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Background: Salinity is one of the most serious threats to world agriculture. An important sugar-yielding crop sugar beet, which shows some tolerance to salt via a mechanism that is poorly understood. Proteomics data can provide important clues that can contribute to finally understand this mechanism.Results: Differentially abundant proteins (DAPs) in sugar beet under salt stress treatment were identified in leaves (70 DAPs) and roots (76 DAPs). Functions of these DAPs were predicted, and included metabolism and cellular, environmental information and genetic information processing. We hypothesize that these processes work in concert to maintain cellular homeostasis. Some DAPs are closely related to salt resistance, such as choline monooxygenase, betaine aldehyde dehydrogenase, glutathione S-transferase (GST) and F-type H+-transporting ATPase. The expression pattern of ten DAPs encoding genes was consistent with the iTRAQ data.Conclusions: During sugar beet adaptation to salt stress, leaves and roots cope using distinct mechanisms of molecular metabolism regulation. This study provides significant insights into the molecular mechanism underlying the response of higher plants to salt stress, and identified some candidate proteins involved in salt stress countermeasures.

Low temperature is a major factor limiting seedling growth and the production of cotton (Gossypium hirsutum L.). However, enhancing chilling tolerance is typically negatively correlated with yield in agricultural production. Here, we demonstrate that transgenic cotton expressing Atriplex hortensis choline monooxygenase (AhCMO) greatly enhanced resistance to chilling stress. The promotion of chilling tolerance is mainly due to an increase in the content of osmoprotectants, especially glycine betaine and proline. The increased chilling tolerance was further verified at the molecular level using genome-wide expression profiling by RNA-sequencing. Further detailed analysis showed that the number of genes involved in scavenging of reactive oxygen species (ROS) was down-regulated and the activity of superoxide dismutase (SOD) and catalase (CAT) were decreased in AhCMO transgenic cotton compared with wild type after low temperture treatment. More importantly, overexpression of AhCMO in cotton moderately improved cotton fiber yield in normal growth condition. These data show that AhCMO transgenic cotton enhances low temperature tolerance via directly accumulating cellular osmoprotectants. Manipulating the expression of AhCMO by biotechnological tools could be a powerful method to enhance chilling tolerance in cotton.

Choline monooxygenase (CMO) is a key enzyme involved in betaine synthesis and our preliminary work has shown that the SlCMO gene promoter (pC5: − 267 to + 128 base pair), cloned from Suaeda liaotungensis, is salt-inducible. In the present study, pC5-SlCMO was transferred into tomato (Solanum lycopersicon L. ‘Micro-Tom’) plants via Agrobacterium mediation. Homozygous transgenic plants were selected using quantitative real-time polymerase chain reaction. The expression of SlCMO in pC5-SlCMO transgenic plants was induced by salinity. Under salt tolerance, betaine content, chlorophyll content, and net photosynthetic rate were higher in transgenic plants than in wild-type (WT) plants. Proline content was lower in transgenic plants than in WT plants. Under normal conditions, seed germination, length of the whole plant, dry weight, and fruit products of transgenic plants were the same as in WT plants. These results demonstrated that the pC5 promoter can drive increased expression of SlCMO in transgenic tomato plants under salt stress and increase salt tolerance without affecting plant growth and yield.

In a changing climate, drought stress accounts for significant crop yield loss. To test the hypothesis that proteins play an important role in plant drought tolerance, we conducted physiological and proteomic analyses to investigate the stress responses of two sugar beet (Beta vulgaris L.) cultivars with contrasting tolerance to drought stress. Under drought, the tolerant cultivar had higher relative water content, root length, dry weight, and root system area than the drought-sensitive cultivar. In addition, the tolerant cultivar had higher antioxidant enzyme activities than the sensitive cultivar to prevent ROS damage under drought stress conditions. Proteomic analysis resulted in identification of 23 and 27 drought-responsive proteins in the sensitive and tolerant cultivars, respectively. Based on their functions, these proteins were classified into nine categories. Many proteins in several different biological processes showed different abundance patterns between the genotypes (for example, oxygen-evolving enhancer proteins, choline monooxygenase, and malate dehydrogenase). Although some proteins changed similarly in trend between the two genotypes, they showed different levels of changes (for example, S-adenosylmethionine synthase). Furthermore, the poor correlation between transcription and protein levels was confirmed in this study. Our findings would lead to an improved understanding of the integrated physiology and proteome responses of the different sugar beet genotypes under drought stress.

