Tissue-specific expression of a plant turgor-responsive gene with amino acid sequence homology to transport-facilitating proteins

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  • Plant Molecular Biology 21: 929-935, 1993. © 1993 Kluwer Academic Publishers. Printed in Belgium. Update section Short communication 929 Tissue-specific expression of a plant turgor-responsive gene with amino acid sequence homology to transport-facilitating proteins Felix D. Guerrero 1, and Lyle Crossland CIBA-GEIGY Agricultural Biotechnology Research Unit, P.O. Box 12257, 3054 Cornwallis Rd, Research Triangle Park, NC 27709, USA; 1present address: USDA Livestock Insects Research Laboratory, 2700 Fredericksburg Rd., Kerrville, TX 78028, USA (* author for correspondence) Received 20 July 1992; accepted in revised form 3 December 1992 Key words: gene promoter, transgenic tobacco, transport, turgor, water stress Abstract We report the isolation of a turgor-responsive gene of pea, trg-31, whose transcription induced within 30 min after the loss of leaf turgor. Structure of the coding region and 1.4 kb of 5' untranslated region was determined by DNA sequencing. A 3 kb promoter fragment from trg-31 was fused to a fl-glucuronidase (GUS) reporter gene in pBI101, tobacco leaf disks and mature plants analyzed for turgor-responsive induction of GUS mRNA. Significant amino acid sequence homology exists between trg-31 and putative transport proteins of bovine, Phaseolus, soybean and Escherichia coli membranes. To study early responses to water deficit at the molecular level, we initiated research whose main objective was the identification of genes regulated by the reduction of cell water potential and tur- gor. We initially identified several poly(A) RNAs whose transcription is induced within 30 min after reduction of leaf turgor [ 5 ]. Three unique cDNAs derived from turgor-responsive mRNAs were cloned and sequenced and the in vivo responses of their corresponding genes to various stimuli char- acterized [6]. We previously reported the cloning and sequencing of the gene for one of these turgor-responsive mRNAs. The gene is desig- nated trg-31 (for turgor-responsive gene) and contains an open reading frame encoding a pu- tative protein of ca. 31 kDa. The trg-31 amino acid sequence possesses homology with a group of proteins believed to be involved in facilitating transport of small molecules across cell mem- branes. Pisum sativum (Progress No. 9) was grown and wilted as reported earlier [6]. Pea genomic DNA was isolated by a method described by Shure etal. [17] and used to synthesize a 2genomic library with the LambdaGEM-11 Xho I Half-Site Arms Cloning System (Promega, Madison, WI). The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number Z18288 (trg-31 gene).
  • 930 The genomic library was screened [7] using the wilting-induced cDNA clone 7a [6]. Inserts from 2 genomic clones were subcloned into Bluescript SK+ plasmid (Stratagene, San Diego, CA) and both strands of a 3297 bp portion of clone pCIB3130 were sequenced using Sequenase VER2.0 (U.S. Biochemicals, Cleveland, OH) and the results shown in Fig. 1. Subclone pCIB3130 contained ca. 3 kb of 5' untranslated sequence, the entire coding region and 0.9 kb of 3' untranslated region. The trg-31 gene has 4 exons of 337, 299, 141 and 90 bp and 3 introns of 81, 89 and 77 bp. The transcription start site was identified by primer extension analysis [ 14] -1397 gtt~tgacca agtaagaata gctcacaacg tgacaccatt ctttacaaga cacacctaaa gtcaataacc ataatatttt gatacacgtt t t t tacgt t t -1297 ttttccccac