Effect of high salinity on tracheary element differentiation in light-grown callus of Mesembryanthemum crystallinum

This study investigated the inhibitory effects of NaCl on tracheary element (TE) differentiation in light-grown callus of ice plant Mesembryanthemum crystallinum L., a halophyte which adaptes well to saline environments. When ice plant callus was grown in a modified Linsmaier-Bednar and Skoog culture medium containing no NaCl (control medium), up to 20% of ice plant cells differentiated into tracheary elements during in vitro culture. Close examination of callus tissues stained with potassium permanganate revealed that tracheary elements were aggregated as discrete nodules. Some strikingly elongated tracheary elements were found in the macerated tissues. Experimental results indicated that adding 200 mM NaCl to the control medium reversibly inhibited the formation of tracheary element in the halophytic cells. The rate of tracheary element formation increased accordingly as the rate of cell growth in control medium. In the presence of high salt, the degree of tracheary element differentation remained low through the growth cycle. The inhibitory effect of salt on tracheary element differentiation was overcome by adding 10 mg l⁻¹ salicylic acid, a known signaling compound that induces a diverse group of defense-related genes, including genes involved in reinforcing the host cell wall. Furthermore, microscopic examination revealed that most tracheary elements formed under this treatment (200 mM NaCl plus 10 mg l⁻¹ salicylic acid) were round shaped. The results suggest that high salt inhibits both the biosynthesis of secondary wall components and cell elongation ice plant in vitro culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

Photoautotrophic Cell Suspension Cultures from Mesembryanthemum crystallinum and their Response to Salt Stress

For the first time, photoautotrophic cell suspension cultures of Mesembryanthemum crystallinum have been established. The cells are growing in a sugar-free culture medium in the presence of 2 % (v/v) CO₂ as the sole carbon source. A 16 h light photoperiod is applied. Increase in fresh and dry weight during a 21 days growth cycle was more than 3-fold. Treatment of the cells with 200 mM NaCl from day 10 to day 21 of subculture stimulated cell culture growth, enhanced CO₂ fixation and elicited an increase in the extractable activities of enzymes related to CO₂ fixation (RubisCO; PEP carboxylase) and malic acid metabolism (NAD / NADP dependent malic enzyme and malic acid dehydrogenase). The cells performed osmotic adjustment to high salinity by uptake of K⁺, Na⁺, Cl^? and formation of proline as well as by a reduction in cell size. Although sugar and starch content of the cells changed during light/dark transition, a CAM-related diurnal fluctuation of malic acid was not observed.     

Salt tolerance, salt accumulation, and ionic homeostasis in an epidermal bladder-cell-less mutant of the common ice plant Mesembryanthemum crystallinum

The aerial surfaces of the common or crystalline ice plant Mesembryanthemum crystallinum L., a halophytic, facultative crassulacean acid metabolism species, are covered with specialized trichome cells called epidermal bladder cells (EBCs). EBCs are thought to serve as a peripheral salinity and/or water storage organ to improve survival under high salinity or water deficit stress conditions. However, the exact contribution of EBCs to salt tolerance in the ice plant remains poorly understood. An M. crystallinum mutant lacking EBCs was isolated from plant collections mutagenized by fast neutron irradiation. Light and electron microscopy revealed that mutant plants lacked EBCs on all surfaces of leaves and stems. Dry weight gain of aerial parts of the mutant was almost half that of wild-type plants after 3 weeks of growth at 400 mM NaCl. The EBC mutant also showed reduced leaf succulence and leaf and stem water contents compared with wild-type plants. Aerial tissues of wild-type plants had approximately 1.5-fold higher Na(+) and Cl(-) content than the mutant grown under 400 mM NaCl for 2 weeks. Na(+) and Cl(-) partitioning into EBCs of wild-type plants resulted in lower concentrations of these ions in photosynthetically active leaf tissues than in leaves of the EBC-less mutant, particularly under conditions of high salt stress. Potassium, nitrate, and phosphate ion content decreased with incorporation of NaCl into tissues in both the wild type and the mutant, but the ratios of Na(+)/K(+) and Cl(-)/NO(3)(-)content were maintained only in the leaf and stem tissues of wild-type plants. The EBC mutant showed significant impairment in plant productivity under salt stress as evaluated by seed pod and seed number and average seed weight. These results clearly show that EBCs contribute to succulence by serving as a water storage reservoir and to salt tolerance by maintaining ion sequestration and homeostasis within photosynthetically active tissues of M. crystallinum.     