Higher plants contain a small, 5-member family of Rieske non-heme oxygenases that comprise the inner plastid envelope protein TIC55, phaeophorbide a oxygenasee (PAO), chlorophyllide a oxygenase (CAO), choline monooxygenase, and a 52 kDa protein (PTC52) associated with the precursor NADPH:protochlorophyllide (Pchlide) oxidoreductase A (pPORA) A translocon (PTC). Some of these chloroplast proteins have documented roles in chlorophyll biosynthesis (CAO) and degradation (PAO and TIC55), whereas the function of PTC52 remains unresolved. Biochemical evidence provided here identifies PTC52 as Pchlide a oxygenase of the inner plastid envelope linking Pchlide b synthesis to pPORA import. Protochlorophyllide b is the preferred substrate of PORA and its lack no longer allows pPORA import. The Pchlide b-dependent import pathway of pPORA thus operates in etiolated seedlings and is switched off during greening. Using dexamethasone-induced RNA interference (RNAi) we tested if PTC52 is involved in controlling both, pPORA import and Pchlide homeostasis in planta. As shown here, RNAi plants deprived of PTC52 transcript and PTC52 protein were unable to import pPORA and died as a result of excess Pchlide a accumulation causing singlet oxygen formation during greening. In genetic studies, no homozygous ptc52 knock-out mutants could be obtained presumably as a result of embryo lethality, suggesting a role for PTC52 in the initial greening of plant embryos. Phylogenetic studies identified PTC52-like genes amongst unicellular photosynthetic bacteria and higher plants, suggesting that the biochemical function associated with PTC52 may have an ancient evolutionary origin. PTC52 also harbors conserved motifs with bacterial oxygenases such as the terminal oxygenase component of 3-ketosteroid 9-alpha-hydroxylase (KshA) from Rhodococcus rhodochrous. 3D-modeling of PTC52 structure permitted the prediction of amino acid residues that contribute to the substrate specificity of this enzyme. In vitro-mutagenesis was used to test the predicted PTC52 model and provide insights into the reaction mechanism of this Rieske non-heme oxygenase.

Several halophytes and a few crop plants, including Poaceae, synthesize and accumulate glycine betaine (GB) in response to environmental constraints. GB plays an important role in osmoregulation, in fact, it is one of the main nitrogen-containing compatible osmolytes found in Poaceae. It can interplay with molecules and structures, preserving the activity of macromolecules, maintaining the integrity of membranes against stresses and scavenging ROS. Exogenous GB applications have been proven to induce the expression of genes involved in oxidative stress responses, with a restriction of ROS accumulation and lipid peroxidation in cultured tobacco cells under drought and salinity, and even stabilizing photosynthetic structures under stress. In the plant kingdom, GB is synthesized from choline by a two-step oxidation reaction. The first oxidation is catalyzed by choline monooxygenase (CMO) and the second oxidation is catalyzed by NAD+-dependent betaine aldehyde dehydrogenase. Moreover, in plants, the cytosolic enzyme, named N-methyltransferase, catalyzes the conversion of phosphoethanolamine to phosphocholine. However, changes in CMO expression genes under abiotic stresses have been observed. GB accumulation is ontogenetically controlled since it happens in young tissues during prolonged stress, while its degradation is generally not significant in plants. This ability of plants to accumulate high levels of GB in young tissues under abiotic stress, is independent of nitrogen (N) availability and supports the view that plant N allocation is dictated primarily to supply and protect the growing tissues, even under N limitation. Indeed, the contribution of GB to osmotic adjustment and ionic and oxidative stress defense in young tissues, is much higher than that in older ones. In this review, the biosynthesis and accumulation of GB in plants, under several abiotic stresses, were analyzed focusing on all possible roles this metabolite can play, particularly in young tissues. Several halophytes and a few crop plants, including Poaceae, synthesize and accumulate glycine betaine (GB) in response to environmental constraints. GB plays an important role in osmoregulation, in fact, it is one of the main nitrogen-containing compatible osmolytes found in Poaceae. It can interplay with molecules and structures, preserving the activity of macromolecules, maintaining the integrity of membranes against stresses and scavenging ROS. Exogenous GB applications have been proven to induce the expression of genes involved in oxidative stress responses, with a restriction of ROS accumulation and lipid peroxidation in cultured tobacco cells under drought and salinity, and even stabilizing photosynthetic structures under stress. In the plant kingdom, GB is synthesized from choline by a two-step oxidation reaction. The first oxidation is catalyzed by choline monooxygenase (CMO) and the second oxidation is catalyzed by NAD+-dependent betaine aldehyde dehydrogenase. Moreover, in plants, the cytosolic enzyme, named N-methyltransferase, catalyzes the conversion of phosphoethanolamine to phosphocholine. However, changes in CMO expression genes under abiotic stresses have been observed. GB accumulation is ontogenetically controlled since it happens in young tissues during prolonged stress, while its degradation is generally not significant in plants. This ability of plants to accumulate high levels of GB in young tissues under abiotic stress, is independent of nitrogen (N) availability and supports the view that plant N allocation is dictated primarily to supply and protect the growing tissues, even under N limitation. Indeed, the contribution of GB to osmotic adjustment and ionic and oxidative stress defense in young tissues, is much higher than that in older ones. In this review, the biosynthesis and accumulation of GB in plants, under several abiotic stresses, were analyzed focusing on all possible roles this metabolite can play, particularly in young tissues.

Proline, glycinebetaine, and trehalose function as compatible solutes and are upregulated in plants under abiotic stress. The uptake and inter-organ transport in plants are largely unknown. We review the current information on the transport of these osmoprotectants under abiotic stress. Proline metabolism involves several subcellular compartments. Proline concentrations are regulated by the interplay of biosynthesis, degradation, and transport processes. Among the proline transporter proteins, both general amino acid permeases and selective compatible solute transporters were identified. The review summarized our current knowledge on proline transport under abiotic stress conditions. Trehalose is a nonreducing disaccharide formed by two glucose molecules. Sugar transporters have essential roles in the appropriate distribution of carbohydrates throughout the plants. Trehalose transporter has been poorly characterized because it is difficult to predict the characteristics of sugar transporters based solely on the amino acid sequences. Transport properties of exogenous applied trehalose for abiotic stress tolerance have been discussed. Glycinebetaine is synthesized by two-step oxidations of choline with enzymes choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH). Different biosynthetic pathways among monocot and dicot plants were discussed. Expression and substrate specificity of betaine/proline transporters from various plants were compared. Exogenous application of glycinebetaine to plants under stress conditions improved abiotic stress tolerance and gained some attentions. Further application using the important plants both in laboratory and field will contribute to increase the crop production under stress environments.