tttttttttt ttgaatatac tttattgaaa atatattctt aaattaattt aattgaaata catcgataat aaaagaattt tacaccttca -1197 gtteatcata accgttggat attaaaataa gtttgatctt tattttaaaa atctataaaa ta~tacaaac ggttaatagt gatgaatcga ttgtataaaa -1097 ttttttatat tgacagtgta tagtaattaa tctcttctta aatcecaaga tttagatatg agattaaagg taatgcattg accgtgtaaa atagttttat -997 acttataccc aataaaaatt tatcaatcaa ccatatcata taactaattt ataaatatca ggatgactta aaagaataaa tgggatgatt gataaacggt -897 gtaaaataat tttacactat caagacatat cttttttctc tttaggtata ttatctaaaa acacttaaaa aaatgagaag tattaatcga aaaattaaat -797 aatctgtgtt cttaaaataa ttaaatgata tatttatttt gtgtgtaaat tgttctcagg tttttccctt tgtaagctat ttgtttcaaa ttactaagca -697 tcttgttgtt tatgggatag aatttattaa cttacaagtc actatacaat aatgtagaaa taatcataat caagcaatca attacatcaa tatactccag -597 tttgagtaaa tcgtaagcta agcaaaaaac aatataaact tagaagtaac acctgttatt aacattagat caaaatcctc atcataatat ataaatcgtg -497 taaattttaa ttcatcccat tattatgaag agaaaaaaaa tatcactttc ggcacatcgc cgtagtagcc accgcctata ataagaaata gcattaccca -397 acctaacctg tgtgcttatg caatgcacaa cacgacaaac tcaagagaca cgtgtcacgt cacagaggaa tcccaatttg acaataacca ccgcccagct -297 taaaatacac gtggaaaatg gaccacaatt tctacactac atttacactt ttacccttcc tatatttctc attcttacag caagtagtcc acgttccagt -197 actacgcgga acatggtggg tcttacctta agtgttagta gaataaagcc agctaggata aaccattcat gaataattgg tcctatcaat agaaaaatga -97 aacatgttac gcatggtagg acccacgttt ccattaactc aaaccaattc ctattataaa tccgcttctt attcttatcc cttcacactc acctcac~ca +4 caaacaagaa accagaacag tctagaaaga aacATGGAAG CCAAGGAACA GGATGTGTCT q~fGGGAGCCA ACAAGTTTCC AGAGAGACAG CCACTCGGGA M E A K E Q D V S L G A N K F P E R Q P L G I +104 TTGCAGCTCA GAGCCAAGAC GAGCCAAAGG ACTATCAAGA GCCACCACCG GCGCCACTTT TTC~AGCCCTC GGAGCTGACT TCATC~T TCTACAGAGC A A Q S Q D E P K D Y Q E P P P A P L F E P S E L T S W S F Y R A +204 CGC~A~AGCC O~AC~ATAG CCAC~T TT'PCCTT'PAT A~AACC~ TAACC~A~ GGC4C~q~ CGAGAAAGT'P CCAAGq~ AACGGq~T G I A E F I A T F L F L Y I T V L T V M G V V R E S S K C K T V G +304 ATTCAAGGAA TCGCTTGGGC TI~fTGGTGGC ATGATATI~fG CTCTCGq~fTA CTGCACCGCT GGAATCI~AG gtatatttct gttttatctt ttggttggtg I Q G I A W A F G G M I F A L V Y C T A G I S G +404 gcactttctg catagctatc aactgaaccg ggttaactga atatcatgta _GGGGGTCATA TAAACCCAGC TGTGACGq'PT (3GGCTATqq~ TGGCGAGGAA G H I N P A V T F G L F L A R K +504 Aq'II~,TCGTTG ACAAGAC~ TATTCTACA~ GGTC~ATGCAA GTGn~G~O~TG CTATATGCGG TGCTGGTGTG GTGAAAGGTT qWGAAC~3TAA ACAAAGGT~ L S L T R A I F Y M V M Q V L G A I C G A G V V K G F E G K Q R F +604 GGAGATCTGA ACGGTGGTC~ C A A ~ GCT~CTGGTT ACACCAAAGG TGATGGAC~ GGTGCTGAAA TTGTI~_~=CAC %~I'fCATTCTT GTATACACAG G D L N G G A N F V A P G Y T K G D G L G A E I V G T F I L V Y T V +704 TT~TCAGC CACTGATGCT AAACGTAGCG CCAGAGACTC TCATGTTCCT gtaagtagat ttttaatata tttgaggtct attcattttt acaagaatag F S A T D A K R S A R D S H V P +804 tataattaat taattgtatg gttaattttt catgagtagA TT'PTGGCACC GTTGCCAATT G(~AT'~CC~C~G TGq'PCq'FC~T GCAq~PTGGCA AC'PATACCAA I L A P L P I G F A V F L V H L A T I P I +904 TTACTGGAAC TGGTAq~PAAC CCTGCCAGAA GTCq~FGGTGC TGCTATTGTC I"PCAACAAGA AAATTGGT~fG GAATGATCAC gtaagttgaa aaattataat T G T G I N P A R S L G A A I V F N K K I G W N D H +1004 ccattaaagt tttttattta aaatattttt gttgaccaat tattattatt gtggcagTGG ATTI~fCTGGG TTGGACCATT TATTGGAGCA GCTCTTGCAG W I F W V G P F I G A A L A A +1104 CACTATACCA TCAAGqq~TT ATCAGAGCCA T~CCCTTCAA GTCTAAGtga ttgaattgaa tcaaacggtt cttgatcaat catatcatgt ttgtggattt L Y H Q V V I R A I P F K S K * +1204 catttcatcat gaaatggaa taatcttatg tatgatattt ttttctgaat ttttaaatgt ttgttttgta atttgtatgt aaagaagtac tatttattat +1304 gattattagg tgtagcatgt