PHYSIOLOGIA PLANTARUM 101: 526-532. 1997Printed in Dentnark - all rights resetted

Copyright © Physiologia Plantarutn 1997

ISSN 0031-9317

Salt-induced changes in protein composition in light-grown callusof Mesembryanthemum crystallinum

H. Emilie Yen, Dianzhong Zhang, Jeng-Horng Lin, Gerald E. Edwards and Maurice S. B. Ku

Yen, H. E., Zhang, D., Lin, J.-H., Edwards, G, E. and Ku, M. S. B. 1997. Salt-inducedchanges in protein composition in light-grown callus of Mesembryanthemum crystal-linum. - Physiol. Plant. 101: 526-532.

The halophyte Mesembryanthemum crystallinum (ice plant) has been suggested as amodel for salt-tolerance in higher plants. To investigate salt-induced changes inpolypeptide pattems at the cellular level, a light-grown callus of M. crystallinum withsubstantial chlorophyll content, was established and the effect of NaCl on the compo-sition of phenol-extracted protein was examined by SDS- and 2D-polyacrylamide gelelectrophoresis (PAGE). SDS-PAGE showed the accumulation of five polypeptideswith estimated molecular masses of 40, 34, 32, 29 and 14 kDa was enhanced by theaddition of 200 mM NaCl to the culture media. The addition of ABA (10 \xM) or man-nitol (400 vaM) did not elicit the same degree of accumulation of these salt-specificproteins. These polypeptides were classified into two groups according to their courseof induction: early-'responsive (40, 34, 29 kDa) and late-responsive (32, 14 kDa) pro-teins. In addition, two polypeptides (20, 18 kDa) were transiently accumulated duringsalt treatment. Further separation of soluble proteins by 2-D gel electrophoresis, eitherisoelectric focusing (IEF) or non-equilibrium pH-gradient electrophoresis (NEPHGE)followed by SDS-PAGE, showed more alterations in accumulation of polypeptides byNaCl than 1-D gel electrophoresis. Overall, levels of more than 30% of basic polypep-tides, detected by NEPHGE/SDS-PAGE, were altered by 200 mM NaCl treatment,while only 10% of neutral and acidic polypeptides, detected by IEF/SDS-PAGE, werechanged. The enhanced expression of these proteins by salt in cultured cells is mostlikely related to the cellular responses to salinity, and not to the mechanism of CAMinduction in this facultative halophyte.

Key words - Ice plant, Mesembryanthemum crystallinum, protein composition, saltstress.

H. E. Yen (corresponding author, e-mail heyen@dragon.nchu.edu.tw) and J.-H. Lin,Dept of Botany, National Chung Hsing Univ., Taichung, Taiwan 40227, ROC; D.Zhang, G. E. Edwards and M. S. B. Ku, Dept of Botany, Washington State Univ., Pull-man, WA 99164-4238, USA.

Introduction

The ice plant, Mesembryanthemum crystallinum, is ahalophyte and requires some salt (e.g. 20 mM NaCl) foroptimal growth of young seedlings (Winter 1973) andcell cultures (Yen et al. 1995). The mode of carbon as-similation in this plant changes from C3 photosynthesisto Crassulacean acid metabolism (CAM) when salt- ordrought-stressed. Such a shift in photosynthetic pathwayis accompanied by marked increases in a set of carbon-metabolism enzymes in the glycolytic and CAM path-ways (Holtum and Winter 1982, Winter et al. 1982,

Forsthoefel et al. 1995). Among them, phospho-enolpyruvate carboxylase (PEPC; EC 4.1.1.31), amarker enzyme for induction of CAM by environmentalfactors, is by far the most thoroughly studied (for review,see Edwards et al. 1996). The shift in the primary car-bon-fixing pathway is thought to be a photosynthetic ad-aptation to water deficit (Bohnert et al. 1995).