Glycine betaine (N,N,N-trimethyl glycine, GB) plays an important role in the response of plants to abiotic stress, mainly hydric stress. The aim of this review is to gather information about biochemical processes in which glycine betaine is involved and their impact in plant growth and development. In plants, GB is synthesized by two choline oxidation steps: the first step is choline oxidation to betaine aldehyde (BA) catalyzed by choline monooxygenase, and the second step is BA oxidation to GB catalyzed by betaine aldehyde dehydrogenase which uses NAD(P)+ as coenzyme. In plants, GB synthesis takes place in chloroplast, peroxisome, and cytoplasm. There is scarce information about GB degradation routes in plants. The role of GB as osmolyte is well known, but only until recently, the participation of GB in several metabolic processes including regulation of gene expression, regulation of the concentration and activity of enzymes, and proper protein folding and association has been studied. GB plays a role in growth and development because it increases photosynthetic capacity and protects the thylakoid membrane and increases antioxidant enzymes activity and concentration. GB synthesis provokes changes in ethylene synthesis and increases expression of auxin responsive IAA gene levels. The modulation mechanisms mediated by GB are described in this work.

BACKGROUND Oat is considered as a moderately salt-tolerant crop that could be used to improve saline and alkaline soil. Previous studies have focused on short-term salt stress exposure (0.5–48 h), while molecular mechanisms of salt tolerance in oat remain unclear. RESULTS Long-term salt stress (16 days) increased the levels of superoxide dismutase activity, peroxidase activity, malondialdehyde content, putrescine content, spermidine content and soluble sugar content and reduced catalase activity in oat roots. The stress also caused changes in protein profiles in the roots. At least 1400 reproducible protein spots were identified in a two-dimensional electrophoresis gel, among which 23 were differentially expressed between treated vs control plants and 13 were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. CONCLUSION These differentially expressed proteins are involved in five types of biological process: (1) two fructose-bisphosphate aldolases, four alcohol dehydrogenases, an enolase, a UDP-glucuronic acid decarboxylase and an F1-ATPase alpha subunit related to carbohydrate and energy metabolism; (2) a choline monooxygenase related to stress and defense; (3) a lipase related to fat metabolism; (4) a polyubiquitin related to protein degradation; (5) a 14-3-3 protein related to signaling. © 2015 Society of Chemical Industry

Glycinebetaine is an important non-toxic osmoprotectant, which is accumulated in higher plants under various stresses. The biosynthesis of glycinebetaine achieved via is a two-step oxidation from choline and betaine aldehyde, catalyzed by choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH), respectively. Up-regulated gene expression of BADH and CMO induced by stress is clearly observed, but the signal transduction is poorly understood. Here, glycinebetaine accumulation in response to osmotic stress and growth recovery induced by exogenous glycinebetaine were observed in a watermelon cell line. When tracing back to the genome sequence of watermelon, it shows that there exists only one member of ClCMO or ClBADH corresponding to glycinebetaine biosynthesis. Both genes harbor a CGTCA-motif in their promoter region which is involved in methyl jasmonate (MeJA)-responsiveness. Amongst MeJA, Ethephon, abscisic acid (ABA), and salicylic acid (SA), MeJA was most effective in gene inducing the expression of ClCMO and ClBADH, and the accumulation of glycinebetaine could also reach an amount comparable to that after osmotic stress by mannitol. Moreover, when ibuprofen (IBU), a JA biosynthesis inhibitor, was pre-perfused into the cells before osmotic stress, glycinebetaine accumulation was suppressed significantly. Interestingly, newly grown cells can keep a high content of glycinebetaine when they are sub-cultured from osmotic stressed cells. This study suggests that osmotic stress induced glycinebetaine biosynthesis occurs via JA signal transduction and not only plays a key role in osmotic stress resistance but also contributes to osmotic stress hardening.

Suaeda maritima(L.) Dumort, an annual halophyte known to be a salt-accumulator is also known for the accumulation of the osmolyte, glycine betaine (GB). This study is an attempt to understand the growth and GB accumulation under optimal concentration of 200 mM NaCl in S. maritima. Salt treatment with 200 mM NaCl showed a significant increase in shoot growth after two weeks. The shoots appeared succulent and turgid after two weeks of salt treatment compared to that of the control which appeared slender and wilted. The treated seedlings also exhibited a significant increase in GB content after two weeks of salt treatment. In order to determine the molecular basis of GB accumulation, qRT PCR of three key genes involved in the pathway, choline monooxygenase, betaine aldehyde dehydrogenase and phospho-ethanolamine N-methyl transferase was performed. Transcript level expression of the three genes revealed a high up-regulation of choline monooxygeanse transcripts when compared to that of the other two transcripts at two days of salt treatment. The results indicate that S. maritima requires salt for its growth and is a natural accumulator of GB. Although all the three genes were salt inducible, the high up-regulation of choline monooxygenase greatly contributes to the accumulation of GB under optimal growth as well as NaCl concentration.AbbreviationsBADHbetaine aldehyde dehydrogenaseCMOcholine monooxygenaseGBglycine betainePEAMTphosphoethanolamine N-methyl transferase

