Limitation of C3–CAM shift in the common ice plant under high irradiance

In the halophytic plant Mesembryanthemum crystallinum salinity or drought can change the mode of photosynthesis from C₃ to crassulacean acid metabolism (CAM). These two stress factors are linked to oxidative stress, however, the induction of CAM by oxidative stress per se is not straightforward. Treatment with high light (HL) did not lead to the induction of CAM, as documented by a low night/day difference in malate level and a low expression of the CAM-related form of phosphoenolcarboxylase (Ppc1), despite causing some oxidative damage (elevated MDA level, malondialdehyde). In contrast to the action of high salinity (0.4 M NaCl), HL treatment did not activate neither the cytosolic NADP-malic enzyme nor the chloroplastic form of NADP-dependent malate dehydrogenase (NADP-MDH). In plastids of HL-treated plants a huge amount of starch was accumulated. This was associated with a weak stimulation of hydrolytic and phosphorolytic starch-degrading enzymes, in contrast to their strong up-regulation under high salinity. It is concluded that HL alone is not able to activate starch degradation necessary for CAM performance. Moreover, in the absence of salinity in C₃M. crystallinum plants an age-dependent increase in energy dissipation from PSII was documented under high irradiance, as illustrated by non-photochemical quenching (NPQ). Obtained data suggest that in this halophytic species several photoprotective strategies are strictly salinity-dependent.     

Starch Degradation and Distribution of the Starch-Degrading Enzymes in Vicia faba Leaves (Diurnal Oscillation of Amylolytic Activity and Starch Content in Chloroplasts)

Subcellular localization of the starch-degrading enzymes in Vicia faba leaves was achieved by an electrophoretic transfer method through a starch-containing gel (SCG) and enzyme activity measurements. Total amylolytic and phosphorolytic activities were found predominantly in the extrachloroplastic fraction, whereas the debranching enzymes showed homogenous distribution between stromal and extrachloroplastic fractions. Staining of end products in the SCG revealed two isoforms of [alpha]-amylase (EC 3.2.1.1) and very low [beta]-amylase activity (EC 3.2.1.2) in the chloroplast preparation, whereas [alpha]- and [beta]-amylase exhibited higher activities in the crude extract. However, it is unclear whether the low [alpha]- and [beta]-amylase activities associated with the chloroplast are contamination or activities that are integrally associated with the chloroplast. Study of the diurnal fluctuation of the starch content and of the amylase activities under a 9-h/15-h photoperiod showed a 2-fold increase of the total amylolytic activity in the chloroplasts concurrent with the starch degradation in the dark. No fluctuation was detectable for the extrachloroplastic enzymes. The possible roles and function of the chloroplastic and extrachloroplastic hydrolytic enzymes are discussed.     

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.     

Effects of lactic acid fermentation and gamma irradiation of barley on antinutrient contents and nutrient digestibility in mink (Mustela vison) with and without dietary enzyme supplement

Abstract

The experiment was conducted to study the effects of fermentation of barley, using two different strains of lactic acid bacteria, a Lactobacillus plantarum/pentosus strain isolated from spontaneously fermented rye sourdough (AD2) and a starch-degrading Lactobacillus plantarum (AM4), on contents of mixed-linked (1 → 3) (1 → 4)-β-glucans, α-amylase inhibitor activity, inositol phosphates, and apparent digestibility of macronutrients in mink. Effects of fermentation were compared with effects of gamma irradiation (γ-irradiation: ⁶⁰Co γ-rays at 25 kGy). The diets were fed to mink with and without a supplementary enzyme preparation. Both lactic acid fermentation and γ-irradiation followed by soaking and incubation, reduced concentrations of soluble β-glucans, phytate and α-amylase inhibitor activity. Dietary enzyme supplementation increased significantly digestibility of crude protein, fat, starch and crude carbohydrate (CHO). Fermentation of the barley increased digestibility of starch and CHO. Fermentation with lactic acid bacteria AD2 resulted in higher starch and CHO digestibility than strain AM4, and had greater effect than γ-irradiation, soaking and incubation. The highest digestibility of starch and CHO was obtained after AD2 fermentation followed by enzyme supplementation. It is concluded that both lactic acid fermentation of barley and enzyme supplementation have positive nutritional implications in the mink by limiting the effects of antinutrients and improving digestibility and energy utilization.     

Starch degradation in the cotyledons of germinating lentils

Starch, total amylolytic and phosphorylase activities were determined in lentil cotyledons during the first days of germination. Several independent criteria show that the amylolytic activity is due mainly to an amylase of the α type. Starch is degraded slowly in the first days; during this time, α- and β-amylase activity are very low, while phosphorylase increases and reach a peak on the 3rd day. On the 4th day, there is a more rapid depletion of starch which coincides with an increase in α-amylase activity. By polyacrylamide gel electrophoresis of the crude starch-degrading enzyme, five bands were obtained: one phosphorylase, three α-amylases, and one β-amylase. Based on their heat lability or heat stability, two sets of α-amylase seem to exist in lentil cotyledons.     

