Comparative transcriptomic profiling reveals differentially expressed genes and important related metabolic pathways in shoots and roots of a Saudi wheat cultivar (Najran) under salinity stress.

High salinity of soil is a threatening constraint for agricultural output worldwide. The adverse effects of salt stress on plants can be revealed in different manners, from phenotypic to genetic changes. A comparative RNA-Sequencing analysis was done in roots and shoots of bread wheat, Najran cultivar between plants grown under unstressed control condition (0 mM NaCl) and salt treatment (200 mM NaCl). More than 135 million and 137 million pair-end reads were obtained from root and shoot samples, respectively. Of which, the mapped reads to Triticum aestivum genome IWGSC_V51 ranged from 83.9% to 85% in the root and 71.6% to 79% in the shoot. Interestingly, a comparison of transcriptomic profiling identified that total number of significantly differentially expressed genes (DEGs) examined in the roots was much higher than that found in the shoots under NaCl treatment, 5829 genes were differentially expressed in the roots whereas 3495 genes in the shoots. The salt-induced change in the transcriptome was confirmed by RT-qPCR using a set of randomly selected genes. KEGG enrichment analysis classified all DEGs in both roots and shoots into 25 enriched KEGG pathways from three main KEGG classes: Metabolism, organismal systems and genetic information processing. According to that, the most significantly regulated pathways in the root and shoot tissues were glutathione metabolism and biosynthesis of secondary metabolites such as phenylpropanoids and galactose metabolism suggesting that these pathways might participate in wheat salt tolerance. The findings highlight the importance of the control of oxidative stress via Glutathione and phenylpropanoids and the regulation of galactose metabolism in the roots and shoots for salt-tolerance in wheat. They open promising prospects for engineering salt-tolerance in this important crop via targeted improvement of the regulation of key genes in the production of these compounds.

Leaf carbohydrates influence transcriptional and post-transcriptional regulation of nocturnal carboxylation and starch degradation in the facultative CAM plant, Mesembryanthemum crystallinum.

Nocturnal degradation of transitory starch is a limiting factor for the optimal function of crassulacean acid metabolism and must be coordinated with phosphoenolypyruvate carboxylase (PEPC)-mediated CO2 uptake to optimise carbon gain over the diel cycle. The aim of this study was to test the hypothesis that nocturnal carboxylation is coordinated with starch degradation in CAM via a mechanism whereby the products of these pathways regulate diel transcript abundance and enzyme activities for both processes. To test this hypothesis, a starch and CAM-deficient mutant of Mesembryanthemum crystallinum was compared with wild type plants under well-watered and saline (CAM-inducing) conditions. Exposure to salinity increased the transcript abundance of genes required for nocturnal carboxylation, starch and sucrose degradation in both wild type and mutant, but the transcript abundance of several of these genes was not sustained over the dark period in the low-carbohydrate, CAM-deficient mutant. The diel pattern of transcript abundance for PEPC mirrored that of PEPC protein, as did the transcripts, protein, and activity of chloroplastic starch phosphorylase in both wild type and mutant, suggesting robust diel coordination of these metabolic processes. Activities of several amylase isoforms were low or lacking in the mutant, whilst the activity of a cytosolic isoform of starch phosphorylase was significantly elevated, indicating contrasting modes of metabolic regulation for the hydrolytic and phosphorylytic routes of starch degradation. Externally supplied sucrose resulted in an increase in nocturnal transcript abundance of genes required for nocturnal carboxylation and starch degradation. These results demonstrate that carbohydrates impact on transcriptional and post-transcriptional regulation of nocturnal carboxylation and starch degradation in CAM.

Light quality modulates metabolic synchronization over the diel phases of crassulacean acid metabolism.