Durum wheat is one of the oldest and most important edible cereal crops and its cultivation has considerable economic importance in many countries. However, adverse conditions, such as high irradiance and increasing salinity of soils, could lead to a decrease in productivity over the next few decades. Durum wheat plants under salinityare able toaccumulate glycine betaine to osmotically balance the cytosol and reduce oxidative stress, especially in young tissues. However, the synthesis of this fundamental osmolyte is inhibited by high light in T. durum even under salinity. Choline monooxygenase is the first enzyme involved in the glycine betaine biosynthetic pathway. Thus, to explain the glycine betaine inhibition, we analyzed the effect of both salinity and high light on the putative TdCMO gene expression. Thirty-eight TdCMO different transcripts were isolated in the young leaves of durum wheat grown in different stress conditions. All translated amino acid sequences, except for the TdCMO1a6 clone, showed a frame shift caused by insertions or deletions. The presence of different transcripts could depend on the presence of duplicated genes, different allelic forms, and alternative splicing events. TdCMO1a6 computational modeling of the 3D structure showed that in durum wheat, a putative CMO-like enzyme with a different Rieske type motif, is present and could be responsible for the glycine betaine synthesis.

Suaeda maritima varieties native to Japan and Egypt were cultured under aseptic conditions. The varieties differed in genetic distance but exhibited similar expression profiles of superoxide dismutase isozyme genes. The expression characteristics of superoxide dismutase (SOD; EC 1.15.1.1) isozyme genes from halophytic Suaeda marit ima plants native to Japan and Egypt were analyzed using young plants grown under aseptic conditions. A phylogenetic tree based on internal transcribed spacer sequences suggested that Egyptian S. maritima is related to European and India S. maritima, while Japanese S. maritima belongs to a separate clade. An in-gel SOD activity staining assay revealed that leaves from both the Egyptian and Japanese varieties showed high levels of CuZn-SOD and Fe-SOD activity, but no Mn-SOD activity; conversely, stems from both varieties showed Mn-SOD activity as well as other SOD isozyme activities. In Japanese S. maritima leaves, SOD activity was increased by incubation in growth medium containing 400 mM NaCl, while Egyptian S. maritima leaves showed elevated SOD activity in the absence of high salt. Genes encoding Mn-SOD and Fe-SOD were isolated from both plant types. RT-PCR analysis revealed that all SOD isozyme-encoding genes were expressed at the same levels in leaves from both plant types grown in normal or high-salt medium. In contrast, the expression of genes encoding choline monooxygenase and betaine aldehyde dehydrogenase, which are involved in betacyanin biosynthesis, was increased in high-salt medium. In leaves of Japanese S. maritima plants, Fe deficiency without high salt exposure preferentially decreased Fe-SOD activity. On the other hand, Fe deficiency with high salt exposure decreased not only Fe-SOD activity but also CuZn-SOD activity, suggesting that Fe availability is involved in the up-regulation of SOD isozymes mediating salt tolerance.

Drought is one of the main environmental stresses and many investigators identified beneficial effects of both silicon and selenium on plant growth and development. To examine the effects of Si and Se on rice (Oryza sativa L.) responses to drought, two cultivars Giza 177 and IET 1444 pretreated with 1.5 mM Si or 0.03 mM Se were then exposed to a water stress until leaf rolling was observed. The enhanced growth of Se or Si pre-treated plants was associated with a significant increase in the content of proline and glycine betaine in both shoots and roots. Furthermore, the transcription factors (TFs), dehydration responsive element-binding protein DREB2A, and NAC5 [no apical meristem (NAM), Arabidopsis thaliana activating factor (ATAF), and cup-shaped cotyledon (CUC)] were over-expressed in the drought stressed rice shoots. Notably, a pretreatment with either Se or Si significantly enhanced the expression of both TFs, DREB2A, NAC5, as well as the expression of the ring domain containing OsRDCP1 gene and some drought specific genes, such as OsCMO coding rice choline monooxygenase and dehydrin OsRAB16b. Expression of TFs and the studied genes was markedly enhanced in the Si-stressed shoots of cv. IET 1444 which favors its drought tolerance.

Sixteen yeasts with sequenced genomes belonging to the ascomycete subphyla Saccharomycotina and Taphrinomycotina were assayed for their ability to utilize a variety of primary, secondary, tertiary and quartenary aliphatic amines as nitrogen sources. The results support a previously proposed pathway of quaternary amine catabolism whereby glycine betaine is first converted into choline, which is then cleaved to release trimethylamine, followed by stepwise demethylation of trimethylamine to release free ammonia. There were only a few instances of utilization ofN-methylated glycine species (sarcosine andN,N-dimethylglycine), which suggests that this pathway is not intact in any of the species tested. The ability to utilize choline as a sole nitrogen source correlated strongly with the presence of a putative Rieske non-haem iron protein homologous to bacterial ring-hydroxylating oxygenases and plant choline monooxygenases. Deletion of the gene encoding the Rieske non-haem iron protein in the yeastScheffersomyces stipitisabolished its ability to utilize choline as the sole nitrogen source, but did not affect its ability to use methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, ethanolamine or glycine as nitrogen sources. The gene was namedCMO1for putative choline monooxygenase 1. A bioinformatic survey of eukaryotic genomes showed thatCMO1homologues are found throughout the eukaryotic domain.

