Transfer of soybean seedlings to low-water-potential vermiculite (~O w = - 0.3 MPa) results in a revers- ible decrease in hypocotyl growth and modulation of ...
Plant Molecular Biology 17: 591-608, 1991. © 1991 Kluwer Academic Publishers. Printed in Belgium.
591
Water deficit modulates gene expression in growing zones of soybean seedlings. Analysis of differentially expressed cDNAs, a new/~-tubulin gene, and expression of genes encoding cell wall proteins Robert A. Creelman and John E. Mullet*
Department of Biochemistry and Biophysics, Texas A &M University, College Station, TX 77843, USA (* author for correspondence) Received 3 December 1990; accepted in revised form 9 May 1991
Key words: hormones, gene expression, soybean, water deficit, tubulin, cell wall proteins
Abstract
Transfer of soybean seedlings to low-water-potential vermiculite (~Ow = - 0.3 MPa) results in a reversible decrease in hypocotyl growth and modulation of several polysomal mRNAs (Plant Physiol 92: 205-214). We report here the isolation of two cDNA clones (pGE16 and pGE95) which correspond to genes whose mRNA levels are increased, and one eDNA clone (pGE23) which corresponds to a gene whose mRNA level is decreased in the hypocotyl zone of cell elongation by water deficit. In well-watered seedlings mRNAs hybridizing to pGE 16 and pGE95 are most abundant in mature regions of the seedling, but in water-deficient seedlings mRNA levels are reduced in mature regions and enhanced in elongating regions. RNA corresponding to soybean proline-rich protein 1 (sbPRP1) shows a similar tissue distribution and response to water deficit. In contrast, in well-watered seedlings, the gene corresponding to pGE23 was highly expressed in the hypocotyl and root growing zones. Transfer of seedlings to low-water-potential vermiculite caused a rapid decrease in mRNA hybridizing to pGE23. Sequence analysis revealed that pGE23 has high homology with fl-tubulin. Water deficit also reduced the level of mRNA hybridizing to JCW1, an auxin-modulated gene, although with different kinetics. Furthermore, mRNA encoding actin, glycine-rich proteins (GRPs), and hydroxyproline-rich glycoproteins (HRGPs) were down-regulated in the hypocotyl zone of elongation of seedlings exposed to water deficit. No effect of water deficit was observed on the expression of chalcone synthase. Decreased expression of fltubulin, actin, JCW1, H R G P and GRP and increased expression of sbPRP1, pGE95 and pGE16 in the hypocotyl zone of cell elongation could participate in the reversible growth inhibition observed in water-deficient soybean seedlings.
The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X60216.
592 Introduction
Plant growth and development depend on the acquisition of water from soil. As a consequence, exposure of plants to water deficit causes a diverse set of metabolic and developmental changes [30]. Responses to mild water deficit include stomatal closure (reducing transpiration, water loss and photosynthesis), osmotic adjustment and growth inhibition [63]. More severe water deficit may result in the accumulation of proline [30], betaine [30] and dehydrins [11]. These compounds may protect cells from damage at very low water potentials, such as that which occurs during seed desiccation [21]. While much is known about plant adaptations to dry environments at the developmental, morphological and physiological levels, less is known about changes in gene expression which may contribute to these responses [30]. In previous studies of plant responses to water deficit, we used etiolated soybean seedlings grown at 100~o RH in low-water-potential vermiculite (~b= - 0.3 MPa) [4, 5, 18, 42, 46, 49, 50]. Use of etiolated material avoids water deficit-induced changes in photosynthesis and transpiration which may complicate analysis of growth inhibition. In this system, when well-watered seedlings are transferred to low-water-potential vermiculite, hypocotyl growth rate rapidly declines [ 18, 43, 46, 49, 50], although root growth continues [ 18]. Exposure of seedlings to low-water-potential vermiculite ( ~ = - 0 . 3 MPa) decreases the rate of hypocotyl cell elongation, but if plants are rewatered within 48 h, no irreversible change in cell size occurs [46]. Transfer of seedlings to lowwater-potential vermiculite induces a transient decrease in cell turgor in the inner cortical cells of the elongating region of the hypocotyl and causes disruption of water potential gradients [49, 50]. By 24 h after transfer turgor pressure is reestablished, however, shoot growth remains inhibited. Growth inhibition is reversible, and growth rate begins to recover 48 to 60 h aftertransfer, reaching about 60~o of the control level. Since turgor has been restored by 24 h, these data suggest that biochemical changes which alter wall extensibil-
ity and hydraulic conductivity [49] may contribute to growth inhibition. Rewatering seedlings grown in water-deficient vermiculite results in growth rate recovery [4, 5, 43, 48, 49]. Growth inhibition in hypocotyls is due in part to abscisic acid (ABA), which increases in waterdeficient soybean seedlings [4, 18, 55]. Gibberellic acids, such as GA1, decrease in seedlings transferred to low-water-potential vermiculite [ 5 ], suggesting that the ABA/GA ratio may play a role in modulating growth in soybean seedlings under these conditions. Based on measurements of indoleacetic acid (IAA) oxidase activity, it has been suggested that auxin levels decrease during water deficit [ 19], and it has been shown that exogenous auxin alters hydraulic conductivity in soybean seedlings [6]. Hence, plant growth substances play a role in modulating some of the processes which control growth rates in plants exposed to water-limiting conditions. In addition to changes in the levels of ABA and GAs [4, 5, 18], soybean seedlings grown in lowwater-potential vermiculite have decreased polysome contents [ 43 ] and