Modulation of Dehydration Tolerance in Soybean ... - Plant Physiology

Plant Physiol. (1 997) 114: 91 7-925

Modulation of Dehydration Tolerance in Soybean Seedlings' Dehydrin Matl 1s lnduced by Dehydration but Not by Abscisic Acid Mark S . Whitsitt, Robert C. Collins, and John E. Mullet* Department of Biochemistry and Biophysics, Texas A & M University, College Station, Texas 77843-21 28 system described by Boyer and co-workers (Meyer and Boyer, 1972, 1981; Boyer, 1988; Mason et al., 1988) employed an MD treatment involving the transfer of 2-d-old well-watered seedlings (soil qw= -0.01 MPa) to low qW soil (Tw = -0.3 MPa) (Nonami and Boyer, 1989). This treatment caused a reversible inhibition of hypocotyl elongation while root growth continued relatively unabated (Mullet, 1990), and hypocotyl elongation recovered between 16 and 24 h after transfer (Mason et al., 1988).During MD, turgor is maintained in the hypocotyl zone of elongation (Nonami and Boyer, 1989) and is correlated with osmotic adjustment of these tissues (Meyer and Boyer, 1981; Creelman et al., 1990). In contrast, osmotic adjustment is reduced and turgor decreases in the more mature, nongrowing portions of the hypocotyl (Meyer and Boyer, 1981; Nonami and Boyer, 1989). MD .also results in the accumulation of ABA, which inhibits stem growth (Creelman et al., 1990) and has been shown to enhance continued root growth at low soil Yw in maize (Saab et al., 1990). This MD treatment does not, however, result in decreased seedling water content, even though water uptake and hypocotyl growth are initially inhibited (Bozarth et al., 1987). Exposure of soybean seedlings to MD results in decreased polysome content and specific changes in mRNA populations in the hypocotyl growing zone (Bensen et al., 1988). Application of ABA to well-watered seedlings also causes hypocotyl growth inhibition, but the changes in mRNA population induced by ABA are not identical to those induced by MD (Creelman et al., 1990).Other studies have identified turgor-responsive genes that do not respond to ABA (Creelman and Mullet, 1991), suggesting, along with other data (Bostock and Quatrano, 1992; Butler and Cuming, 1993; Espelund et al., 1995), that plants employ multiple signal transduction pathways to sense changes in water status and to activate specific changes in gene expression. Furthermore, gene expression modulated by water deficit may be regulated by different pathways depending on the developmental stage of the plant (Finkelstein, 1993). Water deficit often causes significant tissue dehydration, and plants have a wide range of tolerance to dehydration depending on the species and stage of development. In

Cerminated soybean (Glycine max 1. cv Williams 82) seedlings subjected to rapid dehydration begin to lose the ability to recover when the relative water content of the plant decreases below 60%. The expanded cells of the hypocotyl appear more susceptible to dehydration-induced damage than do cells in the hypocotyl zone of cell growth. Pretreatment of seedlings prior to rapid dehydration with nonlethal water deficit or exogenous abscisic acid (ABA) shifts this viability threshold to progressively lower relative water contents, indicating the acquisition of increased dehydration tolerance. lncreased tolerance is associated with osmotic adjustment in the hypocotyl zone of cell growth and with increases in soybean dehydrin M a t l mRNA levels. The accumulation of M a t l mRNA is dehydration dependent but insensitive to ABA. lnduction of M a t l mRNA accumulation by dehydration but not by ABA makes it an unusual member of the dehydrin family.

Exposure of plants to moderate water deficit can reduce growth, inhibit photosynthesis, and cause diverse changes in metabolism (Bradford and Hsiao, 1982; Hanson and Hitz, 1982). Adaptations to moderate water deficit include differential growth of roots versus shoots (Creelman et al., 1990) and osmotic adjustment, which help to re-establish water extraction from soil and turgor required for growth (Westgate and Boyer, 1985). More SD results in tissue dehydration, which, under some conditions, can cause plant death. During seed development most plant embryos acquire the ability to survive extreme desiccation and reduced RWCs of 10 to 15% (Dasgupta et al., 1982; Senaratna and McKersie, 1983; Ellis, 1991).This dehydration tolerance is associated with the accumulation of soluble sugars, such as Suc and stachyose (Koster and Leopold, 1988; Blackman et al., 1992), and LEA or maturation proteins, such as the dehydrins (Dure et al., 1989; Close et al., 1993b; Blackman et al., 1995). These components presumably help to protect plant cells from the damage caused by dehydration and allow growth recovery after imbibition. In previous studies we utilized etiolated soybean (Glycine max L. cv Williams 82) seedlings to analyze plant responses to MD treatment (Mason et al., 1988; Creelman et al., 1990; Mullet, 1990). The etiolated soybean seedling This research was supported by National Research Initiative Competitive Grants Program grant no. 93-37100-9014 and the Texas Agricultura1 Experiment Station. * Corresponding author; e-mail [email protected]; fax 1-409-862-4718.