Temporal compartmentation of carboxylation processes is a defining feature of crassulacean acid metabolism and involves circadian control of key metabolic and transport steps that regulate the supply and demand for carbon over a 24h cycle. Recent insights on the molecular workings of the circadian clock and its connection with environmental inputs raise new questions on the importance of light quality and, by analogy, certain photoreceptors for synchronizing the metabolic components of CAM. The present work tested the hypothesis that optimal coupling of stomatal conductance, net CO2 uptake, and the reciprocal turnover of carbohydrates and organic acids over the diel CAM cycle requires both blue and red light input signals. Contrasting monochromatic wavelengths of blue, green, and red light (i.e. 475, 530, 630nm) with low fluence rates (10 µmol m(-2) s(-1)) were administered for 16 hours each diel cycle for a total treatment time of 48 hours to the obligate CAM bromeliad, Aechmea 'Maya'. Of the light treatments imposed, low-fluence blue light was a key determinant in regulating stomatal responses, organic acid mobilization from the vacuole, and daytime decarboxylation. However, the reciprocal relationship between starch and organic acid turnover that is typical for CAM was uncoupled under low-fluence blue light. Under low-fluence red or green light, the diel turnover of storage carbohydrates was orchestrated in line with the requirements of CAM, but a consistent delay in acid consumption at dawn compared with plants under white or low-fluence blue light was noted. Consistent with the acknowledged influences of both red and blue light as input signals for the circadian clock, the data stress the importance of both red and blue-light signalling pathways for synchronizing the metabolic and physiological components of CAM over the day/night cycle.

Cloning, localization and expression analysis of vacuolar sugar transporters in the CAM plant Ananas comosus (pineapple).

In photosynthetic tissues of the CAM plant pineapple (Ananas comosus), storage of soluble sugars in the central vacuole during the daytime and their remobilization at night is required to provide carbon skeletons for nocturnal CO(2) fixation. However, soluble sugars produced photosynthetically must also be exported to support growth processes in heterotrophic tissues. To begin to address how vacuolar sugar storage and assimilate partitioning are regulated in A. comosus, degenerate PCR and cDNA library screening were used to clone three candidate sugar transporters from the leaves of this species. Subcellular localization of the three transporters was investigated via expression of YFP-fusion proteins in tobacco epidermal cells and their co-localization with subcellular markers by confocal microscopy. Using this strategy, a putative hexose transporter (AcMST1) and a putative inositol transporter (AcINT1) were identified that both localized to the tonoplast, whereas a putative sucrose transporter (AcSUT1) was found to localize to prevacuolar compartments. A cDNA (AcMST2) with high similarity to a recently characterized tonoplast hexose transporter in Arabidopsis was also identified from an A. comosus fruit EST database. Analyses of transcript abundance indicated that AcMST1 was more highly expressed in fruits compared to leaves of A. comosus, whilst transcripts of AcINT1, AcSUT1, and AcMST2 were more abundant in leaves. Transcript abundance of AcINT1, the putative inositol transporter, showed day-night changes comparable to those of other CAM-related transcripts described in Mesembryanthemum crystallinum. The results are discussed in terms of the role of vacuolar sugar transporters in regulating carbon flow during the diel cycle in CAM plants.

Isolation and characterization of mutants of common ice plant deficient in crassulacean acid metabolism.

Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that improves water use efficiency by shifting part or all of net atmospheric CO2 uptake to the night. Genetic dissection of regulatory and metabolic attributes of CAM has been limited by the difficulty of identifying a reliable phenotype for mutant screening. We developed a novel and simple colorimetric assay to measure leaf pH to screen fast neutron-mutagenized populations of common ice plant (Mesembryanthemum crystallinum), a facultative CAM species, to detect CAM-deficient mutants with limited nocturnal acidification. The isolated CAM-deficient mutants showed negligible net dark CO2 uptake compared with wild-type plants following the imposition of salinity stress. The mutants and wild-type plants accumulated nearly comparable levels of sodium in leaves, but the mutants grew more slowly than the wild-type plants. The mutants also had substantially reduced seed set and seed weight relative to wild type under salinity stress. Carbon-isotope ratios of seed collected from 4-month-old plants indicated that C3 photosynthesis made a greater contribution to seed production in mutants compared to wild type. The CAM-deficient mutants were deficient in leaf starch and lacked plastidic phosphoglucomutase, an enzyme critical for gluconeogenesis and starch formation, resulting in substrate limitation of nocturnal C4 acid formation. The restoration of nocturnal acidification by feeding detached leaves of salt-stressed mutants with glucose or sucrose supported this defect and served to illustrate the flexibility of CAM. The CAM-deficient mutants described here constitute important models for exploring regulatory features and metabolic consequences of CAM.

