DevelopmentalControl of Crassulacean Acid Metabolism ... · transcriptional activation of Ppcl by salt stress in 9-week-old plants is no longer observed despite an increase of both

Plant Physiol. (1990) 94, 1137-11420032-0889/90/94/11 37/06/$01 .00/0

Received for publication May 21, 1990Accepted July 12, 1990

Developmental Control of Crassulacean Acid MetabolismInducibility by Salt Stress in the Common Ice Plant1

John C. Cushman*, Christine B. Michalowski, and Hans J. Bohnert

Department of Biochemistry, Biological Sciences West, University of Arizona, Tucson, Arizona 85721

ABSTRACT

Ice plant (Mesembryanthemum crystallinum) is a facultativehalophyte that responds to water stress in the form of drought orhigh salinity by switching from C3 photosynthesis to Crassulaceanacid metabolism (CAM), a physiological adaptation that increaseswater conservation. Although CAM is clearly environmentallycontrolled, and reversible upon removal of water stress, thecompetence to switch is developmentally determined. We havedemonstrated this by measuring three parameters in the expres-sion of a gene encoding a stress-specific isoform of a key enzymeof CAM, phosphoenolpyruvate carboxylase (PEPCase, Ppcl): (a)protein accumulation; (b) steady-state amounts of mRNA; and (3)transcriptional activity in isolated nuclei. Young plants (3 weeksof age) show little induction of PEPCase protein, mRNA, or tran-scription when stressed. In contrast, salt stress elicits a stronginduction at all three levels of expression at 6 weeks of age. By9 weeks of age, plants have already accumulated PEPCaseprotein and mRNA without being stressed. More importantly,transcriptional activation of Ppcl by salt stress in 9-week-oldplants is no longer observed despite an increase of both PpclmRNA and protein. From these results we suggest that a devel-opmental program exists that regulates PEPCase transcriptionand mRNA stability. This program appears to be synchronizedwith the climatic conditions in the plant's native environment.

Developmental status governs many different aspects of a

plant's life cycle, particularly those involved in the transitionfrom vegetative to reproductive growth. Comparatively littleis known, however, about the developmental mechanismsinvolved in ecophysiological adaptations to environmentalstress. We have exploited the phenomenon ofCAM inductionin the common ice plant (Mesembryanthemum crystallinum)as a model for monitoring the hierarchy of stress responsesthat are invoked during plant development. CAM representsan ecological adaptation to and environments that is foundpredominantly, although not exclusively, in succulent plants.CAM plants assimilate external CO2 with minimal water lossby opening their stomata only at night when evaporativewater loss is low (1 1). Nocturnal C02 fixation into principallymalic acid, which is localized to the vacuole, is performed by

' Supported by grants from the U.S. Department of Agriculture(CRGP-89-37264-4711) and Arizona Agricultural Experiment Sta-tion (ARZT- 136334/6) to H.J.B., and a National Science Foundationpostdoctoral fellowship in plant biology to J.C.C. (DMB-8710662)

PEPCase.2 The CO2 released from malic acid decarboxylationduring the following day is reassimilated by ribulose- 1,5-bisphosphate carboxylase (26). Because of the high affinityPEPCase has for CO2, CAM plants, like C4 plants, have lowerCO2 compensation points during the dark and early lightperiod of the diurnal cycle than C3 plants and thus haveconsiderably lower photorespiration rates than C3 plants.Some plants can exhibit either C3 photosynthesis or CAM

depending on a variety of environmental conditions (25).High day and low night temperatures favor CO2 assimilationin the dark in pineapple (15). In other plants, CAM can betriggered by changes in photoperiod (2, 4). The switch toCAM can also be induced by salt or drought stress in Mesem-bryanthemum (28), Portulacaria (7, 24), or Peperomia (23).In the ice plant the transition from C3 to CAM normallyoccurs when environmental conditions change from a moistwinter/spring season to a dry summer season (1, 31). Plantswere found to switch between the two carbon fixation modesas measured by changes in acid fluctuations depending onwater availability. More recently, Vernon et al. (27) reportedthat if salt-stressed M. crystallinum plants were relieved ofstress by flushing soil with distilled water, the levels of PEP-Case mRNA, protein, and activity dropped dramatically,suggesting that CAM induction is a reversible process. Theseresults confirmed earlier reports of the reversibility of CAMbased on measurements of CO2 exchange (30).