altered polysomal m R N A populations [ 18, 43]. These plants also accumulate VSP~ and VSP/~, two antigenically related vegetative storage proteins [42]. Changes in polysome m R N A populations indicate that gene expression is modulated by water deficit [ 18,43]. In addition, water deficit causes a shift in the m R N A population of the cells in the hypocotyl zone of elongation towards that found in the mature cells of the hypocotyl [43]. Most of the changes in m R N A populations caused by transfer of plants to low-water-potential vermiculite were not induced by exogenous ABA sufficient to cause significant inhibition of growth and internal ABA levels similar to that found in water-deficient seedlings [ 18]. These genes probably differ from those modulated by ABA during seed desiccation [13, 22, 25, 22, 31, 34, 39, 41, 47, 53, 62], plant dehydration [8, 12, 18, 27, 32, 47], or low temperature [40, 52]. In previous studies, hypocotyl growth rate and polysome status of the elongating region reached a minimum 24 h after transfer to low-waterpotential vermiculite and began to increase be-
593 tween 24 and 72 h after transplanting [43, 48, 49]. Changes in polysomal translation products behaved similarly [43]. Because these mRNA changes occurred in elongating tissue, there could be corresponding alterations in protein composition which affect growth (maintenance of growth inhibition or recovery of growth). In order to begin to address these possibilities, we constructed a cDNA library from polysomal mRNA isolated from the elongating region of soybean seedlings grown in low-water-potential vermiculite for 24 h. Our goal was to obtain cDNA clones which hybridized to mRNAs whose levels were induced or repressed in the hypocotyl zone of elongation by growth in low-water-potential vermiculite. We report here the isolation of three cDNA clones corresponding to genes whose expression is modulated (two induced, one repressed) in the elongating region of soybean seedlings transplanted to low-water-potential vermiculite. The distribution and changes in abundance of mRNAs hybridizing to these genes was determined in soybean seedlings grown in well-watered and low-water-potential vermiculite. Furthermore, the ability of plant growth substances to modulate these genes was determined. This study was broadened to include analysis of mRNAs corresponding to JCWl, an auxin-responsive gene [1,43], actin [33, 57, 59], three cell wall proteins (glycine-rich proteins (GRPs, [10, 17, 26, 36, 38]), hydroxyproline-rich glycoproteins (extensins or HRGPs [10, 12, 23, 58]), and proline-rich proteins (PRPs [ 10, 12, 20, 35, 60])), and chaicone synthase [51, 54]. In general, genes highly expressed in the hypoctyl zone of elongation of well-watered seedlings (JCWl, GRPs, actin, tubulin) were down-regulated in seedlings exposed to water deficit. In contrast, several genes which showed high expression in non-growing regions of well-watered seedlings exhibited increased expression in the hypocotyl zone of elongation of seedlings exposed to low-water-potential vermiculite.
Materials and methods Plant material and treatments
Soybean (Glycine max [L.] Merr. cv. Williams) seeds were grown in the dark as previously described [4, 5, 18, 43, 46, 48, 49]. After 48 h, seedlings were transplanted into vermiculite saturated with 10 -4 M CaC12(Ww = - 0.01 MPa) or vermiculite containing 1/8 as much CaC12 solution (Ww = -0.3 MPa). In one experiment, plant growth substances were added to the CaC12 solution to give final concentrations of 2 0 # M methyl jasmonate, 10 #M or 1 mM ABA, 10 #M GA3, or 0.5 mg/1 ethrel. For all growth substances except GA3, the vermiculite was saturated with the appropriate solution. For GA3, 1/8 as much solution was added to vermiculite compared with the well-watered treatments. To prevent volatile growth substances (methyl jasmonate and ethylene) from interfering with other treatments, the seedlings were placed in separate covered styrofoam containers for the duration of the experiment. In another experiment, 2-day old plants were transferred from 28 °C to 20 °C for 24 h. Seedlings were harvested at various times after transplanting and dissected into the following sections: hook and elongating region correspond to the first 5 mm and next 15 mm below the cotyledons with the remaining portion of the hypocotyl defined as the mature hypocotyl. The root tip was defined as the terminal 15 mm of the root. This section contains a number of structures, including the elongating region of the root. The remaining portion of the root was defined as the mature root. To determine if any clones were modulated by auxin, the elongating region from 2-day old seedlings was excised and incubated in 10-aM CaC12 for 4 h with buffer changes at 1 and 2 h. At 4 h, sections were incubated for 90 min with 20 #M naphtaleneocetic acid (NAA). In all experiments there were 15 to 20 sections per treatment. Tissue was frozen immediately in liquid N2 and stored at -80 °C until extraction.
594
RNA extraction For cDNA cloning, polysomal RNA was isolated from the elongating regions ofhypocotyls of seedlings exposed to low-water-potential vermiculite for 24 h as previously described [43]. Poly(A) + RNA was isolated from polysomal RNA using two rounds of adsorption to oligo(dT)-cellulose (Pharmacia). Total nucleic acids were prepared by grinding tissue in liquid N2 and homogenizing in 5 volumes of 0.2 M Tris-HCl (pH 8.5), 0.3 M NaC1, 20 mM EDTA, and 1~ SDS. The homogenate was extracted once with phenol/ chloroform/isoamyl alcohol (50:49:1) and nucleic acids were precipitated with 0.8 M LiC1 and 2.5 volumes ethanol. The pellet was dissolved in sterile DEPC-treated H20 and re-extracted with phenol/chloroform/isoamyl alcohol as above. Total nucleic acids were precipitated with 0.3 M NaOAC and 2.5 volumes ethanol, pelleted, dried under vacuum, and dissolved in sterile DEPCtreated H20.
