AU5449301A - Method for controlling water content of plant - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): JAPAN TOBACCO INC.

0 0 Invention Title: METHOD FOR CONTROLLING WATER CONTENT OF PLANT The following statement is a full description of this invention, including the best method of performing it known to me/us: 1

SPECIFICATION

METHOD FOR CONTROLLING

WATER

CONTENT OF PLANT FIELD OF THE INVENTION This invention relates to a method for controlling the water content of a plant in order to provide a plant which is tolerant to water-related stress such as salt stress or drought stress.

PRIOR ART *Plants are continuously exposed to stress even under normal growing conditions. Such stress is caused by various factors such as salt, drought, high temperature, low temperature, strong light, air pollution, etc. From the viewpoint of agriculture, salt and drought stress cause the most serious problems. Salt stress is observed not only in areas which have a soil inherently rich in salt but also in irrigated farms. At present, more than 10% of cultivated land suffers from some degree of salt stress.

To increase food production, it is desired to grow crop in land which has previously been regarded as uncultivatable.

Drought stress is caused by unseasonable weather and geographic location. In the United States, drought causes a fall in crop yield once every several years.

Therefore, it is important to find a plant which is tolerant to water-related stress.

In this regard, it is known that water channel LA

I

proteins (hereinafter sometimes referred to simply as WCH proteins) seemingly have a role to play in "water flux in plants".

These plant WCH proteins are classified into plasma membrane-located types and tonoplast-located types. Some reports have been already published on the genes of these proteins.

In connection with the genes of plasma membranelocated WCH proteins, there have been reported, for example, the cloning of cDNA of a turgor responsible WCH protein derived from pea [Plant Molecular Biology 15, 11-26 (1990)]; the cloning of cDNA of a desiccation responsible WCH protein derived from Arabidopsis thaliana (Plant Cell Physiology 33, 217-224); WCH gene of Arabidopsis thaliana [The Plant Journal 6, 187-199 (1994)]; WCH gene of tomato o* [Plant Molecular Biology 24, 539-543 (1994)]; the introduction of an antisense gene into Arabidopsis thaliana to confirm its function in the water channel [The Plant Journal 7, 87-95 (1995)]; WCH gene of common ice plant 20 (Mesembryanthemum crystallinum) [The plant Cell 7, 1129- 1142 (1995)], etc. In addition, those derived from corn, rice, kohlrabi (Brassica oleracea), soybean, barley, etc.

have been registered at gene data bases, etc.

In connection with the genes of tonoplast-located WCH proteins, there have been reported, for example, one derived from tobacco [Nucleic Acids Research 18, 7449 (1990)]; one derived from common bean [The Plant Cell 2, 525-532 (1990)]; one derived from Arabldopsis thaliana 2 [Plant Physiology 99, 561-570 (1992)], etc. In addition, those of rice, barley, soybean, radish (Raphanus sativa), white clover (Triforium repens), alfalfa (Medicago sativa), etc. have been registered at gene data bases, etc.

As described above, it has been urgently required to provide a plant which is tolerant to water-related stress.

One approach to obtain such a plant is directed to controlling the water content of the plant by, for example, gene manipulation. In general, it is considered that a plant which is tolerant to water-related stress can be obtained by increasing the water content of the plant, namely, enhancing the capability of the plant to retain water.

To obtain a water-related stress-tolerant plant in the above-mentioned manner, WO 96/00789 and Nature 379 (22) *'i eo 683-684 (1996) disclose a method for obtaining a plant tolerant to water-related stress by way of controlling the water content of the plant. The method comprises introducing the gene of an enzyme for trehalose 20 biosynthesis into the plant, thereby to induce the synthesis and accumulation of trehalose in the plant, and to eliminate the loss in water content by way of the waterretaining effect of trehalose. The disclosure of these references is incorporated herein by reference.

However, this method suffers from various problems.

Namely, a certain amount of carbon fixed by photosynthesis is consumed in this method, which is disadvantageous from the viewpoint of energy consumption. In addition, there is 3 a risk that the accumulation of trehalose might exert adverse effects on the qualities of the product and the metabolic system of the plant.

On the other hand, it has been considered that the WCH proteins described above may participate in water flux in a plant. However, no one has suggested so far that the water content of a plant may be controlled by using these proteins.

