Increased Expression of a Gene Coding for NAD:Glyceraldehyde-3-phosphate Dehydrogenase during the Transition from CS Photosynthesis to Crassulacean Acid Metabolism in Mesembryanthemum crystallinum*

We utilized differential plaque hybridization to iden- tify three cDNA clones for transcripts which increase in abundance during the salinity-induced transition from CS photosynthesis to crassulacean acid metabo- lism (CAM) in Mesembryanthemum crystallinum. Al- there in the abundance of these in unstressed tissue, steady-state levels of all three increased within 30 h following with 0.5 M NaCl. One encodes

We utilized differential plaque hybridization to identify three cDNA clones for transcripts which increase in abundance during the salinity-induced transition from CS photosynthesis to crassulacean acid metabolism (CAM) in Mesembryanthemum crystallinum.
Although there are differences in the abundance of these transcripts in unstressed tissue, steady-state levels of all three increased within 30 h following irrigation with 0.5 M NaCl. One cDNA encodes the cytosolic form of glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating) (NAD-GAPDH)), an enzyme involved in the production of phosphoenolpyruvate for CO, fixation at night and the conversion of pyruvate to storage carbohydrate during the day. Coding region and 3'-noncoding sequence probes were used to examine the expression of NAD-GAPDH transcripts in leaf and root tissue. We show that the gene encoding the NAD-GAPDH cDNA is expressed in both leaf and root tissue during CS photosynthesis and CAM. NAD-GAPDH transcript levels increase rapidly in leaf (but not in root) tissue during the transition to CAM. Our data indicate that the predominant NAD-GAPDH transcript expressed during CB photosynthesis and CAM is encoded by a single gene in M crystallinum.
These results imply that the transition to CAM in some cases involves an upward readjustment in the level of a gene product expressed during Cs photosynthesis, rather than the expression of a CAM-specific isoform with unique regulatory or kinetic properties.
A small number of plant species switch from Cs photosynthesis to crassulacean acid metabolism (CAM)' as an adaptive response to water stress. A number of changes accompany this transition.
During CAM, the normal diurnal cycle of stomata1 opening is reversed. Stomata open at night, and the initial carboxylation step utilizes phosphoenolpyruvate carboxylase (EC 4. 1.1.31 to malate and stored temporarily in the cell vacuole. Malate is released into the cytoplasm during the following light period and decarboxylated, and the CO, is refixed by ribulose-1,5bisphosphate carboxylase (EC 4. 1.1.39) and the Calvin cycle (Osmond and . The transition from Cs photosynthesis to CAM has been characterized most thoroughly in the halophyte Mesembryanthemum crystullinum . Water stress brought about by increased salinity in the rooting medium ("salt stress") results in a lo-20 fold increase in the activity of P-enolpyruvate carboxylase and a 3-4 fold increase in the activity of several other enzymes involved in carbon metabolism Winter et al., 1982). Penolpyruvate carboxylase is synthesized de novo in response to salt stress  due at least in part to an increase in P-enolpyruvate carboxylase transcript levels in both fully expanded and rapidly growing leaf tissue (Ostrem et al., 1987). More recent results show that the increased expression of P-enolpyruvate carboxylase is reversible (Vernon et al., 1988) and that stress increases the expression of one member of a multigene P-enolpyruvate carboxylase family .
We are characterizing cDNA clones for transcripts which increase in abundance during CAM induction as a first step toward understanding the mechanisms underlying the shift from CB photosynthesis to CAM Rickers et al., 1989). We have now identified clones for several stressregulated transcripts by differential screening of a cDNA library. One encodes the cytosolic form of glyceraldehyde-3phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD' oxidoreductase (phosphorylating), EC 1.2.1.12) (NAD-GAPDH), an enzyme that catalyzes an essential step in the production of substrate for nighttime CO* fixation in CAM. We report here the sequence of a full-length cDNA and the results from an analysis of the regulation of this gene in M. crystullinum.