gtgtgttgcg taatgggtcc agctctcgtg tgtgaatgtg tattttgcca aatctaaaat aacactcgag tcatcatttc +1404 tgaatttctt attattatgc tattgcttga atttttgtca tcttcccaag agtatatatc aagcttgggt ttctttatcc atactttcgt tcgctatcac +1504 atgttaaggt tttgaaattc gaaatccttt gaggggtcgt gaggaaaagg gtggtgggtt gggtgggggg ctttgttgtt acctatcact aaatgggacc +1604 ccatcctctt cagaataatg gattttgtgt ggtaaaattt ataataactt ttcatcatag cctcgagaaa aaactactac cctttatggt atatgccaac +1704 tagactgtag agacacattt gagaaactaa agttgtaaat ttatgggcca tgcattctat acttaagttt gaaaataaat tttgtttaaa aaacgacaca +1804 cattctcaat ttaaaataca ttctcaattt aaaatgacaa acattctcaa ttttcgaaag ttactttttc aattatattt tataatttat attttac Fig. 1. DNA sequence of trg-31. Putative exons are underlined and capitalized while intron areas are in lower case. The amino acid sequence is in capital letters below the coding region. The transcription start site at + 1 as determined by primer extension analysis is underlined along with the most proximal 'TATA' and 'CAT' motifs. A potential polyadenylation sequence motif at + 1381 is also underlined and the 'g' in bold print at + 1405 was the polyadenylation site previously identified from the cDNA clone (clone 7a, 6).
  • and its position at + 1 is underlined in Fig. 1. The 5' and 3' untranslated regions of the mRNA con- tain 36 and 255 nucleotides, respectively. The 'TATA' box most proximal to the transcriptional start site is at -43 and a potential 'CAAT' se- quence is at -53. The polyadenylation site at + 1405 was determined from the cDNA clone sequence and the most proximal polyadenylation motif is at + 1381. The sequence of the trg-31 gene promoter was examined for repetitive sequences or putative cis- acting promoter elements similar to those found in ABA-inducible genes [8]. A single copy of the sequence CACGTGGA, found in the ABA- and wound-inducible proteinase inhibitor II gene, is present at -290 of the trg-31 promoter. Although this sequence is very similar to the CACGTGGC sequence from the em(la) gene of wheat, no per- fect matches to reported ABA-responsive ele- ments were found in the trg-31 promoter. Perhaps this single imperfect copy of an ABA-responsive sequence element explains the slight increase in 7a mRNA level following uptake of exogenous ABA reported previously [6]. The sequence T-T- TTCTTAA is found at - 1256, - 1067 and -793. GTGTAAAAT is present at - 1014 and -899 of the coding strand and -1203 and -881 of the noncoding strand. The sequence AACCGTT is found at -1188 and + 246 of the coding strand and -1124, -897 (one mismatch with the A at the 5' end), + 300 and + 1175 of the noncoding strand. Two other repeated sequence motifs were found 3' of the gene's coding region. C(A/ T)TTTCAT(C/G)AT is located at + 1204 and + 1651 on the coding strand and + 1219 of the noncoding strand, while A-ACATTCTCAATTT is found at + 1801, + 1819 and + 1842. At this time, the involvement of these motifs in the ex- pression of trg-31 is not known. Genomic DNA copy number analysis showed that one or two copies of the gene were present in the pea genome (data not shown). DNA sequence analysis was performed with MacVector DNA analysis software (Internation- al Biotechnologies, New Haven, CT). The trans- lated coding region was used as a query sequence to search the GenBank Release 63 data bank. 931 The homology search scoring matrix was the MacVector pam250s scoring file, basing scoring of two compared sequences on similarities be- tween amino acid side chain chemical properties rather than strict amino acid identity. Four gene products possessing regions