of older plants, seeds were mixed with vermiculite that was placed on nylon nets and half- strength Hoagland's nutrient solution, in the greenhouse. After four weeks, plants of uniform size were subjected to NiCl2 treatments for two weeks.

Calli were generated from leaves on B5 medium (Gamborg et al., 1968) supplemented with 2-4-D (0.5 g ml) and kinetin (0.2 ig ml), mixed with 0.8 % agar. Tolerance to Ni2+ was studied by transferring calli to B5 medium supplemented with NiCl2 for two weeks and then to Ni-free B5 medium for another two weeks. Statistical analysis of results was by single factor ANOVA.

Metal accumulation analysis : To measure Ni accumulation, plants were grown for four weeks on 0.5 strength Hoagland's solution (pH 5.7) and then transferred to the same medium supplemented with a range of concentrations of NiC12 (0.025,0.050.0.1,0.15 and 0.2 mM). For the analysis of Pb accumulation, plants were grown for 4 weeks on 0.5 strength Hoagland's solution (pH 5.7), transferred to modified Blaydes medium, pH 4.5 (Ca2+ concentration was 100 uM and Pi concentration was 10) in), supplemented with 100 uM Pb. Shoots were harvested at the times indicated, rinsed briefly in deionized water, blotted and dried at 80 °C for 3 days. The dried plant material was digested as described (Kramer et al., 1997) and metal content was measured by Inductively Coupled Plasma Atomic Emission Spectrometer (ICP-AES) with a spectroflame-ICP (Spectro Analytical Instruments, Germany).

Preparation of transgenic plants expressing NtCBP4iC : The DNA construct for the expression of the C-terminal truncated NtCBP4 (NtCBP4AC) was prepared by cloning a PCR-amplified DNA fragment using the gene-specific oligonucleotides 5'- CCGCTGAGCTATGAATCACCGCCAAGACGAG-3' (sense) (SEQ ID NO: 44) and 5'- GAAGGAATTCTTAAGAGGCTACAAGCTTTAAATCAT-3' (antisense) (SEQ ID NO: 45), spanning the coding region Met,-Sers93 of NtCBP4 (Arazi et al., 1999), and containing XhoI and EcoRI restriction sites on the 5'and 3'termini, respectively. The XhoI-EcoRI-digested DNA fragment was ligated into the corresponding sites of the binary vector described by Arazi et al. (1999). Transgenic lines expressing the truncated NtCBP4AC produced very few seeds by self-pollination and were therefore routinely hand pollinated with WT pollen. Transfer of the transgene to progeny was verified by germion kanamycin, and then by Northern hybridizations to confirm the expression of the NtCNBP4AC mRNA.

Analysis of transgene expression by Northern hybridizations and RT-PCR: Total RNA was isolated from 3-week-old seedlings as described (Logemann et al., 1987). RNA fractionation, blotting and hybridisation conditions were as described (Chen et al., 1994). A HindlII fragment from the cDNA of NtCBP4 (nucleotides 1113-2024, GenBank accession AF079872) was labelled with [32P] dCTP by random priming and used as a probe. The same membrane was hybridised with a [32P] dCTP-labelled actin probe prepared by amplifying an actin gene fragment using the tomato actin gene (GenBank accession number U60480) specific primers 5'-

TTGTGTTGGACTCTGGTGATGG-3' (sense) (SEQ ID NO: 46) and 5'- AGCCAAGATAGAGCCTCCAATC-3' (antisense) (SEQ ID NO: 47). Total RNA was passed through an oligo-dT column to enrich for poly-A mRNA, and reverse transcriptase was used to prepare the corresponding cDNA templates. PCR amplification of a 404-bp NtCBP4 full-length-specific sequence was performed with the primers 5'- GACGACTTCACAGTAAGCAGC-3' (SEQ ID NO: 48) and 5'- CACGACTAAAAATGCACTCAATC-3' (SEQ ID NO: 49) (sense and antisense, respectively), which amplify a region spanning nucleotides 2044- 2447 of the NtCBP4 cDNA (GenBank accession number AF079872). The P-ATPase cDNA amplification served as a control for RT-PCR with the primers 5'-CTTACAGGTTTGACCGTGGCTGAGC-3' (sense) (SEQ ID NO: 50) and 5'-TAGTGATCCTCTCCCAAAATGTGAGG-3' (antisense) (SEQ ID NO: 51) designed for the gene from Nicotiana plumbaginifolia (GenBank accession number X02868).

EXPERIMENTAL RESULTS Example 1 Cloning of CaM-binding Channel-Like Proteinsfrom Nicotiana tabacum and characterization based on sequence In order to determine the molecular components involved in Ca2+/CaM-mediated signaling pathways in plants, a tobacco cDNA expression library was screened with radiolabeled recombinant CaM as a probe. Preliminary analysis revealed several partial clones containing a region with a marked similarity to the cyclic nucleotide-binding domains of mammalian cyclic AMP/GMP dependent kinases and cyclic nucleotide- gated cation channels (Shabb et al., 1992). The full-length clones of two such cDNAs were obtained and their sequences revealed open reading frames of 2124 and 2106 nucleotides encoding proteins of 708 and 702 amino acid residues, respectively (see SEQ ID NOs: l, 2,3 and 4). These clones were named NtCBP4 and NtCBP7 (for Nicotiana tabacum CaM- Binding Protein). The occurrence of an in-frame termination codon 5'to the translational initiation codon in each clone indicates that the NtCBP4 and NtCBP7 cDNAs contain the complete coding regions of their respective proteins. A sequence comparison of NtCBP4 and NtCBP7 revealed an overall amino acid sequence homology (identical + similar acids of 62 %.

The highest level of homology (73 %) was found in their C-termini

(corresponding to NtCBP4 amino acids 348-708, SEQ ID NO: 2) and a lower homology (50 %) in their N-termini (corresponding to NtCBP4 amino acids 1-347, SEQ ID NO: 2) (Figure la).

Evidence for homology to ion channels at the amino acid sequence level of NtCBP4 and NtCBP7 came from several types of analyses.

BLASTP searches of all databases, using the amino acid sequence of NtCBP4 and NtCBP7 as query sequences, revealed sequences similar to cyclic nucleotide-gated non selective cation channels and voltage-gated K+ channels, all belonging to a group of ion channels that contain a cyclic nucleotide-binding domain in their C-terminus (Warmke et al., 1994). The rat cyclic nucleotide-gated olfactory channel (Dhallan., 1990) received the most significant score (23 % identity and 48.5 % total homology (similarity + identity) over the whole NtCBP4 sequence. Furthermore, a conserved putative cyclic nucleotide binding site (cNBD) was located at the carboxy termini of NtCBP4 and NtCBP7 (Figures la-b). This domain shows similarities with cyclic nucleotide binding domains of different eukaryotic and prokaryotic proteins (Shabb et al., 1992).

