Characterization and expression of a NADP‐malic enzyme cDNA induced by salt stress from the facultative crassulacean acid metabolism plant, Mesembryanthemum crystallinum

Abstract

The facultative halophyte and crassulacean acid-metabolism plant, Mesembryanthemum crystallinum shifts from C3 photosynthesis to crassulacean acid metabolism when exposed to high-salt or drought conditions. To study the molecular basis of this metabolic transition, the expression of NADP+-dependent malic enzyme (NADP-ME), which catalyzes the decarboxylation of malate to release pyruvate and ( 2 0 2 , has been investigated. The complete nucleotide sequence of a full-length cDNA clone was determined and found to contain a single open reading frame encoding a 585-amino-acid polypeptide of 64284 Da. The ice plant ( M . crystallinurn) NADP-ME shares amino acid identities in the range 72.5-79.0% when compared to other higher-plant enzymes and is more closely related to C3 rather than C, forms of the enzyme. Genomic Southern-blot analysis of ice-plant DNA indicates that NADP-ME is encoded by a small gene family. Steady-state transcript levels increase 8 -10-fold in response to salt stress in the leaves. Transcript levels in roots are extremely low and are unaffected by salt-stress treatment. Nuclear run-on experiments, using isolated nuclei from leaf tissue, confirm that the accumulation of NADP-ME transcripts is, in part, the result of increased transcriphon of this gene during salt stress.Crassulacean acid metabolism (CAM), a specialized mode of photosynthetic carbon assimilation, is a functional adaptation to arid environments and a C0,-concentrating mechanism [l].Phosphoenolpyruvate carboxylase catalyzes the primary, nocturnal fixation of atmospheric COz into oxaloacetate, which is subsequently reduced to malate and stored in the vacuole. During the day, plants close their stomata to avoid evaporative water loss and draw upon the stored malate as a carbon source. Malate is decarboxylated to form pyruvate and C02. Pyruvate is phosphorylated by pyruvate orthophosphate dikinase to phosphoenolpyruvate which enters the gluconeogenic pathway. The released CO, is refixed by ribulosebisphosphate carboxylase/oxygenase to enter the reductive pentose-phosphate pathway.

Two Genes Encode Highly Similar Chloroplastic NADP-Malic Enzymes in Flaveria (Implications for the Evolution of C4 Photosynthesis)

“…The nature of these genes and the number that are involved in C, photosynthesis were not determined. However, it seems likely that at least one of the Flaveria NADP-ME genes encodes a cytosolic enzyme, since cytosolic NADP-MEs have been identified in a number of diverse plant species (Walter et al, 1988;Van Doorsselaere et al, 1991;Cushman, 1992;Franke and Adams, 1995). The proteins encoded by the F. trinervia and F. pringlei NADP-ME cDNAs are highly similar and contain apparent chloroplast transit peptides of identical length.…”

Molecular Genetics of Crassulacean Acid Metabolism

“…An important challenge for the future will be to determine the relative contribution of specific members of multigene families and the interplay of their gene products, particularly when they occupy different subcellular compartments. For example, both mitochondrial NAD-ME and cytosolic NADP-ME are coordinately induced to support daytime malate decarboxylation (Cushman, 1992;Saitou et al, 1994). Similarly, the combined activities of cytosolic NAD-MDH and chloroplastic NADP-MDH (Cushman, 1993) may be necessary for nocturnal malic acid formation.…”

“…Studies concerning the regulation of CAMspecific gene expression have been confined largely to facultative CAM, wherein the shift from C, photosynthesis to CAM can be induced by a number of environmental Thomas et al, 1992 Gehrig et al, Taybi et al, 1995Cushman, 1993Cushman, 1992 Fisslthaler et Forsthoefel et al, 1995aForsthoefel et al, 1995b N. Forsthoefel, personal communication Ostrem et al, 1990 Low et al ., Low et al, 1996 Dietz and Arbinger, 1996 Bartholomew et al, 1996;Low et al, 1996;Tsiantis et al, 1996 Cushman, factors such as photoperiod, drought, or high salinity (see Table I). Although transcriptional and posttranscriptional regulatory mechanisms are clearly involved, the exact regulatory mechanisms important for transcriptional activation and transcript stability and recruitment remain largely unknown.…”

“…Multigene families have also been reported for ME, MDH, enolase, and phosphoglyceromutase in M . crystallinum (Cushman, 1992(Cushman, , 1993Forsthoefel et al, 1995a, 199513) and the H+-ATPase c subunit in M . crystallinum (Tsiantis et al, 1996) and Kalanchoe daigremontiana (Bartholomew et al, 1996).…”

Pathogen-induced changes in malate content and NADP-dependent malic enzyme activity in C3 or CAM performing Mesembryanthemum crystallinum L. plants

“…One of the important malate metabolizing enzyme is NADP-malic enzyme (NADP-ME, L-malate: NADP oxidoreductase [oxaloacetate decarboxylating], EC 1.1.1.40) which produces after malate decarboxylation apart from pyruvate also CO 2 to be used in carbon fixation by ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco). NADP-ME is known as one of CAM-related enzymes and in M. crystallinum at least one CAM-specific isogene for NADP-ME was reported (Cushman 1992).…”