Changes in protein synthesis during drought ...

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roots of jack pine seedlings (Pinus banksiana Lamb.) MICHAEL B. .... sucrose) and centrifuged in an SW28 rotor (Beckman, Palo Alto, CA) at 80,000 g for. 2.5 h.

Tree Physiology 0 1994 Heron

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Publishing-Victoriu,

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Changes in protein synthesis during drought conditioning in roots of jack pine seedlings(Pinus banksiana Lamb.) MICHAEL B. MAYNE,‘,* MOHAN SUBRAMANIAN,‘,2 TERENCE J. BLAKE,2,3 JOHN R. COLEMAN’%* and EDUARDO BLUMWALD’,* ’ Department of Botany, University of Toronto, 25 Willcocks Street, Toronto, Ontario MjS 3B2, Cunudu 2 Center,for Plant Biotechnology, University of Toronto, Ontario M5.Y 3B2, Canada ’ Faculty of Forestry, University of Toronto, 33 Willcocks Street, Toronto, Ontario M.5S 3B3, Canada Received

June 7. 1993

Keywords: drought stress, protein uwumulation, control.

seedling sunsival, transcriptional and trunslutional

Introduction The degree of drought resistance exhibited by a particular species is related to one of four basic responses to drought stress: (1) dehydration tolerance-the ability of cells and tissues to tolerate and withstand reduced tissue water potential during water deficit; (2) dehydration avoidance-the ability of plants to prevent reduction of tissue water potentials; (3) drought escape-the ability of plants to complete their life cycle within the period of water availability thereby not experiencing water deficit; and (4) drought recovery-the ability of plants to resume growth and finish their life cycle after a water-deficit period (O’Toole and Chang 1979, Newton et al. 1991). Of these drought responses, dehydration tolerance and dehydration avoidance are thought to be important components of drought resistance in forest species (Newton et al. 1991). One procedure for improving forest seedling survival is to condition nurserygrown plants by exposure to variable environments before transplantation to the

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The impact of drought conditioning on the ability of eight-week-old jack pine (Pinus hanks&a Lamb.) seedlings to withstand drought was assessed. Two progressive cycles of drought conditioning significantly increased the survival of seedlings subjected to a subsequent prolonged drought. The in vivo accumulation of several root membrane proteins during drought conditioning was correlated with an increase in seedling survival. A group of root proteins, ranging in molecular mass from 43 to 47 kDa, increased accumulation during one cycle of drought conditioning and to a lesser extent during two cycles of drought conditioning. The accumulation of several low molecular mass membrane and soluble proteins also increased during drought conditioning, suggesting that these proteins may play an important role in the enhancement of drought tolerance. In vitro translation studies showed a general increase in the abundance of protein products encoded by mRNAs from drought-conditioned seedlings. Although the majority of the in vitro translation products appeared in both control and drought-conditioned seedlings, one mRNA encoding a 15 kDa translated protein was more prominent during the second cycle of drought conditioning.

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Materials

and methods

Plant material Jack pine seeds were obtained from a northwestern Ontario general seed collection and sown in a 2/l (v/v) peat/vermiculite mixture. The seedlings were grown as previously described (Blumwald et al. 1989) in a greenhouse for eight to ten weeks at 24 “C in an 18-h photoperiod in which natural day length was extended with high pressure sodium vapor lamps (200 pmol m-2 s-l, minimum light intensity). Plant drought conditioning Seedlings were transferred to a Conviron 15 E growth chamber, where conditioning treatments commenced under the same conditions as in the greenhouse. Three degrees of drought conditioning of jack pine seedlings were obtained by exposing the seedlings to cycles of progressive non-lethal drought. Cycle A drought-conditioned seedlings were obtained by withholding water over a 3-day period until the xylem water potential reached -1.1 MPa as determined with a pressure chamber (Arinad 2, ARJ, Israel). To generate Cycle B drought-conditioned seedlings, Cycle A drought-conditioned seedlings were rewatered to full turgor for a period of three days and then water was withheld for an additional three days at which time xylem water potential dropped to -1.1 MPa. Cycle C drought-conditioned seedlings were obtained by subjecting Cycle B drought-conditioned seedlings to an additional cycle of watering to full turgor (three days) followed by withholding water (three days) until xylem water potential reached -1.1 MPa. At the end of each conditioning cycle and before rewatering, some of the seedlings were removed from the soil and prepared for in vivo protein labeling and RNA extraction. The remaining seedlings were used for survival experiments (see below).