In addition to environmental influences, the ability toperform CAM is related to plant or leaf age. Such ontogeneticresponses have been described for several CAM species, suchas Bryophyllum (4, 10, 12, 17), Kalanchoe (2), Portulacaria(8), Peperomia (9, 23), and Mesembryanthemum (29, 30). Wehave reported previously that the inducibility ofCAM by saltstress in M. crystallinum is influenced by plant age (19). Noincrease in PEPCase activity was observed during a 5-d irri-gation with 0.5 M NaCl in plants less than approximately 5weeks of age. In this report, we have examined the expressionof a stress-inducible PEPCase gene over the course of plantdevelopment under both unstressed and stressed conditions.We conclude that C3 to CAM switching is developmentallyprogrammed, but the magnitude of the response is enhancedby environmental stress once the plants have acquired thecompetence to respond. This developmental program occursduring a time interval that is closely synchronized with the

2 Abbreviation: PEPCase, phosphoenolpyruvate carboxylase.1137

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Plant Physiol. Vol. 94, 1990

changing climatic conditions found in the native habitat ofthe ice plant.

MATERIALS AND METHODS

Plant Materials

Ice plants (Mesembryanthemum crystallinum) were grownunder conditions described previously (19). For develop-mental studies, plants 3, 6, and 9 weeks of age were irrigatedwith nutrient solution or nutrient solution containing 0.5 MNaCl each day for 5 d. Unstressed plants were always wellwatered and were never allowed to be stressed accidentally.Plant material for protein and RNA analyses were frozen inliquid N2 and stored at -70°C until extraction. The first andsecond leaf pairs were collected from 3-week-old plants. Thesecond, third, and fourth leaf pairs were collected from 6-week-old plants. For 9-week-old plants, the third, fourth, andfifth leaf pairs and the first and second axillary shoots werecollected. All plant material was harvested at the same time(6 PM) during the dark/light cycle to avoid possible changesin gene expression patterns linked to diurnal rhythmicity.

Protein Blotting

Total soluble proteins were extracted from leaves accordingto the method ofOstrem et al. ( 19), except that the extractionbuffer was supplemented with 20 mM EDTA. Equal amountsof soluble protein were loaded onto a 12.5% SDS-polyacryl-amide gel, and proteins were blotted and probed with anti-PEPCase antibody as described previously (19).

RNA Extraction and Slot Blots

Total leaf RNA was isolated from plants 3, 6, and 9 weeksof age that were irrigated with nutrient solution alone orirrigated each day for 5 d with nutrient solution containing0.5 M NaCl as described previously (19). Equal amounts ofRNA were then blotted onto nitrocellulose as twofold serialdilutions (5.0-0.625 Mg). Filters were hybridized with either aPpc2 (PPC2; constitutively expressed isogene) or a Ppcl(PPC1; stress-induced isoform) specific radiolabeled probe asdescribed previously (3). The blots were quantified with GS300 scanning densitometer (Hoefer Scientific Instruments)coupled to a Shimadzu Chromatapac CR- 1 integrator.

plant, and 18S rDNA (pSRI.2B3) from soybean (5). Tran-scripts synthesized in vitro by isolated nuclei were quantifiedby cutting out slots from nitrocellulose filters and liquidscintillation counting. Radioactive counts were plotted as apercentage of relative hybridization to 18S rDNA. The meanvalues from two independent experiments are shown.

