The Irreversible C3 to CAM Shift in Well-watered and Salt-stressed Plants of Mesembryanthemum crystallinum is under Strict Ontogenetic Control

The shift from C₃ to CAM was investigated as a function of both leaf and plant age in well-watered and salt-stressed (300 mM NaCl solution) plants of Mesembryanthemum crystallinum. Initiation of a night-time accumulation of malic acid, the decisive criterion of CAM, was followed in plants that were continuously stressed at different points in their life cycle. The deinducibility of CAM was examined after the release from stress by extensively rinsing the potting soil with de-mineralized water. Our results show that in M. crystallinum CAM is under strict developmental control, since CAM appeared only when a certain stage of development of the whole plant was reached. CAM was not present in any plant before this threshold, which was the same in salt-stressed as well as in well-watered plants. The metabolic shift coincided with the change from the seedling to the juvenile growth phase, and not with that from vegetative to reproductive growth, represented by the start of branching. The latter is timed to the end of extension growth. In well-watered plants, after this decisive point in development, a weak nighttime accumulation of malic acid could be measured (? 0.05 mol kg_DW^?1) in the oldest, mature leaves but not in young, developing ones. This “CAM capacity” gradually increased up to 0.2 mol kg_DE^?1 with further plant ageing. Leaf senescence, characterized by wilting and yellowing, diminished the CAM activity. In mature leaves salt stress drastically enhanced the magnitude of diurnal fluctuation in malic acid content. Removal of salt stress did not deinduce CAM activity, but diminished the amplitude of malic acid oscillations to some extent in those plants which had been stressed from early in their life cycle. In these plants, salt stress delayed plant development and growth thus retarding the life cycle. Well-watered plants, for example, branched about three weeks earlier than those that had been stressed continuously from one week after germination. After removal of stress a quasi-preserved earlier developmental stage in relation to the control plants determined the weaker CAM expression.     

盐胁迫对露花叶片的脱落酸含量及磷酸烯醇式丙酮酸羧化酶昼夜调节特性的影响

兼性CAM植物在转为CAM型后,CAM代谢的关键酶磷酸烯醇式丙酮酸（PEP）核化酶会出现昼夜调节特性的变化（Osmond1978）。关于PEP梭化酶昼夜调节特性的机理存在两种观点：1．PEPK化酶昼夜聚合度发生了变化,白天为二聚体PEPK化酶,对苹果酸抑制敏感;而夜间为四聚体,对苹果酸抑制不敏感（U和Wedding1985）。2·nsv＄化酶昼夜磷酸化状态发生变化,夜间PEPW化酶磷酸化,对苹果酸抑制不敏感;而白天PEP＄化酶脱磷酸化,对苹果酸抑制敏感（Nimmo等1986）。植物生长调节物质如ABA和细胞分裂素对兼性CAM植物PEP＆化酶的表达有诱…     

The Effect of Irradiance on Carboxylating/Decarboxylating Enzymes and Fumarase Activities in Mesembryanthemum crystallinum L. Exposed to Salinity Stress

Abstract: In Mesembryanthemum crystallinum plants, treated for 9 days with 0.4 M NaCl at low light intensities (80 - 90 or 95 - 100 μE m^-2 s^-1; λ = 400 - 700 nm), no day/night malate level differences (Δmalate) were detected. At high light (385 - 400 μE m^-2 s^-1) strong stimulation of PEPC activity, accompanied by a Δmalate of 11.3 mM, demonstrated the presence of CAM metabolism. This indicates that, to evolve day/night differences in malate concentration, high light is required. Salt treatment at low light induces and increases the activity of NAD- and NADP-malic enzymes by as much as 3.7- and 3.9-fold, while at high light these values reach 6.4- and 17.7-fold, respectively. The induction of activity of both malic enzymes and PEPC (phospo enol pyruvate carboxylase) take place before Δmalate is detectable. An increase in SOD (superoxide dismutase) was observed in plants cultivated at high light in both control and salt-treated plants. However, in salt-treated plants this effect was more pronounced. Carboxylating and decarboxylating enzymes seem to be induced by a combination of different signals, i.e., salt and light intensity. Plants performing CAM, after the decrease of activity of both the decarboxylating enzymes at the beginning of the light period, showed an increase in these enzymes in darkness when the malate pool reaches higher levels. In CAM plants the activity of fumarase (Krebs cycle) is much lower than that in C₃ plants. The role of mitochondria in CAM plants is discussed.     