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forest site. Drought conditioning to enhance drought resistance is a common procedure. Although the mechanism underlying drought conditioning is not known, it has been suggested that when a plant is exposed to a moderate drought stress, an enhanced drought resistance response is triggered (Levitt 1980). Modification of gene expression can occur in response to drought (Bray 1988, Gomez et al. 1988, Guerrero and Mullet 1988, Mundy and Chua 1988). Although there are reports describing the synthesis of low molecular mass proteins during drought in various angiosperms, for example, tomato (Bray et al. 1990) and alfalfa (Luo et al. 1992), there have been few studies on changes in gene expression in gymnosperms during drought and, particularly, following drought conditioning. We have examined the effects of drought conditioning on the ability of jack pine (Pinus banks&a Lamb.) seedlings to survive prolonged water stress. In addition, we have described changes in root-localized polypeptides that occur during drought conditioning and have analyzed the impact of drought conditioning treatments on mRNA abundance by in vitro translation.

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Seedling survival For seedling-survival experiments, eight-week-old jack pine seedlings at the end of Cycle A, B or C were rewatered and maintained at full turgor for seven days. At this time, the seedlings were subjected to prolonged drought by withholding water for varying periods up to nine days. Over this period, xylem water potential values declined from -0.2 MPa at Day 0 to -5.5 MPa at Day 9. Following the prolonged drought, the seedlings were returned to normal greenhouse growing conditions and allowed to recover for three weeks. Seedling survival was then estimated according to Koppenaal et al. (1991).

In vivo labeling of seedlings

Protein extraction and determination of [35S]-methionine incorporation Proteins were extracted from excised root segments and purified as previously described by Blumwald et al. (1989) with the following modifications. The homogenization buffer included a final concentration of 10% insoluble PVP and 1 mol mm3 benzamidine. For isolation of membrane-bound proteins, the microsomal pellet was resuspended and layered onto discontinuous sucrose gradients (20,28 and 34% (w/v) sucrose) and centrifuged in an SW28 rotor (Beckman, Palo Alto, CA) at 80,000 g for 2.5 h. Enriched tonoplast and plasma membrane fractions (Blumwald et al. 1989) were collected from the O-20% and 28-34% interfaces, respectively, and immediately frozen in liquid nitrogen. Soluble proteins were purified from the microsomal supematant by precipitation with TCA followed by centrifugation at 700 g. To solubilize lipids, the collected pellet was washed with 50 ml l/l (v/v) cold (4 “C) 100% acetone and 95% ethanol, and stored overnight at -20 “C. Precipitated proteins were collected by centrifugation, lyophilized and then frozen in liquid nitrogen. To determine [“?S]-methionine incorporation, immediately following the drought conditioning cycles (A, B and C), total protein in the root tissue homogenate was precipitated on ice with 10% TCA (final concentration) and collected by centrifugation at 700 g. Incorporation of radioactive label was calculated per g of total protein. Aliquots containing equal amounts of total protein were spotted onto GFC filter paper, dried, washed once with 10% TCA, and their radioactivity determined by liquid scintillation spectrometry.

mRNA Isolation Root tissue from approximately

200 seedlings was ground in the presence of liquid

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Approximately four hundred intact seedlings (at the end of Cycle A or Cycle B) were removed from the soil and their roots submerged in a 500 ml solution containing 0.4 mol mm3 mannitol (osmotic potential = -1.0 MPa) and 1 g mm3 fertilizer solution (20,20,20 N,P,K) containing 1.42 MBq (specific activity of 41.26 TBq mmol13) L-[35S]-methionine (ICN, Irvine, CA). Incubation of the seedlings in the aerated [35S]-methionine solution was allowed to proceed for 16 h under normal greenhouse growth conditions. Control seedlings were incubated under the same conditions in a fertilizer solution in the absence of mannitol.

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In vitro translation In vitro translation of mRNAs isolated from the seedlings was achieved by the protocol described in the Translation In Vitro Technical Manual (Promega Madison, WI). One pg of mRNA (optical density ratio 260/280 nm = 1.9) was used for each in vitro translation. Reactions were carried out in a volume of 100 ~1 containing 70 pl of rabbit reticulocyte lysate, 600 units ml-’ ribonuclease inhibitor (Promega Madison, WI), 2 pmol mm3 amino acid mixture (minus methionine), 10 mol mm3 magnesium acetate and 6.2 TBq mol mm3 of [35S]-methionine (Amersham, Mississauga, Ontario). Individual reactions were incubated for 90 min at 30 “C.