Some common features of a group of eukaryotic ion channels are the presence of six transmembrane domains (S1-S6) and a pore region (P) (Jan et al., 1992) (Figures 2; 3 a-c). Analysis of the hydrophobic profile of NtCBP4 and NtCBP7 showed the presence of several hydrophobic stretches along the sequence (Figure lb). Only six hydrophobic stretches, which lie in the N-terminal half of the protein, are presumably long enough (19-26 amino acids) to span the membrane and indeed, they are predicted to be transmembrane domains (according to Eisenberg, 1984). In addition, these domains are nicely aligned with the core regions (S1-S6) of cyclic nucleotide-binding domain-containing channels. The equivalent membrane- spanning regions of the Drosophila EAG voltage-activated K+ channel (Warmke et al., 1991) showed the highest similarity (sequence alignments not shown).

A group of ion channels that show intrinsic voltage-induced gating contain five to seven positively charged residues located within a stretch of hydrophobic amino acids that constitute the fourth transmembrane domain (S4) (Liman et al., 1991). In contrast, cyclic nucleotide-gated channels have less positive charges in the S4 domain and are insensitive to voltage (Fin et al., 1996). The fourth hydrophobic domain (S4) of NtCBP4 and NtCBP7 contains only 4 positively charged residues. Hence the S4 domains

of NtCBP4 and NtCBP7 are more related to the S4 domains of cyclic nucleotide-gated channels.

Thus, the predicted channel-like structure of NtCBP4 and NtCBP7 is corroborated by the presence of a putative pore region preceding the S6 hydrophobic domain (Figures 2; 3b-c). This region shares similarities with pore-lining regions of K-selective (Nakamura et al., 1997) and cyclic nucleotide-gated non selective cation channels (Fin et al., 1996) (Figures 3 a-c). However, in contrast to K-selective channels, the NtCBP4 and NtCBP7 pore regions lack the consensus amino acid pair YG (SEQ ID NO: 40) that is essential for K+ selectivity (Nakamura et al., 1997 and Doyle et al., 1998). Moreover, in both NtCBP4 and NtCBP7 the aspartate residue typically following the GYG (acids 1-3 of SEQ ID NO: 43) signature is replaced by leucine, and the cluster of threonine residues that co-determines K+ selectivity (Doyle et al., 1998) is missing. This suggests that their permeability may differ from that of known K-selective channels. A few amino acid differences between the predicted pore regions of NtCBP4 and NtCBP7 (e. g., GQN (SEQ ID NO: 41) versus GQG (SEQ ID NO: 42), respectively) may suggest differences in ion permeability between these two putative channels.

Similar to these channels, NtCBP4 and NtCBP7 contain six putative transmembrane domains, a pore motif between the S5 and S6 domains, and a highly conserved cyclic nucleotide-binding domain in their C-terminus.

Sequence analysis indicated that NtCBP4 and NtCBP7 are most similar to the olfactory cyclic nucleotide-gated channel (Dhallan et al., 1990). Like the olfactory channel, NtCBP4 and NtCBP7 contain a 90 amino acid spacer between the end of S6 and the predicted cyclic nucleotide-binding domains.

Recently, this region was shown to contain amino acid residues important for channel gating (Varnum et al., 1997). In addition, the S4 domains of NtCBP4 and NtCBP7 are more closely related to the S4 domains of non selective cation channels that are voltage-insensitive (Fin et al., 1996).

NtCBP4 and NtCBP7-putative P domains share similarities with pore regions of non selective cation channels and K-selective channels (Nakamura et al., 1997). However, both lack some of the residues found to be important for K+ selectivity such as the GYG (SEQ ID NO: 43) motif (Doyle et al., 1998) and thus may be selective for cations other than potassium.

Therefore, the two full-length tobacco cDNAs designated NtCBP4 and NtCBP7, encode Ca2+/CaM-binding proteins with sequences similar to

K+-selective channels like Drosophila EAG, AtKATI, and AtAKTI (Figures 3a-c) inward rectifiers, as well as to animal cyclic nucleotide-gated non selective cation channels such as the rat olfactory channel (OCNG2).

NtCBP4 has an amino acid sequence which resembles recently cloned ion transporters from Arabidopsis (Kohler et al., 1999; SEQ ID NOs: 17 and 18, AtCNGCI) and Hordeum vulgare (Schuurink et al., 1998; SEQ ID NOs: 15 and 16, HvCBTI). Kohler et al. recently demonstrated that AtCNGC1 (Figure 2, SEQ ID NO: 17) can partially complement a yeast mutant that is deficient in K+ uptake and therefore suggested that this protein belongs to a family of non-selective cation channels. However, neither of these earlier works studied the function of this or other members of this plant protein family in plants. Characterization of the function of NtCBP4, and other family members, is described herein for the first time.

The following Table provides nucleic acid identity and amino acid homology (in brackets, identical + similar acids) between sequences described herein. GENE NtCBP4 NtCBP7 NtCBPIO NtCBP63 StCBPI8 MsCBPIS HvCBTI AtCNGCI NtCBP4 100 (100) 100(100)************************NtCBP769(72) NtCBP10 71 (76) 81 84) 100(100) **** **** **** **** **** NtCBP63 63 (66) 63 (62) 66 (66) 100 (100) StCBP18 68 (71) 76 (72) 84 (83) 61 (57) 100 (100) **** **** **** MsCBP15 72 (81) 69 (72) 70 (77) 64 (69) 67 (71) 100 (100) **** **** HvCBTI 68 (73) 64 (68) 67 (73) 65 (61) 63 (67) 68 (71) 100 (100) **** AtCNGCt72 (82) 68 (72) 71 (78) 64 (64) 66 (73) 72 (81) 68 (72) 100 (100) Analysis was performed using the Bestfit procedure of the DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the university of Wisconsin (gap creation penalty-50, gap extension penalty-3).

Example 2 Discovery of a Family of Proteins Related to NtCBP4 and NtCBP7 in Tobacco and other plants In addition to NtCBP4 and NtCBP7, another related partial clone designated NtCBP10 (SEQ ID NOs: 5 and 6) was isolated from the tobacco library by protein-interaction screening. To explore the possible occurrence

of other tobacco proteins related to NtCBP4 and NtCBP7, RT-PCR with degenerated PCR primers was used to isolate related cDNAs based on similarities among NtCBP4, NtCBP7, and NtCBPIO in the regions flanking the S5 through S6 transmembrane domains. These screenings produced another related cDNA, designated NtCBP63 (SEQ ID NOs: 7 and 8).

Figure 4 shows the sequences of the four related tobacco proteins in the region spanning the S5 through S6 transmembrane domains. The major differences between the clones, including insertions or deletions, occur in the region between S5 and the pore (Figure 4), whereas the S5, pore, and S6 domains are relatively conserved in sequence. Thus tobacco possesses a family of proteins related to NtCBP4 and NtCBP7.

Within this family, two of the four tobacco clones (NtCBP4 and NtCBPIO; Figure 4; SEQ ID NOs: 2 and 6) have a GQN (SEQ ID NO: 41) sequence instead of GYG (acids 1-3 of SEQ ID NO: 43), whereas the two other tobacco proteins (NtCBP7 and NtCBP63; Figure 3; SEQ ID NOs: 4 and 8) have a GQG (SEQ ID NO: 42) sequence at the same position. This, as well as other differences in amino acid sequences (Figure 4), demonstrate potential differences in ion permeability between members of this new tobacco protein family which suggest that different family members may be suited to different future applications.