Efficient plant regeneration of Mesembryanthemum crystallinum via somatic embryogenesis

 An efficient plant regeneration procedure has been established from hypocotyl explants of the common ice plant, Mesembryanthemum crystallinum L, a halophytic leaf succulent that exhibits a stress-induced switch from C3 photosynthesis to crassulacean acid metabolism (CAM). Somatic embryos were initiated and developed up to globular and heart stages in Murashige and Skoog (MS) media supplemented with 3% sucrose, 0.6% bacto-agar, 80 mM NaCl, 5 μM 2,4-D and 1 μM kinetin. High frequency regeneration occurred when somatic embryos were germinated on media that lacked 2,4-D. High cytokinin treatment suppressed normal growth of embryos and favored abnormal embryo proliferation. Without growth regulators, regenerated plants rooted on MS medium with 100% efficiency. Mature, regenerated plants were fertile and morphologically identical to seed-derived plants. Received: 29 April 1999 / Revision received: 2 July 1999 · Accepted: 12 July 1999     

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.     

Evidence that the induction of crassulacean acid metabolism by water stress in Mesembryanthemum crystallinum (L.) involves root signalling

The aim of this study was to investigate whether the root system of Mesembryanthemum crystallinum (L.) plays a role in triggering the induction of crassulacean acid metabolism (CAM) during water stress. Depriving well-irrigated plants of water, by allowing the soil surrounding the roots to dry, caused increased daily losses in leaf relative water content (RVVC) and mesophyll cell turgor pressure. The RWC of the roots also declined. Subsequently plants exhibited physiological characteristics of CAM photosynthesis (i.e. diurnal fluctuations in leaf titratable acidity and nocturnal net CO₂ fixation). When the root system of plants was divided equally between two soil compartments and one half deprived of water, plants exhibited physiological characteristics of CAM without prior changes in leaf RWC content or mesophyll cell turgor pressure. Only the RWC of the water-stressed portion of the roots was reduced. These data suggest that in water-stressed plants daily changes in leaf water relations greater than those observed in well-irrigated plants, are not essential to trigger CAM expression. It is probable that a reduction in soil water availability can be perceived by the roots of M. crystallinum and that this information is conveyed to the leaves triggering the transition from C₃ to CAM photosynthesis.     

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.     

盐条件下不同植物生长调节剂对冰菜种子萌发的影响

冰菜是一种具有极强耐盐性的新型蔬菜,具有极高的营养价值和保健作用。本文针对冰菜种子萌发率低的问题,采用不同浓度的赤霉素(GA_3)、水杨酸(SA)和抗坏血酸(Vc)等3种植物生长调节剂在盐条件下处理冰菜种子。研究结果表明:冰菜种子萌发的最适盐浓度为10 g·L^-1。在此盐度下,若添加10 mg·L^-1的GA_3,冰菜种子的发芽率最高可达77.3%;也可添加20 mg·L^-1的水杨酸或20 mg·L^-1的抗坏血酸,都会对冰菜种子萌发具有明显的促进效果。     

Subcellular localization and stress responses of superoxide dismutase isoforms from leaves in the C3-CAM intermediate halophyte Mesembryanthemum crystallinum L.

BSA, bovine serum albumin
CAM, Crassulacean acid metabolism
DTT, dithiothreitol
EDTA, ethylenediaminetetraacetic acid
FPLCfast protein liquid chromatography
HEPES, N-(2-hydroxyethyl)piperazine-?-(ethanesulphonic acid)
ME, β-mercaptoethanol
NBT, nitro blue tetrazolium
PAGE, polyacrylamide gel electrophoresis
SDS, sodium dodecyl sulphate
SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis
Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39)
SOD, superoxide dismutase (EC 1.15.1.1)
TEMED, N,N,?,?-tetramethylethylenediamine
Tris, Tris (hydroxymethyl) aminomethane
Tricine, N-Tris(hydroxymethyl)methylglycine