RESULTS

We wished to determine how the ability to induce CAM bychanging environmental conditions (i.e. salt stress) was relatedto plant development. The induction of PEPCase was exam-ined at the level of protein accumulation, steady-state tran-script accumulation, and transcriptional activity. The expres-sion oftwo specific isogenes of PEPCase (Ppcl and Ppc2) wasfollowed in 3-, 6-, and 9-week-old plants with and withoutNaCl stress. The Ppcl isogene, encoding a CAM-specific formofPEPCase, is induced by salt stress, whereas the Ppc2 isogeneis constitutively expressed at a low level (3).The magnitude of the stress-induced increase in PEPCase

varies with plant age. PEPCase levels were estimated at threedifferent times during development by protein blotting (Fig.1). Anti-PEPCase antibodies react with four apparent isoformsof PEPCase with only the 110-kD isoform (Fig. 1), beinginduced during stress (13). Accumulation of this proteincorrelates well with increases in enzyme activity (22). Theband at 109 kD corresponds to the Ppc2 gene product on thebasis of molecular weight derived from the predicted aminoacid sequence. Over the course of 9 weeks of growth, the levelof the Ppcl gene product steadily increases in unstressedplants. In 3-week-old plants, salt stress induces the 110-kDaPEPCase isoform slightly. In 6- and 9-week-old plants, how-ever, the induction of this isoform is greatly enhanced. Incontrast, the 109-kD species exhibits no increase at any stageof development in either stressed or unstressed plants. Twoother bands of higher molecular weight also cross-react withthe antibodies. The origin of these cross-reacting species iscurrently being investigated and may represent other mem-

3 wks 6 wks Y wks

US 5 day US 5 day U-S s dady

110 kDa -t --_

In Vitro Transcription Analyses

Nuclei were isolated and in vitro transcription run-on ex-periments were performed as described previously (3). Radi-olabeled in vitro transcripts synthesized by nuclei were hy-bridized to DNA slot blots containing 5 mg of DNA fromPpcl (p2C-9.0), Ppc2 (p1 lC-3.9) (3), a small subunit of ribu-lose 1,5-bisphosphate carboxylase/oxygenase (pC3-3) from iceplant (DeRocher, unpublished observations), ferredoxinNADP+ reductase (pFNR- 1) (14) specific clones from ice

- 97 .4 kDa.

68A.1'. kDa

Figure 1. Accumulation of PEPCase isoforms under unstressed andstressed conditions. Plants were grown for 3, 6, and 9 weeks andirrigated with nutrient solution (US) or nutrient solution containing 0.5M NaCI each day for 5 d (5 day). Equal amounts (20 ,ug) of solubleprotein were loaded onto a 12.5% polyacrylamide gel, blotted, andprobed with anti-PEPCase antibodies.

1138 CUSHMAN ET AL.

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DEVELOPMENTAL COMPETENCE TO RESPOND TO SALT STRESS

A PPC2

us 5 day

II

PPC1

uS 5 day

.. .: .*. a* . ....... I 3 weeks

I 6 weeks

I 9 weeks

B

response to stress, Ppcl transcripts increase fourfold at 3weeks, and about 10-fold at both 6 and 9 weeks (Fig. 2B).Quantitation of Ppc2 transcripts by densitometry revealedthat they decline between two- and threefold by 9 weeks ofage in both unstressed and stressed plants (Fig. 2B).To understand the mechanism by which steady-state levels

ofmRNA for Ppcl are being regulated, in vitro transcriptionrun-on experiments were performed with nuclei isolated fromleaves. The relative rates of transcription for Ppcl and Ppc2were measured with respect to the rate of 18S rRNA synthesis(Fig. 3A). Along with Ppc2, the rates of transcription for thesmall subunit of ribulose-1,5-bisphosphate carboxylase (E.J.DeRocher, unpublished observations) and ferredoxinNADP+ reductase (14) were followed as internal controls.These genes showed little overall change in transcription ratesover the course of development or when plants were stressed.At 3 weeks, Ppcl transcription rates increased about threefoldafter 5 d of stress (Fig. 3B). At 6 weeks, Ppcl transcriptionrate increased more than 1 -fold in stressed plants relative to