Construction and differential screening of cDNA library in 2gtlO Synthesis of double-stranded cDNA was performed as described [27] except that secondstrand synthesis was performed according to Ausubel et al. [2]. Double-stranded cDNAs were methylated with Eco RI methylase (BRL) and ligated to Eco RI linkers (BRL). After digestion with a large excess of Eco RI, the cDNAs were size-fractionated on a Biorad Bio-gel A-50m column and those > 500 bp in length were cloned into the Eco RI site of ;tgtl0. Recombinant lambda DNA was packaged in vitro using a commercial packaging extract (Gigapack Plus: Stratagene) according to the manufacturer's instructions and plated on Escherichia coli C600 hfl. The cDNA library was differentially screened with labeled first-strand cDNA synthesized from poly(A) + RNA from the elongating region of well-watered and water-deficient soybean seedlings as previously described [27]. About 30000 phage were screened. Inserts from phage which
passed the primary and secondary screens were subcloned into the Eco RI site of pGEM7zf( + ) (Promega). Of seven clones which showed differential expression with regard to water deficit, three were selected for further characterization (pGE16, pGE23, and pGE95).
Isolation of cell wall protein, chalcone synthase, and JCW1 cDNAs The polymerase chain reaction (PCR) was used for the isolation of GRP-rich (pCRGRP) and JCWl (pCRJWl) cDNAs. Primers (ca. 1/~M) for GRP-rich (5'-TTAGGGGGTGGAGGAGGTGCAGGAGGAGGCTTTGGT-3 ' (forward), 5'-ACCAACACCAACTCCTATACC-3' (reverse)) and JCWl (5'-AGGAAGAGGGGTTTCTCTGA-3' (forward), 5'-CCATGGGACATCACCGACAA-3' (reverse)) were mixed with 50 ng of cDNA from the elongating region of water-deficient seedlings. PCR mixture conditions were as described from the manufacturer (Perkin-Elmer). Cycle conditions were: 1 min at 92 °C, 1.5 min at 50 °C, and 2 min at 72 °C for 40 cycles. There was an additional 15 min at 72 °C followed by cooling to 4 °C. The PCR product was cloned into pCR1000 according to the manufacturer's instructions (Invitrogen). A soybean HRGP cDNA clone (pGEEXT) was obtained by radiolabeling GRP-rich PCR product and probing the 2g10 cDNA library from the elongating region of the soybean hypocotyl as described above. The insert from the 2gtl0 clone was cloned into pGEM7zf(+) as described above. A soybean chalcone synthase cDNA (pGECHS) was isolated by hybridizing 32p. labeled pCHS 1 (ref) insert with the 2gtl0 waterdeficient cDNA library and cloned into pGEM7zf( + ) as described above for HRGP.
Agarose gel electrophores&, blotting and hybridization of RNA Total nucleic acid (10 ktg/lane) was electrophoresed in 1~o or 1.5~o (cell wall protein mRNAs)
595 agarose gels containing 2% formaldehyde and transferred onto GeneScreen (Dupont) with 0.25 M sodium phosphate (pH 6.5) overnight. Prehybridization and hybridization were performed at 42 ° C (65 ° C for RNA probes) in 50 ~o formamide, 5 × SSC (1 x SSC is 0.15 M NaCI, 15 mM sodium citrate), 1 × PE (0.2% BSA, 0.2% PVP-40, 0.2% Ficoll, 5 m M Tris-HCl pH7.5, 0.1% sodium pyrophosphate) and 150 #g//fl salmon sperm D N A (hybridization solution also contained 500000 cpm/ml of probe). Blots were washed twice in 2 × SSC, 0.1% SDS and twice in 0.1 × SSC, 0.1% SDS at 55 °C (65 °C for RNA probe) for 15min. For p33 probe, prehybridization, hybridization and washing were performed as described. Dried filters were exposed to Kodak XAR film at -80 °C using an intensifying screen. The m R N A levels were quantified using either a Computing Densitometer model 300A (Molecular Dynamics) or a Betascope 603 blot analyzer (Betagen). Relative responses were similar between machines. RNA standards (0.16-1.77 kb, 0.24-9.5 kb; BRL)were visualized on blots using methylene blue staining [45].
Labeling methods Preparation of labeled cDNA probes. Poly(A) + RNA (3.5 ~tg) was used to generate labeled firststrand c D N A using MMLV reverse transcriptase according to Guerrero and Mullet [27] except that the final dCTP concentration was 2 ~tM and 100 laCi [~-32p]dCTP (3000 Ci/mmol) was used. Unincorporated nucleotides were removed using a spun column containing Sephadex G-25 (Pharmacia).
Labeling of cDNA inserts. For use in northern blots, c D N A clones were digested with Eco RI and separated in 0.8 ~o low-melting-point agarose (BRL). Inserts were cut out, melted at 65 ° C, and an equal amount of TE added. To generate probe, agarose containing the appropriate insert was melted at 65 ° C. From the melted agarose, 20 Ixl was removed, boiled for 5 min, and labeled ac-
cording to instructions in a commercial random primer labeling kit (BRL). Unincorporated nucleotides were removed as described above.
Labeling of JCW1 probe. An oligonucleotide homologous to JCW1 ([61; corresponding to gene aux28 [1]) was synthesized using appropriate primers, c D N A from well-watered elongating regions, and the polymerase chain reaction (PerkinElmer). The oligonucleotide was purified from unincorporated nucleotides through a spun column and labeled with a commercial random primer kit as described above.
Labeling of pCR GRP probe. To generate labeled antisense RNA probe, pCRGRP was cut with
Eco RI and used with T7 RNA polymerase according to kit instructions (Stratagene). In addition to 5 ~tCi [a-32p]UTP (800 Ci/mmol), the reaction mixture contained 10 ~tM UTP.
Sequencing of cDNA clones The D N A sequences were determined by the dideoxy chain termination method using the Sequenase version 2 sequencing kit (USB) and m13 reverse and -20 primers. For pGE23, a nested series of deletions from both directions was constructed using ExoI/S 1 nuclease with a commercial kit (Promega). If needed, primers were synthesized to cover gaps. D N A sequences were analyzed using Intelligenetics computer programs (Intelligenetics, Mountain View, CA).