Ascorbate oxidase-dependent changes in the redox state of the apoplast modulate gene transcript accumulation leading to modified hormone signaling and orchestration of defense processes in tobacco.

The role of the redox state of the apoplast in hormone responses, signaling cascades, and gene expression was studied in transgenic tobacco (Nicotiana tabacum) plants with modified cell wall-localized ascorbate oxidase (AO). High AO activity specifically decreased the ascorbic acid (AA) content of the apoplast and altered plant growth responses triggered by hormones. Auxin stimulated shoot growth only when the apoplastic AA pool was reduced in wild-type or AO antisense lines. Oxidation of apoplastic AA in AO sense lines was associated with loss of the auxin response, higher mitogen-activated protein kinase activities, and susceptibility to a virulent strain of the pathogen Pseudomonas syringae. The total leaf glutathione pool, the ratio of reduced glutathione to glutathione disulfide, and glutathione reductase activities were similar in the leaves of all lines. However, AO sense leaves exhibited significantly lower dehydroascorbate reductase and ascorbate peroxidase activities than wild-type and antisense leaves. The abundance of mRNAs encoding antioxidant enzymes was similar in all lines. However, the day/night rhythms in the abundance of transcripts encoding the three catalase isoforms were changed in response to the AA content of the apoplast. Other transcripts influenced by AO included photorespiratory genes and a plasma membrane Ca(2+) channel-associated gene. We conclude that the redox state of the apoplast modulates plant growth and defense responses by regulating signal transduction cascades and gene expression patterns. Hence, AO activity, which modulates the redox state of the apoplastic AA pool, strongly influences the responses of plant cells to external and internal stimuli.

Are the metabolic components of crassulacean acid metabolism up-regulated in response to an increase in oxidative burden?

In the halophytic species Mesembryanthemum crystallinum, crassulacean acid metabolism (CAM) may be induced by a range of abiotic factors including drought, salinity, high light intensity, low temperature, and anoxia. A key biotic consequence of all these environmental changes is the generation of reactive oxygen species in planta that can elicit potentially damaging oxidative reactions and/or act as signals for engaging mechanisms that alleviate oxidative stress. However, induction of CAM per se also has the potential for increasing the oxidative burden via the enhanced internal O2 concentrations that develop behind closed stomata during daytime decarboxylation. The aim of this paper was to test two hypotheses. The first one, that reactive oxygen species are key signals for up-regulating the major genes and proteins required for the operation of CAM as part of an integrated strategy for alleviating oxidative burden, was tested using gaseous ozone to increase the oxidative burden at a cellular level. The second hypothesis, that CAM potentially increases oxidative load, was tested using a CAM-deficient mutant of M. crystallinum. The data indicate that ozone, like salinity, elicits an increase in the transcript and protein abundance of myo-inositol o-methyl transferase (a key enzyme of cyclitol synthesis), together with phosphoenolpyruvate carboxylase and other 'CAM-related' enzymes. However, ozone, unlike salinity, does not induce functional CAM, implying that the various metabolic components required for CAM respond to different signals. Comparing the activities of different subcellular isoforms of superoxide dismutase in wild-type and CAM-deficient mutants of M. crystallinum suggests that the induction of CAM potentially curtails the oxidative load in planta.

Autophosphorylation and subcellular localization dynamics of a salt- and water deficit-induced calcium-dependent protein kinase from ice plant.

A salinity and dehydration stress-responsive calcium-dependent protein kinase (CDPK) was isolated from the common ice plant (Mesembryanthemum crystallinum; McCPK1). McCPK1 undergoes myristoylation, but not palmitoylation in vitro. Removal of the N-terminal myristate acceptor site partially reduced McCPK1 plasma membrane (PM) localization as determined by transient expression of green fluorescent protein fusions in microprojectile-bombarded cells. Removal of the N-terminal domain (amino acids 1-70) completely abolished PM localization, suggesting that myristoylation and possibly the N-terminal domain contribute to membrane association of the kinase. The recombinant, Escherichia coli-expressed, full-length McCPK1 protein was catalytically active in a calcium-dependent manner (K0.5 = 0.15 microm). Autophosphorylation of recombinant McCPK1 was observed in vitro on at least two different Ser residues, with the location of two sites being mapped to Ser-62 and Ser-420. An Ala substitution at the Ser-62 or Ser-420 autophosphorylation site resulted in a slight increase in kinase activity relative to wild-type McCPK1 against a histone H1 substrate. In contrast, Ala substitutions at both sites resulted in a dramatic decrease in kinase activity relative to wild-type McCPK1 using histone H1 as substrate. McCPK1 undergoes a reversible change in subcellular localization from the PM to the nucleus, endoplasmic reticulum, and actin microfilaments of the cytoskeleton in response to reductions in humidity, as determined by transient expression of McCPK1-green fluorescent protein fusions in microprojectile-bombarded cells and confirmed by subcellular fractionation and western-blot analysis of 6x His-tagged McCPK1.