Abbreviations: LEA, late embryogenesis abundant; MD, mild water deficit; qP,turgor potential; Vr,, osmotic potential; Tw, water potential; RD, rapid dehydration; RWC, relative water content; SD, severe water deficit. 917

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plants such as mosses (Bewley and Oliver, 1992), Craterostigma pluntagineum (Gaff, 1971), and some grasses (Doggett, 1986), vegetative tissues, as well as seeds, are highly desiccation tolerant. In general, however, the desiccation tolerance acquired during seed development is lost soon after seed imbibition and germination. Koster and Leopold (1988) found that soybean axes retain dehydration tolerance until approximately 18 h postimbibition, coinciding with emergence of the axis from the seed coat. The loss of tolerance was correlated with decreased high-affinity water binding (Vertucci and Leopold, 1987), the loss of soluble sugars (Blackman et al., 1992), turnover of LEA proteins (Blackman et al., 1991), and an increase in sensitivity to dehydration-induced plasma membrane damage (Senaratna and McKersie, 1983). LEA proteins have been proposed to facilitate water retention, membrane stability, and ion sequestration (Dure, 1993b), in addition to protecting cytoplasmic constituents during dehydration (Blackman et al., 1995). One class of LEA proteins, the dehydrins or group 2 LEAs, are characterized by the presence of the consensus amino acid sequence EKKGIMDKIKEKLPG at or near the carboxyl terminus, which may be repeated one or more times (Close et al., 1993b). Many dehydrins also contain a series of contiguous Ser residues near the amino-most consensus tract. Like most other LEA proteins, dehydrins are highly hydrophilic, remain soluble after heat treatment, and have a predicted structure that is mostly random coil, with the consensus tracts forming putative a-helices (Close et al., 199313). In soybean the dehydrin Matl was first identified as a polypeptide with an apparent molecular mass of 31 kD that is synthesized de novo during the desiccation phase of embryo maturation (Chyan, 1992). Matl contains only one Lys-rich motif at the carboxyl terminus and lacks the upstream tract of Ser residues. It is also a member of the family of soybean maturation proteins correlated with the acquisition of desiccation tolerance in the embryo and the progression of seed germination to vegetative growth (Rosenberg and Rinne, 1986; Blackman et al., 1991). In this study we have analyzed the responses of soybean seedlings to dehydration. The results show that nonelongating hypocotyl tissues are more sensitive to dehydration-induced damage, and that pretreatment of seedlings with MD, ABA, or partia1 dehydration enhances the plant's ability to survive subsequent RD. Improved dehydration tolerance is associated with osmotic adjustment in the hypocotyl's growth zone and with accumulation of soybean dehydrin mRNA. In addition, we report that the soybean Matl gene, which encodes a dehydrin, is induced by dehydration but not by ABA. MATERIALS A N D M E T H O D S Plant Material and Growth

Soybean (Glycine mux L. Merr. cv Williams; Illinois Foundation Seed, Champaign, IL) seedlings were grown in the dark at 100% RH and 29"C, as described previously (Creelman et al., 1990). After 48 h control seedlings were transferred to vermiculite watered to runoff (well-watered)

Plant Physiol. Vol. 114, 1997

with 1 O p 4 M CaCl, solution (medium qW= -0.01 MPa). MD treatment was imposed by transferring seedlings into vermiculite containing one-eighth as much CaC1, solution as in well-watered conditions (240 mL/ 12 L vermiculite; Y w = -0.3 MPa). Seedlings subjected to ABA treatment were transferred to well-watered vermiculite containing 10-3 M (L)-ABA (Sigma). Seedlings used in the SD treatment were transferred to dry vermiculite without the addition of CaC1, solution. Control, ABA-, and SD-treated seedlings were suspended by the hypocotyl hook on a square plastic grid of sixty-four 14-mm square openings on top of square plastic containers. Vermiculite was added through the open grid squares and the appropriate watering solution, if any, was added. In the control, ABA, and MD treatments, transferred seedlings were returned to the dark at 100% RH and 29°C for up to 72 h. Seedlings in the SD treatment were incubated at 29°C and 89% RH for up to 72 h. At various times during the treatments, seedlings were harvested and dissected into segments corresponding to three regions of seedling tissue: (a) the hypocotyl growth zone (zones of cell division and elongation extending from the cotyledons to 15 mm below the hook of the hypocotyl); (b) the nonelongating portion of the hypocotyl; and (c) the root, as per Mason et al. (1988). Excised tissue sections were immediately frozen in liquid N, for RNA isolation or were immediately used for Twmeasurements. A11 manipulations except the measurement of Y w were done under a green safelight, as described previously (Meyer and Boyer, 1981).