Results
Isolation of cDNAs corresponding to genes modulated by water deficit A complementary D N A library was constructed from m R N A isolated from the elongating region of soybean seedlings exposed to low water potential vermiculite (q~w = - 0.3 MPa) for 24 h. By differential screening, we isolated and have characterized three c D N A clones whose mRNAs are
596 modulated by water deficit (Fig. 1). Two clones were obtained which correspond to mRNAs which are induced (pGE16 and pGE95) and one clone to an m R N A which is repressed (pGE23) in the zone of elongation in response to water deficit. Under high-stringency conditions, 2 mRNAs (0.53, 1.03 kb) were detected when RNA blots were probed with pGE16. In some blots, an additional m R N A band could be detected (1.5 kb; Fig. 2). RNAs of 1.25 and 1.7 kb hybridized to pGE95 and pGE16, respectively.
Water deficit modulation of mRNA levels
Fig. 1. Time course showing changes in mRNA isolated from the elongating region of soybean hypocotyls which hybridizes to pGE95, pGE16, pGE23, or JCW1. Seedlings were rehydrated 24 h after transfer to low water potential vermiculite by saturating the vermiculite with 10-4M CaC12. Sizes of mRNAs hybridizing to pGE16 are 0.53, 1.03, and 1.5 kb, to pGE23 1.7 kb, and to pGE95 1.25 kb. In this figure (and Figs. 2 and 3), in the histogram for pGE16 the open and cross-hatched bars correspond to mRNA sizes 0.53 kb and 1 kb, respectively.
Levels of mRNAs hybridizing to pGE16 and pGE95 in the hypocotyl zone of elongation increased slowly with time in well-watered seedlings (Fig. 1). For the gene corresponding to pGE23, m R N A levels reached a maximum at 24 h after transfer to well-watered vermiculite which correlates with maximum hypocotyl growth rate. When seedlings were transferred to low-waterpotential vermicufite, the level of m R N A hybridizing to pGE16 and pGE95 increased. RNA hybridizing to pGE95 increased without reaching any apparent plateau. In contrast, m R N A hybridizing to pGE16 began to decline between 24 to 48 h after transfer. When water-deficient seedlings were rewatered, m R N A levels corresponding to either pGE16 or pGE95 declined such that by 24 h after rewatering, levels were similar to those observed in well-watered seedlings of similar age. RNA hybridizing to pGE23 rapidly declined and remained low up to 48 h after transfer to low-water-potential vermiculite, reaching a minimum 24h after transfer. Rewatering of water-deficient seedlings induced pGE23 m R N A levels reaching a maximum by 6 h after rewatering, then declining by 24 h. Changes in m R N A hybridizing to JCW1 were similar to pGE23, except that JCW1 m R N A levels declined gradually in well-watered seedlings and water deficit accelerated the rate of decline.
597
Expression of water deficit-modulated genes in various tissues of intact seedlings RNA which hybridized to pGE95, pGE16, pGE23, and JCW1 in tissues of 2-day old seedlings transferred to well-watered and low-waterpotential vermiculite for 24 h was quantified (Fig. 2). RNA hybridizing to pGE95 was detected in mature portions of the hypocotyl and root and root tip in well-watered seedlings. In water-limited seedlings, m R N A levels were increased in the elongating region and reduced in the mature portions of the seedling. In well-watered seedlings, a 1.03 kb transcript hybridizing to pGE 16 was most abundant in cotyledons, while the 0.53 kb transcript was detected in the hook and elongating regions of the hypocotyl. In the mature sections of the hypocotyl and root, both the 0.53 and 1.03 kb transcript could be detected. In waterdeficient seedlings, the 0.53 and 1.03 kb transcripts were induced in elongating tissue, while in the mature regions the level of these RNAs was reduced. No other changes in transcript level were detected in other portions of the seedling. RNA which hybridized to pGE23 was most abundant in sections of well-watered seedlings containing cells undergoing division and elongation. In seedlings grown in low-water-potential vermiculite for 24 h, m R N A levels were strongly repressed in the hypocotyl growing zones and to a smaller extent in root tips (Fig. 2). RNA hybridizing to JCW1 declined in both growing zones to about the same extent.
Effects of plant growth substances on expression of water deficit-modulated genes Fig. 2. Water deficit-modulated mRNAs detected in various sections of 3-day-old soybean seedlings after growing in either well-watered vermiculite or water-deficient vermiculite for 24 h using either pGE95, pGE16, pGE23, or JCW1 as probe. C, cotyledon; H, hook; E, elongating; M, mature (stem or root); T, root tip.