Treatment of Mesembryanthemum crystallinum for several days with 0·4 kmol m^–3 NaCl in the root medium, in parallel to an increase of the cell sap osmolarity enhances activity of important antioxidative enzymes, such as superoxide dismutases (SODs). M. crystallinum is equipped with three SOD isoforms. These isoforms were identified as Mn-, Fe-, and Cu/Zn-SODs, respectively. Mn-SOD was found in the mitochondrial fraction, Fe-SOD in the chloroplast fraction, and Cu/Zn-SOD is probably localized in the cytosol. The Fe-SOD found in M. crystallinum is the first iron-containing SOD enzyme to be characterized in the plant family Aizoaceae. Salt treatment increases the activity of this isoform earlier than the other SODs. Molecular masses of SOD isoforms were determined as 82, 48 and 34 kDa for Mn-, Fe-, Cu/Zn-SODs, respectively. Native Mn-SOD seems to be a tetramer, while Fe-SOD and Cu/Zn-SOD are dimers. All SOD isoforms show high thermal stability. Mn-SOD is active even after short heating at 90 °C and Fe-SOD at 70 °C. Moreover, high concentrations of β-mercaptoethanol used as a reducing agent did not destroy the function of all isoforms. With the salinity treatment in M. crystallinum, Crassulacean acid metabolism (CAM) is induced. Build-up of large stationary O₂ concentrations in the leaf air spaces is associated with the photosynthetic CO₂ reduction behind closed stomata in phase III of CAM. This illustrates why M. crystallinum may require higher antioxidative activities under NaCl stress and also explains earlier findings that CAM plants are more resistant than C₃ plants to environmental stress as imposed by, for example, SO₂ and O₃.     

Influence of long photoperiods on plant development and expression of Crassulacean acid metabolism in Mesembryanthemum crystallinum

Abstract. In the natural habitat plants of Mesembryanthemum crystallinum are induced to perform Crassulacean acid metabolism (CAM) after 3 months, and reproductive growth begins after 5 months (Winter, Liittge & Winter, 1978, Oecologia (Berlin), 34, 225-237). The life cycle of M. crystallinum and the extent of growth required prior to induction of enzymes of Crassulacean acid metabolism (CAM) are dramatically shortened by growing seedlings with a long photoperiod (3=16h/8h light/dark). Reproductive growth begins as soon as five weeks after germination when plants are grown in continuous light (under 600μmol quanta m⁻² s⁻¹, 30°C). In plants grown under well-watered conditions, the activities of PEP carboxylase and NADP-malic enzyme begin increasing markedly 2 weeks after germination, with plants grown under longer photoperiods having higher enzyme activities. After 3 weeks of growth, leaves accumulated a large amount of malate, but the microequivalents of malate present were up to nine times greater than the total titratable acidities. Interestingly, plants from a 24h/0h or a 20h/4h photo-period showed no diurnal fluctuation of malate, but did produce malate in the light as a major photosynthetic end product. That is, under these environmental conditions, principal enzymes of CAM can be induced without the plants performing CAM. However, plants grown in a 16h/8h photoperiod did exhibit nocturnal accumulation of malate after 3 weeks of growth. In plants of all three growth conditions, the activities of NADP-malic enzyme and PEP carboxylase were further increased two- to live-fold by irrigating 3-week-old-plants with 350mol m⁻³ NaCl. Such early enhancement of these enzymes by salt and the shortened life cycle may be due to an accelerated development under the long photoperiods.     

Stress-dependent accumulation of spermidine and spermine in the halophyte Mesembryanthemum crystallinum under salinity conditions

We studied the effects of chloride salinity (300 and 500 mM NaCl) on the content of free polyamines (PAs) from putrescine (Put) family in Mesembryanthemum crystallinum L. leaves and roots. The contents of Put and spermidine (Spd) in leaves increased temporarily, achieving the highest values on the third day of salinity treatment; thereafter (by days 7–14), they dropped sharply. The content of spermine (Spm) increased gradually, and its high level was maintained until the end of experiment. The dynamics of Spm accumulation in leaves under salinity conditions resembled that of phosphoenolpyruvate carboxylase (PEPC), a key enzyme of the water-saving CAM pathway of photosynthesis. This indicates indirectly the involvement of Spm in the common ice plant adaptation to salinity. A decrease in the molar ratios of Spd to Spm in the leaves under salinity conditions could point to the acceleration of Spm biosynthesis (accumulation) during plant adaptation, whereas the levels of Spm precursors, Put and Spd, did not increase. This phenomenon could be explained by an accelerated conversion of Spd into Spm, an active liberation of free Spm from its conjugates, or changes in the rates of studied PA biosynthesis and degradation under salinity. At the same time, the intracellular concentration of ethylene rose under these conditions. It was supposed and then demonstrated, that the pathway of ethylene biosynthesis and that of the synthesis of Put family PAs compete under severe salinity conditions. This competition might be based on the disturbances in sulfur metabolism and a decrease in the methionine content, an immediate precursor of S-adenosyl-L-methionine.     