R D Treatment and Pretreatments

Seedlings to be subjected to RD treatment were removed from their respective soils and placed on a plastic grid on the bench. Seedlings were dehydrated by exposing them to a stream of cool air from a blow-dryer with its nozzle 40 cm above the grid for up to 180 min. MD- and ABA-pretreated seedlings were subjected to 24 h of MD or ABA treatment, respectively, prior to RD treatment. SD-pretreated seedlings were subjected to 8 h of SD treatment and subsequently placed at 29°C and 100% RH for 16 h prior to RD. Seedlings were harvested and stored as described above or were used immediately for Twanalysis.

Seedling Recovery and R W C Measurement

At various times during the SD treatment, groups of 20 seedlings in dry vermiculite were watered to runoff and placed at 100%RH and 29°C in the dark for 24 h. After 24 h seedling recovery was scored by counting the number of seedlings exhibiting hypocotyl growth. Seedlings subjected to RD were removed from the stream of air at various times, transferred to well-watered vermiculite, then scored for growth recovery. Water contents of samples of 20 seedlings were determined by subtracting dry tissue weights from fresh weights at each time point. Percent RWC was calculated by dividing the water content at any time during treatment by the water content prior to treatment and multiplying by 100. To determine tissue dry weights, rep-

Soybean Dehydrins and Dehydration Tolerance resentative whole seedlings (including cotyledons) were dried at 90°C under a vacuum for 24 h and weighed.

Ww Measurements The growth zones of seedlings subjected to RD stress and the associated pretreatments were excised and used for qw measurements. Yw was measured in triplicate by the psychrometric method (Bayer and Knipling, 1965) using C-52 sample chambers and an HR33T microvoltmeter (Wescor, Logan, UT). Excised seedling sections were immediately sealed in the chambers and allowed to come to vapor and temperature equilibrium for 90 to 180 min. After equilibration a cooling voltage was applied to chamber thermocouples for 8 to 24 s and the resulting induced voltage was recorded 22 s later. Tissue used in qWdetermination was immediately frozen in liquid N, and stored at -70°C until used for Tsmeasurement. Previously frozen tissue sections were allowed to thaw, and were then placed in the sample chambers and equilibrated for 90 to 180 min. Ts was then determined in triplicate as described for Tw.The sample chambers were calibrated using filter paper discs saturated with 0.0, -0.5, -0.9, and -1.8 MPa standards according to the manufacturer’s (Wescor) instructions. Each standardization was performed in triplicate. Microvoltmeter data were collected by computer and analyzed using a data-acquisition system (Axiom Chromatography, Piscataway, NJ). Hypocotyl Tissue RWC and Cellular Leakage Measurement

The hypocotyls of 20 seedlings subjected to SD treatment for up to 48 h were dissected into nonelongating and elongating tissue segments. The cut ends of excised segments were blotted on filter paper and then weighed. Immediately after weighing, all 20 segments were placed in 15-mL polyethylene tubes containing 10 mL of distilled water. The tubes were incubated at room temperature for 40 min on a rocking aliquot mixer. The supernatants were decanted and the tissue segments were subjected to dryweight analysis. RWCs for each group were determined as previously described. The of each supernatant was immediately measured in triplicate. Absorbances from a11 samples were normalized on a dry-weight basis, and the average A,,, per gram of dry weight was determined.

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being washed the blots were autoradiographed for 16 h at -70°C with intensifying screens. RNA molecular weight standards (0.16-1.77 kb; BRL) were visualized on blots by methylene blue staining (Monroy, 1988). Probes used for hybridization were synthesized from cDNA inserts of soybean Matl (pMAT1) (Chyan, 1992), soybean P-tubulin (pGE23) (Creelman and Mullet, 1991), and soybean LoxA (Bell and Mullet, 1991). The cDNA inserts were purified and used as templates for probe labeling by randomprimer synthesis (Feinberg and Vogelstein, 1984). RESULTS SD and Dehydration Tolerance

An SD treatment was developed to study seedling responses to dehydration. Figure 1A shows that seedling RWC decreased rapidly during the first 24 h of SD treatment, and then more slowly over the next 48 h, reaching approximately 16% RWC by 72 h. Because dehydration tolerance in developing embryos is normally assayed in terms of germination efficiency after imbibition of dry seed (Senaratna and McKersie, 1983), the ability of dehydrated seedlings to recover hypocotyl elongation after water was restored was used to measure the effect of dehydration treatment. Seedling viability, as measured by the fraction of seedlings recovering hypocotyl elongation after stress alleviation, remained above 90% for the first 8 h of SD treatment, decreased to 28% recovery by 24 h of treatment, and then decreased gradually to 0% recovery by 72 h (Fig. 1B). When seedling recovery was examined as a function of water-deficit severity ~-(Fig. 2), seedling viability was unaffected by SD treatment until seedling RWC docreased to