Glycine betaine (GB) is an important osmoprotectant, which improves plant tolerance to various abiotic stresses. In higher plants, GB is synthesized through two-step oxidations of choline, catalyzed by choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH), respectively. Choline, the precursor of GB, is synthesized by phosphoethanolamine N-methyltransferase (PEAMT). Rice is known as a typical non-GB-accumulated species. However, the underlying mechanism related to GB accumulation remains elusive. Here, we determined whether the endogenous accumulation of choline is sufficient to GB biosynthesis in rice and whether the rice CMO protein has the function of oxidizing choline to generate betaine aldehyde. The results showed that overexpression of the rice PEAMT1 gene (OsPEAMT1) resulted in increased levels of choline, while GB content remained unchanged in the transgenic rice plants overexpressing OsPEAMT1. However, the intracellular GB level and the tolerance to salt stress of the transgenic lines overexpressing OsCMO were significantly enhanced. Immunoblotting analysis demonstrated that abundant functional OsCMO proteins with correct size were detected in OsCMO-overexpressing transgenic rice plants, but rarely accumulated in the wild type. Collectively, these results implicated that the endogenous accumulation level of choline is not the major factor leading to non-GB accumulation in rice. Instead, the defective expression of OsCMO resulted in non-GB accumulation.

The halophyte Suaeda salsa displayed strong resistance to salinity. Up to date, molecular mechanisms underlying tolerance of S. salsa to salinity have not been well understood. In the present study, S. salsa seedlings were treated with 30‰ salinity and then leaves and roots were subjected to Illumina sequencing. Compared with the control, 68,599 and 77,250 unigenes were significantly differentially expressed in leaves and roots in saline treatment, respectively. KEGG enrichment analyses indicated that photosynthesis process, carbohydrate, lipid and amino acid metabolisms were all downregulated in saline treatment, which should inhibit growth of S. salsa. Expression levels of Na+/H+ exchanger, V-H+ ATPase, choline monooxygenase, potassium and chloride channels were upregulated in saline treatment, which could relieve reduce over-accumulation of Na+ and Cl-. Fe-SOD, glutathione, L-ascorbate and flavonoids function as antioxidants in plants. Genes in relation to them were all upregulated, suggesting that S. salsa initiated various antioxidant mechanisms to tolerate high salinity. Besides, plant hormones, especially auxin, ethylene and jasmonic acid signaling transduction pathways were all upregulated in response to saline treatment, which were important to gene regulations of ion transportation and antioxidation. These changes might comprehensively contribute to tolerance of S. salsa to salinity. Overall, the present study provided new insights to understand the mechanisms underlying tolerance to salinity in halophytes.

Soil salinity and drought severely affect all aspects of plant physiology, leading to significant losses of crop pro- ductivity and native biodiversity. A key to sustainable land use in such areas is to cultivate well-adapted native plants that are also commercially important and have the appropriate gene pool. Glycine betaine (GB) is an osmoprotectant that im- parts salt and drought tolerance to some plants. It is also shown separately to provide significant health benefits to animals and humans. We investigated whether Australian saltbushes, which are extremely salt and drought tolerant and also impart health benefits to grazing animals, may have the genetic basis for GB biosynthesis, explaining the two different observa- tions. Complementary DNAs encoding the two key enzymes of the plant GB biosynthesis pathway, choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH), were identified and analysed from Atriplex nummularia and Atriplex semibaccata. The sequences showed the putative CMO proteins exhibited all functionally important features including the Reiske-type cluster (2Fe-2S) and mononuclear non-heme Fe cluster, and the putative BADHs exhibited conservation of active site residues. The expression of both genes was found to be significantly up-regulated in leaf tissues under salt stress. The leaf tissues also showed accumulation of very high levels of GB, at 29.69 mmol/kg fresh weight for A. nummularia and 42.68 mmol/kg fresh weight for A. semibaccata, which is several times higher than in cereal crops. The results demonstrate a strong potential of cultivation of saltbushes for re-vegetation and as a perennial fodder in salinity and drought-affected areas.

The effects of extracts of the brown marine alga (Ascophyllum nodosum, ANE) on growth and biochemical and molecular changes in spinach were studied. Overall increases in biomass, chlorophyll, and antioxidant activity were observed at an application rate of 0.1 g L –1 ANE. Shoot fresh weight, dry-matter content, and total soluble protein showed 1.6-, 1.2-, and 1.5-fold increases, respectively. Total chlorophyll increased by 30% and total antioxidant capacity, phenolics, and flavonoid content increased by at least 33%. A 1.4-fold increase in chalcone isomerase activity was observed, whereas the activity of phenylalanine ammonia lyase was not affected. The ANE affected the transcript abundance of genes that affect sucrose and glycine betaine metabolism. The transcript abundance of cytosolic glutamine synthetase (GS1), betaine aldehyde dehydrogenase (BADH), choline monooxygenase (CMO), and glutathione reductase (GR) increased in plants treated with 0.1 g L –1 ANE.

Choline monooxygenase (CMO) is the first regulatory enzyme in the biosynthetic pathway for glycine betaine, an effective osmoprotectant in Kochia scoparia, a highly drought- and salt-tolerant species. In seedlings, CMO transcript levels are rapidly increased in response to both NaCl and osmotic stress treatments. The mRNA level in shoots was substantially higher than in roots. The rapid induction seen in whole plants was in contrast to the apparent down-regulation observed in suspension-cultured K. scoparia cells in response to the same salt stress. Treatment with exogenous abscisic acid (ABA) or fluridone shows that CMO induction proceeds via an ABA-independent signal transduction pathway. Examination of the CMO upstream regulatory region reveals a number of stress response-related elements, some of which may be involved in the stress tolerance shown by this species.