Changes in the abundance of mRNAs hybridizing to c D N A clones pGE95, pGE16, pGE23, and JCWl were determined in response to various hormone treatments (Fig. 3). The influence of auxin was determined by excising the elongating region of the hypocotyl and incubating this tissue in buffer for 4 h to allow endogenous auxin levels to decline. This treatment was followed by application of 20/aM NAA for 1.5 h [61]. Inter-
598 estingly, excision caused a dramatic reduction in mRNA levels for pGE95, pGE16, pGE23 and JCW1. However, only JCW1 (and to a lesser extent pGE23) appeared to be modulated by a 1.5 h NAA treatment. Although we have not measured endogenous methyl jasmonate levels in soybean seedlings, this growth substance may increase in water-limited soybean seedlings since it has been demonstrated that vspA/B mRNA levels increase in response to water deficit and methyl
jasmonate treatment. Growing seedlings in the presence of 20/~M methyl jasmonate caused no change in growth, and increased mRNA levels hybridizing to pGE16. Since ABA [4, 18] levels increase with water deficit in soybean seedlings, we grew 2-day old well-watered seedlings in the presence of 10/IM ABA for 24 h [18]. This treatment caused a 34~o reduction in growth, but did not appear to affect mRNA levels for JCW1 or pGE95. For pGE16 and pGE23, mRNA levels were slightly increased or decreased, respectively. Seedlings transplanted into 1 mM ABA exhibited greater than 50~o growth rate and had a corresponding reduction in the level of mRNA hybridizing to pGE23 (data not shown). Ethylene levels increase when soybean seedlings are transferred into low-water-potential vermiculite (R.A. Creelman and J.E. Mullet, unpublished). Therefore, we grew 2-day old well-watered seedlings in the presence of 0.5 mg/1 ethrel for 24 h. This treatment caused a dramatic decrease in growth (86~o) by causing a shift from cell elongation growth to isodiametric cell enlargement (the zone of cell enlargement increased in size radially). This treatment caused a reduction in mRNA levels hybridizing to pGE16, pGE95, pGE23, and JCW1. Since GA levels decrease with water deficit [4], we enriched low-waterpotential vermiculite with G A 3. This treatment caused a slight increase in the growth of treated seedlings compared with seedlings grown in lowwater-potential vermiculite (42~o growth inhibiFig. 3. Effect of plant growth substances on expression of water deficit-modulated mRNAs hybridizing to pGE95, p G E 16, pGE23, or JCW 1. The elongating region of 2-day old seedlings was excised and incubated in buffer of 20 ~tM NAA as described in Materials and methods. Other seedlings were transplanted into well-watered (C), water-deficient (S), wellwatered plugs 20 p M methyl jasmonate (J), well-watered plus 10 ~tM ABA (A), well-watered plus 0.5 mg/1 ethrel (E), or water-deficient plus 10 ~tM GA3,vermiculite (G) and the elongating region of the hypocotyl removed after 24 h. The initial length of the hypocotyl at 2 days was 36.4 + 0.5 mm and the treatments affected growth in the following manner: + 24 h well-watered, 85.8 + 6.9 mm; + 24 h water-deficient, 50.7 + 3.9 mm; + 20 ~tM methyl jasmonate, 84.7 + 4.6 mm; + 10/~M ABA, 69.1 + 8.0 mm; + 5 mg/1 ethre144.1 + 5.7 mm; + 10 p M GA, 57.0 _+3.2 mm.
599 tion versus 30~o inhibition). For pGE16 and pGE95, GA 3 appeared to partially counteract the effects of water deficit. No effect was seen on mRNA hybridizing to pGE23 and JCW1.
cDNA sequence analysis of pGE23 The cDNA insert in pGE23 was close to the size expected for a full-length copy of the mRNA. To obtain further information concerning the gene corresponding to pGE23, DNA sequence analysis was carried out. A nested series of deletions was obtained and completely sequenced. The deduced amino acid sequence (Fig. 4) contained 411 codons, assuming the first ATG which yielded an open reading frame was the_start of translation initiation. The deduced amino acid sequence was compared to sequences compiled in the Intelligenetics Protein Identification Resource. The 411 amino acids of the deduced amino acid sequence had 75~o and 73~o overall residue identity with soybean fl-2 and fl-1 tubulin [28], respectively.
Isolation and sequencing cDNAs encoding cell wall proteins, chalcone synthase and JCW1 The results described above show that some genes (fl-tubulin) which are highly expressed in wellwatered seedlings are down-regulated in waterdeficient seedlings. Others which show high expression in mature regions are induced by water deficit in growing regions. To extend these studies, we obtained several known genes (cell wall proteins, chalcone synthase, actin) to determine the expression pattern of the corresponding genes in well-watered and water-deficient seedlings. Use of the PCR was successful in isolating cDNAs coding for H R G P (pGEEXT), GRP (pCRGRP), and JCW1 (pCRJW1; Fig. 5). Partial sequence analysis ofpGEEXT revealed a high tyrosine composition, with a repeat (SPxY3K, x = 4,6) similar to that described for HRGPs isolated from rape (extB, SPaY2 K [23 ]) and tomato (pTOM17-1, SP4Y3K [58]). Complete sequence analysis of the insert in pCRGRP revealed the
typical glycine repeat (GX)n(X --- G,A,V,S,I,K,F) similar to that described for a variety of plants [ 10,17,26,36,38]. Another larger repeat (consisting of (GGIGGIGK)2) was also detected. Comparison of partial sequences from pGECHS and pCRJW 1 showed strong similarity with published sequences (pLF15 [51]; aux28 [1, 61].