of significant amino acid similarity to TRG-31 were identified. Fig- ure 2 shows an optimal alignment of the amino acid sequences from these genes with TRG-31. The gene products are listed by decreasing amount of amino acid sequence relatedness to TRG-31 and identified as TRG, BOV, TIP, SOY and ECO representing pea TRG-31, bovine lens fiber major intrinsic protein [4], intrinsic tono- plast protein of Phaseolus vulgaris [12], soybean nodulin-26 [ 3 ] and E. coli glycerol facilitator pro- tein [ 15], respectively. Colons indicate identities with the TRG-31 sequence and boxes enclose areas where at least 3 of the 4 aligned amino acids are either identical to or conservative replace- ments of the TRG-31 amino acid. By that crite- rion, 110 of a possible 243 aligned amino acids are in conserved regions. The amino acid sequence homology between TRG-31 and E. coli glycerol facilitator protein (GlpF) is very interesting and suggests that TRG-31 is involved in small molecule transport. By analyzing the sequence alignment in Fig. 2 to compare TRG-31 and the glycerol facilitator pro- tein, 48~o of the amino acids were found to be either identical or conservative replacements (28~o identical). The GlpF is known to be re- sponsible for the channel-type transport of glyc- erol and other small molecules in E. coli [9]. Johnson etal. [12] noted sequence similarities between bovine lens fiber major intrinsic protein, intrinsic tonoplast protein of Phaseolus vulgaris (TIP), soybean nodulin-26 and E. coli glycerol fa- cilitator protein. They found a 32~o sequence identity between glycerol facilitator protein and TIP, with the greatest homology occurring in the putative trans-membrane domains of TIP. The authors also discussed that since TIP was located in the tonoplast of seed reserve tissues, it would be a candidate for metabolite transport during seed development. The amino acid sequence sim- ilarities between TRG-31 and TIP are also strik-
  • 932 BOV T IP SOY ECO TRG BOV T IP 8OY ECO B , , o BOV T Ip SOy ECO 1 M E A K E Q D V S L G A N K F P E R Q P L G I A A 25 1 M A D Y S A G T 9 E : : N V V V N V T K N T S E T I Q R S D S 33 1 MS 4 ~V 7 A : :W: : IC : : :F :S~[ I FY FG- : 30 T IP 17 PDSM SL : : :AS : I : AGEG 4~ SOY 34 VP LQK V ; :AVG: FL AGC 57 ECO S STLKGQ ~: : :LG: : L FG G 29 n : N : N 7 - M l i l T IF IP I : : : l I IV lWl : h V I lO SOY 58 Y N ECO 30 A K V A G A S r G Q W~EU-~S V I~ : G~JL I- : V I S~ , .o .0. . . . BOV $2 AV H : : : A : V : : : : : : FL 76 T IP 70 A S H V : V 95 : : : : : : A 5 SOY 81 ~.~T~: T y G..~H V F ; : : : : I F A 10S ECO 53 M : L A L : : : : : I : W 76 BOV 77 .S~i I!I li i i l I{I I!I I.~. LL5 {I I!I I~ I S Q M : L C : A : A I01 T IP 96 G R I : I Y W A S V 121 SOY 106 : R F P I A V A S L 130 I co 77 A c ~ D~RKVtK /_HP~IUS~:~: AL~_j 1.2 TRG 151 K F E G K Q R F G D S N A N BOV 102 Y V T P P A V R : N : : - T H 124 T IP 122 R V T N N M : - - P H 141 SOY 131 M : N H D Q F 158 ECO 103 D F E : T H H I V V E 129 " "°° 12S : T : L Q : L : A Y 142 V V H M F V M G M : - : 165 130 T Y P N P F Q I T A L M L 157 BOV 150 : : - - G G S A : S T 170 T IP 166 D P G A S N A L I G 189 SOY 174 - D A A S T h 194 ECO 158 I A L T V N .~_~." : : : ~ - LL_ I J L L A 184 ECO 185 I G S M G L F A P K 208 BOV 195 T R N F N : : : : : G : G S : 217 T IP 214 G W Q ~ Q : : L L 237 : : : : : : : SOY 219 H G E G I L V : : I A G ; V 242 moo 2o9 A W h A ~v I- A : I T I~ : R~D[ : P ~ ~ : v ~ :1 ~ V R 289 BOV 218 F F P R L : : V 230 T IP "~38 ~IGY~TA I E : ? P H H 251 SOY "~43 D K L S E I "~56 ECO "233 : I V G A F A Y 2~6