Hydrogen sulfide (H2S) is a newly appreciated participant in physiological and biochemical regulation in plants. However, whether H2S is involved in the regulation of plant responses to drought stress remains unclear. Here, the role of H2S in the regulation of drought stress response in Spinacia oleracea seedlings is reported. First, drought stress dramatically decreased the relative water content (RWC) of leaves, photosynthesis, and the efficiency of PSII. Moreover, drought caused the accumulation of ROS and increased the MDA content. However, the application of NaHS counteracted the drought-induced changes in these parameters. Second, NaHS application increased the water and osmotic potential of leaves. Additionally, osmoprotectants such as proline and glycinebetaine (GB) content were altered by NaHS application under drought conditions, suggesting that osmoprotectant contributes to H2S-induced drought resistance. Third, the levels of soluble sugars and polyamines (PAs) were increased differentially by NaHS application in S. oleracea seedlings. Moreover, several genes related to PA and soluble sugar biosynthesis, as well as betaine aldehyde dehydrogenase (SoBADH), choline monooxygenase (SoCMO), and aquaporin (SoPIP1;2), were up-regulated by H2S under drought stress. These results suggest that H2S contributes to drought tolerance in S. oleracea through its effect on the biosynthesis of PAs and soluble sugars. Additionally, GB and trehalose also play key roles in enhancing S. oleracea drought resistance.

Water stress (WS) predisposes peanut plants to fungal infection resulting in pre-harvest aflatoxin contamination. Major changes during water stress including oxidative stress, lead to destruction of photosynthetic apparatus and other macromolecules within cells. Two peanut cultivars with diverse drought tolerance characteristics were subjected to WS, and their leaf proteome was compared using two-dimensional electrophoresis complemented with MALDI-TOF/TOF mass spectrometry. Ninety-six protein spots were differentially abundant to water stress in both cultivars that corresponded to 60 non-redundant proteins. Protein interaction prediction analysis suggests that 42 unique proteins showed interactions in tolerant cultivar while 20 showed interactions in the susceptible cultivar, activating other proteins in directed system response networks. Four proteins: glutamine ammonia ligase, chitin class II, actin isoform B, and beta tubulin, involved in metabolism, defense and cellular biogenesis, are unique in tolerant cultivar and showed positive interactions with other proteins. In addition, four proteins: serine/threonine protein phosphate PP1, choline monooxygenase, peroxidase 43, and SNF1-related protein kinase regulatory subunit beta-2, that play a role as cryoprotectants through signal transduction, were induced in drought tolerant cultivar following WS. Eleven interologs of these proteins were found in Arabidopsis interacting with several proteins and it is believed that similar mechanisms/pathways exist in peanut. Significance Peanuts ( Arachis hypogaea L.) are a major source of plant protein grown in subtropical and tropical regions of the world. Pre-harvest aflatoxin contamination is a major problem that affects peanut crop yield and food safety. Poor understanding of molecular and cellular mechanisms associated with aflatoxin resistance is largely responsible for the lack of progress in elucidating a process/methodology for reducing aflatoxin contamination in peanuts. Drought perturbs the invasion of the aflatoxin producing fungus and thus affects the quality and yield of peanut. Therefore, more studies involving the effects of drought stress to determine the molecular changes will enhance our understanding of the key metabolic pathways involved in the combined stresses. The changes associated with the biotic and abiotic interactions within the peanut will be used to determine the metabolic pathways involved in the stress tolerance. This research would be beneficial in identifying the tolerant molecular signatures and promoting food safety and consumer health through breeding superior quality peanut cultivars.

Halophyte Suaeda salsa is native to the saline soil in the Yellow River Delta. Soil salinity can reduce plant productivity and therefore is the most important factor for the degradation of wetlands in the Yellow River Delta. In this work we characterized the salinity-induced effects in S. salsa in terms of metabolic profiling, antioxidant enzyme activities, and gene expression quantification. Our results showed that salinity inhibited plant growth of S. salsa and upregulated gene expression levels of myo-inositol-1-phosphate synthase (INPS), choline monooxygenase (CMO), betaine aldehyde dehydrogenase (BADH), and catalase (CAT), and elevated the activities of superoxide dismutase (SOD), peroxidase (POD), CAT, and glutathione peroxidase (GPx). The significant metabolic responses included the depleted amino acids malate, fumarate, choline, phosphocholine, and elevated betaine and allantoin in the aboveground part of S. salsa seedlings as well as depleted glucose and fructose and elevated proline, citrate, and sucrose in root tissues. Based on these significant biological markers, salinity treatments induced clear osmotic stress (for example, INPS, CMO, BADH, betaine, proline) and oxidative stress (for example, SOD, POD, CAT, GPx activities), disturbed protein biosynthesis/degradation (amino acids and total protein) and energy metabolism (for example, glucose, sucrose, citrate) in S. salsa.