In addition to environmental stimuli, the induction ofCAM in the ice plant is developmentally regulated, i.e., itrequires developmental competence for CAM inducibility(Cushman et al. 1990, Cheng and Edwards 1991, Herp-pich et al. 1992). Young ice plants are incapable of per-

Received 19 February, 1997; revised 28 May, 1997

526 Physiol, Plant. 101,1997

forming CAM until the onset of secondary growth, at the medium), as previously described (Yen et al. 1995).stage where auxiliary branches are initiated. Undifferenti- Likewise, callus maintained in the control medium is re-ated cultured ice plant cells exhibit limited CAM inducib- ferred to as control callus. Treatments of NaCl and man-lity. Indeed, heterotrophic cell suspension cultures of ice nitol were made by the addition of concentrated stocksplant have failed to induce PEPC enzyme activity (Yen et to the solid media before autoclaving, whereas treatmental. 1995), accumulation of PEPC protein, and expression with abscisic acid was made by addition from 10 mMof PEPC genes by high salt (Thomas et al. 1992a). Re- sterile ABA stock [(±)-2-cis-4-trans isomers dissolvedplacement of sucrose by starch as the carbon supply in the in 95% ethanol] to cooled, sterilized culture media. Forculture media not only increases the photosynthetic ability the desalting experiment, callus grown for 3 weeks in abut also increases the PEPC activity of cultured cells (Yen medium containing 200 mM NaCl was subcultured to aet al. 1995). However, the degree of induction as mea- fresh medium lacking NaCl. Samples from two or threesured by PEPC activity is far less than that reported for in- replicates were combined at each experimental point,tact plants (Holtum and Winter 1982, Chu et al. 1990).The fully-functional CAM pathway apparently requiresmorphologically organized mature tissues or organs. Protein extraction and determination

While the major survival strategy of cultured cells to Total soluble protein from different treatments was ex-high NaCl is different from that of organized plant tis- tracted following the method of Schuster and Daviessues, proline, a nitrogen-containing compatible solute (1983). Briefly, frozen callus tissues were rapidlywas found to accumulate up to 13-fold when the cells ground at 0°C in 0.5 M Tris buffer, pH 7.5, containingwere stressed with 200 mM NaCl (Yen et al. 1995). 0.1 M KCl, 0.7 M sucrose, 50 mM EDTA and 2% (v/v)However, sugar alcohol osmolytes, which accumulated 2-mercaptoethanol. Preliminary experiments showedto over 9% of the dry matter in stressed leaves, did not that no protein degradation was observed even whenincrease in salt-stressed suspension cells (Adams et al. protease inhibitors, such as leupeptin, were not present1992). Nevertheless, a cell culture of ice plant has been in the grinding medium. After centrifugation for 25 min,shown to retain a moderate degree of salt tolerance (200 35 000 g, supernatant was extracted twice with an equalmM NaCl), as measured by an increase in fresh weight volume of water-saturated phenol. After a phase separa-and the ability to accumulate proline (Yen et al. 1995). tion by centrifugation, the protein in the phenol phaseThe reason why the accumulation of nonreducing sugars was precipitated with 4 volumes of 100% ice-cold meth-requires organized tissues, as in the induction of CAM, anol containing 100 mM ammonium acetate and 2% (v/is not clear but may be linked to photosynthesis. v) 2-mercaptoethanol. The protein precipitate was col-

Changes in the protein pattem in leaves of the ice lected by centrifuging at 5 000 g for 20 min, washedplant have been examined under salt stress (Micha- twice with the methanol solution and once with 100%lowski et al. 1989, Chu et al. 1990) and the most dra- ice-cold acetone containing 2% (v/v) 2-mercaptoetha-matic were associated with the induction of several key nol, air dried and then resuspended in SDS or IEE lysisenzymes for the CAM pathway. Since CAM pathway buffer (Laemmli 1975). Protein content was determinedwas not induced in cultured ice plant cells (Thomas et al. using Pierce's Coomassie Plus protein assay reagent1992a, Yen et al. 1995), we would expect to find differ- (Pierce, Rockford, IL, USA) and an equal amount ofent salt-induced changes in the protein pattem in these protein was loaded on each lane (40 |ag for SDS-PAGEhalophytic cells. The emphasis of this study is on the ex- and 350 |ig for 2-D gel),amination of changes in protein composition before andafter salt treatment in a light-grown callus culture of iceplant. Without the complication of CAM induction or SDS-PAGE and 2-D gel electrophoresis

other tissue-specific responses associated with the whole For SDS-PAGE, the phenol-extracted total soluble pro-plant system, we have found that the levels of several teins were separated electrophoretically in a 7.5-15%polypeptides, especially some basic proteins, were stim- acrylamide linear gradient gel, stablized by a 5-15% su-ulated by addition of NaCl to the culture medium. These crose gradient. Two methods were used for the separa-salt-responsive proteins may be valuable for further tion of protein by 2-D electrophoresis. For the separationanalysis of the general cellular adaptive mechanism to by isoelectric focusing (IEF), protein was dissolved inabiotic stresses in this halophyte. Laemmli's lysis buffer containing 4% (v/v) pH 5-7 am-

pholytes and 1% (v/v) pH 3-10 ampholytes (Biolytes,Abbreviations - IEF, isoelectric focusing; NEPHGE, non-equi- Bio-Rad) and separated in 4% cylindrical gels by elec-hbrium pH-gradient electrophoresis; pi, isoelectric point. trofocusing for 16 h at 300 V. The final pH range of the