  • 933 ing. Using the alignment of Fig. 2 in conjunction with the putative topological model of TIP in Johnson's report, we found a sequence identity of 42~o, 43~, 40~o, 74~o and 48~o between TRG-31 and the putative transmembrane do- mains C, D, E and F and the TIP extramembrane region 2, respectively. The percentages are higher when conservative amino acid replacements are taken into consideration. Figure 2 also shows the sequence alignment with bovine lens fiber major intrinsic protein (BOV), a junctional membrane protein able to induce voltage-dependent chan- nels when added to preformed lipid films [ 19] and soybean nodulin-26 (SOY), a major protein of the peribacteroid membrane in soybean root nodules. To learn more about the trg-31 promoter spec- ificity, transcriptional fusions to a fl-glucuronidase (GUS) gene were constructed and transformed into tobacco. Nicotiana tabacum var. Havana 38 was transformed by a leaf disk Agrobacterium- mediated transformation-regeneration method [7] using the transformation plasmid vector pBI101 (Clontech, Palo Alto, CA). This plasmid contains a promoter-less GUS cassette [ 11 ] fused to the nopaline synthase polyadenylation region in the binary vector pBIN19 [1]. A 3 kb Xba I- Xba I restriction fragment was isolated from the genomic clone pCIB3130 and subcloned into the Xba I polylinker site of pBI101 in both orienta- tions with respect to the GUS coding sequences. The promoter fragment contains 18 bp at its 5' end carried over from the Xba I-Sst I Bluescript polylinker, ca. 3 kb that precedes the transcrip- tion start site and 29 of the 36 bases from the 5' untranslated region of the transcript. The 3' end of the promoter fragment is located at + 29 which is 7 bases before the putative AUG translation start codon. The GUS copy number of 25 independent KAN r transformants was determined by South- ern analysis of genomic DNA (data not shown). To establish the transcriptional pattern and time course of induction of the GUS RNA, one single- copy To individual from each transcriptional fu- sion was selected for RNA analysis. For these experiments, an entire leaf was excised from the stem, the midrib and large veins rapidly separated from the mesophyll, and the mesophyll and mid- rib wilted in a room temperature air stream for 0, 0.1, 0.5 and 1 h followed by immediate immersion in liquid nitrogen. A 4 h time-point sample was obtained by following the 1 h air stream wilting treatment by a 3 h incubation period in still air at room temperature. Roots were quickly excised from a mature flowering plant, soil rinsed off in a stream of water, tissue blotted dry onto filter paper and wilted as described above. Total RNA was isolated [ 18], analyzed by formaldehyde gel elec- trophoresis and Northern blots probed with radiolabelled GUS DNA [ 13]. To verify the in- tegrity of the RNA samples and insure that equiva- lent amounts of RNA were applied to each lane shown in Fig. 3, a replica RNA gel was run in TBE buffer and stained with ethidium bromide (data not shown). Figure 3 indicates that tran- scription of the 3 kb trg-31 promoter fragment- GU S coding region fusion is significantly induced by the 0.5 hour treatment in leaf and midrib tis- sue, reaching a peak at the 1 h point but remain- ing elevated above non-wilted levels for 4 h. Al- though in Fig. 3 root expression of GUS RNA is not evident, on a longer autoradiogram exposure GUS RNA is detected in both wilted and un- wilted root tissue (data not shown). No GUS transcript was detected from the 3126 plant RNA which was transformed with the