Polyacrylamide gel electrophoresis and autoradiography Preparation of protein samples of in vivo and in vitro labeled polypeptides for two-dimensional SDS-PAGE were carried out as previously described (Barkla and Blumwald 1991). The accumulation of root-localized proteins synthesized during a 16-h labeling period in Cycle A and Cycle B drought-conditioned seedlings was analyzed by two-dimensional PAGE. Protein profiles obtained from tonoplast, plasma membrane, and soluble fractions, were also compared with labeled polypeptides isolated from control seedlings. Samples were heated at 70 “C for 2 min and quickly chilled on ice for 2 min before loading onto the gel. To analyze polypeptides that accumulated in vivo, equal amounts of protein (100 pg) were loaded onto 2-D PAGE gels (12% acrylamide). To analyze in vitro translation protein products, 500,000 counts per minute of TCA-precipitable protein products from mRNAs from control and drought-conditioned seedlings were loaded onto 2-D PAGE gels (12% acrylamide). Isoelectric points were estimated as described by Barkla and Blumwald (1991). Autoradiograms were obtained by exposing the dried gels to Hyperfilmpmax (Amersham, Mississauga, Ontario) for six (in vivo labeled polypeptides) or three weeks (in vitro labeled polypeptides) at -70 “C.

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nitrogen and stored at -70 “C. Extraction of total RNA from the frozen samples was carried out according to Schneiderbauer et al. (1991) with some modifications. Following phenol/chloroform/isoamyl alcohol extraction (24/24/l, v/v), total RNA was precipitated from the aqueous phase with 2.5 volumes of ethanol in the presence of a final concentration of 30 mol me3 sodium acetate (pH 5.2) at -70 “C for 1 h. Excess polyphenols were removed by extracting the RNA solution twice with 30% (w/v) PEG 6000. Cesium chloride was added to the RNA solution to a final concentration of 2.4 kmol m-3 and 3 ml of this solution was layered onto a 0.8 ml cushion of 5.7 kmol mm3 cesium chloride and centrifuged at 376,000 g (rotor TLA100.4, Beckman, Palo Alto, CA) in an Optima TL centrifuge (Beckman, Palo Alto, CA) for 2.75 h. The total RNA pellet was dissolved in RNase-free water and purified by precipitation with 2.5 volumes of 4 kmol mm3 sodium acetate (pH 7.0) at -70 “C for 2 h. Poly (A+) RNA was obtained from the total RNA preparation by the protocol described for the PolyATtract mRNA Isolation System (Promega Biotech, Madison, WI).

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Results Seedling survival Eight-week-old jack pine seedlings were grown under normal greenhouse growth conditions and then drought conditioned. The drought conditioning treatments did not result in seedling mortality. Conditioned seedlings were subjected to prolonged drought stress over a nine-day period. When control and Cycle A drought-conditioned seedlings had water withheld for eight or more days (xylem water potential I-4.8 MPa) followed by a recovery period, more than 50% of the seedlings died (Figure 1). In contrast, Cycle B drought-conditioned seedlings exhibited enhanced survival compared with control plants. Three cycles of drought conditioning did not further enhance seedling survival (Figure 1).

The amount of [35S]-methionine uptake was determined by measuring the radioactivity in aliquots of TCA-precipitated, total-seedling, root protein. Incorporation of [Sj5]-methionine by Cycle A, B and C drought-conditioned seedlings was increased twofold compared with control seedlings (Figure 2).

In vivo protein labeling The 2-D PAGE and autoradiographic analyses revealed an increased accumulation of tonoplast- and plasma-membrane-associated proteins during both the Cycle A (Figures 3b and 4b, respectively) and Cycle B drought-conditioning treatments (Figures 3c and 4c, respectively). A prominent group of proteins (43 to 47 kDa, p1 ranging from 5.7 to 6.5) associated with the tonoplast (Figure 3b) and plasma membrane fractions (Figure 4b) from roots of Cycle A drought-conditioned seedlings and to a lesser extent from Cycle B drought-conditioned seedlings (Figures 3c and 4c) were absent from the autoradiograms of the control roots (Figures 3a and 4a).