Additional screenings of Potato guard cell cDNA library and Alfalfa root cDNA library produced additional NtCBP4 family members (SEQ ID NOs: 9,10,11, and 12), showing that the family is widespread in the plant kingdom.

Example 3 Demonstration of the Ability of CaM to Bind to the Full-Length NtCBP4 Elucidation of the potential role of CaM in regulating NtCBP4 and NtCBP7 focused first on NtCBP4. Initially, examination of whether the full-length NtCBP4 binds CaM was undertaken. The baculovirus-infected Sf9 insect cell system (Lockow et al., 1988) was employed to express NtCBP4. As predicted, the expressed NtCBP4 was found solely in the insect cell membrane fraction (data not shown). Membrane proteins were solubilized with 0.1 % LPC as described hereinabove and loaded on a CaM- agarose column in the presence of 1 mM Ca2+. After washing the column, CaM binding proteins were eluted by exchanging Ca2+ with 2 mM EGTA.

An equal volume from each fraction was analyzed by SDS-PAGE. Staining

with Coomassie-Blue revealed two major bands at a relative mobility of approximately 80 kDa (Figure 5A), consistent with the predicted Mr of NtCBP4 (81 kDa). Western blot analysis with anti-NtCBP4 antibodies confirmed that these bands represent the recombinant NtCBP4 (Figure 5B).

Proteins from control insect cells infected with wild type baculovirus showed no cross reactivity with anti-NtCBP4 antibodies (data not shown).

These data indicate that the full-length NtCBP4 is capable of binding CaM in its phospholipid-solubilized form.

To test the capacity of NtCBP4 to bind CaM in vivo, the heterologous yeast two-hybrid system described hereinabove was employed.

A full-length CaM coding region was used as the prey with NtCBP4 C- terminal (amino acids 401-708, SEQ ID NO: 2) as bait. Protein-protein interactions were identified by yeast growing on galactose plates lacking leucine and by the appearance of blue on the galactose-X-gal plates as opposed to white on glucose-X-gal plates (Figure 6). The results indicate that a CaM-binding domain is present in the C-terminal part of NtCBP4. In addition, these results show the ability of NtCBP4 to interact with CaM in vivo, in the yeast system.

Example 4 Demonstration that NtCBP4 Contains a Single CaM-Binding Domain CaM-binding domains are not conserved in amino acid sequences (O'Neil et al., 1990). Therefore, to determine the number and positions of CaM binding sites in NtCBP4, different regions of NtCBP4 were fused to GST (Figure 7B constructs Nos. 2,3,4,5,6 and 7) and their binding to 35S-CaM was tested using a CaM-overlay assay (Arazi et al., 1995). A minimal region of 66 amino acids (SEQ ID NO: 2 residues Q573-R639.

Figure 6B: construct No. 6) was sufficient for CaM binding on a blot (Figure 7A; lane 5). This binding was Ca2+ dependent as evidenced by the fact that it could be blocked with 2 mM EGTA (data not shown). Thus, the NtCBP4 Q573-R639 domain contains the CaM binding site.

CaM binds to peptides that tend to form amphipathic a helices with one face of the helix positively charged (O'Neil et al., 1990). Sequence analysis of the 66-amino acid CaM binding sequence, revealed a region with typical CaM binding characteristics between amino acid residues Phe595-Gly617. When drawn in the form of an a-helical wheel (Devereux et al., 1984), it exhibits an amphipathic structure with a positively charged binding face and an opposite hydrophobic face (Figure 8A). Subsequently,

a synthetic peptide corresponding to NtCBP4 amino acids Phe595-Trp617 (SEQ ID NO: 2; acids 595-617) was synthesized, incubated with bovine CaM and complex formation was determined by non-denaturing PAGE (Arazi et al., 1995) with or without 0.1 mM Ca2+. In the presence of a peptide derived from the N-terminal part of NtCBP4 (SEQ ID NO: 2; amino acids 1-13), which was used as a negative control (Figure 8B, control lane), or in the absence of the peptide (Figure 8B, lane 0), only a single band corresponding to free CaM was apparent. When the NtCBP4 peptide corresponding to amino acids Phe595-Gly617 was added in the presence of Ca2+, another band of slower mobility appeared, representing a peptide/CaM complex. When the ratio of peptide to CaM was unity, most of the free CaM was no longer apparent, and the intensity of the peptide/CaM complex increased. At a peptide-to-CaM molar ratio of 2, very low free CaM was detected. At a still higher ratio of 4, no new band appeared on the gel, nor did the peptide/CaM complex change its intensity.

The conclusion is that multivalent complexes did not form. No mobility shift of CaM was apparent in the absence of free Ca2+ (data not shown).

These results demonstrate that this peptide is capable of forming a stable complex with CaM in the presence of Ca2+ and suggest that NtCBP4 binds Ca2+/CaM with a one-to-one stochiometry.

As mentioned above, a protein (designated HvCBTI, Figure 2, SEQ ID NO: 16) which is similar to NtCBP4 was isolated recently from barley (Schuurink et al., 1998). In that study the CaM-binding site was not determined experimentally and, based on sequence analysis, it was assumed to be closer to the C-terminal end of the protein (Schuurink et al., 1998), where no CaM-binding activity occurs in NtCBP4. The full-length NtCBP4 can bind CaM in a Ca2-dependent manner and this binding is via a single CaM-binding domain of approximately 23 amino acid (Phe595-Trp617).

This domain is characterized by aromatic and long-chain aliphatic residues separated by positively charged amino acids. These features are present in known CaM-binding sites of several proteins such as myosin light chain kinase, and peptides such as melittin and mastoparan (O'Neil et al., 1990).

However, except for NtCBP4 and its family members, the olfactory channel (SEQ ID NO: 34) is the only identified protein containing both CaM-and cyclic nucleotide-binding domains. In contrast to NtCBP4, in which the CaM-binding domain coincides with a-helix C of its cyclic nucleotide binding domain, in the olfactory channel the CaM-binding domain is found in the N-terminal part of the protein, separated from the cyclic nucleotide

binding domain by 377 amino acid residues. Binding of Ca2+/CaM to this domain in the olfactory channel reduces the effective affinity for cyclic nucleotides by up to 20 fold (Liu et al., 1994).

Example 5 NtCBP4 Binds Ca2+lCaM witlz an Apparent Kd in the Low nMRange Demonstrating high affinity interactions between CaM and a protein in vitro can indicate its physiological importance. Therefore, investigations of the affinity between the NtCBP4 CaM-binding peptide and Ca2+/CaM using fluorescence measurements of dansyl-CaM (Liu et al., 1994) with or without the NtCBP4 CaM binding peptide were undertaken. Without the peptide, the photoexcited emission spectrum of dansyl-CaM (300 nM) peaked at 500 nm (Figure 9A). Adding 300 nM peptide, which should convert all CaM to the bound form, increased the fluorescence intensity of dansyl-CaM 1.96 times, and shifted the emission peak to 482 nm (Figure 9B). These experiments demonstrate that the dansyl moiety enters a more hydrophobic environment upon binding of the peptide to dansyl-CaM (Kinkaid et al., 1982). With the dansyl-CaM concentration at 200 nM, the fraction of bound CaM increased linearly with total peptide concentration until the signal saturated at 300 nM peptide, 1.5 times the concentration of dansyl-CaM (Figure 9B). These data are consistent with the non-denaturing gel results estimating a one-to-one binding ratio between CaM and NtCBP4 (Figure 8B). An apparent Kd of 7.9 2.75 nM was calculated from fitting these data using a method described hereinabove (Faiman et al., 1998).