120z0

80 -

LU UPpcl1

LUc= 20-

0lIS 5 DAY VJS 5DAY UJS 5 DAYTHREE SIX NINE

TIME IN WEEKS

Figure 2. Steady-state levels of mRNA for PEPCase isogenes duringice plant development under unstressed and stressed conditions. A,total leaf RNA was isolated from plants 3, 6, and 9 weeks old thatwere irrigated with nutrient solution alone (US) or irrigated each dayfor 5 d (5 day) with nutrient solution containing 0.5 M NaCI and slotblotted onto nitrocellulose as twofold serial dilutions (5.0-0.625 sg)and probed with either a Ppc2 (PPC2) or a Ppcl (PPC1) specificprobe. B, blots were scanned with a laser densitometer and therelative hybridization intensity of Ppc2 or Ppc1 transcripts was plottedfor plants 3, 6, and 9 weeks old.

bers of the M. crystallinum PEPCase gene family. At least fivemembers of the PEPCase gene family have been found in theC4 plant, Zea mays (6).We next measured the steady-state mRNA levels of PEP-

Case isogenes in 3-, 6-, and 9-week-old plants to determine iftheir expression pattern paralleled that observed at the proteinlevel. Slot blots of total RNA isolated from unstressed orstressed plants were hybridized with either Ppcl or Ppc2specific DNA probes. Steady-state Ppcl transcripts are specif-ically induced during salt stress, whereas Ppc2 transcripts arenot induced (Fig. 2A) (see ref. 3). Ppcl transcripts increasesteadily in unstressed plants, so that by 9 weeks they are morethan threefold more abundant than in 3-week-old plants. In

3 weeks 6 weeks

US 5 day Us 5 day-m_m

0*

...... .. . t.a.¢ .....-

.....*

z0

Er

m

ULJ

F-

120

, '~

6 -

20 -

9 weeks

US 5 day

.6 ElM*..

B

a Ppc2

Ppcl

SIX

IMF 1.% E: KS

Figure 3. In vitro run-on transcription by nuclei isolated from iceplants 3, 6, and 9 weeks old. A, autoradiogram of DNA-RNA slot blothybridizations. Radiolabeled in vitro transcripts synthesized by nucleiisolated from unstressed plants 3, 6, or 9 weeks old, or stressed for5 d with 0.5 M NaCI were hybridized to 5 mg of DNA from Ppcl (p2C-9.0), Ppc2 (p11C-3.9), small subunit of ribulose-1,5-bisphosphatecarboxylase/oxygenase (SSU; pC3-3) from ice plant, ferredoxinNADP+ reductase (FNR; pFNR-1) specific clones from ice plant, and18S rDNA (pSRI.2B3) from soybean. B, quantitation of transcriptssynthesized in vitro by isolated nuclei. Slots from nitrocellulose filterswere cut out and quantitated by liquid scintillation counting. Radio-active counts were plotted as a percentage of relative hybridizationto 18s rDNA. The mean values from two independent experimentsare shown.

A

PPc I

PPC2

ssu

FNR

rDNA

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Plant Physiol. Vol. 94, 1990

3-week-old unstressed plants. By 9 weeks, the rate of Ppcltranscription in unstressed control plants has risen aboutsixfold when compared with the transcription rate in 3-week-old unstressed plants. The relative transcription rate of Ppclis not, however, induced in 9-week-old plants after stress,despite the salt-inducible increase in steady-state Ppcl tran-script levels (Fig. 2B). A reasonable explanation for this ob-servation would be an increase in transcript stability in olderplants.