SUMMARY OF THE INVENTION The present invention, which has been accomplished under the above-mentioned circumstances, provides a method for controlling the water content of a plant by introducing a plant WCH protein gene into the plant.

The present invention further provides a plant which is produced by the above method and hence possesses a oooeo controlled water content.

The present invention further provides a plant which has an improved tolerance to drought stress and/or salt stress due to the controlled water content.

DETAILED DESCRIPTION Now, the present invention will be described in greater detail.

The present invention is characterized in that a plant WCH protein gene is introduced into a plant to thereby control the water content of the plant thus transformed.

The plant WCH proteins to be used in the present invention include plasma membrane-located WCH proteins and 4 tonoplast-located ones. Although genes encoding proteins of both of these types may be used in the present invention, genes of WCH proteins which are located in plasma membrane are preferred.

As described above, there have been known genes encoding plasma membrane-located WCH proteins of various origins such as pea, Arabidopsis thaliana, tomato, Mesembryanthemum crystallinum, corn, rice, kohlrabi, soybean, barley, etc. These known genes may be used in the present invention. However, WCH protein genes to be used in the present invention are not limited thereto, provided that those of WCH proteins located in plasma membrane are preferred.

As a method of obtaining a gene of a plasma membrane-located WCH protein, the Examples hereinbelow will illustrate a method to obtain a gene from Mesembryanthemum crystalllnum. Further, genes from other plants can be obtained by reference to the following literatures.

Pea (Pisum sativum) [Plant Molecular Biology 20 11-26 (1990)].

Arabldopsis thaliana [Plant Cell Physiology 33, 217-224 (1992); Plant Molecular Biology 23, 1187-1198 (1993); The Plant Journal 6, 187-199 (1994)].

Tomato [Plant Molecular Biology 24, 539-543 (1994)].

The gene encoding a plant WCH protein to be used in the present invention may be operatively introduced into a plant in such a manner that the genetic code will be 5 translated either in the sense direction or in the antisense direction. In order to enhance the waterretaining capacity of the plant under water stress, it is preferred in the present invention that the gene is introduced into the plant in the sense direction. However, in certain cases, reduced water flux in plants is expected to be beneficial for them and hence, introduction of the gene in the antisense direction will be desired, depending on the strength, timing or kind of stress.

It is preferred in the present invention that the gene encoding a plant WCH protein, which is to be introduced into a plant in the sense direction, is one derived from a plant which is tolerant to salt stress or drought stress, still preferred is one derived from Mesembryanthemum crystallinum.

When the gene is introduced into the plant in the antisense direction, on the other hand, it is preferred that the species of the plant to be transformed is as close as possible to the species from which the gene is obtained.

20 It is still preferred that the gene is of the same species as the plant to be transformed. When the gene is introduced in the antisense direction, it is not always necessary to introduce the whole gene. Namely, sufficient effects will be achieved in some cases by using a segment of the gene. Therefore, when the gene is introduced in the antisense direction in the present invention, it is required that at least a portion of the gene is used.

As an expression promoter to be used in the present 6 invention, any known ones may be employed. For example, use can be made of those having intense transcriptional function and being capable of inducing the expression of a gene in any cells (35S, 19S, nos, etc.), those responsive to light (rbc, etc.), those sensitive to temperature (hsp, etc.), those responsive to hormones and those reactive in specific tissues. Of particular preferred is a powerful promoter such as In the present invention, it is not necessary to employ any special method for introducing a WCH protein gene into the plant to be transformed either in the sense 9 o direction or in the antisense direction. That is, any method commonly employed to transform plants may be used.

By way of example, the leaf disk method utilizing bacteria of the genus Agrobacterium will be described herein.

oooo9 A WCH protein gene is inserted into an appropriate plant expression vector either in the sense direction or in the antisense direction and then the vector is introduced into bacteria belonging to the genus Agrobacterium. Then, 20 leaf disks taken from a germ-free leaf of the plant to be transformed are immersed in a culture medium containing the bacteria. After the formation of calluses, one in which transformation has occurred is selected and grown to thereby give a transformed plant. The selection of transformed plants may be performed, for example, by adding an appropriate antibiotic to the culture medium for callus formation and selecting calluses which are tolerant to the antibiotic. The transformation method utilizing bacteria 7 of the genus Agrobacterium is applicable not only to dicotyledonous plants but also to monocotyledonous plants as disclosed in WO 94/00977 which is incorporated herein by reference.