These data indicate that NtCBP4 binds CaM with a binding constant that is in the physiological range known for other plant- (Snedden et al., 1996) and animal- (James et al., 1995) CaM-regulated proteins.

Example 6 Demonstration that the CaM-Binding Domain of NtCBP4 Coincides with a Conserved Helix of its Cyclic Nucleotide Binding Domain Proteins that bind cyclic nucleotides (cAMP or cGMP) share a structural domain of about 100-130 residues. The best studied is the prokaryotic catabolite gene activator (CAP). Other proteins that are known to contain these domains are animal cAMP-and cGMP-regulated protein kinases, vertebrate cyclic nucleotide-gated ion channels (Shabb et al., 1992) and several EAG-related K+ channels (Warmke et al., 1994). X-ray crystallography of CAP showed that this domain is composed of three a-

helices and has a distinctive eight-stranded, antiparallel beta-barrel structure (Weber et al., 1987). In addition, in the cyclic nucleotide-binding domains of all eukaryotic and prokaryotic proteins, there are six invariant amino acid residues, three of which are Gly residues, thought to be essential for maintaining the structural integrity of the ß-barrel. The cyclic nucleotide binds within a pocket formed by the a-helix C and the P-barrel, where the remaining invariant residues are located (Weber et al., 1987). The NtCBP4 and NtCBP7 cyclic nucleotide-binding domains contain all the invariant amino acid residues and they fit perfectly the PROSITE data bank cAMP/cGMP binding-domain signature motif (Figure 10). The only variation from the signature is the number of amino acids separating NtCBP4 Leu553 and Arg570 (i. e., 16 amino acids instead of 5 or 11 in the signature motif). However, this region is included in a loop that is not important for the binding of the cyclic nucleotide (Shabb et al., 1992).

When the NtCBP4 and NtCBP7 putative cyclic nucleotide binding sites are aligned with the olfactory channel cyclic nucleotide binding site (Figure 10) and both are superimposed with elements of a secondary structure taken from the E. coli CAP crystal structure (Weber et al., 1987), a unique feature of the NtCBP4 cyclic nucleotide binding domain is revealed. The stretch of amino acids that form the a-helix C (ARG597- Thr616 ; see Figure 1A), a key element in the structure of cyclic nucleotide- binding domains, is exactly the region that forms the NtCBP4 CaM-binding domain (Figure 10, bold letters). Thus, the same structure functions both as a CaM-binding helix and as a key structure in binding cyclic nucleotides in NtCBP4 and NtCBP7.

In summary, Examples 1 through 6 demonstrate identification of a channel-like protein family in plants with a new structural motif not previously described in any other protein. In this motif, a phylogenetically conserved helix in the cyclic nucleotide-binding domain has the ability to bind CaM with high affinity, and thus makes NtCBP4 and related proteins a potential communication point for cross-talk between Ca2+ and cyclic nucleotide signal transduction pathways in plants.

Example 7 Demonstration that NtCBP4 is Associated with the Tobacco Plasma Membrane Ion channels have been discovered in various intracellular membrane compartments of plant cells (Tester, 1990). In order to study the function of

NtCBP4 in planta, its subcellular localization was determined. To facilitate this determination, polyclonal antibodies against the recombinant NtCBP4 were prepared as described hereinabove. Confirmation that these antibodies detect the full-length NtCBP4 came from both a heterologous insect-cell expression system (Figure 11A) and from plant membranes (Figure 11B).

In insect-cell extracts, anti-NtCBP4 antibodies (Figure 11A) detected two close protein bands (80-81 kDa). The intensity of the immunoreactive proteins increased with time post-infection (Figure 11A). No protein was detected with the anti-NtCBP4 antibodies in the control extract infected with the WT baculovirus vector (Figure 11A). Incubation of the same blot with [35S]calmodulin confirmed that the full-length recombinant NtCBP4 protein binds calmodulin (Figure 11A). Moreover, a protein of the same electrophoretic mobility as the full-length recombinant NtCBP4 was detected in membranes of transgenic plants expressing NtCBP4 under the transcriptional control of the Cauliflower Mosaic Virus (CaMV) 35S gene- promoter (Figure 11B). No protein was detected in the soluble fraction of these plants. Thus, this analysis confirmed the specificity of the antibodies against NtCBP4.

Subsequently, the subcellular localization of NtCBP4 in wild type (WT) and in transgenic plants (transgenic line designated 49-79 was chosen for this analysis) was determined. First, microsomal membranes were fractionated on sucrose gradients and analyzed by Western blots using antibodies against NtCBP4 and against known marker proteins from plasma membrane (P), vacuole (V), and endoplasmic reticulum (ER) (Figure 12A).

NtCBP4 co-fractionated with the plasma membrane markers as indicated by sedimentation profiles overlapping with P-ATPase (Figure 12A). In contrast, the vacuolar marker peaked at the 20/30 % sucrose fraction. BiP, a commonly used ER marker (Haas, 1994) also peaked at the 34/38 % sucrose fraction; however, unlike NtCBP4, it was also present in the 20/30 % fraction. These results demonstrate that NtCBP4 is associated with the plasma membrane and not with the vacuole or ER. Immunodetection of NtCBP4 in membrane fractions from transgenic tobacco overexpressing NtCBP4 showed a similar pattern of distribution on the sucrose gradient, except that NtCBP4 levels were higher (Figure 12A). This indicates that the overexpressed NtCBP4 is localized in same membrane compartment as the endogenous NtCBP4 in WT plants.

To confirm the localization of NtCBP4 in the plasma membrane, separation of tobacco root plasma membranes from intracellular membranes

using the aqueous two-phase partitioning method (Larson, 1985) was undertaken. Membrane purity was confirmed by immunodetection of the marker proteins. As shown in Figure 12B, NtCBP4 in both WT and transgenic plants was highly enriched in the upper-phase fraction (U) and largely depleted from lower-phase fraction (L). A similar pattern was detected for P-ATPase. The purity of the plasma membrane preparation was also confirmed by the relative absence of intracellular membrane markers like V-ATPase (Ward et al., 1992) and mitochondrial prohibitin (Snedden et al., 1997), which were highly enriched in the lower-phase fraction (Figure 12B). The ER marker was also detected in the upper-phase fraction, but unlike the P-ATPase, was also present in the lower phase.

These results are consistent with analysis by sucrose gradients and confirm that NtCBP4 is a plasma membrane protein.

The nature of the interaction of NtCBP4 with the plasma membrane was also investigated using various detergents and non-detergent chemicals (data not shown). Specifically, NtCBP4 was removed from WT and transgenic plant membranes only by detergents that completely solubilize the membranes (e. g., 1 % Triton X-100) and not by treatments known to remove membrane-associated or peripheral proteins (e. g., 1 M Urea, 0.1 M Na2C03 pH 11.5, and 0.75 M NaCI). This confirms the results of immunologic assays and sequence analysis that predicted six transmembrane domains. Together, these data constitute a demonstration that NtCBP4 is an integral plasma membrane protein.