We show that transcript levels for NAD-GAPDH, P-enolpyruvate carboxylase, and two cDNAs coding for unidentified proteins increase in leaf tissue at similar but not identical rates following irrigation with 0.5 M NaCl. NAD-GAPDH transcript levels increase more rapidly in leaf tissue than in root tissue during the transition to CAM. These results support previous work (Ostrem et al., 1987;, Michalowski et al., 1989a showing that CAM induction involves a coordinate increase in steady-state transcript levels for a select subset of genes in M. crystallinum Corp.). The filters were screened by differential plaque hybridization (Maniatis et al., 1982) using single-stranded cDNA probes generated from poly(A+) RNA isolated from control plants and from plants which had been irrigated with 0.5 M NaCl for-5 days. Hybridization was carried out in 6 X SSC (1 X SSC consists of 150 mM NaCl, 15 mM sodium citrate, pH 7.0), 0.25% nonfat dry milk, 50% formamide at 42 "C (Johnson et al., 1984) in the presence of l-3 x lo6 cpm of labeled probe. Filters were washed twice for 15 min at room temperature in 2 x SSC, 0.1% (w/v) SDS (low stringency) and then twice for 15 min at 60 "C in 0.1 x SSC, 0.1% (w/v) SDS (high stringency) and exposed to film for 24-48 h.

Construction of Subclones and Nucleotide
Sequencing-Phage DNA was digested with EcoRI and separated in 1.2% low gelling temperature agarose (Seaplaque, FMC Bioproducts). EcoRI restriction fragments-were ligated into the plasmid vector Bluescript KS+ (Stratagene) and used to transform Escherichia coli strain XLl-Blue (Bullock &al., 1987) or K12 strain 71-18 (Messinget al., 1977). Single-stranded DNA was produced with Ml3 K07 helper phage, and its nucleotide sequence was determined by dideoxy sequencing (Sanger et al., 1977). Open reading frames were identified using version 5.07 of the Mount-Conrad-Meyers program (Williams, 1988). A cDNA clone (McUAl) containing a partial sequence of NAD-GAPDH was used to screen aliquots of an amplified cDNA library (Rickers et al., 1989) in X-ZAP (Stratagene in 25 mM sodium phosphate buffer, pH 5.6. The filter was hybridized overnight at 42 "C with the 3'-specific A/X probe and then washed at high stringency and exposed to film as described above. For slot blot analysis, P-fold serial dilutions of total RNA were blotted onto nitrocellulose (BA85, Schleicher & Schuell) as described (Vernon et al., 1988), with the exception that the dilution series began with 2.5 rg of total RNA al., 1988) with EcoRI, HindIII, or SacI. The digests were split into two 50-~1 aliquots and electrophoresed overnight at 25 V in a 0.8% (w/v) agarose gel in TAE buffer (Maniatis et al., 1982). The gels were transferred overnight onto 0.45-pm nitrocellulose by capillary blotting (Southern, 1975). Filters were baked and hybridized at 42 "C as described above with either a coding region (H/S) or a 3'noncoding region (A/X) probe. Hybridization and washing steps were conducted as described in the legend of Fig. 4.

RESULTS
We identified several cDNA clones for transcripts which either increase or decrease in abundance during CAM induction. One clone (McUAl) contained an open reading frame coding for the carboxyl terminus of NAD-GAPDH.
The  Rickers et al., 1989) to compare the kinetics of the induction of NAD-GAPDH and the genes encoded by the two unidentified cDNAs with that of P-enolpyruvate carboxylase. As shown in Fig. 1 (upper), the steady-state levels of all four transcripts increased within 30 h following irrigation with 0.5 M NaCl. Differences were apparent, however, both in transcript levels present in control plants (Fig. 1, 0 h) and in the rate at which the steady-state levels of these transcripts increased during the course of the experiment (Fig. 1, lower). Transcripts hybridizing with the partial NAD-GAPDH cDNA (McUAl) were relatively abundant in unstressed tissue in contrast to McUB4, McUB5, and P-enolpyruvate carboxylase. Transcripts hybridizing with McUB4 were present at very low levels in unstressed tissue and showed the most rapid increase during the initial 4-28 h following irrigation with 0.5 M NaCl. All four transcripts increased to similar levels by the end of the fifth light period (Fig. 1,100 h).