The plant species to be transformed by the present invention, namely, plants the water content of which is to be controlled by the present invention are not particularly limited. Examples thereof include soybean, corn, potato, tomato, tobacco, etc. Among all, soybean and corn are preferred.

The improved water content of the plant transformed in accordance with the present invention may be determined by various methods. For example, the plant grown to a certain stage is placed under stressful conditions, for example, in a climate chamber, for a certain period o .of time. Then the aerial part of the plant is harvested and weighed. The plant is subsequently dried at 600C for several days and then the dry weight is measured. Thus, the water content may be expressed as the ratio of the fresh weight to the dry weight.

To further illustrate the present invention in greater detail, the following Examples will be given.

However, it is to be understood that the present invention is not limited thereto.

Example 1: Isolation of the aene of plasma membranelocated WCH protein from Mesembryanthemum crystalllnum A total RNA fraction was extracted from a root tissue specimen of Mesembryanthemum crystalllnum in 8 accordance with the method of Ostrem et al. [Plant Physiology 84, 1270-1275 (1987)], with modifications made as follows: 1) RNA was extracted from the plant which had been subjected to a salt stress-treatment with 400 mM NaCl.

2) The plant was ground and shaken on ice for 1 hour together with the extraction buffer and phenol.

3) After centrifugation, the supernatant was shaken on ice for 1 hour with chloroform.

poly(A) RNA was purified by the oligo dT cellulose column method. The WCH protein gene was obtained by the a differential screening of a cDNA library which was prepared by using the poly(A) RNA thus purified and ZAP-cDNA synthesis kit in accordance with the manufacturer's instructions. In the differential screening, two different single-stranded DNA were used as probes, namely, one synthesized from the above-mentioned poly(A)+RNA and the other synthesized from poly(A) RNA isolated and purified in the same manner from a plant not subjected to the salt stress treatment. Thus a plasmid was obtained which contained, the WCH protein gene McMipA of Mesembryanthemum crystalllnum at the restriction enzyme site EcoRI/XhoI in plasmid pBSK (purchased from STRATAGENE). The nucleotide sequence of this gene encoding the plasma membrane-located WCH protein derived from Mesembryanthemum crystalllnum and the deduced amino acid sequence are shown respectively in SEQ ID NO:1 and SEQ ID NO:2. In SEQ ID NO:1, the reading frame extends from the start codon ATG at positions 225-227 9 to the stop codon TGA at positions 1068-1070. The details of differential screening are described in the Plant Cell, 7. 1129-1142, 1995, "A family of transcripts encoding water channel proteins: Tissue-specific expression in the common ice plant" Shigehiro Yamada, Maki Katsuhara, Walter B.

Kelly, Cristine B. Michalowski, and Hans J. Bohnert.

Example 2: Construction of gene for the transformation of Nicotiana tabacum The plasmid obtained in Example 1, which carried the WCH protein gene McMipA from Mesembryanthemum crystallinum at the restriction enzyme site EcoRI/XhoI of plasmid pBSK, was digested with restriction enzyme XbaI to isolate the McMipA gene fragment. This gene fragment was inserted into the XbaI site of binary vector pBIl21 (purchased from Clontech) in such a direction that mRNA was transcribed in the positive direction by the promoter 35S to thereby give the gene construct pBI4C. The beta-glucuronidase gene contained in pBIl21 was deleted with restriction enzymes "S SmaI and SacI.

20 Example 3: Transformation of Nicotiana tabacum A green leaf of Nicotiana tabacum, which had been aseptically grown in a test tube, was cut into pieces (1 cm 1 cm) and the tissue pieces were immersed in a culture medium (Murashige and Skoog basal medium with Gamborg's vitamins; purchased from SIGMA) in which Agrobacterlum tumefaclens LBA4404 carrying the gene construct pBI4C obtained in Example 2 was suspended. Then the pieces were left to be infected with A. tumefaclens in dark for 2 days.

10 Subsequently, the pieces of tissue were transplanted into an agar medium (the above-mentioned culture medium containing 0.8% of agar) containing antibiotics (100 mg/L of kanamycin and 250 mg/L of cefotaxime) and a plant hormone (0.5 mg/L of 6-benzylaminopurine) and thus redifferentiated plants were obtained. The aerial part of each redifferentiated plant was transplanted into the above-mentioned agar medium containing the antibiotics (100 mg/L of kanamycin and 250 mg/L of cefotaxime) and allowed to root therein. Rooted individuals were transplanted in a closed greenhouse and thus self-pollinated progenies of the transformants were obtained.