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Analysis of mRNA

Total RNA was isolated from harvested seedling sections by the method of Shirras and Northcote (1984). Total RNA samples were suspended in 15% formaldehyde and 50% formamide and fractionated (10 mg/lane or 2.5 mg/lane) on a 1%agarose gel in lx Mops running buffer (0.02 M Mops, 8 mM NaOAc, and 1 miv EDTA). RNA was transferred to GeneScreen membranes (New England Nuclear) by capillary blotting, and UV-cross-linked in a Stratalinker (Stratagene). Blots were prehybridized 1.5 to 2 h in 6 mL of Rapid-Hyb buffer (Amersham) at 65°C in bottles placed in a rotary incubation oven. 32P-labeledprobe was added to the prehybridization buffer at 106 cpm/mL buffer and the blots were hybridized for 2.5 h at 65°C. Stringent washes were performed with 0.1 X SSC and 0.1% SDS at 65°C. After

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d s

Time (h)

Figure 1. Time courses of RWC loss and seedling recovery during SD treatment. A, RWC as a function of SD treatment duration. Data are means i SE of three experiments. B, Seedling recovery as a function of SD treatment duration. Data are means ? SE of three experiments.

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below 60%. Beyond this point, the rate of decrease in seedling recovery per unit decrease in RWC was fairly constant until RWC reached l6%, at which point none of the seedlings was able to recover. A RD treatment lasting only 3 to 4 h was also developed to minimize the time allowed for seedling adjustment during dehydration. When 72-h-old seedlings (or 48-h-old seedlings, data not shown) were subjected to RD treatment (Fig. 3), recoveries remained above 90% until RWC decreased to below 60%. To examine the effects of MD, ABA, and SD treatments on dehydration tolerance, 48-h postimbibition seedlings were subjected to RD treatment with or without 24 h of prior exposure to these standard treatments (Fig. 3). Whereas nonpretreated seedlings rapidly lost viability when the RWC of the plants decreased to below 6O%, MD-, ABA-, and SD-pretreated seedlings maintained recoveries above 90% until their RWCs decreased to below 53, 49, and 44%, respectively. It is important to note that the SD pretreatment was modified to ensure that seedling viabilities remained near 100% before RD (see "Materials and Methods"). The shift of the viability loss threshold to lower RWCs indicates that the dehydration tolerance of these seedlings was increased by each pretreatment. Osmotic Adjustment 1s Correlated with Seedling Dehydration Tolerance

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Figure 3. Seedling recovery after RD treatment as a function of RWC.

0, M D pretreatment; O, ABA pretreatment; O , SD pretreatment; O, control. RWCs and seedling recoveries were determined as in Figure 1. Dashed line from the ordinate to the first SD pretreatment data point indicates that the RWC of these seedlings was less than 100% at the beginning of RD treatment.

ABA-pretreated tissues decreased to -1.5 and -1.1 MPa, respectively.

To investigate the potential role of osmotic adjustment in the observed increase in dehydration tolerance, the qS of hypocotyl growth zones of pretreated and nonpretreated seedlings before and after dehydration of plants to approximately 60% RWC were measured (Table I). Before dehydration the T, of the hypocotyl growth zone of nonpretreated and ABA-pretreated plants were approximately the same (-0.5 MPa), whereas MD-pretreated tissues exhibited a lower Ts(-0.9 MPa). SD-pretreated tissues had a significantly lower W, (-1.8 MPa) before dehydration. After dehydration the Tsof nonpretreated plants decreased to -0.7 Mpa, whereas the W, of MD- and

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Dehydrin mRNA Accumulation during Dehydration

Accumulation of dehydrins has been associated with the acquisition of dehydration tolerance in both developing seeds and in vegetatively growing plants (Close et al., 1993b). Therefore, we were interested in the potential role of the soybean dehydrin Matl in the increased dehydration tolerance of soybean seedlings. The accumulation of Matl mRNA in the hypocotyl growth zone was analyzed first during the SD treatment (Fig. 4A). The Matl probe hybridized with an mRNA of approximately 1.7 kb, which was detectable after 8 h in the dividing and elongating tissues of SD-treated plants, but transcripts were not detected in the growth zone of untreated, MD-, or ABA-treated plants. Matl transcripts continued to accumulate throughout the 48-h experiment and decreased in abundance by 24 h after rewatering.

Table 1. The effect of pretreatment on qsin the hypocotyl growth

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zone before and after RD treatment q, is presented for each pretreatment regimen at the beginning (to) and near 60% RWC ( t J . Values are means t SD of three replicates. Corresponding RWCs are indicated in parentheses. VI, (RWC) Pretreatment

% Relative Water Content

Figure 2. Seedling recovery after SD treatment as a function of RWC. Seedling recovery data from Figure 1A were associated with RWC data in Figure 1 B by measurement time point and recast as a function of RWC. RWC error bars were removed for the sake of clarity. Data are means t SE of three experiments.