Under natural conditions or in the field, plants are often subjected to a combination of different stresses such as salt stress and heat shock. Although salt stress and heat shock have been extensively studied, little is known about how their combination affects plants. We used proteomics, coupled with physiological measurements, to investigate the effect of salt stress, heat shock, and their combination on Suaeda salsa plants. A combination of salt stress and heat shock resulted in suppression of CO(2) assimilation and the photosystem II efficiency. Approximately 440 protein spots changed their expression levels upon salt stress, heat shock and their combination, and 57 proteins were identified by MS. These proteins were classified into several categories including disease/defense, photosynthesis, energy production, material transport, and signal transduction. Some proteins induced during salt stress, e.g. choline monooxygenase, chloroplastic ATP synthase subunit beta, and V-type proton ATPase catalytic subunit A, and some proteins induced during heat shock, e.g. heat shock 70kDa protein, probable ion channel DMI1, and two component sensor histidine kinase, were either unchanged or suppressed during a combination of salt stress and heat shock. In contrast, the expression of some proteins, including nucleoside diphosphate kinase 1, chlorophyll a/b binding protein, and ABC transporter I family member 1, was specifically induced during a combination of salt stress and heat shock. The potential roles of the stress-responsive proteins are discussed.

Glycine betaine (GB) is a compatible solute accumulated by many plants under various abiotic stresses. GB is synthesized in two steps, choline → betaine aldehyde → GB, where a functional choline-oxidizing enzyme has only been reported in Amaranthaceae (a chloroplastic ferredoxin-dependent choline monooxygenase) thus far. Here, we have cloned a cDNA encoding a choline monooxygenase (CMO) from barley (Hordeum vulgare) plants, HvCMO. In barley plants under non-stress condition, GB had accumulated in all the determined organs (leaves, internodes, awn and floret proper), mostly in the leaves. The expression of HvCMO protein was abundant in the leaves, whereas the expression of betaine aldehyde dehydrogenase (BADH) protein was abundant in the awn, floret proper and the youngest internode than in the leaves. The accumulation of HvCMO mRNA was increased by high osmotic and low-temperature environments. Also, the expression of HvCMO protein was increased by the presence of high NaCl. Immunofluorescent labeling of HvCMO protein and subcellular fractionation analysis showed that HvCMO protein was localized to peroxisomes. [(14)C]choline was oxidized to betaine aldehyde and GB in spinach (Spinacia oleracea) chloroplasts but not in barley, which indicates that the subcellular localization of choline-oxidizing enzyme is different between two plant species. We investigated the choline-oxidizing reaction using recombinant HvCMO protein expressed in yeast (Saccharomyces cerevisiae). The crude extract of HvCMO-expressing yeast coupled with recombinant BBD2 protein converted [(14)C]choline to GB when NADPH was added as a cofactor. These results suggest that choline oxidation in GB synthesis is mediated by a peroxisomal NADPH-dependent choline monooxygenase in barley plants.

Choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) catalyze the first and second steps in the biosynthesis of glycine betaine in betaine-accumulating plants. Over-expression of the Spinacia oleracea chloroplast choline monooxygenase (SoCMO) and betaine aldehyde dehydrogenase (SoBADH) genes has not been reported in Lolium perenne. In this investigation, the SoCMO and SoBADH genes have been used to generate transgenic L. perenne plants via particle bombardment. Transgenic plants have been confirmed with PCR, Southern blot, and Northern blot analyses. Enhanced salt stress tolerance has been observed from SoBADH–SoCMO transgenic L. perenne plants. The dwarf phenotype was first observed 3 months after transgenic plants were established in soil and was to be stably inherited. Height of transgenic plants was decreased by 63% compared to the control. Measurement of endogenous GAs content demonstrated that the content of endogenous GA1 was decreased by 75.2%, and the content of endogenous GA4, GA12, GA19, and GA53 of transgenic plants was increased by 200%, 221%, 105%, and 108%, respectively, compared to the control plants. Dwarf trait of SoBADH–SoCMO transgenic L. perenne plants can be recovered by application of exogenous GAs. These results demonstrated that simultaneous expression of the SoCMO and SoBADH genes enhanced salt stress tolerance and induced dwarfism in transgenic L. perenne. Dwarfism induced by expression of the SoCMO and SoBADH genes was associated with synthesis of endogenous GAs and it could be recovered by application of exogenous GAs. This is the first report on dwarfism induced by expression of the SoCMO and SoBADH genes in a species in turfgrass.

It has been reported that glycinebetaine (betaine) is synthesized in response to abiotic stresses via a two-step oxidation of choline in which choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) are involved. Here we show that significant amounts of betaine, > 20 micromol/gFW, accumulated in young leaves of Beta vulgaris even under normal growth conditions, whereas levels in old leaves, cotyledons, hypocotyls, and roots were low. Under the same conditions, CMO accumulates exclusively in old leaves and is difficult to be detected in young leaves. By contrast, the levels of BADH were high in all tissues. Exogenously supplied choline was converted into betaine in old leaves, but levels were significantly lower in young leaves under the same conditions. When d(11)-betaine was applied exogenously to old leaves, it was translocated preferentially into young leaves and roots. In response to salt stress, betaine levels increased in all tissues, but most significantly increased in young leaves. The levels of CMO increased in various tissues, but were low in young leaves. A betaine transporter gene was isolated. Its expression was more strongly induced in old leaves than in young leaves. Based on these data, we discussed the role of CMO and betaine transporter under stress and non-stress conditions.