Example 4: Measurement of change in water content under stressful conditions Seeds of the transformants obtained in Example 3 were germinated in the above-mentioned agar medium containing 100 mg/L of kanamycin and grown until 2 true leaves developed. Then plants not bleached at this time point were transplanted into pots in a closed greenhouse.

20 After being grown until 4 true leaves developed, the plants were transplanted in a mixture of equivalent amounts of vermiculite and Hydroball and grown in this state for 2 weeks while watering Hoagland's solution (purchased from SIGMA), diluted 4-fold, at a rate of 50 ml/day. Then the plants were transferred into a climate chamber (temperature: 23 0 C, humidity: 70%, lighting period for 12 hours/dark period for 12 hours). After being grown for several days for acclimation, the stress treatment was 11 performed. The plants in the salt stress group were watered with 100 ml (on the first day) or 50 ml (on the second day and thereafter) of Hoagland's solution (purchased from SIGMA), diluted 4-fold and containing 250 mM of NaCl. In the drought stress group, watering was suspended. After the end of the experiment period, the aerial part of each plant was harvested and weighed.

These plants were then dried at 60 0 C for several days and weighed. Based on the results, the water content of each plant exposed to the stress was determined.

Table 1: Salt stress treatment

S

Sao# 0 604 0 099 *515 so *:Go S 9 0 *5S9@ 4 *j.

0

OS

9. 5 Dry weight/fresh Water weight Ratio* content control 0.1505380 1.000 84.92 individual No. 1 0.1287313 0.854 87.12 individual No. 2 0.1215596 0.808 87.84 The relative dry weight/fresh weight ratio of each transformant calculated when the dry weight/fresh weight ratio of the control plant is taken as 1.

Table 2: Dry stress treatment Dry weight/fresh Water weight Ratio* content control 0.1602564 1.000 83.97 individual No. 1 0.1274725 0.795 87.25 individual No. 2 0.1349073 0.842 86.51 The same meaning as the one indicated in Table 1.

12 The data in Tables 1 and 2 show that the plants transformed with WCH protein gene have a greater water content than the control plants.

Plants having a greater ability to retain water or a greater efficiency to utilize water are more tolerant of drought refer to Lincoln Taiz and Eduardo Zeiger, Plant Physiology, Chapter 14 Stress Physiology, The Benjamin/Cummings Publishing Company, Inc.). Further, it is known that the ability to retain water content is an issue of importance for creatures in general which live in dry or high salt areas refer to Kent F. McCue et al., 9 S. TIBTECH-December 8, 358-362). Prior attempts to solve the problem have focused on a method wherein plants were allowed to accumulate an osmoprotectant in their body to enhance a water retaining ability, whereby making them tolerant to both salt and drought stress (Kent F. McCue et al., ibid). One such attempt includes the introduction of trehalose synthase gene into a plant.

In contrast, the present invention is unique in that water-retaining capability of a plant is improved by a substantially different approach, by means of WCH protein gene. It is expected that plants which acquire an improved water-retaining ability in accordance with the method of the invention will be more tolerant to salt stress and drought stress.

Example 5: Isolation of the gene of plasma membranelocated WCH protein from Nicotiana tabacum A total RNA fraction was extracted from a leaf 13 tissue specimen of Nicotiana tabacum in accordance with the method of Ostrem et al. [Plant Physiology 84, 1270-1275 (1987)], with modifications made as follows: 1) The plant was ground and shaken on ice for 1 hour together with the extraction buffer and phenol.

2) After centrifugation, the supernatant was shaken for 1 hour with chloroform on ice.

poly(A) RNA was purified by using QuickPrep mRNA purification Kit (purchased from Pharmacia) in accordance with the manufacturer's instructions.

To isolate the cDNA encoding the WCH protein, "primers were prepared by a DNA synthesizer (manufactured by Applied Biosystems) by reference to the nucleotide sequence (SEQ ID NO:1) of the WCH protein cDNA derived from Mesembryanthemum crystallinum described in Example 1.

The sequences of the primers were as follows.