IEF gel was 4.2 (acidic end) to 7.5 (basic end). For theMaterials and methods detection of very basic proteins, non-equilibrium pH-f. „ , gradient electrophoresis (NEPHGE) was used (O'FarrellCallus culture ^^ , 1^'7^^ T-u , • J TT J-

et al. 1977). The protem was separated m a pH gradientLight-grown callus of Mesembryanthemum crystallinum from 3 to 10, established by 5% (v/v) pH 3-10 am-L. was established and maintained in a medium (control pholytes for 4 h at 400 V. In order for basic proteins

Physiol. Plant. 101, 1997 5 2 7

leading in the separation, electrophoresis was toward thecathode (-) with the acidic reservoir (10 mM H3PO4) onthe top and the basic reservoir (20 mM NaOH) on thebottom. After separation by charge in the first dimen-sion, the cylindrical gels were equilibrated in SDS bufferand overlaid on top of the linear gradient SDS gels forseparation by size in the second dimension. All theacrylamide gels presented in this study were run at leasttwice using the same protein extracts and identical pro-tein pattems were observed.

Results

The light-grown callus tissue was maintained in the con-trol medium or with the addition of 200 mM NaCl, 400

mM mannitol, or 10 \iM ABA for three weeks. As ex-pected, all the treatments showed very similar proteinpattems although specific differences were apparentamong some treatments, with the most significant asso-ciated with the 200 mM NaCl treatment (Fig. 1, lane 2).The accumulation of several polypeptides correspondingin size to 40, 34, 32, 29 and 14 kDa was increased in thesalt-treated cells but not in the control or the other exper-imental treatments (Fig. 1, arrowheads). The effect ofsalt stimulation was most clearly noticed for the 34- and32-kDa polypeptides, and its effect on the other threepolypeptides was less apparent. The accumulation ofthese polypeptides increased with NaCl concentrationsfrom 50 mM to 300 mM but 25 mM NaCl treatment didnot cause any significant increases (data not shown).

Time-course experiments were performed to determinethe response kinetics of these salt-stimulated polypeptidesto salt stress. When a control callus was transferred to amedium containing 200 mM NaCl, up-regulation of the

Days of 200 mM NaCl treatment

0 10 15 20 25

97

80

66

45

36

29

14

40

343229

14

Fig. 1. SDS-PAGE analysis of total soluble protein extractedfrom 3-week-old callus of control, 200 mM NaCl, 400 mM man-nitol, or 10 \iM ABA treatments. Total soluble protein was phe-nol-extracted, separated by 7.5-15% gradient SDS-PAGE, andstained with Coomassie brilliant blue R-250. Correspondingmolecular masses of protein standards are indicated on the left.Arrowheads on the right refer to polypeptides (with estimatedmolecular masses of 40, 34, 32, 29 and 14 kDa, respectively)apparently up-regulated by salt treatment.

97

40

343229

2018

14

Fig. 2. Time-course of induction of salt-regulated polypeptidesduring salt treatment. Callus was grown in control medium for 3weeks and then transferred to medium containing 200 mMNaCl, and samples were collected 5, 10, 15, 20, and 25 days af-ter transfer. Total soluble protein from harvested cells was phe-nol-extracted, separated by 7.5-15% gradient SDS-PAGE andstained with Coomassie brilliant blue R-250. The salt-upregu-lated polypeptides (40, 34, 32, 29, 20, 18 and 14 kDa, arrow-heads on the right) exhibited differential accumulations duringthe course of salt induction. For the 40-, 34- and 29-kDapolypeptides, increased accumulations were found 5 days aftersalt treatment; for the 32- and 14-kDa polypeptides, 15 days;and the 20- and 18-kDa polypeptides were transiently increasedbetween 5 and 10 days and decreased thereafter.