M Pa

None MD ABA

SD

-0.5 2 0.1 (100%) -0.9 ? 0.2 (1 00%) -0.5 i 0.3 (100%) -1.8 ? 0.1 (63.9%)

-0.7 -1.5 -1.1 -1.8

t 0.1 (62.8%) t 0.2 (60.1'/o) i 0.2 (61.8%) -t

0.1 (63.9%)

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Soybean Dehydrins and Dehydration Tolerance

Matt RW

Hours Treatment 0

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of MD treatment. /3-tubulin mRNA levels were relatively unaffected by ABA treatment. In SD-treated seedlings, however, /3-tubulin transcripts were undetectable by 8 h and did not reappear until 24 h after rewatering. LoxA transcripts (2.7 kb; Fig. 4C) were detected in the hypocotyl growth zone of 48-h control seedlings and exhibited a slight increase by 8 h in all treatments. In MD- and ABAtreated tissues LoxA transcript levels then decreased, becoming almost undetectable by 48 h. LoxA mRNA also decreased after 24 to 48 h of SD treatment, but recovered when plants were rewatered. Mat 1 mRNA accumulation was also analyzed in the nonelongating portions of the hypocotyl (Fig. 5A) and root (Fig. 5B). Significant levels of Mat! transcripts were present only in SD-treated plants. Treatment of seedlings with ABA did not induce Matl transcript levels. However, MD did cause a small accumulation of Matl mRNA by 8 h, but transcripts were again undetectable by 24 h. The same pattern of expression was also seen in mature root tissues, but the transient accumulation of Matl mRNA appeared to be greater than that seen in nonelongating hypocotyl tissues.

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ABA Figure 4. Accumulation of mRNA corresponding to Mat (A), j3-tubulin (B), and LoxA (C) during SD, MD, and ABA treatments. Total RNA was extracted from the hypocotyl growing zones of 48-h postimbibition seedlings (0-h, control; lanes 1) and SD-, MD-, or ABA-treated seedlings (8-48 h; lanes 2-5, all panels). RNA was also extracted from the growing zones of seedlings that had been rewatered after 24 h of SD treatment and allowed to recover for 24 additional h (RW 24; lanes 6). Identical blots of total RNA (10 mg per lane) were hybridized with random-primed DNA probes synthesized using soybean dehydrin (pMatl), /3-tubulin (pCE23), and LoxA cDNAs as the templates. Approximate molecular masses of detected transcripts are indicated in kilobars to left of the SD blot in each panel.

The levels of mRNAs corresponding to soybean /3-tubulin and LoxA were analyzed to provide additional information on changes in gene expression in response to dehydration (Fig. 4, B and C). A 1.3-kb transcript from the hypocotyl growth zone hybridized to a /3-tubulin probe in 48-h postimbibition seedlings (Fig. 4B; 0 h all treatments). Levels of this transcript were significantly reduced by 8 h

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Figure 5. Mat! mRNA accumulation in nonelongating hypocotyl and root tissues during MD, ABA, and SD treatments. Total RNA was extracted from nonelongating hypocotyl and root tissues of 48-h postimbibition (0 h; lane 1), SD-treated (lanes 2 and 3), MD-treated (lanes 4 and 5), and ABA-treated (lanes 6 and 7) soybean seedlings at 8 h (lanes 2, 4, and 6) and 24 h (lanes 3, 5, and 7). Identical blots of total RNA (10 mg per lane) were hybridized with a random-primed DNA probe synthesized using Mat'1 cDNA as a template.

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The accumulation of Mat! rnRNA during RD treatment in pretreated and control seedlings was also characterized (Fig. 6). Matl transcripts were not detected in the elongating zones of control, MD-, or ABA-pretreated seedlings prior to RD treatment (lane 1), and were only slightly detectable in nonpretreated tissues after 60 min. In both MD- and ABA-pretreated plants Mail transcripts were readily detected by 30 min of treatment (72.0 and 67.4% RWC, respectively), and transcript levels continued to increase until 180 min. Because of the nature of the SD pretreatment, Matl transcripts were detected at 0 min (63.9% RWC), and levels increased slightly after 30 min, remaining relatively constant throughout the remainder of the experiment. At 180 min accumulation of Matl mRNA was the greatest in the MD- and ABA-pretreated seedlings. Sensitivity of Nonelongating Hypocotyl Tissue to Dehydration Injury

Visual inspection of seedlings during the 72-h SD treatment showed greater apparent tissue damage to the nonelongating zones of the hypocotyl and root than the growth zones at moderate levels of dehydration. This led us to investigate the effect of dehydration on shoot tissues located in the growth zone and the nonelongating portions of the hypocotyl. First, we determined the rates of water loss in the two tissues (Fig. 7A) and found that during the first 6 h of SD treatment, nonelongating hypocotyl tissue lost water more rapidly than tissue in the growth zone. After 6 h the apparent rates of water loss between the two tissues were similar, but nonelongating tissues lost more water overall, reaching 7% RWC by 48 h, compared with 20% RWC in the zone of cell growth. Possible differences in plasma membrane damage due to dehydration was assessed next by measuring the leakage of cytoplasmic solutes, particularly macromolecules, in a manner similar to that described by Schoettle and Leopold (1984). Figure 7B shows that tissues from nonelongating Minutes Rapid Dehydration 0