Primer 1: GAAGATCTATGATCTTTGCCCTTGTTTACTGC. (SEQ ID NO:4) Primer 2: GTCAGATCTAGCACGCGACTTGAATGGAATAGCC. (SEQ ID The cDNA template used in the reverse transcription polymerase chain reaction (RT-PCR) was synthesized by StrataScript" RT-PCR+Kit; (purchased from STRATAGENE) by *employing 50 ng of poly(A)*RNA extracted and purified from Nicotiana tabacum and the oligo dT primer in accordance with the manufacturer's instructions.

The RT-PCR was performed by using the abovementioned cDNA template, 20 pmol of the above-mentioned primers 1 and 2, 200 mmol each of dATP, dGTP, dCTP and dTTP, 1 x PCR buffer (purchased from Takara Shuzo Co., Ltd.) and 14 U of AmpliTaq DNA polymerase (purchased from Takara Shuzo Co., Ltd.) (in a total reaction volume of 100 ml. The reaction was performed for 32 cycles, the cycle consisting of 1 minute at 91 0 C, 1 minute at 42 0 C and 2 minutes at 72 0 C. The PCR product was separated on a 1% agarose gel and stained with ethidium bromide. Thus the product was detected at the position of about 550 bp.

The PCR fragment was recovered from the gel and subcloned into plasmid pCRII by using TA Cloning Kit (purchased from Invitrogen) in accordance with the manufacturer's instructions.

The nucleotide sequence of the resulted PCR fragment was identified by DNA Sequencer Model 373A (manufactured by Applied Biosystems). The reaction was performed by using S 15 Taq Dye Terminator Cycle Sequencing Kit (purchased from :Applied Biosystems) in accordance with the manufacturer's guidance. In this Example, a partial cDNA fragment of the gene of the plasma membrane-located WCH protein having the nucleotide sequence shown in SEQ ID NO: 3 was obtained.

Example 6: Construction of gene for the transformation of Nicotlana tabacum By using the gene fragment obtained in Example 5, an antisense gene to be introduced into Nicotiana tabacum was constructed. The PCR fragment subcloned into the plasmid pCRII was digested with restriction enzymes SacI and XbaI and purified. This fragment was inserted into plasmid pBI121 at the site formed by digestion with SacI and XbaI.

Thus the gene construct pA121 was obtained.

15 Example 7: Transformation of Nicotiana tabacul Nicotiana tabacum was transformed with pAl21 by substantially the same method as the one described in Example 3.

In accordance with the present invention, which provides a method for controlling the water content of a plant by way of the introduction therein a gene which encodes a plant water channel protein, the water content of the plant can be controlled and hence, the plant is made tolerant to water-related stress without any disadvantages from the viewpoint of energy due to the consumption of carbon fixed by photosynthesis or problems due to the accumulation of saccharides which may exert unpredictable effects on the qualities of products and the metabolic S 15 system of the plant.

It is to be understood that a reference herein to a prior art publication does not constitute an admission that the publication forms a part of the common general knowledge in the art in Australia, or any other country.

o*oo 16 Page(s) ~3~iare claims pages they appear after the sequence listing SEQUENCE LISTING INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 1272 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: Mesembryanthemum crystallinum.

(ix) FEATURE: OTHER INFORMATION: plasma membrane-located

WCH

protein cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

GAGAGAACTA

AACATACAAC

AACTCAAACC

TTTCTAGAGA

AGGATGTGAG

AGGACAGAGA

GGTCGTTCTA

CTATCTTGAC

AGGGTATTGC

TTTCAGGAGG

CCTTGACAAG

GTCTCGAGTT

AACAACATAT

ACCACAGTCT

GAGAAACAAG

ACTAGGAGCC

CTACAGGGAG

CAGAGCTGGG

TGTTATGGGG

TTGGTCTTTT

TCACATTAAC

GGCAGTCTTC

TTTTTTTTTT

TTCACAAATC

CACAGAGCAG

AGAGAGAGAG

AACAAGTTCT

CCACCGCGCG

ATTGCTGAGT

GTTAATAGGA

GGTGGCATGA

CCAGCAGTCA

TACATGGTCA

TTTTTTTTTA

GAAATTTACA

CACCAAAAAG

AAACAAGAGA

CGGAGAGGCA

GCCTCTTTGA

TCATTGCTAC

GTCCCTCAAA

TCTTTGCCCT

CATTTGGGCT

TGCAATGCTT

17 TTCATAAGAG TTTAATGAAT GCAATGATGT AATTAACCTC AGCATAGCTC ACTGCCTTTC AGCAATGGAG GGGAAGGAAG GCCGCTGGGG ACGGTGGCGC GGCCGGCGAG CTGACGTCAT CTTCTTGTTC CTCTACATCT GTGTGCCAGT GTTGGAATTC TGTTTACTGC ACTGCTGGAA ATTCTTGGCA AGGAAATTGT GGGTGCCATT TGTGGTGCTG 120 180 240 300 360 420 480 540 600 660 GTGTTGTCAA GGGCTTCCAG