528 Physiol. Plant. 101, 1997

Days after desalting

10 15 20 25

97

29

14

40

343229

14

Fig. 3. Time-course of disappearance of salt-regulated polypep-tides during desalting. Callus was grown in 200 mA/ NaCl for 3weeks and then transferred to control medium, and sampleswere collected 5, 10, 15, 20, and 25 days after transfer. Total sol-uble protein from harvested cells was phenol-extracted, sepa-rated by 7.5-15% gradient SDS-PAGE and stained with Coo-massie briUiant blue R-250. For the 40-, 34- and 29-kDapolypeptides, decreased accumulations were found 5 days aftertransfer; for the 32- and 14-kDa polypeptides, 15 days; and forthe 20- and 18-kDa polypeptides were transiently increased be-tween 10 and 15 days and decreased thereafter.

40-, 34- and 29-kDa polypeptides appeared within 5 days(referred to as early-responsive) of the treatment and con-tinued increasing with culture times up to 25 days (Fig. 2).Accumulation of the 32- and 14-kDa polypeptides did notappear until after 15 days of 200 mM NaCl treatment(late-responsive). Furthermore, by transferring callusgrown in 200 mM NaCl back to the control medium (de-salting), those polypeptides which accumulated quicklyduring induction (e.g. 40, 34 and 29 kDa) also decreasedquickly. Those which took longer time to accumulate (e.g.32 and 14 kDa) also decreased more slowly (Fig. 3). Ac-cumulation of two additional polypeptides with molecularmasses of 20 and 18 kDa was found to be transiently en-hanced by salt between 5 and 10 days of treatment and de-creased thereafter. Changes in these two polypeptides inresponse to salt treatments were not apparent in the earlierexperiments since the proteins were extracted from 3-week-old callus. The results of this experiment emphasizethe importance of examining stress responses in a time-dependent manner.

Analyses of protein composition by SDS-PAGE pre-sented thus far suggest that the polypeptide profiles ofthe salt-grown callus differ in several respects fromthose of the control callus. However, the resolution oftotal cellular protein by SDS-PAGE is somewhat limitedbecause not all polypeptides having the same relativemolecular mass are salt-responsive. Therefore, a furtherseparation of phenol-extracted protein by 2D-PAGE wasmade to clarify the differences. Isoelectric focusing(IEF) followed by SDS-PAGE was used to separate pro-teins extracted from the control callus and callus grownin the presence of 200 mM NaCl. A comparison of thepolypeptide pattems was made on the Coomassie blue-stained 2-D gels (Fig. 4). Overall, more than 200 spotswere identified and pattems of protein compositionlooked very similar between control and salt treatment.Salt treatment increased the amount of 7 clear spots (Fig.4, small arrows) and decreased the amounts of more than17 observable polypeptides (Fig. 4, small arrowheads).The number of polypeptides (with pi between 4.2 and7.5) induced by salt was far less than the number ofpolypeptides suppressed as revealed in this IEF/SDS-PAGE system. Up-regulation of 34- and 14-kDa poly-peptides identified by SDS-PAGE was also found in thecorresponding position in the IEF/SDS-PAGE (Fig. 4,arrowheads), but it is unlikely that the amounts accumu-lated contributed to the altered protein profile observedby SDS-PAGE.

The differences in the protein profiles between SDS-PAGE and IEF/SDS-PAGE were noticeable. Since thefinal pH gradient in the IEF gels ranged from pH 4.2 to7.5, proteins with pi higher or lower than this range wereexcluded from this one-dimensional separation. Manysalt-induced polypeptides are known to have pi valueshigher than 8.0 (Bol et al. 1990, Koiwa et al. 1994).Therefore, a non-equilibrium pH-gradient electrophore-sis (NEPHGE) in the first dimension followed by anSDS-PAGE in the second dimension were performed todetect changes in basic polypeptides. The results fromthe NEPHGE/SDS-PAGE showed that, within 90 detect-able spots, salt induced the accumulation of 17 basicpolypeptides (Fig. 5, small arrows), while 12 noticeabledecreases in polypeptide level were observed (Fig. 5,small arrowheads). Under salinity, the most significantaccumulation was observed for 3 very basic polypep-tides of 40, 39 and 27 kDa. The salt-upregulated proteinsobserved by the SDS-PAGE (e.g. 40, 34, 32, 29 and 14kDa) were all found in the corresponding positions in theNEPHGE/SDS-PAGE system (Fig. 5, arrowheads).Overall, a comparison of Figs 4 and 5 shows that salt al-ters 30% of the levels of basic polypeptides but affectsonly 10% ofthe levels of acidic and neutral polypeptidesin cultured ice plant cells.