Induction of Crassulacean Acid Metabolism in the Facultative Halophyte Mesembryanthemum crystallinum by Abscisic Acid

The facultative halophyte, Mesembryanthemum crystallinum, shifts its mode of carbon assimilation from the C₃ pathway to Crassulacean acid metabolism (CAM) in response to water stress. In this study, exogenously applied abscisic acid (ABA), at micromolar concentrations, could partially substitute for water stress in induction of CAM in this species. ABA at concentrations of 5 to 10 micromolar, when applied to leaves or to the roots in hydroponic culture or in soil, induced the expression of CAM within days (as indicated by the nocturnal accumulation of total titratable acidity and malate). After applying ABA there was also an increase in phosphoenolpyruvate carboxylase and NADP-malic enzyme activities. The degree and time course of induction by ABA were comparable to those induced by salt and water stress. Electrophoretic analyses of leaf soluble protein indicate that the increases in phosphoenolpyruvate carboxylase activity during the induction by ABA, salt, and water stress are due to an increase in the quantity of the enzyme protein. ABA may be a factor in the stress-induced expression of CAM in M. crystallinum, serving as a functional link between stress and biochemical adaptation.     

Investigation of phosphoeno/pyruvate carboxylase (PEPCase) in Mesembryanthemum crystallinum L. in C3 and CAM photosynthetic states

When exposed to osmotic stress, Mesembryanthemum crystallinum plants switch from C₃ to CAM photosynthesis. Phosphoenolpyruvate carboxylase (PEPCase) is a key enzyme in CAM plants, being responsible for the initial fixation of CO₂. In C₃ plants the enzyme has been shown to be involved in the replenishing of TCA cycle intermediates and in the operation of stomatal guard cells. Multiple PEPCase isoforms were observed in C₃-performing leaves with four isoelectric points of 5.2, 5.5, 5.6 and 5.9 and four apparent subunit molecular masses of 105, 108, 113 and 116 kDa. In some instances, subunits of different size possessed exactly the same pI. The induction of CAM led to the predominance of a new isoform of pI 6.5 with subunit molecular mass of 108 kDa, but in addition, changes were observed in some of the isoforms present in the C₃ plant. PEPCase subunits were purified from the C₃ and CAM forms of M. crystallinum and subjected to pep-tide mapping. Two distinct though similar sets of maps were obtained, one from the CAM isoform (pI 6.5) and C₃-associated subunits of pi 5.9 and another for C₃ subunits of pI 5.2 and 5.5. It was inferred from these data that the C₃ isoforms expressed in the leaf were derived from at least two genes. The C₃ isoform (pI 5.9) showing greatest similarity to the CAM isoform in terms of peptide mapping also increased in response to salt stress. It is speculated that the CAM isoform may have evolved from this enzyme.     

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

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 Imtl, which encodes a methyl transferase involved in the biosynthesis of a putative osmoprotectant, pinitol. Imtl 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 Imtl 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.     

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.     

A starch-accumulating mutant of Arabidopsis thaliana deficient in a chloroplastic starch-hydrolysing enzyme

The aim of this work was to identify enzymes that participate in the degradation of transitory starch in Arabidopsis . A mutant line was isolated by screening leaves at the end of the night for the presence of starch. The mutant had a higher starch content than the wild-type throughout the diurnal cycle. This accumulation was due to a reduction in starch breakdown, leading to an imbalance between the rates of synthesis and degradation. No reduction in the activity of endo-amylase (α-amylase), β-amylase, starch phosphorylase, maltase, pullulanase or D-enzyme could be detected in crude extracts of leaves of the mutant. However, native PAGE in gels containing amylopectin revealed that a starch-hydrolysing activity, putatively identified as an endo-amylase and present in wild-type chloroplasts, was absent or appreciably reduced in the mutant. This is the first time that a specific enzyme required for starch degradation has been identified in leaves.     

Activity of enzymes of carbon metabolism during the induction of Crassulacean acid metabolism in Mesembryanthemum crystallinum L.