trg-31 promoter Fig. 2. Amino acid sequence alignment of TRG-31 and related gene products. A search of translated sequences in the GenBank DNA sequence database and recent literature reports identified several gene products having significant amino acid sequence similarity to TRG-31. BOV, TIP, SOY and ECO represent the gene products from bovine lens fiber major intrinsic protein, in- trinsic tonoplast protein of Phaseolus vulgaris, soybean nodulin-26 and E. coli glycerol facilitator protein, respectively. Amino acid identifies with the TRG-31 residue are indicated by a colon while a dash indicates an introduced gap to optimize an alignment. When the DNA analysis software caused amino acid residues to be removed from a sequence to improve its alignment with TRG-31, this is indicated by underlining the amino acids which precede and follow the adjusted region. Boxed areas indicate regions where at least 3 of the 4 aligned amino acids are identical to, or conservative replacements of, the corresponding amino acid of TRG- 31.
  • 934 Fig. 3. RNA blot analysis of GUS expression directed by the trg-31 promoter in transgenic tobacco. Expression of the trg-31-GUS transcriptional fusions in wilted and control transgenic tobacco plants was analyzed by probing total RNA Northern blots with radiolabelled GUS probe. Tissue was excised from greenhouse plants and wilted for 0, 0.1, 0.5, 1 or 4 h prior to RNA isolation. L, MR, or R: RNA from mature deveined leaf, leaf midrib or root tissue, respectively. 3125: individual transformed with pCIB3125 containing the trg-31 promoter in the 5' to 3' orientation with respect to the GUS coding region. 3126: Individual transformed with pCIB3126 containing the trg-31 promoter in the 3' to 5' orientation. Approximately 2 #g of total RNA was loaded per lane. A second gel replicating these loading conditions was run in TBE and stained with ethidium bromide. This verified the RNA of each sample was intact (indicated by discrete ribosomal RNA bands) and that equivalent amounts of total RNA was loaded per lane (data not shown). in the reverse orientation with respect to the GUS coding region. Given its relatedness to putative membrane transport proteins (Fig. 2) and promoter activity in the plant stem, it is possible that the trg-31 gene product is involved in the transport of ions or sugars during osmoregulation, as this is one mechanism by which plants attempt to restore turgor [10]. The amino acid sequence similarity between TRG-31 and the E. coli glycerol facilita- tor protein is particularly interesting since Fisch- barg etal. [2] provided evidence that glucose transporters from mammalian cells serve as water channels. Xenopus oocytes expressing injected mRNA for rat glucose transporters had signifi- cantly increased osmotic water permeability com- pared to noninjected oocytes, while cells tested in the presence of glucose transport inhibitors had decreased water permeability. The root expres- sion pattern of trg-31 in Pisum sativurn showed trg-31 poly(A) RNA present in both wilted and unwilted roots [6]. This would be consistent with a role facilitating water transport during normal plant growth. The induction of TRG-31 in stem tissue during water stress would assist rapid os- moregulation in sensitive tissues. Resurrection plants (Craterostigma platagineum) are capable of tolerating severe water loss and yet recover upon rewatering. Osmoregulation plays an important role in their survival during periods of water shortage. During slow desiccation of these plants, polysaccharides are hybridized and