Discussion

To begin to evaluate the mechanisms of salt tolerance inthe halophyte M. crystallinum (ice plant), the effect of

Physiol. Plant. 101, 1997 529

Control

pH 7.5

200 mM NaCl

4.2 pH 7.5

— 97

~ . 80

— 66

— 45— 36

29 —

J T— 14

->1EF

Fig. 4. Effects of salt on polypeptide profile separated by IEF/SDS-PAGE gel electrophoresis. Total cellular protein, extracted from3-week-old control (left panel) and 200 mM NaCl-grown callus (right panel), was separated by isoelectric focusing (equilibrium pH)gel electrophoresis in the horizontal direction followed by 7.5-15% gradient SDS-PAGE in the vertical direction. The final pH rangein the IEF gels was 4.2 to 7.5. Proteins were stained with Coomassie brilliant blue R-250. The pH values for the first dimension gelare indicated at the top and the molecular mass markers used in the second dimension gel in the middle of the figure. Polypeptidelevels that were increased by salt treatment are marked by small arrows, while those decreased by salt treatment are marked by smallarrowheads. Unidentifiable polypeptides, including vague spots and long stretches, were not considered.

salt stress on alterations of the cellular protein profile stress) or ABA, which is known to induce a shift in thewas examined in light-grown callus of the plant by SDS- photosynthetic mode in this plant (Edwards et al. 1996).PAGE. These salt-specific changes were not found in The time-course experiments demonstrated that salt-spe-callus treated with mannitol (mimicking water-deficit cific polypeptides respond differently and reversibly to

Control

Acidic Basic

200 mM NaClAcidic Basic

978066

45

36

29

14

C34t32t29t27

V

NEPHQE

Fig. 5. Effects of salt on polypeptide pattem separated by NEPHGE/SDS-PAGE. Total cellular protein, extracted from 3-week-oldcontrol (left panel) and 200 mM NaCl-grown callus (right panel), was separated by non-equilibrium pH-gradient (NEPHGE) gelelectrophoresis in the horizontal direction followed by 7.5-15% gradient SDS-PAGE in the vertical direction. Proteins were stainedwith Coomassie brilliant blue R-250. The acidic and basic ends of the first dimension gel are indicated at the top and the molecularmass markers used in the second dimension gel are indicated in the middle of the figure. Polypeptide levels that were increased bysalt treatment are marked by small arrows, while those decreased by salt treatment are marked by small arrowheads. Unidentifiablepolypeptides were not considered.

530 Physiol. Plant. 101, 1997

the same degree of salt stress, with some of them (40, 34 above 100 mM (Yen et al. 1995). A combined electro-and 29 kDa) accumulating right after the addition of salt, phoretic system using non-equilibrium pH-gradient elec-some (32 and 14 kDa) accumulating at the later stage as trophoresis (NEPHGE) followed by SDS-PAGE enabledthe stress persisted, while others (20 and 18 kDa) accu- us to separate proteins containing a high ratio of nega-mulated transiently during the treatment. The results tively-charged amino acid residues. All of the fivesuggest that the regulation of these salt-specific polypep- polypeptides, detected by the 1-D system, were found totides differs in accumulation kinetics and may not all be accumulate to high levels in the NEPHGE gel (Eig. 5).triggered by the same inductive pathway in this halo- The 14- and 29-kDa bands, whose accumulation was in-phyte. duced by salt as shown in SDS-PAGE, were further sep-