Expression of cell wall protein, actin and chalcone synthase genes in various tissues of intact seedlings RNA which hybridized to pGEEXT, pCRGRP, p33, pSac3, and pGECHS in tissues of 2-day old seedlings transferred to well-watered and lowwater-potential vermiculite for 24 h was quantified (Figs. 6 and 7). Three RNA transcripts hybridizing to pGEEXT (5.8, 4.0 (major band) and 2.6 kb; in other northerns (data not shown), there were additional minor bands at 7.8 and 1.3 kb) were detected primarily in root tips and mature root [23] although slight hybridization was also detected in the elongating and mature regions of the hypocotyl in well-watered seedlings [35]. In water-limited seedlings, mRNA levels decreased in the elongating and mature hypocotyl regions and remained high in roots. In well-watered seedlings, several transcripts (1.2, 1.4, 1.7, 2.2 kb) which hybridized to pCRGRP were detected. The size distribution was similar to that described for GRPs in petunia [17] and bean [36]. The strongest hybridization signal occurred with the 2.1 kb transcript. Expression of the 2.1 kb transcript was highest in the hook and elongating region of the hypocotyls in well-watered seedlings, and declined dramatically during water deficit. Similarly, in bean, expression of the GRP1.8 gene was highest in young hypocotyl compared with old hypocotyl (no tissue distribution given [36]). Of the three transcripts which hybridized to p33, the strongest signal corresponded to the tissue distribution previously described for sbPRP1 (1.2 kb) [20, 35, 60]. Transcripts corresponding to sbPRP2 (1.05 kb), and sbPRP3 (0.6 kb) [20, 35, 60] were also detected (data not shown). Strong hybridization to p33 occurred in root tips corresponding to the transcript for sbPRP1. In
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Soybean ~ - 2 Soybean B-1
N N
pGE23 pGE23
ACA CCG AAA GCG CCG ACG AGC TCA TCG ACC TCC GTC CTC GAC GTC GTA CGC GCA AAG AAG CCG AGA -475 T P K A P T S S S T S V L D V V R A K K P R -129
Soybean B-2 Soybean #-1
Y Y
pGE23 pGE23
ATT GCG ACC TGC TTG CAA GGG TTT CAA GTG TGC CAT TCG CTT TGG TGG AGG AAC GGG GGT TCC GGC -541 ! A T C L Q G F Q V C H S L W W R N G G S G -151
Soybean ~ - 2 Soybean ~-1
N N
pGE23 pGE23
ATG GGG CCG CTT CTG ATC TCG AAG ATT CGG GAG GAG TAT CCG GAT CGG ATG ATG TTG ACT TTT TCC -607 M G P L L ] S K ! R E E Y P D R M M L T F S -173
Soybean ~ - 2 Soybean #-1
M M
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GTG TTT CCT TCT CCC AAG GTT TCT GAT ACC GTT GTG GAG CCT TAC AAT GCT ACT CTT TCT GTT CAC -673 V F P S P K V S D T V V E P Y N A T L S V H -195 *
*
Soybean ~ - 2 Soybean B-1
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pGE23 [oGE23
-739 CAG CTT GTT GAG AAT GCT GAT GAG TGT ATG GTT TTG GAC AAT GAG GCT CTC TAT GAC ATC TGT TTC Q L V E N A D E C M V L D N E A L Y D ! C F -217
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601 pGE23 pGE23 Soybean ~-2 Soybean p-1
AGG ACT CTC AAG CTC GCT ACG CCC ACG TTT GGT GAC CTT AAC CAC CTG ATA TCT GCC ACC ATG AGT
R
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pGE23 pGE23
GGA GTT ACT TGT TGT CTA CGT TTC CCT GGG CAA CTG AAC TCT GAT CTT CGC AAG CTT GCT GTT AAT
G
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pGE23 I~E23
CTT ATC CCA TTC CCC CGG CTC CAT TTC TTC ATG GTT GGG TTT GCA CCC TTG ACA TCA AGA GGA TCC -937 L I P F P R L H F F N V G F A P L T S R G S -283
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CAG CAG TAC CGG GCT TTG ACT GTT CCC GAA TTG ACT CAG CAA ATG TGG GAT GCT /tAG AAC ATG ATG -1003 Q Q Y R A L T V P E L T Q Q N W D A K N M M -305 •
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Fig, 4. Nucleotide and deduced amino acid sequence of the insert in pGE23. Also shown are the sequences for soybean [3-2 and soybean ~-1 tubulin cDNAs. Dashes indicate that space was introduced to maximize the alignment. Asterisks indicate amino acids found in all three pep*ides, while a vertical bar indicates amino acids found in pGE23 and either [3-2 or [3-1,
602 water-deficient seedlings, expression of sbPRP1 was induced in the elongating region and reduced in the root tip. RNA which hybridized to pSac3 was most abundant in sections of well-watered seedlings containing cells undergoing division and elongation. In seedlings grown in low-waterpotential vermiculite for 24 h, m R N A levels were strongly repressed in the hypocotyl growing zones and to a smaller extent in root tips. No effect of water deficit was seen on the expression of the gene corresponding to pGECHS in any tissue section. Expression was highest in roots.
pression may reflect modulation of overall rates of cell growth and maturation while others may indicate the activation of specific responses to water deficit such as osmotic adjustment and reversible modulation of cell wall extensibility. The isolation and characterization of the three cDNAs described in this paper is a first step towards understanding the role of specific genes which are modulated in the hypocotyl zone of cell elongation in response to water limitation.
Discussion
Several mRNAs were previously found to increase in abundance in the hypocotyl growing zone when soybean seedlings were transferred to low water potential vermiculite (qJ = - 0.3 MPa) [18, 43]. Differential screening identified two cDNAs which corresponded to mRNAs which show this behavior (pGE16, pGE95). In wellwatered plants, the genes corresponding to these cDNAs were expressed highest in mature sections of stems and roots. Increased expression of these genes in hypocotyl growing zones during water deficit suggests that some facets of cell maturation may be occurring in the cells of this tissue. Changes in polysome status [43], polysome translation products [43 ], and specific gene transcripts (pGE16, pGE95) suggest that cells in the elongating region are shifting towards a develop-
Genes which are up-regulated in the hypocotyl zone of elongation in response to water deficit
Transfer of 2-day old soybean seedlings to lowwater-potential vermiculite (qJ = - 0.3 MPa) resuits in a reversible decrease in hypocotyl growth rate [48, 49]. The causes of decreased growth rate include a decrease in the water potential gradient which provides water to the growing zone [49], increases in ABA [4, 18], and decreases in GA level [5]. Growth inhibition is accompanied by reduced cell wall extensibility and hydraulic conductivity [50], changes in cell wall protein composition [7], and osmotic adjustment [18]. Furthermore, water deficit results in reduced polysome content [18,43] and altered m R N A populations in the hypocotyl zone of cell elongation [18, 43]. Some of these changes in gene ex-
pGEEXT
GLLLPSPTPDYYKSPPPPSPTPYYKSPPPPPPYYYKSPPPPSPAPYYYKSPPPPPPYYYK
pCRGRP
GGGGGAGGGFGGGVGGGSGGGIGGGIGKGGGIGGG]GKGGGIGVGVG
pGECHS
RPKLGKEAATKAIKEMC~PKSKITHLIFCTTSGVDNPGADYQLTKLLGLRPSVKRYNNY~GCFAGGTVLRLAKDLAENN
pLF15
VPKLGKEAAS~t~KE~GQPKS~ITHL]FCTT$GVD~PGADYQLTKLLGLRPSVKRFNNY~GCFAGGTVLR]AKDLAENN
pCRJWI
KSVGEENEKNSSPNASFVKVSMDGAPYLRKVI)VKSVQELPRLSDSLG
aux28
KSVGEESEKNSSPNASFVK'VSI4DGAPYLRKVI)LIOIYKSYRELSDSLG
IIIIIIII IIIIIIII
IIIIII IIIIII
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IIIIIIIIIIIIIIIIIIIIIIIII IIIIIIIIIIIIIIIIIII1|1111
I I
IIIIIIIIIIIIIII IIIIIIIIIIIIIII
IIIIIIII IIIIIIII
IIIIII IIIIII
Fig. 5. Deduced amino acid sequences for inserts in p G E E X T (partial), pCRGRP, p G E C H S (partial) and pCRJW1. For those sequences where a comparison was made, a vertical bar indicates amino acids found in both sequences.