These results concur with earlier findings of Schuurink et al.

(Schuurink et al., 1998), which demonstrated that an overexpressed GFP/HvCBTI fusion protein was associated with the plasma membrane of barley aleurone cells. In conclusion, these data support the postulated function of NtCBP4 as a component of a plasma membrane ion channel.

Example 8 Transgenic Seedlings Exhibit Improved Tolerance to Ni2+ To elucidate the function of NtCBP4 in planta, several transgenic tobacco lines were established that express the NtCBP4 RNA in the sense orientation under the transcriptional control of the promoter of the CaMV 35S gene. Twenty-five independent transgenic lines were screened for the presence of NtCBP4 RNA, and these showing relatively high levels were selected for further investigation at the protein level. As a control, transgenic tobacco lines were prepared with the NtCBP4 cDNA in the

antisense orientation. Several independently derived transgenic lines were obtained, differing in their level of NtCBP4. Figure 13A shows an example of some of these lines. Two lines (49-79 and 10-2) had about double the amount of NtCBP4 protein compared to the WT plants. One line (13-93) had intermediate levels of NtCBP4 and one line (15-89) exhibited levels of NtCBP4 similar to WT (Figure 13A). The transgenic line with the antisense construct (AS) had about the same levels of NtCBP4 as WT and the 15-89 transgenic line. Transgenic lines overexpressing NtCBP4 and control plants were then subjected to different physiological studies.

Under normal growth conditions in the greenhouse, none of the transgenic plants showed any aberrant phenotype compared to the WT plants, and NtCBP4 overexpression was genetically transmitted to the progeny. Because NtCBP4 was assumed to function as a plasma membrane cation channel, the WT and transgenic plants were challenged with exposure to different toxic metals including Na+, Ba2+, Cd2+, Co, Cu, La3 and Mn2+ as chloride salts; Pb2+ as Pb (N03) 2 and Zn2+ as ZnS04.

Interestingly, transgenic plants overexpressing NtCBP4 were specifically more tolerant to NiCl2 but more sensitive to Pb (NO3) 2. No differences were found between WT and the transgenic lines in their responses to the other metal salts. This demonstrates that transgenic plants that overexpress NtCBP4 have an improved tolerance to Ni2+ and a hypersensitivity to Pb2+. These observations were followed by more detailed analysis of transgenic plants, as described hereinbelow.

The relationship between the levels of NtCBP4 in transgenic lines and the effects of Ni on early seedling development was investigated.

Without Ni2+, seedling development was similar in WT and in the transgenic lines (Figure 13B). Increasing NiCl2 concentrations adversely affected seed development of WT, AS, and line 15-89, in which the NtCBP4 protein levels were indistinguishable. Specifically, with increasing NiC12 concentrations, seedlings of these lines became more chlorotic and their development was severely retarded. These symptoms are known to occur in plants with phytotoxic levels of Ni2+ (Woolhouse, 1983; Brune et al., 1995; and Brune et al., 1995b). In contrast, the seedlings of transgenic lines 49-79 and 10-2 that expressed higher levels of NtCBP4 (Figure 13A) showed fewer toxic symptoms, and grew and developed even at a concentration of 200 pM NiC12 (Figure 13B and 13C). Four additional transgenic lines with relatively high levels of NtCBP4 had phenotypes similar to that of lines 49-79 and 10-2 (not shown). Seedlings of line 13-93,

which had intermediate level of NtCBP4 (Figure 13A), showed intermediate symptoms of toxicity (Figure 13B). 2+ For a quantitative evaluation of Ni tolerance, three parameters typically used to describe metal toxicity in plants were monitored: chlorophyll content, root length and seedling biomass. In the presence of NiC12, chlorophyll content was the highest in seedlings of transgenic lines that expressed higher levels of NtCBP4 (i. e., lines 49-79 and 10-2; Figure 14A). Chlorophyll levels in these two lines were double that of WT or in transgenic seedlings with the NtCBP4 antisense construct (AS; Figure 14A).

These differences were statistically significant atp<0. 001. Root length of 12-day-old seedlings grown in the presence or absence of 100 µM NiC12 was also measured. WT root growth was inhibited by 50 % (compared to the control without NiC12), whereas root growth of lines 49-79 and 10-2 was inhibited by only 30 % and 15 %, respectively (Figure 14B). Analysis of whole seedling fresh weight (Figure 14C) revealed a 50 % inhibition (LC50) at 0.09 mM Ni2+ for WT, versus 0.20 mM for transgenic lines 10-2 and 49-79 (Figure 14C). These differences were statistically significant (p<0.001) at Ni2+ concentrations above 0.05 mM. Taken together, these results show that transgenic lines with the highest levels of NtCBP4 were the most tolerant to Ni2+ stress.

Example 9 Demonstration that Tolerance to Ni2+ is not Dependent on the Developmental Stage of the Plant In order to demonstrate that Ni tolerance is not stage specific, the Ni2+ tolerance of 4-week-old plants was tested. After the plants had been grown in a Ni2+ free medium for 4 weeks, they were transferred to the same medium supplemented with 200 pM NiC12. WT plants showed severe toxicity symptoms after two weeks of exposure; they became necrotic and ceased to grow (Figure 15A). In contrast, transgenic plants showed attenuated symptoms and continued to grow (Figure 15A). Without NiC12, both WT and transgenic plants grew at the same rate (data not shown). 2+ The response of calli derived from WT and transgenic plants to Ni stress was also examined. Calli were exposed to different concentrations of NiCl2 for two weeks, and then transferred to a Ni-free medium for two weeks to recover. Without NiCl2, both WT and transgenic calli grew to the same size (Figure 15B). After an exposure to 0.5 mM NiCl2, the recovery of WT calli was poor compared with the transgenic calli, which showed

only slight growth retardation. WT calli exposed to 1.0 mM NiC12, became brown and could not recover on the Ni-free medium (Figure 15B), whereas transgenic calli exposed to the same concentration of NiC12 showed only about 50 % growth retardation compared with unstressed calli (Figure 15B).

Western blot analysis confirmed that transgenic calli had high levels of NtCBP4 compared with that in WT calli (Figure 15C). Therefore, Ni2+ tolerance is not dependent upon a specific developmental stage of the plant and is exhibited even in undifferentiated transgenic cells with high levels of NtCBP4.

Example 10 Demonstration that Ni Tolerance is Associated with Reduced Ni2+ Accumulation in Transgenic Plants One of the possible explanations for the improved tolerance of transgenic plants to Ni2+ may be the reduced uptake rates and slower accumulation of Ni. To test this hypothesis, the concentration-dependent accumulation of Ni in shoots of WT and transgenic plants was determined.

Plants were exposed to different concentrations of NiC12, and Ni content in shoots was measured by ICP-AES (Experimental procedures). At concentrations up to 100 uM NiC12, shoots of WT and transgenic plants accumulated Ni at about the same rate. However, at higher concentrations of NiC12 (150 and 200 pM), Ni2+ accumulated more rapidly in the WT compared with transgenic plants (Figure 16). At even higher concentrations (300 uM and above) WT but not transgenic plants wilted after one day.