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% Relative Water Content Figure 7. Differential effects of SD treatment on the elongating and nonelongating zones of soybean hypocotyls. A, RWC as a function of SD treatment duration. O, Elongating hypocotyl segments; D, nonelongating hypocotyl segments. B, Cellular leakage as measured by increased A2bo during SD treatment. O, Elongating hypocotyl segments; D, nonelongating hypocotyl segments. Absorbances were normalized on a dry weight basis and are the means ± SE of three replicates.

hypocotyl sections exhibited more apparent leakage at all time points, beginning at 30.7 A 260 /g dry weight at 100% RWC, and increasing to approximately 70 A 260 /g dry weight at 7% RWC. In contrast, tissues in the hypocotyl zone exhibited much lower apparent leakage initially (9.4 ^260/g dry weight), and dehydration caused only a small change in apparent leakage (17.1 A 260 /g dry weight). It is interesting to note that leakage in nonelongating tissues increased significantly from 47.0 A 260 /g dry weight at 63% RWC to 75.8 A2fa/S dry weight at 53% RWC. This change is correlated with the decrease in seedling viability during dehydration, which occurs at 60% RWC.

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DISCUSSION MD

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Figure 6. Matl mRNA accumulation during RD treatment. Total RNA was extracted from the elongating and dividing hypocotyl zone of seedlings subjected to RD treatment for various times, with or without pretreatment, as indicated. Identical blots of total RNA (10 mg per lane) were hybridized with a random-primed DNA probe synthesized using the Matl cDNA as a template.

Two-day-old seedlings subjected to dehydration begin to lose their ability to recover from stress when seedling RWC is reduced to below 60%. Brief exposure to nonlethal water deficit or ABA prior to RD increases the dehydration tolerance of seedlings, but not to the level acquired during seed development (Senaratna and McKersie, 1983). A similar phenomenon has been observed in the case of heat shock, in which brief exposure to nonlethal high temperatures increases the thermotolerance of plants subsequently subjected to normally lethal temperatures (for review, see Lindquist [1986]).

Soybean Dehydrins and Dehydration Tolerance The increase in dehydration tolerance that occurs in soybean seedlings exposed to nonlethal water deficit or ABA is associated with increased osmotic adjustment in the soybean seedling hypocotyl growth zone. Previous studies showed that sugars and amino acids accumulate in the soybean hypocotyl growth zone when seedlings are transferred to 1 0 w - T ~vermiculite (Meyer and Boyer, 1981; Sharp et al., 1990). The accumulation of solutes aids the maintenance of water content and cell turgor in this tissue. In addition, compatible solutes can also increase dehydration tolerance by acting as cellular protectants (Yancey et al., 1982). Analysis of cell leakage suggests that the growth zone of soybean hypocotyls is well protected from dehydration-induced damage (Fig. 7). In contrast, reduction of the RWC of soybean seedlings below 60% caused an increase in the leakage of cells in the lower, nongrowing portion of the hypocotyl (Fig. 7). The dehydration-induced damage in this tissue may be the cause of decreased seedling recovery below 60% RWC. Plasma membrane damage has been suggested to be a major cause of viability loss in water-deficit-stressed plants (Bewley and Krochko, 1982). Microscopic analysis of desiccation-intolerant soybean axis cells showed extensive damage, including vesiculation of membranes and coagulation of cytoplasmic contents (Blackman et al., 1995). It is possible that because of the reduced capacity for osmotic adjustment in more mature hypocotyl tissues (Meyer and Boyer, 1981), compatible solute concentrations are not high enough to provide sufficient protection to the plasma membrane. Reduced osmotic adjustment would also adversely affect water retention in this tissue. Alternatively, the less developed and more flexible cell walls of tissues in growth zones may reduce damage from plasmolysis. The increase in dehydration tolerance caused by water deficit or ABA pretreatments could be due in part to the accumulation of protective proteins such as the dehydrin class of LEA proteins. LEA proteins have been proposed to contribute to dehydration tolerance at the cellular level by sequestering ions (Dure, 1993a), replacing water that hydrates membranes and proteins (Dure, 1993b), and by tightly binding water, preventing its loss (McCubbin et al., 1985). Accumulation of the soybean dehydrin MatZ during seed development and its turnover after germination are closely associated with the acquisition and loss of dehydration tolerance in embryos (Rosenberg and Rinne, 1988; Chyan, 1992; Blackman et al., 1995). In fully germinated seedlings we found that MD or ABA treatment was unable to induce Matl mRNA accumulation (Fig. 4A). Only treatments resulting in a net loss of water from the seedlings (SD and RD) were able to induce Matl transcript accumulation (Figs. 4A, 5, and 6). MD and ABA pretreatment, however, did allow more rapid and greater accumulation of MatZ mRNA when plants were subjected to RD (Fig. 6). The observation that ABA pretreatment alters the accumulation of MatZ transcripts when combined with RD suggests that the effects of ABA and dehydration may be the result of two parallel regulatory pathways acting in concert. Bostock and Quatrano (1992) found that another Lea gene, the Em gene in wheat, is controlled by

