Increased Expression of a myo-lnositol Methyl Transferase in Mesembryanthemum crystallinum Is Part of a Stress Response Distinct from Crassulacean Acid Metabolism

The facultative halophyte Mesembryanthemum crystallinum responds to osmotic stress by switching from C3 photosynthesis to Crassulacean acid metabolism (CAM). This shift to CAM involves the stress-initiated up-regulation of mRNAs encoding CAM enzymes. The capability of the plants to induce a key CAM enzyme, phosphoenolpyruvate carboxylase, is influenced by plant age, and it has been suggested that adaptation to salinity in M. crystallinum may be modulated by a developmental program that controls molecular responses to stress. We have compared the effects of plant age on the expression of two salinity-induced genes: Gpdl, which encodes the photosynthesis-related enzyme glyceraldehyde 3-phosphate dehydrogenase, and ImtI, which encodes a methyl transferase involved in the biosynthesis of a putative osmoprotectant, pinitol. Imt! mRNA accumulation and the accompanying increase in pinitol in stressed Mesembryanthemum exhibit a pattern of induction distinct from that observed for CAM-related genes. We conclude that the molecular mechanisms that trigger Imt! and pinitol accumulation in response to salt stress in M. crystallinum differ in some respects from those that lead to CAM induction. There may be multiple signals or pathways that regulate inducible components of salinity tolerance in this facultative halophyte. The common ice plant (Mesembryanthemum crystallinum) is a facultative halophyte that switches from C3 photosynthesis to CAM in response to drought or high salinity. CAM is an alternate photosynthesis pathway that serves as a water conservation mechanism (1, 9). During CAM, stomatal opening and primary carbon fixation occur at night, allowing for closure of stomata during the day and, consequently, decreased evaporative water loss. CAM induction in the ice plant has been well characterized at the biochemical level. Exposure to high salinity has been shown to trigger the accumulation of a number of mRNAs encoding CAM enzymes (5, 6, 8), resulting in increased expression and activity of these proteins (4). 1 Supported by U.S. Department of Agriculture and Arizona Agricultural Experimental Station. One such stress-induced enzyme is PEPCase2, which catalyzes the primary carbon fixation step in CAM. Ppcl and protein expression have been used as markers to study the stress response in M. crystallinum at the molecular level. Although PEPCase expression in the ice plant is primarily environmentally regulated (11), a number of recent studies have noted that the capacity of the plants to induce PEPCase in response to stress and the rate of Ppcl induction increase with plant age (1, 3, 5, 12). Three-week-old ice plant seedlings exhibit no significant increase in PEPCase activity (5) and only a modest increase in Ppcl mRNA levels during a 5-d stress with 500 mm NaCl, whereas plants 6 or 9 weeks of age induce Ppcl to maximum levels during such a stress (3). Also, gradual increases in Ppcl transcription and PEPCase activity are observed in unstressed plants of increasing age (2, 3, 12). Such observations have led to the proposal that the stress response in M. crystallinum is modulated by a developmental program that allows maximum expression of salinity-responsive genes only after a certain plant age (approximately 5 weeks under our growth conditions) is reached. To determine whether other salt stress-responsive mRNAs exhibit expression patterns similar to that of Ppcl, we have examined the influence of plant age on the expression of two additional salinity-induced genes. One, Gpdl, encodes GAPdH (6), an enzyme that has important housekeeping functions and photosynthetic carbon reduction roles in C3 plants. GAPdH is thought to be up-regulated during salt stress in the ice plant to fulfill an increased need for the enzyme in carbon flux during the large diurnal fluctuations in organic acids and starch that are characteristic of CAM. For this reason, the increased expression of GAPdH in stressed M. crystallinum is considered 'CAM-related.' The second salinity-induced gene, Imtl, encodes a myo-inositol 0methyl transferase that catalyzes the first step in the biosynthesis of pinitol (10), a methylated cyclic sugar alcohol that is thought to serve as an intracellular osmolyte and/or os2 Abbreviations: PEPCase, phosphoenolpyruvate carboxylase; GAPdH, glyceraldehyde-3-phosphate dehydrogenase; Imtl, gene encoding myo-inositol 0-methyl transferase; Gpdl, gene encoding GAPdH; Ppcl, gene encoding PEPCase.