CONTROL

CYCLE A

CYCLE B

CYCLE C

Figure 1. Seedlings were drought-conditioned, allowed to recover and then severely drought stressed as described in Materials andmethods. Percentage survival of drought-conditioned seedlings was estimated according to Koppenaal et al. (199 1). Cycle A is one cycle of drought conditioning. Cycle B and Cycle C are two and three cycles of drought conditioning, respectively. Statistical analysis showed that the means of the controls and Cycle A (a) and Cycle B and C (b) were significantly different (P = 0.05). Values are means k SD (n = 50).

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Radiolabel uptake

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Figure 3. Two-dimensional PAGE of tonoplast membrane polypeptides synthesized in roots during Cycle A and Cycle B drought conditioning. Proteins were radiolabeled with [35S]-methionine as described in Materials and methods. The abundance of proteins enclosed by circles increased during Cycle A or Cycle B drought conditioning, whereas the abundance of proteins enclosed by squares decreased during Cycle A or Cycle B drought conditioning. (a) Non-conditioned seedlings; (b) Cycle A droughtconditioned seedlings; (c) Cycle B drought-conditioned seedlings. Figure is representative of five independent experiments.

Two 31 kDa polypeptides were prominent in the tonoplast fractions obtained from roots of Cycle A (~15.5) and Cycle B (~15.3) drought-conditioned seedlings but were absent in control tissue (Figure 3). The accumulation of a 40 kDa (~15.2) tonoplastassociated polypeptide was repressed during Cycle A and Cycle B drought conditioning (Figure 3). Several smaller polypeptides (molecular mass less than 25 kDa) were detected only in the tonoplast and plasma membrane fractions from Cycle B droughtconditioned seedlings (Figures 3 and 4). In contrast with the membrane proteins, soluble proteins isolated from roots of control seedlings appeared to accumulate at a greater rate during the labeling period

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Figure 2. Incorporation of [%I-methionine into root proteins during the labeling period as outlined in the Materials and methods. Total root protein was extracted and precipitated as described in the Materials and methods. Incorporation rates were calculated based on equal amounts of protein, and radioactivity was determined by liquid scintillation spectrometry. Values are means * SD (n = 3).

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than soluble root proteins of Cycle B drought-conditioned seedlings (Figure 5). It should be noted that, although many differences were not apparent, autoradiographic analysis following 2-D PAGE of equal amounts of soluble protein showed increased accumulation of some soluble proteins ranging in molecular mass from 14 to 18 kDa (p1 ranging from 5.0 to 6.4) and molecular masses of 30 to 32 kDa (~15.8) in roots of seedlings subjected to the second cycle of drought conditioning (Figure 5b) compared with the control seedlings.

In vitro translation In vitro translation studies showed a general increase in the abundance of protein products encoded by mRNAs from Cycle B drought-conditioned seedlings compared with the control seedlings. In particular, several low molecular weight translation products (p1 ranging from 5.0 to 5.6) encoded by mRNAs from drought-conditioned seedlings increased in abundance relative to translation products obtained from control seedling mRNAs (Figure 6b). Although the majority of the in vitro translation products were present in both control and drought-conditioned seedlings, an mRNA encoding a 15 kDa product (p1 6.5) was more prominent in seedlings subjected to the second cycle of drought conditioning.

Discussion Jack pine seedlings exhibited enhanced survival of prolonged drought after two conditioning cycles, suggesting that the seedlings modified their physiology during the drought-conditioning cycles and that these modifications resulted in enhanced drought resistance. It is generally thought that total protein accumulation is decreased by drought (Hanson and Hitz 1982, Hulbert et al. 1988, Valluri et al. 1988, 1989, Bray 1990).

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Figure 4. Two-dimensional PAGE of plasma membrane polypeptides synthesized in roots during Cycle A or Cycle B drought conditioning. Proteins were radiolabeled with [3’S]-methionine as described in Materials and methods. The abundance of proteins enclosed by circles increased during Cycle A or Cycle B drought conditioning. (a) Non-conditioned seedlings; (b) Cycle A drought-conditioned seedlings; (c) Cycle B drought-conditioned seedlings. Figure is representative of five independent experiments.