603
Fig. 7. Water deficit-modulated mRNAs detected in various sections of 3-day old soybean seedlings after growing in either well-watered vermiculite or water-deficient vermiculite for 24 h using either pGECHS, or pSac3 as probe. C, cotyledon; H, hook; E, elongating; M, mature (stem or root); T, root tip. The most abundant transcript (of those with multiple transcripts) is quantified in the histogram.
Fig. 6. Water deficit-modulated mRNAs detected in various sections of 3-day old soybean seedlings after growing in either well-watered vermiculite or water-deficient vermiculite for 24 h using either pGEEXT, pCRGRP, or p33 (sbPRP1 transcript shown) as probe. C, cotyledon; H, hook; E, elongating; M, mature (stem or root); T, root tip. The most abundant transcript (of those with multiple transcripts) is quantified in the histogram.
mental state observed in mature tissue [19, 43]. However, although cell elongation rates are reduced in water-limited seedlings, cell growth is not irreversibly inhibited if seedlings are rewatered within 48 h [46]. It is possible that changes in gene expression contribute to a reversible decrease in cell wall extensibility or hydraulic conductivity. Further analysis of the function of these genes will be required to clarify these possibilities. The increase in m R N A hybridizing to pGE16
and pGE95 in the hypocotyl zone of cell elongation could be due to decreased growth rate per se or to changes in plant growth regulator level. Application of methyl jasmonate to well-watered seedlings increased m R N A levels hybridizing to pGE 16 with little effect on mRNAs hybridizing to pGE95, pGE23 or JCWl. VspB, which encodes a soybean vegetative storage protein, is similarly induced by water deficit and jasmonate [42]. ABA, ethylene and GA treatments also modulated the level of m R N A which hybridizes to pGE95 and pGE16 to a small extent. However, these effects could not be separated from the influence of these compounds on growth. Based on properties similar to other cell wall proteins (highly basic, high levels of pro, lys, and tyr, and having a regular repeat) it has been suggested that PRPs are also localized in cell walls [ 10, 60]. In water-deficient soybean seedlings, the
604 expression of sbPRP1 was induced in the hypocotyl elongating region and repressed in the root tip. Increased expression in the elongating region during water deficit may reflect a maturation phenomenon [35] similar to that observed with pGE16, pGE23, and pGE95. If PRPs serve to increase the rigidity of the cell wall, then the prediction is that wall extensibility should decrease in the hypocotyl zone of elongation and increase in root tips.
Genes which are down-regulated in the hypocotyl zone of elongation by water deficit
The abundance of a group of in vitro translation products (50-55 kDa found in region 'E') decreased in the hypocotyl growing zone when seedlings were transferred to low-water-potential vermiculite [ 18, 43]. This group of in vitro translation products also declined in the growing zone of roots of water-deficient seedlings but to a smaller extent [ 18, 43]. The c D N A clone pGE23 hybridizes to m R N A which exhibits similar behavior. Previous analysis of poly(A) + RNA populations in soybean seedlings using two dimensional separations of in vitro translation products revealed that some mRNAs were more abundant in the root and shoot growing zones than in nongrowing sections of the hypocotyl and root [18,43]. RNA hybridizing to pGE23 is most abundant in the growing zones of the root and hypocotyl and water deficit results in decreased m R N A levels in both zones although m R N A levels decline more in the hypocotyl growing zone. The predicted amino acid sequence of the protein encoded by pGE23 has 75~o and 73~o overall residue identity with soybean/3-2 and fl-1 tubulin respectively. High expression of the genes which encode/3tubulin in growing zones is reasonable due to the involvement of microtubules in cell elongation and division [29]. In hypocotyls, water deficit results in reduced rates of cell division and cell elongation [44 ]. Therefore it is not unexpected that water deficit would decrease the rate of/3-tubulin synthesis and utilization. Decreased synthesis of fl-
tubulin could be accomplished by reducing fl-tubulin m R N A levels. Consistent with this possibility, we found that treatments which reduce growth such as water deficit, application of ABA or ethrel or low temperature (data not shown), resulted in reduced fl-tubulin m R N A levels. In addition, it was previously shown that illumination of soybean seedlings reduced hypocotyl elongation and fl-tubulin m R N A levels [9 ], suggesting that fl-tubulin levels could be regulated like phytochrome [ 16 ]. However, while decreased growth rates and reduced fl-tubulin m R N A levels always correlated, the percent decrease in fl-tubulin m R N A was usually less than the percent growth inhibition. It may be that some of the genes encoding fl-tubulin in soybean are not responsive to changes in growth rate and it is probable that our fl-tubulin probe cross hybridizes with m R N A produced from several fl-tubulin genes. Modulation of fl-tubulin m R N A levels could be accomplished by altering gene transcription or RNA stability. In Chlamydomonas, tubulin m R N A levels are controlled at the level of transcription and RNA stability [3, 37]. In animal cells, tubulin m R N A stability is coupled to m R N A translation and modulated by free tubulin levels [ 14]. The recognition event for this selective RNA destabilization is the binding of tubulin dimers to the nascent amino terminal fi-tubulin tetrapeptide (MREI) just after it emerges from the ribosome. Translation of fl-tubulin m R N A in this state leads to RNA degradation [ 14, 24]. It is interesting to note that the gene corresponding to pGE23 and fl-1 contain this tetrapeptide, whereas fl-2 contains a similar but not identical sequence (MRES). A second gene which is down-regulated in soybean hypocotyl growing zones in response to water deficit is actin. Actin performs many functions, ranging from cytoplasmic streaming and organelle orientation to cytoplasmic structure [59]. Plant tissues undergoing division contain large amounts of actin in the phragmoplast. Since water deficit results in reduced rates of cell division and cell elongation [44], it is not unexpected that water deficit would decrease the amount of actin gene expression.