Stress-induced NAD-GAPDH Transcript Is Present in Both Control and Stressed Leaf and Root Tissue-The cDNA clone McUAl encoded -125 amino acids of the carboxyl-terminal domain of NAD-GAPDH as well as a 303-nucleotide 3'noncoding region ending with a 14-bp poly(A+) tail. This clone was used to screen a cDNA library enriched for CAMrelated sequences (Rickers et al., 1989).  Upper, 2-fold serial dilutions of total RNA isolated from leaf tissue at the times indicated (hours) were blotted onto nitrocellulose filters and hybridized with 2 x 10' cpm of nick-translated cDNA inserts prepared from McUAl, McUB4, McUB5, or P-enolpyruvate carboxylase cDNA clone 12r1 (PEPC; Rickers et al., 1989). The filters were washed at high stringency (see "Experimental Procedures") and exposed to film overnight. Lower, the film images were quantitated by densitometry. Peak areas in dilutions which fell within the linear range of the film (as estimated by linear regression) were used to calculate transcript abundance as The results from Northern analysis of total RNA probed with the 3'-noncoding region probe (A/X) are shown in Fig.  3. The probe hybridized with a 1.4-kb transcript present in total RNA isolated from control plants (Fig. 3, 0 h). Steadystate levels of the 1.4-kb transcript increased -IO-fold in abundance following irrigation with 0.5 M NaCl (Fig. 3, upper). The relative abundance of this transcript in control leaf tissue and the kinetics of its increase following irrigation with 0.5 M NaCl were virtually identical to that observed previously using a probe consisting of both coding and 2'-noncoding sequences (Fig. 3, lower). These results suggested that GAPal encodes the predominant NAD-GAPDH transcript present in M. crystallinum leaf tissue during both CZ photosynthesis and CAM.
To make a more quantitative estimate of NAD-GAPDH transcript levels during C1 photosynthesis and CAM, we compared hybridization signals obtained from total RNA with those obtained from known quantities of RNA prepared by transcription of plasmid templates containing either the fulllength cDNA (GAPal) or the majority of the coding region for NAD-GAPDH (GAPCOD). We estimated the total amount of NAD-GAPDH transcript present in leaf and root tissue RNA by hybridization of the coding region probe (H/ S) to total RNA. The 3'-noncoding region probe (A/X) was used to determine the proportion of the total NAD-GAPDH hybridization signal that could be attributed to the stressinduced transcript encoded by GAPal. Both probes give equivalent hybridization signals in serial dilutions of total RNA from three separate experiments. NAD-GAPDH transcripts were present at -100 ng/pg of total RNA in root tissue from unstressed plants and at 40 ng/wg of total RNA in unstressed leaf tissue. Transcript levels in root tissue decreased within 6 h following irrigation with 0.5 M NaCl, but increased to levels equal to or slightly above those present in unstressed root tissue at 30-and 54-h time points. In contrast, NAD-GAPDH transcript levels in leaf tissue increased to -125 ng/pg of total RNA at 30 h and 160 ng/pg of total RNA at 54 h following irrigation with 0.5 M NaCl. After 12 days of irrigation with 0.5 M NaCl, NAD-GAPDH transcript levels (as determined by hybridization with both coding region and 3'-specific probes) were -IO-fold greater in stressed than in unstressed leaf tissue. Hybridization to ribosomal RNA dilutions included on each blot, and to coding region transcripts probed with A/X, was insignificant (data not shown).
The results from Southern analysis of genomic DNA using the H/S and A/X probes are presented in Fig. 4. The blot probed with H/S was washed at low stringency to identify, if possible, all NAD-GAPDH gene family members. This blot was compared with the hybridization pattern from an identical blot probed with A/X and washed at high stringency to identify restriction fragments hybridizing with the 3'-portion of the stress-regulated NAD-GAPDH transcript.
The hybridization patterns of these two blots are remarkably similar (Fig. 4). The gene encoding NAD-GAPDH (including the 3'untranslated region of GAPal) is located on a 15-kb EcoRI fragment, whereas an adjacent 15-kb Hind111 fragment contains the 3'-portion of GAPal, but none of the coding region. A 15-kb genomic clone derived from a partial Mb01 digest of M. crystallinum DNA contains the entire NAD-GAPDH cDNA described here." Preliminary analysis of this clone shows that there is an EcoRI site located -700 bp upstream of the first exon-containing coding sequence, a 2.0-kb Sac1 fragment containing the majority of the coding region (Fig. 4, Sac1 digest probed with H/S), and an adjacent 1.5-kb Sac1 fragment containing the 3'-noncoding region (faintly visible in Fig. 4, Sac1 digest probed with A/X). We believe that the 3.5-kb fragment hybridizing with both A/X and H/S probes in the Sac1 digest is due to incomplete digestion of the Sac1 site linking these adjacent Sac1 fragments. These data provide evidence that NAD-GAPDH transcripts expressed in leaf and root tissue during C3 photosynthesis and CAM are encoded by a single gene in M. crystallinum. DlSCUSSION CAM conserves plant water resources in arid environments by allowing CO, fixation to occur primarily at night when water loss due to transpiration is minimal.  Z. moys (Brinkmann et al., 1987). N. tubacum (Shih et al., 1986), andS. albu (Martin andCerff, 1986). The numbering system used is that of Harris and Waters (1976 matal opening and diurnal fluctuation in malate levels) throughout their lifespan, other species adapt to seasonal changes in water availability by switching between Cs photosynthesis and CAM (Ting and Hanscom, 1977;Winter et al., 1978;Guralnick and Ting, 1986). The salinity-induced transition to CAM in M. crystallinum provides a simple and reproducible system for investigating the mechanisms underlying this response.