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Figure 6. Two-dimensional PAGE of in vitro translation products from mRNAs extracted from roots of non-conditioned or Cycle B drought-conditioned seedlings. Products enclosed by circles increased in abundance during two cycles of drought conditioning. (a) Translation products using mRNAs of non-conditioned seedlings; (b) Translation products using mRNAs from Cycle B drought-conditioned seedlings. Figure is representative of three independent experiments.

However, we observed that the incorporation of [3sS]-methionine into a TCA-precipitable fraction doubled during Cycle A drought conditioning and remained high during Cycle B drought conditioning (Figure 2). The increase in label incorporation into the TCA-precipitable fraction during drought conditioning was not due to changes in [“?S]-methionine uptake because both control and drought-conditioned seedlings displayed comparable rates of radiolabel uptake (63,000-70,000 cpm mm-‘).

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Figure 5. Two-dimensional PAGE of soluble polypeptides synthesized in roots during the second cycle of drought conditioning (Cycle B). Proteins were radiolabeled with [35S]-methionine as described in Materials and methods. Abundance of proteins enclosed by circles increased during Cycle B drought conditioning. (a) Soluble proteins from non-conditioned seedlings; (b) Soluble proteins from Cycle B drought-conditioned seedlings. Figure is representative of five independent experiments.

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Analysis of labeled proteins indicated that the drought-conditioning treatments resulted in the accumulation of several tonoplast and plasma membrane polypeptides, and the reduction of many soluble proteins (Figure 5) in both Cycle A and Cycle B drought-conditioned seedlings (Figures 3 and 4). Some polypeptides were expressed solely in response to drought in both Cycle A and Cycle B drought-conditioned seedlings. The accumulation of membrane-associated 43 to 47 kDa proteins is of particular interest, because polypeptides of a comparable molecular mass are expressed in slash pine hypocotyl sections when exposed to mannitol-induced drought (Valluri et al. 1989). In our study, these polypeptides were observed in both tonoplast and plasma membrane fractions. Although the membrane fractions were enriched in tonoplast and plasma membranes (Blumwald et al. 1989), these fractions are contaminated by proteins associated with other endo-membranes (in particular Golgi). The proteins expressed in response to the drought treatments did not accumulate to concentrations sufficient for detection by silver or Coomassie stain. Several membrane-bound and soluble proteins that increased during Cycle A drought conditioning also remained high during Cycle B drought conditioning. It is possible that proteins synthesized in response to a mild drought are also required to elicit an enhanced drought tolerance response. For example, a group of plasma membrane proteins within the molecular mass range of 43 to 47 kDa (p1 ranging from 5.7 to 6.5) showed increased accumulation in both Cycle A and Cycle B drought-conditioned seedlings. Drought conditioning may enhance drought tolerance through gene regulation thereby changing mRNA populations. Although few changes in mRNA translation products were observed during drought conditioning, an abundant 15 kDa in vitro translation product was obtained with mRNAs isolated from Cycle B drought-conditioned seedlings, but not with mRNAs isolated from control seedlings (Figure 6b). Stress-induced in vitro translation products of similar molecular weight have also been identified in tomato (Bray 1988, Cohen and Bray 1990, Ho et al. 1991) and a protein of similar molecular weight, encoded by abscisic acid-responsive genes (rub genes), has been reported (Yamaguichi-Shinozaki et al. 1989, Vilardelli et al. 1990, Plant et al. 1991). Joshi et al. (1992) suggested that seedlings store mRNAs when fully watered and only utilize these transcripts to enhance drought tolerance. A result supporting this possibility was recently reported by Reviron et al. (1992) who identified a 22 kDa protein that accumulated in drought-hardened leaves of Brussica naps. The transcript for this protein was present in control and drought-hardened leaves. The lack of many new in vitro translation products from Cycle B drought-conditioned seedlings suggests that the acclimation of drought tolerance could rely partly on posttranslational modifications of proteins; however, the abundant 15 kDa in vitro translated product obtained with mRNA from Cycle B drought-conditioned seedlings may be indicative of induced transcriptionally regulated mechanisms. In summary, progressive cycles of drought conditioning significantly increased the survival of jack pine seedlings when subjected to a subsequent severe drought, and

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the synthesis of several root proteins during drought conditioning increased survival of the seedlings.

ET AL.

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Acknowledgments Research Canada.

was supported

by a Strategic

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Sciences

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References

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