Therefore, no measurements were taken at these concentrations. This analysis revealed slower accumulation of Ni in transgenic plants compared with WT. These results are consistent with previous reports on the critical concentrations of Ni necessary to confer toxic symptoms in plant tissues (Chaney et al., 1997) and validate the hypothesis that increased tolerance to high concentrations of environmental Ni is due to reduced rates of Ni uptake and accumulation.

Example 11 Demonstration that Pb2+ Hypersensitivity is Associated with Enhanced Accumulation in Transgenic Plants As indicated previously, preliminary analysis showed that transgenic seedlings overexpressing NtCBP4 have a greater sensitivity to Pb2+ than do WT plants. In the presence of 1 mM Pb (N02) 2, transgenic seedlings (line

49-79) were chlorotic and their development was retarded compared with WT seedlings (Figure 17A), whereas the growth and chlorophyll content in WT and transgenic plants were indistinguishable in the absence of Pb (N02) 2 (Figure 17A). Subsequently, chlorophyll was measured in WT and different transgenic lines after 12-days of growth in the presence of 1 mM Pb (N02) 2. The chlorophyll content was the lowest in the transgenic lines with relatively high levels of NtCBP4 (e. g., lines 49-79 and 10-2; Figure 17B). These values were significantly different (p<0.001) from these of WT or AS. Dose-response growth studies based on fresh weight (data not shown) revealed that transgenic line 49-79 had a 50 % greater sensitivity to Pb (N02) 2 than did WT (LC50 of 0.6 and 0.9 mM, respectively).

One explanation for the Pb2+ hypersensitivity of transgenic plants is enhanced Pb2+ accumulation. To test this possibility, 4-week-old WT and transgenic lines were exposed to 0.1 mM Pb (N02) 2 for 1-2 days and determined Pb content in shoots by ICP-AES. This analysis revealed that Pb accumulated faster in shoots of the transgenic plants (Figure 17C). This observation confirms the hypothesis that increased Pb sensitivity is due to increased uptake and accumulation. This means that transgenic plants overexpressing NtCBP4 are uniquely suited to phytoremediation of lead from soil.

Example 12 Transgenic plants expressing a truncated NtCBP4 exhibit improved tolerance to Pb2+ The C-terminal half of NtCBP4 contains a structurally conserved putative cyclic nucleotide binding domain and a high-affinity calmodulin- binding site (Arazi et al., 2000). In contrast to the mammalian cyclic nucleotide-gated channel, where the calmodulin-binding domain is near the N-terminus of the protein (Varnum and Zagotta, 1997), in NtCBP4 the calmodulin-binding site coincides with the predicted aC-helix structure of the cyclic nucleotide-binding domain (Arazi et al., 2000). This helix is essential for cyclic nucleotide binding in related proteins from other organisms (Shabb and Corbin, 1992). It is reasoned that removal of this important helix should disrupt the binding of both cyclic nucleotides and calmodulin and could possibly render the channel protein inactive.

Moreover, if plant NtCBP4-containing channels function as tetramers, like the mammalian cyclic nucleotide-gated channels (Liu et al., 1996),

expression of an inactive subunit might have inhibitory effects on endogenous native NtCBP4-associated channel activities. As a first step to test this possibility, a truncated version of NtCBP4 was constructed from which a major part of the C-terminal half including the aC helix, was removed (designated NtCBP4AC; Figures 18A-B).

Transgenic plants expressing NtCBP4AC were prepared as described in Experimental procedures, selected on kanamycin, and transferred to the greenhouse for seed production. Most of the putative transgenic lines were found to be male sterile and were therefore pollinated manually with pollen from WT plants. Seeds were collected from the maternal plants and plated on kanamycin-containing medium to select for progeny carrying the transgene. Apparently, all lines that were resistant to kanamycin were derived from male sterile plants. These plants expressed the truncated NtCBP4AC transgene, as will be shown. This suggests that NtCBP4AC had detrimental effects on functions crucial for plant reproduction.

To investigate the possible effects of NtCBP4AC expression on plant response to Pb2+, NtCBP4AC transgenic lines were germinated in the presence of Pb and their growth was compared with that of WT plants and of the transgenic line 49-79, which overexpresses the full-length NtCBP4 (designated herein NtCBP4FL or NtCBP4) and is associated with Pb2+ hypersensitivity (Arazi et al., 1999). Several independent NtCBP4AC lines designated 42-11,22-11,29,33-11,16-6,45-16,52-36,55-16,9-2, 36-16,55-61 were found to be more tolerant of Pb2+ than WT plants.

Figures 19A-B show three of these NtCBP4AC lines, with the WT and NtCBP4FL control lines, grown with or without Pb2+. The NtCBP4AC plants were clearly more tolerant of the toxic metal than the WT seedlings, whereas the NtCBP4FL plants were more sensitive, as previously reported (Arazi et al., 1999). The extent of tolerance was not identical in all NtCBP4AC transgenic lines, which could be attributed to differences in expression levels of the transgene or other reasons. Nevertheless, several NtCBP4AC independent transgenic lines exhibited a phenotype contrasting that of NtCBP4FL transgenic plants with respect to their response to pb2.

No differences were found between WT and the transgenic lines in their response to other metals including Na+, Zn2+, Mn2+, Cd2+ and La Although the expression of NtCBP4FL and NtCBP4AC in transgenic plants is driven by the same 35S CaMV promoter, it was desired to confirm that their expression levels were comparable. In addition, it was desired to exclude the possibility that the expression of the NtCBP4AC mRNA

abolished the expression of the endogenous native gene (e. g., by a gene- silencing mechanism). Northern hybridisations with total-RNA samples were used for analysing the steady-state levels of the NtCBP4FL and NtCBP4AC mRNAs in representative transgenic lines (Figure 20A). The mRNAs of both transgenes were easily detected and distinguishable by their different lengths (727 bases difference) in transgenic lines expressing the full-length mRNA versus that in NtCBP4AC lines (AC-42-11, AC-22-11 and AC-29). These results indicate that the steady-state levels of the mRNAs for both transgenes are comparable, although some variation in mRNA levels between lines is apparent. Thus, the contrasting phenotypes of the NtCBP4FL and NtCBP4AC transgenic plants are certainly not the result of differences in expression levels. The endogenous native NtCBP4 mRNA in WT plants was not detected in the total RNA sample due to its very low abundance (T. Arazi and H. Fromm, unpublished results) (Figure 20A).

To ascertain that the expression of the endogenous native gene in NtCBP4AC transgenic plants was not silenced, RT-PCR amplification was performed with a set of primers that amplify a 404-bp region of the full- length NtCBP4 mRNA but not of the truncated mRNA (see, Experimental procedures). The RT-PCR results (Figure 20B) show that a 404-bp DNA band was amplified from mRNA of the NtCBP4AC plants, indicating that the endogenous native NtCBP4 mRNA was indeed present in these plants.