605 A third gene which is down-regulated in soybean hypocotyl growing zones in response to water deficit is JCWl. The expression of this gene has been shown to be modulated by auxin [61], and consistent with earlier studies, JCWl m R N A levels were highest in growing zones. The kinetics of the m R N A decrease for JCWl in response to the transfer of seedlings to low-waterpotential vermiculite was slower than pGE23. This may indicate that auxin active in modulating gene expression or the sensitivity to auxin declines gradually with time in plants exposed to water deficit. Based on published data [ 19], it is probable that auxin levels decline during water deficit. This change in auxin level could contribute to reduced hypocotyl elongation rates [6, 61 ]. Changes in the composition of structural proteins of cell walls, such as HRGPs, GRPs and PRPs, could play a role in modulating cell wall extensibility. Sequence analysis of p G E E X T reveals a high tyrosine content. Therefore, this protein could form isodityrosine crosslinks and correspond to a tightly wall-bound HRGP. Genes encoding GRPs have been isolated in many plants, including petunia [17], bean [36], maize [26] and rice [38, 47]. The bean GRP1.8 gene was highly expressed in young hypocotyl and its product has been localized in cell walls of vascular tissue [36]. Keller et aL [36] speculated that, by analogy with silk fibroin, GRPs could provide the vascular tissue with strong tensile strength combined with flexibility. These qualities may be required for wall structural proteins in dividing and elongating regions. Following maturation of vascular tissue, there may be no need for additional synthesis of this protein. During maturation, GRPs could be replaced or reinforced with a more rigid matrix with less flexibility. As most GRPs contain tyrosine, this could be accomplished by forming isodityrosine crosslinks with HRGPs. In the hypocotyl of well-watered soybean seedlings, there is an inverse expression pattern of GRPs (abundant in elongating and dividing) and extensins (abundant in mature). In water-deficient seedlings, expression of GRPs and HRGPs are reduced in hypocotyl tissue. If GRPs provide flexibility (wall extensibility), then
the large decline in GRP expression combined with increased sbPRP 1 gene expression may contribute to decreased extensibility observed in water-deficient seedlings. Differential inhibition of shoot growth relative to root growth is an important adaptive response of plants to water deficit [18]. At present, we know that water status [48, 49, 50] and ABA [4, 18] play a role in this response and that changes in gene expression [43] occur in the hypocotyl zone of cell elongation when growth is inhibited. Some of the changes in gene expression may be the result of growth inhibition. For example, decreased expression of fl-tubulin may result from decreased utilization offl-tubulin subunits in growth-inhibited cells and an autogamous feedback system which down-regulates fl-tubulin gene expression. Changes in actin gene expression may operate in a similar fashion. It has been demonstrated that soluble H R G P accumulation in the wall of growing pea epicotyls was coincident with the cessation of growth [56]. A buildup in unincorporated cell wall carbohydrates or proteins which occurs due to the decrease in growth rate could down-regulate GRPs and HRGPs gene expression. Other changes in gene expression may contribute to selective growth inhibition. Increased expression of genes normally activated during cell maturation in the zones of cell elongation of water-limited plants may serve these functions. Expression of genes involved in selective growth inhibition would be expected to be at a maximum when growth is at a minimum, such as that observed with pGE16. Some changes in gene expression might be involved in ameliorating the effects of water deficit. Expression of these genes would parallel growth recovery, such as that observed with pGE95. In summary, exposure of soybean seedlings to water deficit results in decreased hypocotyl growth rates and to decreased levels of m R N A which hybridize to JCW1, pGE23, actin, H R G P and GRP, genes which are highly expressed in growing zones. In addition, we have identified three genes (pGE16, pGE95, and sbPRP1) which are highly expressed in non-growing tissues of well-watered seedlings, which show elevated ex-
606 pression in the hypocotyl zone of cell elongation in response to water deficit. Analysis of these genes may provide insight into the modulation of plant growth in response to water deficit.
Acknowledgements We thank Mr Mark Whitsitt for isolation of the poly(A) ÷ RNA used in cDNA synthesis and differential cloning and gratefully acknowledge the assistance of Ms Sara A. Kaplan during the initial steps of the primary screening of the 2gtl0 libraries. Thanks also go to Mary Tierney for p33, Christopher Lamb for pCHS1, and Richard Meagher for pSac3. We also thank Mrs Sharyll Pressley for her assistance in preparing this manuscript. This research was supported by Grant 90-37280 from the United States Department of Agriculture.
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