In addition to enzymes involved in the CAM pathway, arated into three or four polypeptides with different pithere are several low molecular mass salt-responsive values in the NEPHGE/SDS-PAGE, and particularly thepolypeptides with no known enzymatic function, which level of the most basic one was enhanced by salt. Sev-have been identified in the ice plant, either via analysis eral stress-related proteins, such as the basic pathogene-of in vitro translation products (Michalowski et al. 1989) sis-related proteins, have pi values >8 (Bol et al. 1990,or through analysis of extracted cellular protein (Chu et Koiwa et al. 1994). The dramatic increase in the accu-al. 1990, Yen et al. 1994). These include osmotin-like mulation of certain basic proteins is noteworthy, since(Yen et al. 1994) and germin-like proteins (Michalowski they may serve as a more stable form in the presence ofand Bohnert 1992). The accumulation of osmotin or ger- a high concentration of Na"" in the cytoplasm (Singh etmin has been shown to be sensitive to the changes in tur- al. 1989).gor pressure in the cells (Singh et al. 1987, Hurkman et Salt-induced changes in polypeptide patterns haveal. 1991). Other known salt-induced polypeptides found been noticed for almost every plant species examined,in other plants do have similar molecular masses, such but the roles of these salt-induced polypeptides under sa-as salT, a 15-kDa protein isolated from rice (Claes et al. Unity stress are largely undetermined (for review see1990), group 3 Lea proteins, a family of proteins with Hurkman 1990). The complexity is mainly associatedmolecular masses around 30 kDa (Dure et al. 1989), and with different levels of salt tolerance and/or differenta 40-kDa osmotically-induced protein that is immuno- strategies adopted by various plant species (even differ-logically related to dehydrin (Close and Lammers 1993). ent parts of a plant) in coping with salinity stress. SomeThe accumulation of these proteins is not salt-specific plant proteins that are induced by salt, such as osmotinbecause they can also be induced by chilling or dehydra- or germin, may be suppressed by the same concentrationtion stresses. of NaCl in other species.

The term "salt-specific" used here refers to polypep- This study has identified specific proteins whose ac-tides that accumulate only under prolonged 200 mM cumulation is significantly up-regulated by salt. TheseNaCl treatment, but not to water deficit (400 mM manni- proteins could be useful targets for identifying the possi-tol) or ABA (10 |iM). Other factors, mainly associated ble mechanisms of salt tolerance in this halophyte. Thiswith the tissue culture techniques, may also affect their report also shows the importance of using different gelaccumulation. Biotic stresses, such as wounding, are systems (IEE vs NEPHGE) to search for stress-relatedknown to alter the overall protein accumulation with an proteins. The ice plant cell line is also a suitable materialincrease in the numbers of pathogenesis-related proteins for studying the signal transduction pathways for saltin cultured tobacco cells (Takeda et al. 1990). Exoge- stress because of the synchronous induction of the pro-nous plant growth regulators such as kinetin and 2,4-D, teins by salt, and the recovery of a large amount of mate-which promote growth and division of ice plant cells in rial in a relatively short time,culture, can have a combined effect with NaCl. Cytoki-nin alone is an efficient elicitor of PEPC activities, os- Acknowledgments - Thanks are due to Mr Shyi-kae Yen and Mrmolyte accumulation and an increase of osmotin-like Yen-Shun Chen for help in the preparation of manuscript and^^. • • 1 J • 11 • 1 «. /T i- ^ figures. This work was supported in part by National Scienceproteins in hydropomcally-grown ice plant (Thomas et council of Taiwan grant NSC 86-2311 B-005-013 to H. E. Yal. 1992b, Dai et al. 1994). Whether the 0.5 [iM kinetinin our culture medium had an additive effect to 200 mMNaCl on the accumulation of these "salt-specific" Referencespolypeptides requires further investigation. Adams, P, Thomas, J. C, Vemon, D. M., Bohnert, H. J. &

The results from the IEF/SDS-PAGE (Eig. 4) showed Jensen, R. G. 1992. Distinct cellular and organismic re-that salt treatment resulted in decreased accumulation of sponses to salt stress. - Plant Cell Physiol. 33: 1215-1223.many proteins in the pi range between 4.2 and 7.5. Simi- ^°h"ert, H. B., Nelson D. E. & Jenseri R G. 1995. Adaptationsi n , , ^ , , , , , .^^^^ to environmental Stresses.-Plant Cell 7: 1099-1111.lar results have been reported by Meyer et al. (1990), BOI, J. F, Linthorst, H. J. M. & Comelissen, B. J. C. 1990. Plantnamely, that levels of several transcripts decreased in pathogenesis-related proteins induced by virus infection. -salt-stressed plants. These proteins are possibly cellular Annu. Rev. Phytopathol. 28: 113-138.protdns with strucural or enzymatic funcons, because '^'Xi^!';,^^^^^^^^^^^ K Scellular metabohsm can slow down and the growth rates lacean acid metabolism in Mesembryanthemum crystalli-become reduced as the NaCl concentration is raised «Mm.-Plant Cell Environ. 14: 271-278.

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Edited by C. H. Bomman

532 Physiol. Plant. 101, 1997