This amplified band had the same mobility as the control band amplified from the NtCBP4 cDNA clone. Although this RT-PCR analysis did not allow quantitative assessment, the results show that with the same amount of mRNA used for each sample and with the same primers, the DNA band amplified from the mRNA of NtCBP4AC plants was similar in intensity to that amplified from the WT plants (Figure 19B), and both were substantially less intense than the amplified band from the NtCBP4FL plants. Where reverse transcriptase was omitted (-RT), no amplification was detected, indicating that the observed amplifications described above originated from mRNA templates rather than from residual genomic DNA or from contaminating plasmid DNA in the samples. Amplification with ß-ATPase- specific primers indicated that similar amounts of poly-A+ mRNA were used in all reactions. Thus, it is concluded that expression of NtCBP4AC does not result in silencing of the endogenous NtCBP4 gene. Rather, the apparent phenotype conferred by NtCBP4AC occurs in the presence of the full-length native endogenous NtCBP4.

Example 13 Quantitative assessment of Pb tolerance in NtCBP4dC transgenic seedlings, and Pb accumulation The extent of growth inhibition of seedlings by Pb2+ was evaluated by determining the fresh weight of seedlings grown in different concentrations of Pb (N03) 2 (Figure 21A). Whole-seedling fresh weight analysis revealed 50% inhibition at 0.34 mM, 0.47 mM, 0.58 mM, 0.62 mM Pb2+ for NtCBP4FL, WT, NtCBP4AC-22-11 and NtCBP4AC-42-1 1, respectively (Figure 21A). Moreover, statistical analysis revealed that the two NtCBP4AC lines were significantly more tolerant (p<0.05) than the WT seedlings at Pb2+ concentrations of 0.1 mM and more.

One of the possible explanations for the Pb2+-tolerant phenotype of the NtCBP4AC plants may be the attenuated uptake of the metal, since is has already been demonstrated that Pb2+ hypersensitivity in NtCBP4FL plants is associated with enhanced accumulation of Pb2 (Arazi et al., 1999).

To test this possibility, Pb2+ accumulation was determined by ICP-AES in 12-day-old seedlings grown in the presence of 0.2 mM Pb (N03) 2.

NtCBP4AC plants (lines AC-42-11 and AC-22-11) showed a marked reduction in Pb2+ accumulation compared with WT seedlings, whereas NtCBP4FL plants accumulated substantially more Pb (p<0.05) than WT (Figure 21B).

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art.

Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents, patent applications and sequences identified by an accession number mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent, patent application or sequence was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

REFERENCES: 1. Arazi, T., Baum, G., Snedden, W. A., Shelp, B. J., and Fromm H. (1995) Plant Physiol. 108,551-561.

2. Arazi, T., Sunkar R., Kaplan, B. and Fromm, H. (1999) A tobacco plasma membrane calmodulin-binding transporter confers Ni2+ tolerance and Pb2+ hypersensitivity in transgenic plants. Plant J. 20,171-182.

3. Arazi, T., Kaplan, B. and Fromm, H. (2000) A high-affinity calmodulin- binding site in a tobacco plasma membrane channel protein coincides with a characteristic element of cyclic nucleotide-binding domains.

Plant Mol. Biol. (in press) 4. Arnon, D. I. (1949) Plant Physiol. 24,1-15.

5. Ayres, R. U. (1992) Proc. Natl. Acad. Sci. USA 89,815-820.

6. Baker, A. J. M. (1987) New Phytol. 106,93-111.

7. Baum, G., Chen, Y., Arazi, T., Takatsuji, H. and Fromm, H. (1993) J.

Biol. Chem. 268,19610-19617.

8. Baum, G., Lev-Yadun, S., Fridmann, Y., Arazi, T., Katsnelson, H., Zik, M., and Fromm, H. (1996) EMBO J. 15,2988-2996 9. Bei, Q. and Luan, S. (1998). Plant J. 13,857-865.

10. Bowler, C., and Chua, N. H. (1994) Plant Cell 6,1529-1541 11. Bowler, C., Neuhaus, G., Yamagata, H., and Chua, N.-H. (1994) Cell 77,73-81 12. Braam, J. and Davis, R. W. (1990). Cell, 60,357-364.

13. Brune, A. andDietz, K-J. (1995) J. PlantNutr. 18,85-868.

14. Brune, A., Urbac, W. and Dietz, K-J. (1995) New Phytol. 129,403-409.

15. Bush, D. S. (1995) Annu. Rev. Plant Physiol. Plant Mol. Biol. 46,95- 122 16. Chaney, R. L., Malik, M., Li, Y. M., Brown, S. L., Brewer, E. P., Angle, J. S. and Baker, 52. A. J. M. (1997) Curr. Opin. Biotechnol. 8,279-283.

17. Chen Y, Baum G. and Fromm, H. (1994) The 58-kilodalton calmodulin- binding glutamate decarboxylase is a ubiquitous protein in petunia organs and its expression is developmentally regulated. Plant Physiol.

106,1381-1387.

18. Clapham, D. E. (1995) Cell 80,259-268.

19. Cunningham, S. D. and Ow, D. W. (1996) Plant Physiol. 110,715-719.

20. Cuozzo, M., O'Connell, K. M., Kaniewski, W., Fang, R. X., Chua, N. H. and Tumer, N. E. (1988) Biotech. 6,549-557.

21. Dhallan, R. S., Yau, K. W., Schrader, K. A., and Reed, R. R. (1990) Nature 347, 184-187 22. Devereux, J., Haeberli, P., and Smithies O. (1984) Nucl. Acids Res. 12, 387-395 23. Doyle, D. A., Cabral, J. M., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T., and Mackinnon, R. (1998) Science 280,69-77 24. Ehsan, H., Reichheld, J.-P., Roef, L., Witters, E., Lardon, F., Van Bockstaele, D., Van Montagu, M., Inze, D., and Van Onckelen, H.

(1998) FEBS Lett. 422,165-169 25. Eisenberg, D. (1984) Annu. Rev. Biochem. 53,595-623 26. Faiman G. A., Levy R., Anglister J., and Horovitz A. (1998) J. Biol.

Chem. 271,13829-13833 27. Foy, C. D., Chaney, R. L. and White, M. C. (1978) Ann. Rev. Plant Physiol. 29,511-566.

28. Fin, J. T., Grunwald, M. E., and Yau, K. W. (1996) Annu. Rev. Physiol.

58,395-426 29. Fromm, H., and Chua, N. H. (1992) Plant Mol. Biol. Rep. 10,199-206 30. Gamborg, O. L., Miller, R. A. and Ojima, K. (1968) Exp. Cell Res. 50, 151-158.

31. Gaymard, F., Cerutti, M., Horeau, C., Lemaillet, G., Urbach, S., Revallec, M., Devauchelle, G., Sentenac, H., and Thibaud, J. B. (1996) J.

Biol. Chem. 271,2283-2287 32. Golemis, E. A., Gyuris, J., and Brent, R. (1997) In: Current Protocolsin Molecular Biology. (Chanda, V. B., ed.) Chapter 20, unit 20.1. Wiley, New York 33. Guille, H., Dudley, R., Jonard, G., Balas, E. and Richards, K. (1982) Cell 30,763-773.

34. Haas, I. G. (1994) Experientia 50,1012-1020.

35. Heginbotham, L., Lu, Z., Abramson, T., and Mackinnon, R. (1994) Biophys. J. 66,1061-1067 36. Hoagland, D. R. and Arnon, D. I. (1950) California Agricultural Experiment Station Circular 371. The College ofAgriculture, University of California, Berkeley, CA.