soluble sug- ars accumulate [ 16]. It is possible that a similar mechanism occurs in other plants involving gene products such as TRG-31. Acknowledgements We thank George Lankford and Susan Jayne for greenhouse assistance with transformed tobacco plants and Danielle Brost and Hope Thompson- Taylor for DNA oligomer synthesis. John Mullet provided valuable discussions concerning gene expression and possible functions of trg-31. References 1. Bevan M: Binary Agrobacterium vectors for plant trans- formation. Nucl Acids Res 12:8711-8721 (1984). 2. Fischbarg J, Kuang K, Vera JC, Arant S, Silverstein SC, Loike J, Rosen OM: Glucose transporters serve as water channels. Proc Natl Acad Sci USA 87:3244-3247 (1990). 3. Fortin MG, Morrison NA, Verma DPS: Nodulin-26, a peribacteroid membrane nodulin is expressed indepen- dently of the development of the peribacteroid compart- ment. Nucl Acids Res 15:813-824 (1987). 4. Gorin MB, Yancey SB, Cline J, Revel JP, Horwitz J: The major intrinsic protein (MIP) of the bovine lens fiber membrane: Characterization and structure based on cDNA cloning. Cell 39:49-59 (1984). 5. Guerrero FD, Mullet JE: Reduction of turgor induces rapid changes in leaf translatable RNA. Plant Physiol 88: 401-408 (1988). 6. Guerrero FD, Jones JT, Mullet JE: Turgor-responsive
  • gene transcription and RNA levels increase rapidly when pea shoots are wilted. Sequence and expression of three inducible genes. Plant Mol Biol 15:11-26 (1990). 7. Guerrero FD, Crossland L, Smutzer GS, Hamilton DA, Mascarenhas JP: Promoter sequences from a maize pollen-specific gene direct tissue-specific transcription in tobacco. Mol Gen Genet 224:161-168 (1990). 8. Guiltinan MJ, Marcotte Jr WR, Quatrano RS: A plant leucine zipper protein that recognizes an abscisic acid response element. Science 250:267-271 (1990). 9. Heller KB, Lin ECC, Wilson TH: Substrate specificity and transport properties of the glycerol facilitator of Es- eherichia coli. J Bact 144:274-278 (1980). 10. Hsiao TC: Plant responses to water stress. Annu Rev Plant Physiol 24:519-570 (1973). 11. Jefferson RA, Burgess SM, Hirsh D: /~-glucuronidase from Escherichia coli as a gene-fusion marker. Proc Natl Acad Sci USA 83:8447-8451 (1986). 12. Johnson KD, Hofte H, Chrispeels MJ: An intrinsic tono- plast protein of protein storage vacuoles in seeds is struc- turally related to a bacterial solute transporter (GlpF). Plant Cell 2:525-532 (1990). 13. Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: 935 A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1982). 14. Metraux JP, Burkhart W, Moyer M, Dincher S, Midd- lesteadt W, Williams S, Payne G, Carnes M, Ryals J: Isolation of a complementary DNA encoding a chitinase with structural homology to a bifunctional lysozyme/ chitinase. Proc Natl Acad Sci USA 86:896-900 (1989). 15. Muramatsu S, Mizuno T: Nucleotide sequence of the region encompassing the glpKF operon and its upstream region containing a bent DNA sequence of Escherichia coli. Nucl Acids Res 17:4378 (1989). 16. Schwab KB, Gaff DF: Sugar and ion contents in leaf tissues of several drought tolerant plants under water stress. J PLant Physiol 125:257-265 (1986). 17. Shure M, Wessler S, FederoffN: Molecular identification and isolation of the waxy locus in maize. Cell 35:225-233 (1983). 18. Verwoerd TC, Dekker BMM, Hoekema A: A small-scale procedure for the rapid isolation of plant RNAs. Nucl Acids Res 17:2362 (1989). 19. Zampighi GA, Hall JE, Kreman M: Purified lens junc- tional protein forms channels in planar lipid films. Proc Natl Acad Sci USA 82:8468-8472 (1985).