DISCUSSION

We have demonstrated that the ability of stressed M. crys-tallinum to induce CAM as assessed by PEPCase gene expres-sion is intimately related to the developmental status of theplant. We had previously noted that 5-week-old plants didnot exhibit an increase in PEPCase activity when stressedwith 0.5 M NaCl, whereas 6-week-old plants did (19). The in-depth characterization ofa stress-induced isoform ofPEPCase(3, 13, 21) provided a tool to study this apparent age depend-ence of CAM inducibility in more detail. In this study,younger plants displayed a limited response to salt stress asmeasured by increases in Ppcl expression, whereas olderplants displayed a much greater response.The limited response to stress by young plants is based

primarily at the level of transcription. Possible mechanismsto explain this marginal induction include a lack of recogni-tion or activation of the Ppcl promoter due to the presenceof repressors, DNA methylation, the lack of a specific tran-scription factor(s), or to inactivating modifications of pre-existing factors. We are currently dissecting the Ppcl 5' up-stream regulatory region to ascertain which regions are im-

004u2

A:

E U_/_

0 3

portant for transcriptional induction during salt stress. Inaddition, we have begun to characterize DNA-binding pro-teins associated with this promoter in order to elucidatepossible developmental mechanisms of transcriptional acti-vation (J.C. Cushman, manuscript in preparation). In C4plants, development of Kranz anatomy seems to be a prereq-uisite for the efficient accumulation of C4 enzymes (16). Asimilar situation may exist in CAM plants. Since CAM isassociated with cells that have large vacuoles, it has beensuggested that the presence of such large vacuoles enables cellsto store the C4 acids produced during the dark period. Supportfor this idea comes from a recent report by Nishio and Ting(18), who observed that Peperomia tissues with cells havingsmall vacuole size exhibited lower C4 acid fluctuations andenzyme activities (including PEPCase), than did tissues withmuch larger vacuoles. The expression of CAM enzymes mayrequire the presence of cells with large vacuoles for malatestorage, which may not be present in young tissues.We have clearly shown that a preformed developmental

gradient exists in M. crystallinum that controls the expressionof a key CAM enzyme. The pattern of Ppcl steady-statetranscript levels parallels the accumulation of PEPCase pro-tein at all three age classes, indicating that enzyme inductionis likely controlled at the level ofmRNA amounts and not atthe level of translation. Transcripts, however, accumulate bya factor that is even higher than transcription rate increases,which leads us to assume that not only transcription, but alsomRNA stability is regulated. Old plants exhibit increasedPpcl transcription rates even without stress, and the rate doesnot increase further upon stress. This observation is a strongindication for the regulation ofmRNA stability as stress leads

Stressed

_ _ Unstressed

I 1

6 9Time in Weeks

1 2

________

Vegetative- -

Reproductive

Figure 4. CAM responsiveness during plant development under both unstressed and stressed conditions. The velocity with which CAM isreached is dependent upon the developmental status of the plant. Under stressed conditions this velocity is accelerated dramatically. Theapproximate vegetative and reproductive phases of the plant's life cycle are indicated.

i a 0 0 11

1140 CUSHMAN ETAL.

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DEVELOPMENTAL COMPETENCE TO RESPOND TO SALT STRESS

to a further increase in mRNA amount in these plants.Increased transcript stability might obviate the need for fur-ther transcriptional induction. We have summarized thesetrends in CAM gene expression in Figure 4, which presentsan interpretation of our results in graphical form. The devel-opmental progression of CAM is accelerated or enhanced byenvironmental stress.We propose that the competence of Mesembryanthemum

to initiate CAM is part of a developmental program that issynchronized with seasonal variations in moisture availabilityto which the plants have adapted. The correlation betweenlocal climatic conditions and fluctuations in titratable acidity,as an indicator of CAM, has been recognized previously (1,31). Ice plants normally germinate after winter rains and thenencounter increasing drought conditions as they enter the dryseason. Under continuous stress, plants remain small and,depending on the severity of the conditions, start floweringwithin 2 to 3 months and die. Unstressed plants may live formuch longer periods, although flowering will be delayed.While PEPCase gene expression is clearly induced by salt anddrought stress, well-watered mature plants grown under con-