ACCCCGGCTA

CTACACCGTC

TGGCTCCATT

CTGGCACTGG

ATGCTTGGGC

CCCTGTACCA

TCGAGTGATG

AAGATTGGAC

CTTTTGTACT

TGTTTCATGT

CACCAAGGGA

TTCTCCGCCA

GCCAATTGGG

CATCAACCCA

TGACCATTGG

TGTAGTAGTG

ATGAATGATC

CCCCACGTGT

GTACTAGTTT

TT

CACCCCCTAC

TCAGGCCTTG

CTGACGCCAA

TTCGCTGTGT

GCCAGGAGTC

ATTTTCTGGG

ATAAGGGCAA

ATCGGACGGC

CATTTTCCCT

GTAAAGTTAT

CAGCTCTTGG

CGCTGAGATT

GCGAACGTAG

TCTTGGTTCA

TTGGTGCTGC

TGGGACCCTT

TTCCATTCAA

CAAGATTAAT

AGTTATTTTT

GGTGTTTTGG

GCGGCGGGGC

ATCGGCACTT

GGAGTCCCAT

CTTGGCCACC

TATCATTTAC

CATCGGTGCA

ATCCAAGTGA

TGTCGAGGTC

ATCTCTCCTT

GGTCTCAGAA

AACTCTGTGA

TTGTTCTTGT

GTTCCTATCT

ATCCCCGTTA

AACAGGCCCC

GCACTTGCAG

TGATAAGATT

TCTAGATGAT

CTGTGTTTGT

GAACGTGGGA

720 780 840 900 960 1020 1080 1140 1200 1260 1272 18 INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 284 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: Mesembryanthemum crystallinum (ix) FEATURE: OTHER INFORMATION: plasma membrane-located WCH protein: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Glu Gly Lys Glu Glu Asp Val Arg Leu Gly Ala Asn Lys Phe 1 5 10 Ser Glu Arg Gin Pro Leu Gly Thr Val Ala Gin Asp Arg Asp Tyr 25 Arg Glu Pro Pro Arg Gly Leu Phe Glu Ala Gly Glu Leu Thr Ser 40 Trp Ser Phe Tyr Arg Ala Gly Ile Ala Glu Phe Ile Ala Thr Phe 55 Leu Phe Leu Tyr Ile Ser Ile Leu Thr Val Met Gly Val Asn Arg 70 Ser Pro Ser Lys Cys Ala Ser Val Gly Ile Gin Gly Ile Ala Trp 85 Ser Phe Gly Gly Met Ile Phe Ala Leu Val Tyr Cys Thr Ala Gly 100 105 Ile Ser Gly Gly His Ile Asn Pro Ala Val Thr Phe Gly Leu Phe 110 115 120 19 Leu Ala Arg Met Gin Cys Phe Gin His Asn Pro Gly Ala Leu Leu Ser Giu Arg Lys Leu 125 Leu Gly 140 Pro Leu 155 Tyr Thr 170 Phe Leu 185 Arg Glu 200 Ala Val 215 Gly Ile 230 Arg Pro 245 Phe Ile 260 Arg Ala 275 Ser Leu Thr Arg Ala Ile Cys Gly Pro Ala Leu Gly Lys Gly Ser Gly Ser Thr Pro Ser Ala Val Phe Tyr Met Val 130 135 Ala Gly Val Val Lys Gly 145 150 Arg Arg Gly Asn Ser Val 160 165 Leu Ala Leu Arg Leu Ser 175 180 Ser Pro Pro Leu Thr Pro 190 195 Ile Leu Ala Pro Leu Pro 205 210 Leu Ala Thr Ile Pro Val 220 225 Ser Leu Gly Ala Ala Ile 235 240 Asp His Trp Ile Phe Trp 250 255 Ala Ala Leu Tyr His Val 265 270 Ser His Val Pro Ile Gly Phe Thr Gly Thr Ile Tyr Asn Val Gly Pro Phe Leu Val His Asn Pro Ala Arg His Ala Trp Ala Gly Ala Ala Leu Val Val le Ile Pro Phe Lys Ser Lys 280 20 INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 587 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: Nicotiana tabacum (ix) FEATURE: OTHER INFORMATION: plasma membrane-located

WCH

protein cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GAAGATCTAT GATCTTTGCC CTTGTTTACT GCACTGCTGG TATCTCAGGA GGACACATTA

S.