Suaeda aralocaspica and Bienertia sinuspersici (Chenopodiaceae), which have unusual mechanisms of C4 photosynthesis by dimorphic chloroplasts within individual chlorenchyma cells, grow in saline semi-arid desert regions in Central Asia and around the Persian Gulf. Their response to salinity was studied, along with those of the related C3 Suaeda heterophylla and Kranz (dual cell) type C4 Suaeda eltonica in subfamily Suaedoideae. Light response curves for CO2 fixation with salt treatment (200 mM NaCl) indicated these species have high tolerance to salinity. All accumulated glycine betaine (GB) which is known to protect against abiotic stress. Western blots showed that the protein levels of choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH), enzymes catalyzing synthesis of GB from choline, increased under salt stress in both single-cell type C4 species. Their cDNAs for CMO and BADH were isolated, sequenced and phylogenetic analyses showed that CMO for both species, and BADH for B. sinuspersici, are in a clade in subfamily Suaedoideae. Results of in situ immunolocalization experiments with BADH antibody show that both chloroplast types in the single-cell C4 species and in the Kranz type C4 S. eltonica function in GB synthesis. Scanning electron microscopy (SEM) and X-ray microanalysis showed B. sinuspersici has very prominent salt glands, which accumulate sodium chloride under salt stress, while S. aralocaspica lacks salt glands. The bases for salt tolerance in these species are discussed considering anatomical and biochemical features.

Glycine betaine is an osmoprotectant that plays an important role and accumulates rapidly in many plants during salinity or drought stress. Choline monooxygenase (CMO) is a major catalyst in the synthesis of glycine betaine. In our previous study, a CMO gene (AhCMO) cloned from Atriplex hortensis was introduced into cotton (Gossypium hirsutum L.) via Agrobacterium mediation to enhance resistance to salinity stress. However, there is little or no knowledge of the salinity tolerance of the transgenic plants, particularly under saline-field conditions. In the present study, two transgenic AhCMO cotton lines of the T3 generation were used to study the AhCMO gene expression, and to determine their salinity tolerance in both greenhouse and field under salinity stress. Molecular analysis confirmed that the transgenic plants expressed the AhCMO gene. Greenhouse study showed that on average, seedlings of the transgenic lines accumulated 26 and 131% more glycine betaine than those of non-transgenic plants (SM3) under normal and salt-stress (150 mmol l−1 NaCl) conditions, respectively. The osmotic potential, electrolyte leakage and malondialdehyde (MDA) accumulation were significantly lower in leaves of the transgenic lines than in those of SM3 after salt stress. The net photosynthesis rate and Fv/Fm in transgenic cotton leaves were less affected by salinity than in non-transgenic cotton leaves. Therefore, transgenic cotton over-expressing AhCMO was more tolerant to salt stress due to elevated accumulation of glycine betaine, which provided greater protection of the cell membrane and photosynthetic capacity than in non-transgenic cotton. The seed cotton yield of the transgenic plants was lower under normal conditions, but was significantly higher than that of non-transgenic plants under salt-stressed field conditions. The results indicate that over-expression of AhCMO in cotton enhanced salt stress tolerance, which is of great value in cotton production in the saline fields.

Glycine betaine (GlyBet), a quaternary ammonium compound, functions as an osmoprotectant in many organisms including plants. Previous research has shown that over-expression of enzymes for GlyBet biosynthesis in transgenic plants improved abiotic stress tolerance, but so far no study on the effects of plastid-expression of choline monooxygenase, the enzyme that catalyzes the conversion of choline into betaine aldehyde, has been reported. In the present study, tobacco (Nicotiana tabacum L. cv Wisconsin 38) plants were transformed with a gene for choline monooxygenase (BvCMO) from beet (Beta vulgaris) via plastid genetic engineering. Transplastomic plants constitutively expressing BvCMO under the control of the ribosomal RNA operon promoter and a synthetic T7 gene G10 leader were able to accumulate GlyBet in leaves, roots and seeds, and exhibited improved tolerance to toxic level of choline and to salt/drought stress when compared to wild type plants. Transplastomic plants also demonstrated higher net photosynthetic rate and apparent quantum yield of photosynthesis in the presence of 150 mM NaCl. Salt stress caused no significant change on the maximal efficiency of PSII photochemistry (Fv/Fm) in both wild type and transplastomic plants, but a decrease in the actual efficiency of PSII (PhiPSII) was observed, and such a decrease was much greater in wild type plants. Our results demonstrate the feasibility of improving salt and drought tolerance in plants through plastid transformation with BvCMO gene.

The genes encoding the two enzymes choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) of glycine betaine synthesis in Suaeda salsa were cloned and fused with the 2A region of foot-and-mouth disease virus in a single open reading frame. The fused genes were placed under the control of the alcohol oxidase (AOX1) promoter in pPIC3B and transformed into P. pastoris GS115. The expression of the fused genes in P. pastoris and the ability of recombinant yeasts to tolerate environmental stresses were studied. The results showed that induced with 0.5% methanol for 96 h, the maximal activities of CMO and BADH in the tested recombinant yeasts were 45- and 44-fold higher than those in the control yeast transformed empty vector only, respectively; the content of glycine betaine in the recombinant yeasts was 28- to 35-fold higher than that in the control. The fused genes linked by 2A region of foot-and-mouth disease virus were expressed in P. pastoris successfully and the polyprotein was 'cleaved' to each functional protein. The yeasts transformed the fused genes, which were more resistant to salt, methanol, and high temperature stresses than the control as result of glycine betaine synthesis genes introduced.