Starch-degrading enzymes during the induction of CAM in Mesembryanthemum crystallinum

Mesembryanthemum crystallinum plants were irrigated with 400 mol m^?3 NaCl to induce CAM and levels of leaf starch, and activities of starch-degrading enzymes were measured. During Crassulacean acid metabolism (CAM) induction, daily starch turnover gradually became more pronounced and was three- to four-fold greater than in leaves of C₃ plants after 3 weeks. Activities of α- and β-amylase, D-enzyme and starch phosphorylase all increased 10- to 20-fold within 3 weeks of the start of salt treatment. Activities of α- and β-amylase increased more than fourfold within the first 24 h of salt treatment, which is the fastest increase in enzyme activities so far measured during the induction of CAM with salt solution in intact plants of this species. Most enzyme activities were partially chloroplastic; however, the principal starch-degrading activity was constituted by an extra-chloroplastic β-amylase. CAM starch-phosphorylase activity, which was mainly chloroplastic, exhibited a two- to three-fold diurnal change in parallel with starch content. CAM induction in M. crystallinum is clearly associated with greater starch turnover and enhanced starch-degrading enzyme activities, which as catalysts of the initial reaction to release carbon for synthesis of phosphoenolpyruvate (PEP) appear highly significant for the functioning of the CAM pathway. The diurnal rhythm of phosphorylase activity may be of particular significance.     

Membrane Particles,Proteins and ATPase Activity of Tonoplast Vesicles of Mesembryanthemum crystallinum in the C-3 and CAM State*

Dimensions and area densities of membrane particles were studied by electron microscopy of replicas of freeze-fractured suspensions of tonoplast vesicles of Mesembryanthemum crystallinum L. in the C-3 state and after induction of crassulacean acid metabolism (CAM) by salinity. The results are compared with the relative contribution of tonoplast-ATPase protein to total membrane protein obtained from integration of elution profiles in size-exclusion chromatography. Coverage of tonoplast area by globular membrane particles was 20% and 36 % and ATPase in relation to total membrane protein was 33 % and 35 % in C-3 and CAM M. crystallinum, respectively. Thus, by order of magnitude, it is most likely that the particles represent the ATPase. In Kalanchoë daigremontiana Hamet et Perrier de la Bâthie the ATPase also constituted 36% of total tonoplast protein. Induction of CAM in M. crystallinum was associated with an increase in specific ATPase activity of the tonoplast and an increase of the size and area coverage of tonoplast particles, whereas the relative contribution of ATPase protein to total tonoplast protein and the molecular mass of the ATPase holoenzyme, as seen in size-exclusion chromatography, remained almost unchanged.     

The Activity of the Peroxidase System in the Course of Stress-Induced CAM Development

The activity of the peroxidase system in Mesembryanthemum crystallinum L. plants in relation to the shift from C₃ to CAM photosynthesis was studied. In detached leaves of the fourth and fifth stories treated with NaCl (0.3 M), a rapid (after 30 min) transient induction of the ionically bound peroxidase (the first maximum) was observed followed by a second weak increase in the enzyme activity (90 min after salt treatment). In the leaves of intact plants, which received a longer treatment with NaCl, a two-phase change in the enzyme activity was also observed. It was most pronounced at the early stages of the NaCl-induced plant shift from C₃ to CAM photosynthesis. In this case, in both detached and intact leaves of juvenile plants, the activity of soluble peroxidase was at a low steady-state level. The situation changed dramatically when M. crystallinum plants transitioned to the reproductive developmental phase and photosynthesis switched from C₃ to CAM. The time dependence of the activities of both peroxidase types, the soluble ones in particular, was characterized by marked diurnal oscillations (light–dark), which coincided with the fluctuations of the total titratable acidity. In this case, the activity of the soluble enzyme was several orders of magnitude higher than the activity of the ionically bound peroxidase, even though the optimum pH for both isoforms was similar (pH 5.0). Three acid isoforms of soluble peroxidases, which operated more actively when the cytoplasm had a higher acidity, were distinguished by isoelectrofocusing. Their activity increased under salinity. Alkaline and neutral components were predominant in more than 30 molecular forms of the soluble peroxidase detected. We concluded that the operation of the peroxidase system changed substantially when plants shifted from the juvenile to the reproductive state and switched from C₃ to CAM photosynthesis: the activity of stress-induced ionically bound peroxidase was drastically inhibited with a concurrent increase in the activity of soluble peroxidase and a change in the spectrum of its molecular forms.     

Spectral dependence of flavonol and betacyanin accumulation in Mesembryanthemum crystallinum under enhanced ultraviolet radiation

Mesembryanthemum crystallinum L. (Aizoaceae) is a drought‐ and salt‐tolerant halophyte that is able to endure harsh environmental conditions. Upon irradiation with high light irradiance (1200–1500 µmol m^?2 s^?1) it displays a rapid cell‐specific accumulation of plant secondary metabolites in the upper leaf epidermis; a phenomenon that is not detectable with salt or drought treatment. The accumulation of these compounds, the betacyanins and acylated flavonol glycosides, increases if the plants are exposed to polychromatic radiation with a progressively decreasing short‐wave cut‐off in the ultraviolet range. The response is localized in the epidermal bladder cells on the tips of young leaves and epidermal layers of fully expanded leaves. It is demonstrated that the accumulation of flavonols and betacyanins can be described by a weakly sigmoid dose function in combination with an exponential decrease of the response function of the plant with increasing wavelength.