The maximum extractable activities of twenty-one photosynthetic and glycolytic enzymes were measured in mature leaves of Mesembryanthemum crystallinum plants, grown under a 12 h light 12 h dark photoperiod, exhibiting photosynthetic characteristics of either a C₃ or a Crassulacean acid metabolism (CAM) plant. Following the change from C₃ photosynthesis to CAM in response to an increase in the salinity of in the rooting medium from 100 mM to 400 mM NaCl, the activity of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) increased about 45-fold and the activities of NADP malic enzyme (EC 1.1.1.40) and NAD malic enzyme (EC 1.1.1.38) increased about 4- to 10-fold. Pyruvate, Pi dikinase (EC 2.7.9.1) was not detected in the non-CAM tissue but was present in the CAM tissue; PEP carboxykinase (EC 4.1.1.32) was detected in neither tissue. The induction of CAM was also accompanied by large increases in the activities of the glycolytic enzymes enolase (EC 4.2.1.11), phosphoglyceromutase (EC 2.7.5.3), phosphoglycerate kinase (EC 2.7.2.3), NAD glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), and glucosephosphate isomerase (EC 2.6.1.2). There were 1.5- to 2-fold increases in the activities of NAD malate dehydrogenase (EC 1.1.1.37), alanine and aspartate aminotransferases (EC 2.6.1.2 and 2.6.1.1 respectively) and NADP glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13). The activities of ribulose-1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39), fructose-1,6-bisphosphatase (EC 3.1.3.11), phosphofructokinase (EC 2.7.1.11), hexokinase (EC 2.7.1.2) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) remained relatively constant. NADP malate dehydrogenase (EC 1.1.1.82) activity exhibited two pH optima in the non-CAM tissue, one at pH 6.0 and a second at pH 8.0. The activity at pH 8.0 increased as CAM was induced. With the exceptions of hexokinase and glucose-6-phosphate dehydrogenase, the activities of all enzymes examined in extracts from M. crystallinum exhibiting CAM were equal to, or greater than, those required to sustain the maximum rates of carbon flow during acidification and deacidification observed in vivo. There was no day-night variation in the maximum extractable activities of phosphoenolpyruvate carboxylase, NADP malic enzyme, NAD malic enzyme, fructose-1,6-bisphosphatase and NADP malate dehydrogenase in leaves of M. crystallinum undergoing CAM.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - RuBP ribulose-1,5-bisphosphate     

Starch depletion in germinating wheat,wrinkled-seeded peas and senescing tobacco leaves

The residual starches of germinating wheat and barley grains show similar structural changes. Germinating wheat grains produce malto-oligosaccharides and dextrins. The starch of wrinkled-seeded peas showed some structural changes during germination, but the starch from senescing tobacco leaves showed none. Neither peas nor tobacco produced malto-oligosaccharides or dextrins at any stage. Wrinkled-seeded peas showed some differences to smooth-seeded peas in enzyme content, and starch was probably degraded by phosphorylase initially with α-amylase acting after 3 days. In senescing tobacco leaves the only significant enzyme activities were α-amylase and maltase. Wheat closely resembled barley in showing amylolytic breakdown.     

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.     

Separation and purification of the tonoplast ATPase and pyrophosphatase from plants with constitutive and inducible Crassulacean acid metabolism

Tonoplast vesicles were isolated from Kalanchoe daigremontiana Hamet et Pierrer de la Bâthie and Mesembryanthemum crystallinum L., exhibiting constitutive and inducible crassulacean acid metabolism (CAM), respectively. Membrane-bound proteins were detergent-solubilized with 2% of Triton X-100. During CAM induction in M. crystallinum, ATPase activity increases four-fold, whereas pyrophosphatase activity decreases somewhat. With all plants, ATPase and pyrophosphatase could be separated by size-exclusion chromatography (SEC, Sephacryl S 400), and the ATPase was further purified by diethylaminoethyl-ion-exchange chromatography. Sodium-dodecyl-sulfate electrophoresis of the SEC fractions from K. daigremontiana containing maximum ATPase activity separates several protein bands, indicating subunits of 72, 56, 48, 42, 28, and 16 kDa. Purified ATPase from M. crystallinum in the C₃ and CAM states shows a somewhat different protein pattern. With M. crystallinum, an increase in ATP-hydrolysis and changes in the subunit composition of the native enzyme indicate that the change from the C₃ to the CAM state is accompanied by de-novo synthesis and by structural changes of the tonoplast ATPase.Abbreviations CAM Crassulacean acid metabolism - DTT dithiothreitol - kDa kilodalton - PAGE polyacrylamide gel electrophoresis - PPiase pyrophosphatase - SEC size exclusion chromatography - SDS sodium dodecyl sulfate - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

A STARCH-AGAR GEL METHOD FOR THE LOCALIZATION OF STARCH HYDROLYZING ENZYMES IN THE COTYLEDONS AND HYPOCOTYLS OF BEANS

A starch-agar gel assay was developed for the localization of enzyme activity in the cotyledons and hypocotyls of beans. The four major starch hydrolyzing enzymes, α-amylase, β-amylase, debranching enzyme and phosphorylase, were assayed with this technique using gels containing different substrates and buffers. Hypocotyls and cotyledons were sectioned by hand and the sections were incubated on the gel for varying lengths of time. Only α-amylase was observed to increase significantly. While virtually absent in ungerminated axes and hypocotyls from young seedlings, it increased first in the basal region and subsequently in progressively higher portions of the hypocotyl. High activities were first observed in the pith and later in the cortex. The increased activity was most obviously associated with the degradation of the pith, suggesting a regulation relative to cellular autolysis.