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independent ABA and water-deficit pathways that may work synergistically to boost Em transcript levels above those seen in either treatment alone. Similar results with Em homologs from other species have also been observed in ABA-deficient and -insensitive mutants (McCarty et al., 1991; Butler and Cuming, 1993). Yamaguchi-Shinozaki et al. (1994) have identified a novel, dehydration-specific, cis-acting element in the rd29A gene from Arabidopsis, which they have called the DRE for dehydrationresponsive element. This element appears to be involved in the ABA-independent regulation of rd29A by water deficit and salt stress (Yamaguchi-Shinozaki et al., 1995). Although the A . thaliana rd29A DRE has not yet been found in the Matl gene, a similar mechanism appears to exist in soybean seedlings. The MD treatments used in this study and previously do not reduce turgor in the growth zone, even though turgor reduction occurs in more mature hypocotyl tissues (Nonami and Boyer, 1989). We observed a low level of Matl transcripts in nonelongating hypocotyl sections at 8 h of MD treatment (Fig. 7), but the mRNA disappeared by 24 h, well before the re-establishment of turgor in this tissue. Therefore, turgor loss appears to be insufficient to fully induce Matl transcript accumulation. The small amount of mRNA accumulated under MD conditions may be explained by the withdrawal of water from the nonelongating tissues as the growing zones continue to expand for a short time after transfer to MD treatment (Cavalieri and Boyer, 1982). The insensitivity of MatZ transcription to ABA treatment is unusual because the dehydrins are generally ABA-inducible (Close et al., 1993b). Exceptions to this rule include ecp40 in carrot (Kiyosue et al., 1993) and xerol in Arabidopsis (Welin et al., 1994). Our observation is consistent with that of Hsing et al. (1990), who reported that expression of the Matl gene product does not appear to be regulated by ABA during seed development. Differences in Lea gene expression between embryos and seedlings, however, have also been reported. Finkelstein (1993) reported evidence for differential ABA regulation of an Em homolog in Arabidopsis, in which the choice of signal pathway used is dependent on developmental stage. A set of 23- to 25-kD proteins from maize have been found to be ABA-inducible in embryos but only water-deficitinducible in vegetative tissues (Pla et al., 1989). Espelund et al. (1992) described a family of Em homologs in barley that are similarly regulated in embryos but are differentially regulated by ABA and osmotic stresses in vegetative tissues. Hughes and Galau (1991) suggest that, because embryos generally become tolerant before dehydration begins, these developmental differences in gene expression may be modulated by some maternal factor. Alternatively, differential regulation of gene expression might be explained by developmentally specific patterns of genome methylation (Cedar, 1988). Whatever the mechanism, developmental differences in gene regulation may cause some of the genes required for the extreme dehydration tolerance exhibited by embryos to be unavailable in fully germinated seedlings.

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Whitsitt et al.

The physiological basis of the viability loss threshold at 60% RWC in nonpretreated seedlings remains to be elucidated, although we did find that cell leakage in the nonelongating hypocotyl increased significantly when plants reached 60% RWC. Osmotic adjustment and Matl transcript accumulation were associated with increased dehydration tolerance in seedlings, and both processes were activated before plants reached 60% RWC. These data indicate that some detrimental physiological change occurs in seedlings near this point and that osmotic adjustment and Matl accumulation may ameliorate its effects. Rosenberg and Rinne (1986) found that the accumulation of the Matl gene product and dehydration below 60% RWC during seed development were necessary for the progression of germination to vegetative seedling growth. The loss of embryonic desiccation tolerance in germinating soybean axes is associated with the loss of Matl protein as seedlings begin vegetative growth (Blackman et al., 1991). Therefore, although accumulation of compatible and potentially protective solutes most likely acts to ameliorate the effects of cellular dehydration, the effects of Matl expression may be more important during the recovery of growth than during the dehydration stress. Severa1 mRNAs preserved during slow desiccation of the highly tolerant moss Tortula ruralis are rapidly recruited into active protein synthesis after rewatering, and have been implicated in the protection and repair of cellular structures during recovery (Oliver and Bewley, 1984). It is possible, then, that dehydrationassociated damage near 60% RWC becomes too great for seedlings to recover from unless compatible solutes are already present to reduce damage and some form of repair system (possibly involving M a t l ) is poised for reactivation before rehydration. In summary, we have shown that the dehydration tolerante of fully germinated soybean seedlings can be increased by brief, nonlethal exposure to water deficit and ABA prior to dehydration. This increase in tolerance is associated with the increased accumulation of osmotically active solutes and the accumulation of Matl transcripts prior to dehydration below 60% RWC. Matl mRNA accumulation is dehydration dependent and insensitive to ABA, making it a somewhat unique member of the dehydrin family. The dehydration tolerance attained by pretreatments, however, remains significantly lower than that seen in embryos. It appears that overall seedling dehydration tolerance may be predicated on the sensitivity of nonelongating tissues to dehydrationinduced damage, as opposed to the resistance of the growing zone. Future experiments should, therefore, focus on physiological responses and changes in gene expression in these tissues during dehydration. Preliminary analyses of Matl protein accumulation in seedlings in response to dehydration using antibodies specific for maize dehydrin (Bradford and Chandler, 1992) and for a synthetic dehydrin peptide (Close et al., 1993a) have to date been inconclusive (data not shown). The relationship between Matl mRNA accumulation and protein synthesis in seedlings, therefore, remains uncertain. We have begun work on the bacterial expression of Matl protein for antibody production and for further biochemical and functional analysis.