Compartmentation of Cadmium and Iron in Mesembryanthemum crystallinum Plants during the Adaptation to Cadmium Stress

The common ice plants (Mesembryanthemum crystallinum) at the stage of five leaf pairs were exposed to cadmium chloride solutions (1, 0.1, and 0.01 mM) under the conditions of water culture. After five days, the partition of cadmium and iron in the plant organs and in the cell structures of the apical root region were investigated. Plant adaptation to excess cadmium in the environment was assessed by an increase in the leaf and root weight, a change in peroxidase activity, and an accumulation of proline. The common ice plant accumulated cadmium mainly in the root system. At a high concentration of cadmium in the nutrient solution (1 mM), its content in the root exceeded 2 g/kg fr wt, while at a concentration of 0.01 mM, it was as low as 10 mg/kg. Dithizone staining of transverse sections of the root apical region showed that, after a 48-h-long exposure of plants to 0.1 mM cadmium chloride, cadmium was localized in the cell walls of endodermis and metaxylem. The level of cadmium in leaves varied from 0.5 to 18 mg/kg fr wt. However, there was only a weak correlation between cadmium accumulation and the extent of a biomass decrease in the leaves of various stories, when cadmium concentration in the medium (1 mM cadmium chloride) was toxic. This fact could be related to a marked efflux of endogenous iron from old leaves into the young ones and to a change in the cadmium/iron ratio in the tissues. Proline accumulation in the third leaf pair and in the roots occurred at a relatively low cadmium content (10–12 mg/kg fr wt) in these organs. Maxima of activity of all three forms of peroxidase, viz., soluble, ionically-bound, and covalently-bound peroxidases, in roots were found at a high accumulation of cadmium in these organs (45 mg/kg fr wt). These maxima exceeded 3–4-fold the activity in aging leaves containing 5 mg cadmium/kg fr wt. A decrease in peroxidase activity in leaves was accompanied by a 3.3-fold decrease in iron content; thus, it could be caused by a deficiency of available iron necessary for the enzyme functioning. It was concluded that the resistance of Mesembryanthemum crystallinum, a halophyte, to excess cadmium content in the medium was achieved by its predominant accumulation in roots, where excess cadmium is compartmentalized in the apoplast and seems to be subjected to detoxification through pectate formation. Moreover, the leaves and, particularly, the roots are characterized by a high activity of the antioxidant systems, such as guaiacol-dependent peroxidases, and an occurrence of proline at modest cadmium concentrations.     

Effect of hypermethylation of CCWGG sequences in DNA of Mesembryanthemum crystallinum plants on their adaptation to salt stress

Under salt stress conditions, the level of CpNpG-methylation (N is any nucleoside) of the nuclear genome of the facultative halophyte Mesembryanthemum crystallinum in the CCWGG sequences (W = A or T) increases two-fold and is coupled with hypermethylation of satellite DNA on switching-over of C3-photosynthesis to the crassulacean acid metabolism (CAM) pathway of carbon dioxide assimilation. The methylation pattern of the CCWGG sequences is not changed in both the 5'-promoter region of the gene of phosphoenolpyruvate carboxylase, the key enzyme of C4-photosynthesis and CAM, and in the nuclear ribosomal DNA. Thus, a specific CpNpG-hypermethylation of satellite DNA has been found under conditions of expression of a new metabolic program. The functional role of the CpNpG-hypermethylation of satellite DNA is probably associated with formation of a specialized chromatin structure simultaneously regulating expression of a large number of genes in the cells of M. crystallinum plants on their adaptation to salt stress and switching-over to CAM metabolism.     

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.