Expression of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxylase kinase genes. Implications for genotypic capacity and phenotypic plasticity in the expression of crassulacean acid metabolism.

In plants with crassulacean acid metabolism (CAM), dark CO2 uptake is mediated by phosphoenolpyruvate carboxylase (PEPC), an enzyme that can be regulated at transcriptional and posttranslational levels. Reversible phosphorylation of PEPC is catalyzed by a dedicated PEPC kinase, which in turn is regulated at the transcriptional level over the 24-h cycle in CAM plants. PEPC kinase controls the day/night regulation of PEPC during the CAM cycle, thus facilitating plasticity for optimizing CO2 uptake under different environmental conditions. To understand the importance of PEPC kinase in relation to its target PEPC in terms of CAM performance, the expression of the genes encoding the two enzymes was investigated in four species of Clusia that have photosynthetic patterns ranging from C3 photosynthesis to constitutive CAM. By linking changes in the expression of PEPC and PEPC kinase to day/night patterns of leaf gas exchange, organic acid, and soluble sugar contents under different environmental conditions, the genetic and metabolic limitations to CAM plasticity were assessed. The results indicate that PEPC expression is a major factor underpinning the genotypic capacity for CAM and that PEPC kinase expression does not appear to limit CAM. The day/night regulation of Ppck transcript abundance was found to be a consequence of CAM and the day/night cycling of associated metabolites, rather than the primary controlling factor for the temporal separation of carboxylation processes.

Synchronization of metabolic processes in plants with Crassulacean acid metabolism.

In plants with Crassulacean acid metabolism, a diel separation of carboxylation processes mediated by phosphoenolpyruvate carboxylase (PEPC) and Rubisco optimizes photosynthetic performance and carbon gain in potentially limiting environments. This review considers the mechanisms that synchronize the supply and demand for carbon whilst maintaining photosynthetic plasticity over the 24 h CAM cycle. The circadian clock plays a central role in controlling many of the metabolic, transport and physiological components of CAM. The level of control exerted by the clock can range from transcriptional through to post-translational regulation, depending on the genes, proteins, and even plant species under consideration. A further layer of control is provided by metabolites, including organic acids and carbohydrates, which show substantial reciprocal fluctuations in content over the diel cycle. Mechanisms responsible for the sensing of metabolite contents are discussed, together with signalling requirements for the co-ordination of carbon fluxes. Evolutionary implications are considered in terms of how circadian and metabolic control of the CAM cycle may have been derived from C3 plants.

Expression, purification, and initial characterization of a recombinant form of plant PEP-carboxylase kinase from CAM-induced Mesembryanthemum crystallinum with enhanced solubility in Escherichia coli.