Plant Physiol. Vol. 114, 1997 ACKNOWLEDCMENTS

We would like to thank Dr. Alan Kriz for graciously providing the pMAT7 cDNA clone, Dr. Robert Creelman for pGE23, and Dr. Erin Bell for the LoxA clone. Received January 17, 1997; accepted March 25, 1997. Copyright Clearance Center: 0032-0889/97/ 114/0917/09.

LITERATURE ClTED

Bell E, Mullet JE (1991) Lipoxygenase gene expression is modulated in plants by water deficit, wounding, and methyl jasmonate. Mo1 Gen Genet 230: 456462 Bensen RJ, Boyer JS, Mullet JE (1988) Water deficit-induced changes in abscisic acid, growth, polysomes, and translatable RNA'in soybean hypocotyls. Plant Physiol 88: 289-294 Bewley JD, Krochko JE (1982)Desiccation-tolerance.In OL Lange, PS Nobel, CB Osmond, H Ziegler, eds, Physiological Plant Ecology 11, Vol 128. Springer-Verlag, Berlin, pp 325-378 Bewley JD, Oliver MJ (1992) Desiccation tolerance in vegetative plant tissues and seeds: protein synthesis in relation to desiccation and a potential role for protection and repair mechanisms. In CB Osmond, G Somero, eds, Water and Life: A Comparative Analysis of Water Relationships at the Organismic, Cellular and Molecular Levels. Springer-Verlag, Berlin, pp 187-224 Blackman SA, Obendorf RL, Leopold AC (1992) Maturation proteins and sugars in desiccation tolerance of developing soybean seeds. Plant Physiol 100: 225-230 Blackman SA, Obendorf RL, Leopold AC (1995) Desiccation tolerance in developing soybean seeds: the role of stress proteins. Physiol Plant 93: 630-638 Blackman SA, Wettlaufer SH, Obendorf RL, Leopold AC (1991) Maturation proteins associated with desiccation tolerance in soybean. Plant Physiol 96: 868-874 Bostock RM, Quatrano RS (1992) Regulation of Em gene expression in rice. Interaction between osmotic stress and abscisic acid. Plant Physiol 98: 1356-1363 Boyer JS (1988) Cell enlargement and growth-induced water potentials. Physiol Plant 73: 311-316 Boyer JS, Knipling EB (1965) Isopiestic technique for measuring leaf water potential with a thermocouple psychrometer. Proc Natl Acad Sci USA 5 4 1044-1051 Bozarth CS, Mullet JE, Boyer JS (1987) Cell wall proteins at low water potentials. Plant Physiol 85: 261-267 Bradford KJ, Chandler PM (1992) Expression of "dehydrin-like" proteins in embryos and seedlingsof Zizania palustris and Oryza sativa during dehydration. Plant Physiol 99: 488494 Bradford KJ, Hsiao TC (1982)Physiological responses to moderate water stress. Tn OL Lange, PS Nobel, CB Osmond, H Ziegler, eds, Physiological Plant Ecology 11, Vol 128. Springer-Verlag, Berlin, pp 263-324 Butler WM, Cuming AC (1993) Differential molecular responses to abscisic acid and osmotic stress in viviparous maize embryos. Planta 189: 47-54 Cavalieri AJ, Boyer JS (1982)Water potentials induced by growth in soybean hypocotyls. Plant Physiol 69: 492496 Cedar H (1988) DNA methylation and gene activity. Cell 53: 3 4 Chyan Y-J (1992)Analysis of maturation-specific genes in soybean seeds: gene structure, desiccation induction and abscisic acid responsiveness. PhD thesis. Universiw of Illinois, UrbanaChampaign Close Tl,