S S

S

S

S

S

S S SS S

S

ACCCAGCAGT

TCTACATGGT

TGGTGGGTCC

AAGGTGATGG

CTGCCACTGA

CTATTGGATT

TCAACCCCGC

ATCACTGGAT

AGATAATCAT

GACATTTGGT

GATGCAGTGC

ATACCAGAGA

ACTTGGTGCT

TGCCAAGAGA

CGCGGTGTTC

CAGGAGCCTT

CTTCTGGGTT

CAGGGCTATT

CTGTTTTTGG

CTTGGTGCAA

CTTGGTGGTG

GAGATTATTG

AATGCTAGAG

TTGGTTCATT

GGAGCTGCTA

GGACCATTCA

CCATTCAAGT

CAAGAAAGTT

TCTGTGGTGC

GGGCCAACAT

GGACCTTTGT

ATTCTCATGT

TGGCCACCAT

TCATCTTC.AA

TTGGAGCTGC

CGCGTGCTAG

GTCCTTAACA

TGGTGTGGTT

GGTTCAACCT

CCTAGTTTAC

CCCTATTTTG

CCCAATCACC

CCAAGACCAG

ACTTGCTGCA.

ATCTGAC

AGGGCTCTGT

AAAGGTTTCA

GGCTACACAA

ACTGTTTTCT

GCACCTCTTC

GGAACCGGTA

GCATGGGATG

GTTTACCACC

120 180 240 300 360 420 480 540 587 21 INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: GAAGATCTAT GATCTTTGCC CTTGTTTACT GC INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 34 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 15 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GTCAGATCTA GCACGCGACT TGAATGGAAT AGCC 22

Claims (13)

1. A method for controlling the water content of a plant-characterized in that a gene encoding a plant water channel protein is operatively introduced into the plant.

2. A method for controlling the water content of a plant as claimed in Claim 1 characterized in that said gene encodes a plant water channel protein of the type located in plasma membrane.

3. A method for controlling the water content of a plant as claimed in Claim 1 or 2 characterized in that said plant water channel protein gene is introduced into the plant in such a manner that the genetic code will be translated in the sense direction.

4. A method for controlling the water content of a plant as claimed in Claim 3 characterized in that said plant water channel protein gene is derived from a plant which is tolerant to salt stress or drought stress.

A method for controlling the water content of a plant as claimed in Claim 4 characterized in that said plant water channel protein gene is derived from Mesembryanthemum crystalllnum.

6. A plant which has a gene encoding a plant water channel protein, wherein said gene has been operatively- introduced into said plant so that the plant will have either a greater or smaller water content than the original plant which does not have said gene when these plants are grown under the same condition.

7. A plant as claimed in Claim 6, wherein said gene 23 a encodes a plant water channel protein of the type located in plasma membrane.

8. A plant as claimed in Claim 6 or 7, wherein said plant water channel protein gene has been introduced into the plant in such a manner that the genetic code will be translated in the sense direction.

9. A plant as claimed in Claim 8, wherein said plant water channel protein gene is derived from a plant which is tolerant to salt stress or drought stress.

A plant as claimed in Claim 9, wherein said plant water channel protein gene is derived from Mesembryanthemum c crystallinum.

11. A plant as claimed in Claim 10 selected from the group consisting of soy bean, maize, potato, tomato and tobacco.

12. A method for controlling the water content of a plant substantially as hereinbefore described with reference to any one of Examples 3, 4 and 7.

13. A plant which has a gene encoding a plant water channel protein, wherein said gene has been operatively S" introduced into said plant so that the plant will have either a greater or smaller water content than the original plant substantially as hereinbefore described with reference to any one of Examples 3, 4 and 7. Dated this 19 t h day of July 2001 JAPAN TOBACCO INC. By their Patent Attorneys GRIFFITH HACK 24