Plant phosphoenolpyruvate-carboxylase kinase (PEPC-kinase [PpcK]) is the smallest Ser/Thr kinase identified to date, having a molecular mass of approximately 32,000. This novel, monomeric kinase is dedicated to the phosphorylation of plant PEPC, thereby regulating this target enzyme's activity and allosteric properties. Although several recombinant, non-fusion PpcK proteins have been produced recently in Escherichia coli, these are plagued by their high degree of insolubility. Here, we report the use of the native, E. coli NusA protein and a related E. coli expression vector (pET-43a(+) [Novagen]) for enhancing the solubility of this recalcitrant Ser/Thr kinase at least 10-fold by its production as a dual 6xHis-tagged NusA/McPpcK1 fusion protein, which accounts for approximately 10% of the soluble protein fraction from induced cells. Capture of this fusion protein from the centrifuged cell extract by immobilized metal (Ni(2+)) affinity-chromatography, its "on-bead" cleavage by thrombin, and subsequent elution yielded milligram quantities of a "free," approximately 36-kDa form of PpcK for further purification by fast-protein liquid chromatography on blue dextran-agarose or preparative SDS-PAGE. Steady-state kinetic analysis of the former, active preparation revealed that this dedicated kinase discriminates against neither various isoforms of plant PEPC nor certain mutant forms of recombinant C(4) PEPC. Alternatively, the latter, electrophoretically homogeneous sample of the approximately 36-kDa polypeptide was used as antigen for polyclonal-antibody production in rabbits. The antibodies against the recombinant McPpcK1 from Mesembryanthemum crystallinum cross-reacted on Western blots with an enriched preparation of the maize-leaf kinase, but not with the parent crude extract, thus directly documenting this protein's extremely low abundance in vivo. However, these antibodies were effective in immunoprecipitating 32P-based PpcK activity from crude, desalted extracts of maize leaves and soybean root-nodules.

Integrating diel starch metabolism with the circadian and environmental regulation of Crassulacean acid metabolism in Mesembryanthemum crystallinum.

The diel (24-h) Crassulacean acid metabolism (CAM) cycle in Mesembryanthemum crystallinum (L.) requires rhythmic patterns of transitory starch degradation to produce carbon skeletons for phospho enolpyruvate (PEP) synthesis during the nocturnal Phase I, when PEP carboxylase (PEPc) mediates CO(2) fixation. Under a normal light-dark cycle, nocturnal malate accumulation and nocturnal CO(2) uptake were observed for CAM-induced, but not C(3), M. crystallinum. In both C(3) and CAM plants, transcripts encoding beta-amylase and starch phosphorylase accumulated during the afternoon and declined nocturnally. Under a continuous light regime, ribulose-1,5-bisphosphate carboxylase/oxygenase activity remained co-ordinated with the CAM phases, and circadian abundance patterns were observed for transcripts encoding starch degradative enzymes. Despite circadian PEPc kinase expression, the accumulation of vacuolar malate ceased under continuous light. Exposure to CO(2)-free air for 24 h inhibited starch accumulation over the photoperiod, but re-fixation of respiratory CO(2) resulted in the overnight accumulation of malate to levels comparable to those of control plants. Upon return to normal air, the depleted starch concentration led to stoichiometric decreases in Phase-I CO(2) uptake and malate accumulation. The up-regulation of PEPc kinase transcripts under these conditions was ineffective at sustaining Phase-I CO(2) uptake under starch-limited conditions. We conclude that starch turnover regulates and limits carbon flux through the diel CAM cycle.

Environmental, hormonal and circadian regulation of crassulacean acid metabolism expression.

This paper originates from a presentation at the IIIrd International Congress on Crassulacean Acid Metabolism, Cape Tribulation, Queensland, Australia, August 2001. Expression of crassulacean acid metabolism (CAM) is characterized by the extreme plasticity observed within and between species. Switches between C3 photosynthesis and CAM, and subsequent 24-h patterns of day/night CO2 uptake, are tightly controlled by a variety of environmental and metabolic factors that optimize the response of CAM plants to the most challenging environments over seasonal and daily time scales. Regulation of the genes and enzymes involved in CAM and connected metabolic pathways occurs at a number of levels (transcriptional through to post-translational). Such multiple levels of control are considered to be the key to the photosynthetic plasticity of CAM. Here, we review some of the primary environmental and hormonal factors controlling CAM plasticity in different CAM-inducible species, with emphasis on the regulatory signalling circuits responsible for this control. We also examine the inherent circadian regulation of the pathway, mainly in the context of the diel regulation of phosphoenolpyruvate carboxylase and the dedicated kinase that modulates its activity. We then consider the role of secondary signals, with emphasis on changes in cytosolic [Ca2+]i and the downstream signalling pathways, based on studies conducted on Mesembryanthemum crystallinum L. Besides representing an important metabolic adaptation, CAM provides an intriguing paradigm for studying the complex signalling mechanisms that control and coordinate the expression of genes under a variety of short- and long-term environmental perturbations.