37. Horsch, R. B., Fry, J. E., Hoffman, N. L., Eichnoltz, D., Rogers, S. G. and Fraley, R. T. (1985) Science 227,1229-1231.

38. Huang, J. W., Grunes, D. L. and Kochian, L. V. (1994) Proc. Natl. Acad.

Sci. USA 91,3473-3477.

39. Huang, J. W. and Cunningham, S. D. (1996) New Phytol. 134,75-84.

40. Ichida, A. M., Pei, Z. M., Baizabal-Aguirre, V. M., Turner, K. J. and Scroeder, J. I. (1997) Plant J. 9,1843-1857.

41. Jan, L. Y., and Jan, Y. N. (1992) Annu. Rev. Physiol. 54,537-555 42. James, P., Vorherr, T., and Carafoli, E. (1995) Trends Biochem. Sci. 20, 38-42 43. Kincaid, R. L., Vaughan M., Osborne J. C. Jr., and Tkacheck, V. A.

(1982) J. Biol. Chem. 257,10638-10643 44. Kohler, C., Merkle, T. and Neuhaus, G. (1999) Plant J. 18,97-104.

45. Knight, H., Brandt, S. and Knight, M. R. (1998) Plant J. 16,681-687.

46. Kramer, U., Smith, R. D., Wenzel, W. W., Raskin, 1. and Salt, D. E.

(1997) PlantPhysiol. 115,1641-1650.

47. Kurosaki, F., Kaburaki, H., and Nishi, A. (1994) FEBSLett. 340,193- 196 48. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157,105-132 49. Larson, C. H. (1985) Modern Methods in Plant Analysis. (Linskens, H. F. and Jackson, J. F., eds). Berlin: Springer-Verlag.

50. Leng, Q., Mercier, R. W., Yao, W. and Berkowitz, G. A. (1999) Cloning and first functional characterization of a plant cyclic nucleotide-gated cation channel. Plant Physiol. 121,753-761.

51. Liman, E. R., Hess, P., Weaver, F., and Koren, G. (1991) Nature 353, 752-756.

52. Liu, M., Chen, T. Y., Ahamed, B., Li, J., and Yau, K. W. (1994) Science 266,1348-1354 Lockow, V. A, and Summers, M. D. (1988) BiolTechnology 6,47-55.

53. Liu, D. T., Tibbs, G. R. and Siegelbaum, S. A. (1996) Subunit stochiometry of cyclic nucleotide-gated channels and effects of subunit order on channel function. Neuron 16,983-90.

54. Logemann, J., Schell, J. and Willmitzer, L. (1987) Improved method for the isolation of RNA from plant tissues. Anal Biochem. 163,16-20.

55. Lundblad, V. (1997) In: Current Protocols in Molecular Biology.

Chanda VB, ed. Chapter 13 unit 13.6. Wiley, New York.

56. McAinsh, M. R., and Hetherington, A. M. (1998) Trends Plant Sci. 3, 32-36.

57. Moffat, A. S. (1995) Science, 269,302-303.

58. Nakamura, R. L, Anderson, J. A., and Gaber, R. F. (1997) J. Biol. Chem.

272,1011-1018.

59. Neuhaus, G., Bowler, C., Kern, R., and Chua, N.-H. (1993) Cell 73, 937-952.

60. Nriagu, J. O. and Pacyna, J. M. (1988) Nature 333,134-139.

61. 0'Neil, K. T., and DeGrado, W. F. (1990) Trends Biochem. Sci. 15,59- 64.

62. Norman, T. and Banuelos, G. (1999) Phytoremediation of contaminated soil and water. CRC Press, Boca Raton, FL.

63.0'Reilly, D. R., Miller, L. K. and Luckow, V. A. (1992) Baculovirus Expression Vectors, a Laboratory Manual. New York: WH Freeman and Co.

64.0uyang, H. and Vogel, H. J. (1998) Metal ion binding to calmodulin: NMR and fluorescence studies. BioMetals 11,213-222.

65. Parrot,-W. A. and Bouton, J. H. (1990) Crop Sci. 30, 387-389.

66. Raskin, I. (1996) Proc. Natl. Acad. Sci. USA 93,3164-3166.

67. Rengel, Z. (1996) New Phytol. 134,389-406.

68. Rubio, F., Grassmann, W. and Scroeder, J. I. (1995) Science 270,1660- 1663.

69. Schachtman, D. P., Kumar, R., Schroeder, J. I. and Marsh, E. L. (1997) Proc. Natl. Acad. Sci. USA 94,11079-11084.

70. Schuurink, R. C., Shartzer, S. F., Fath, A. and Jones, R. L. (1998) Proc.

Natl. Acad. Sci. 95,1944-1949.

71. Shabb, J. B., and Corbin, J. D. (1992) J Biol. Chem. 267,5723-5726 72. Simons, T. J. B. and Pocock G. (1987) J. Neurochem. 48,383-389.

73. Snedden, W. A. and Fromm, H. (1997) Plant Mol. Biol. 33,753-756.

74. Snedden, W. A., and Fromm, H. (1998) Trends Plant Sci. 3,299-304 75. Snedden, W. A., Koutsia, N., Baum, G., and Fromm, H. (1996) J. Biol.

Chem. 271,4148-4153 Tester, M. (1990) New Phytol. 114,305-340.

76. Tomsig, J. L. and Suszkiw, J. B. (1991) Biochim. Biophys. Acta 1069, 197-200.

77. Toshi, T. (1995) J. Gen. Physiol. 105,309-328 78. Van der Zaal, B. J., Neuteboom, L. W., Pinas, J. E., Chardonnens, A. N., Schat, H., Verkleij, 82. J. A. C. and Hooykaas, P. J. J. (1999) Plant Physiol. 119,1047-1055.

79. Van Eldik, L., and Watterson, M. D. (1998) Calmodulin and Signal Transduction. Academic Press, New York..

80. Varnum, M. D, and Zagotta, W. N. (1997) Science 278,110-113.

81. Volotovski, 1. D., Sokolovsky, S. G., Molchan, O. V., and Knight, M. R.

(1998) Plant Physiol. 117,1023-1030.

82. Ward, J., Reinders, A., Hsu, H. and Sze, H. (1992) Plant Physiol. 99, 161-169.

83. Warmke, J., and Ganetzky, B. (1994) Proc. Natl. Acad. Sci. 91,3438- 3442.

84. Warmke, J., Drysdale, R., and Ganetzky, B. (1991) Science 252,1560- 1562.

85. Weber, 1. T., Gilliland, G. L., Harman, J. G., and Peterkofsky, A. (1987) J. Biol. Chem. 262,5630-5636.

86. Welch, R. M. (1995) Crit. Rev. Plant Sci. 14,49-82.

87. Woolhouse, H. W. (1983) Physiological Plant Ecology, III. Responses to the chemical and biological environment. Volume 12C (Lange, O. L., Nobel, P. S., Osmond, Osmond, B. and Zeigler, Zeigler, eds). Berlin Berlin Springer- Verlag, pp. 245-300.

88. Zielinski, R. N. (1998) Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 697-725.