trolled conditions exhibit increased PEPCase protein, tran-script accumulation, and increased transcription rates. Bothdevelopmental and environmental control of PEPCase geneexpression operate at the level oftranscriptional enhancementand apparently, at the level ofmRNA stability. Having abun-dant amounts of PEPCase (and presumably other CAM en-

zymes) transcripts and protein present before the onset ofdrought would allow the plant to perform CAM without a

significant lag phase. The transcriptional and posttranscrip-tional mechanisms controlling PEPCase gene expression, andthe expression of other CAM genes (20), during developmentoperate to ensure the establishment of the enzymatic machin-ery required for CAM well before nocturnal stomatal openingoccurs. In this respect, the expression patterns ofCAM genesare like those involved in C4 photosynthesis where the expres-sion patterns ofkey C4 enzymes (i.e. PEPCase) are establishedprior to the appearance of Kranz anatomy (16). Furtherinsight into the control of CAM gene expression shouldprovide a better understanding ofthe mechanisms that enablethis facultative CAM species to occupy habitats that non-

CAM plants find inhospitable.

ACKNOWLEDGMENTS

We would like to thank E. Jay DeRocher and Daniel M. Vernonfor critical review of the manuscript.

LITERATURE CITED

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13. Michalowski CB, Olson SW, Piepenbrock M, Schmitt JM,Bohnert HJ (1989) Time course of mRNA induction elicitedby salt stress in the common ice plant (Mesembryanthemumcrystallinum). Plant Physiol 89: 811-816

14. Michalowski CB, Schmitt JM, Bohnert HJ (1989) Expressionduring salt stress and nucleotide sequence of cDNA for ferre-doxin-NADP+ reductase from Mesembryanthemum crystal-linum. Plant Physiol 89: 817-822

15. Neales TF (1973) Effect of night temperature on the assimilationofcarbon dioxide by mature pineapple plants Ananas comosusL. Merr. Aust J Biol Sci 26: 539-546

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19. Ostrem JA, Olson SW, Schmitt JM, Bohnert HJ (1987) Saltstress increases the level of translatable mRNA for phosphoen-olpyruvate carboxylase in Mesembryanthemum crystallinum.Plant Physiol 84: 1270-1275

20. Ostrem JA, Vernon DM, Bohnert HJ (1990) Increased expres-sion of a gene coding for NAD:glyceraldehyde-3-phosphatedehydrogenase during the transition from C3 photosynthesis toCrassulacean acid metabolism in Mesembryanthemum crystal-linum. J Biol Chem 265: 3497-3502

21. Rickers J, Cushman JC, Michalowski CB, Schmitt JM, BohnertHJ (1988) Expression of the CAM-form of phos-pho(enol)pyruvate carboxylase and nucleotide sequence of afull length cDNA from Mesembryanthemum crystallinum. MolGen Genet 215: 447-454

22. Schmitt JM, Hofner R, Abou-Mandour AA, Vazquez-Moreno L,Bohnert HJ (1988) CAM induction in Mesembryanthemumcrystallinum.protein expression. In GS Singhal, ed, Photosyn-thesis-Molecular Biology and Bioenergetics. Springer India andNaroda Publishing, New Dehli, pp 259-268

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23. Sipes DL, Ting IP (1985) Crassulacean acid metabolism andCrassulacean acid metabolism modifications in Peperomiacamptotricha. Plant Physiol 77: 59-63

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25. Ting IP, Rayder L (1982) Regulation of C3 to CAM shifts. In IPTing, and M Gibbs, eds. Crassulacean Acid Metabolism. Amer-ican Society of Plant Physiologists, Rockville, MD, pp193-207

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28. von Willert DJ, Treichel S, Kirst GO, Curdts E (1976) Environ-mentally controlled changes of phosphoenolpyruvate carbox-ylases in Mesembryanthemum. Phytochemistry 15: 1435-1436

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