CAM induction entails a complex metabolic readjustment in carbon flow within the plant cell. During CAM, glycolysis plays a major role in providing substrate for P-enolpyruvate carboxylase activity at night; during the day, gluconeogenesis regenerates storage carbohydrates which are utilized during the subsequent period of nighttime COZ fixation . Our finding that stress increases the steadystate level of NAD-GAPDH transcripts is not surprising in light of the increased importance of glycolysis/gluconeogenesis during CAM. Previous work has shown that the activity of several enzymes involved in carbon metabolism increases in CAM leaf tissue from M. crystallinum Winter et al., 1982;Fahrendorf et al., 1987). Although it is tempting to speculate that CAM induction is regulated primarily via transcriptional control of the genes encoding these enzymes, post-transcriptional controls may also help regulate the balance between C, photosynthesis and CAM. We have shown recently that P-enolpyruvate carboxylase transcript levels decline rapidly (tH = 2.5 h) in salt-stressed leaf tissue when the rooting medium is flushed with distilled HZ0 (Vernon et al., 1988). This indicates that control of transcript stability may play a major role in maintaining CAM.
Does stress alter the level of existing enzymes or induce the expression of CAM-specific isotypes? The P-enolpyruvate carboxylase gene family in M. crystallinum is made up of at least two members, one of which responds to salt stress and another which is not regulated in response to stress . Our data show that stress acts primarily by altering the level of expression of a NAD-GAPDH gene expressed during both CB photosynthesis and CAM, rather than by inducing the expression of a new CAM-specific isotype. In support of this interpretation, we show that a probe (A/X) unique to a 148-bp sequence in the 3'-noncoding region of GAPal hybridized with transcripts which were present in unstressed tissue (Fig. 3, upper). These transcripts increased in abundance with kinetics identical to those obtained with a probe consisting of coding region and 3'-noncoding sequences in the relative abundance of the transcript hybridizing with the 3'-noncoding probe (*) in this Northern blot is compared with the increase in transcripts which hybridized with a probe consisting of both coding and 3'-noncoding regions (0) in Fig. 1. ( Fig. 3, lower). Furthermore, equivalent quantities of transcripts were detected using either a coding region probe (H/ S) that should hybridize with all NAD-GAPDH-related mRNA or a 3'-specific probe that should hybridize only with the stress-regulated NAD-GAPDH transcript described here. The nucleotide sequences of two P-enolpyruvate carboxylase genes from M. crystallinum show very little similarity outside the coding region Bohnert, 1989a, 1989b). These results suggest that a single gene encodes the primary NAD-GAPDH transcript present in leaf and root tissue during C1 photosynthesis as well as during the stress-induced shift to CAM. Increased expression of this gene occurs primarily in leaf tissue, where glycolysis and gluconeogenesis are essential for nighttime CO, fixation. The cDNA for NAD-GAPDH described here has two interesting features. First, the 55-bp untranslated leader sequence is CT-rich (44% C, 43% T, 11% A, 2% G) compared with the 3'-noncoding region (15% C, 38% T, 24% A, 23% G) and is characterized by CT and CTT repeats throughout its length. CT-rich leader sequences are present in other cDNAs from M. crystallinum (Michalowski et al., 1989b).3 The possible functions of these leader sequences in regulating mRNA translation or stability are unknown at present. Second, there is a striking difference in cysteine content among the plant NAD-GAPDH enzymes. The cDNA from M. crystallinum encoded 7 cysteine residues. In the other two full-length sequences (Zea mays and Sinapis alba), cysteine residues are present only in a highly conserved region which contains the catalytically active Cys14'. Four of the additional cysteines in