Identification and characterization of a plastid-localized Arabidopsis glyoxylate reductase isoform: comparison with a cytosolic isoform and implications for cellular redox homeostasis and aldehyde detoxification

Enzymes that reduce the aldehyde chemical grouping (i.e. H-C=O) to its corresponding alcohol could be crucial in maintaining plant health. Recently, recombinant expression of a cytosolic enzyme from Arabidopsis thaliana (L.) Heynh (designated as glyoxylate reductase 1 or AtGR1) revealed that it effectively catalyses the in vitro reduction of both glyoxylate and succinic semialdehyde (SSA). In this paper, web-based bioinformatics tools revealed a second putative GR cDNA (GenBank Accession No. AAP42747; designated herein as AtGR2) that is 57% identical on an amino acid basis to GR1. Sequence encoding a putative targeting signal (N-terminal 43 amino acids) was deleted from the full-length GR2 cDNA and the resulting truncated gene was co-expressed with the molecular chaperones GroES/EL in Escherichia coli, enabling production and purification of soluble recombinant protein. Kinetic analysis revealed that recombinant GR2 catalysed the conversion of glyoxylate to glycolate (K(m) glyoxylate=34 microM), and SSA to gamma-hydroxybutyrate (K(m) SSA=8.96 mM) via an essentially irreversible, NADPH-based mechanism. GR2 had a 350-fold higher preference for glyoxylate than SSA, based on the performance constants (k(cat)/K(m)). Fluorescence microscopic analysis of tobacco (Nicotiana tabacum L.) suspension cells transiently transformed with GR1 linked to the green fluorescent protein (GFP) revealed that GR1 was localized to the cytosol, whereas GR2-GFP was localized to plastids via targeting information contained within its N-terminal 45 amino acids. The identification and characterization of distinct plastidial and cytosolic glyoxylate reductase isoforms is discussed with respect to aldehyde detoxification and the plant stress response.     

An osmotin from the resurrection plant Tripogon loliiformis (TlOsm) confers tolerance to multiple abiotic stresses in transgenic rice

Osmotin is a key protein associated with abiotic and biotic stress response in plants. In this study, an osmotin from the resurrection plant Tripogon loliiformis (TlOsm) was characterized and functionally analyzed under abiotic stress conditions in T. loliiformis as well as in transgenic Nicotiana tabacum (tobacco) and Oryza sativa (rice) plants. Real‐time PCR analysis on mixed elicitor cDNA libraries from T. loliiformis showed that TlOsm was upregulated a 1000‐fold during the early stages of osmotic stresses (cold, drought, and salinity) in both shoots and roots but downregulated in shoots during heat stress. There was no change in TlOsm gene expression in roots of heat‐stressed plants and during plant development. The plasma membrane localization of TlOsm was showed in fluorescent‐tagged TlOsm tobacco plants using confocal laser scanning microscopic analysis. Transgenic rice plants expressing TlOsm were assessed for enhanced tolerance to salinity, drought and cold stresses. Constitutively expressed TlOsm in transgenic rice plants showed increased tolerance to cold, drought and salinity stress when compared with the wild‐type and vector control counterparts. This was evidenced by maintained growth, retained higher water content and membrane integrity, and improved survival rate of TlOsm‐expressing plants. The results thus indicate the involvement of TlOsm in plant response to multiple abiotic stresses, possibly through the signaling pathway, and highlight its potential applications for engineering crops with improved tolerance to cold, drought and salinity stress.     

Molecular cloning and characterization of the glutathione reductase gene from Stipa purpurea

Reactive oxygen species (ROS) are a key factor in abiotic stresses; excess ROS is harmful to plants. Glutathione reductase (GR) plays an important role in scavenging ROS in plants. Here, a GR gene, named SpGR, was cloned from Stipa purpurea and characterized. The full-length open reading frame was 1497 bp, encoding 498 amino acids. Subcellular localization analysis indicated that SpGR was localized to both the plasma membrane and nucleus. The expression of SpGR was induced by cold, salt, and drought stresses. Functional analysis indicated that ectopic expression of SpGR in Arabidopsis thaliana resulted in greater tolerance to salt stress than that of wild-type plants, but no difference under cold or drought treatments. The results of GR activity and GSSG and GSH content analyses suggested that, under salt stress, transgenic plants produced more GR to reduce GSSG to GSH for scavenging ROS than wild-type plants. Therefore, SpGR may be a candidate gene for plants to resist abiotic stress.     

拟南芥高迁移率族蛋白B（AtHMGB）族基因启动子的转录激活功能分析

为了解高迁移率族蛋白B族（high mobility group protein B,HMGB）基因调控植物响应低温、高盐和干旱等外源胁迫的表达调控方式, 本文克隆了拟南芥AtHMGB前5个家族成员的启动子区域（PAtHMGB1,PAtHMGB2,PAtHMGB3,PAtHMGB4和PAtHMGB5）.运用基因重组技术将其分别替换表达载体上35S启动子区域获得重组表达载体,利用农杆菌介导法侵染烟草获得稳定表达的转基因烟草. 运用实时定量PCR检测上述5种启动子的转基因烟草,观察在外源胁迫（低温、高盐和干旱）处理前后gusA基因的表达差异,同时检测转基因烟草种子在不同外源胁迫条件下的萌发状况. 检测结果证实,在低温胁迫下,PAtHMGB2,PAtHMGB3和PAtHMGB4正调控gusA基因的表达,而在干旱或盐胁迫下,gusA基因的表达被PAtHMGB2和PAtHMGB3负调控. 种子萌发结果表明,在干旱胁迫下,PAtHMGB2调控下的转基因烟草比野生型烟草萌发及生长迟缓|在低温胁迫下,PAtHMGB2调控的转基因烟草长势明显强于野生型. 本研究克隆了拟南芥AtHMGB家族前5个成员启动子,分析其生物学功能发现,PAtHMGB2在响应低温和干旱胁迫方面效果尤为显著.     

Mutants of GABA transaminase (POP2) suppress the severe phenotype of succinic semialdehyde dehydrogenase (ssadh) mutants in Arabidopsis

Background

The γ-aminubutyrate (GABA) shunt bypasses two steps of the tricarboxylic acid cycle, and is present in both prokaryotes and eukaryotes. In plants, the pathway is composed of the calcium/calmodulin-regulated cytosolic enzyme glutamate decarboxylase (GAD), the mitochondrial enzymes GABA transaminase (GABA-T; POP2) and succinic semialdehyde dehydrogenase (SSADH). We have previously shown that compromising the function of the GABA-shunt, by disrupting the SSADH gene of Arabidopsis, causes enhanced accumulation of reactive oxygen intermediates (ROIs) and cell death in response to light and heat stress. However, to date, genetic investigations of the relationships between enzymes of the GABA shunt have not been reported.

Principal Findings

To elucidate the role of succinic semialdehyde (SSA), γ-hydroxybutyrate (GHB) and GABA in the accumulation of ROIs, we combined two genetic approaches to suppress the severe phenotype of ssadh mutants. Analysis of double pop2 ssadh mutants revealed that pop2 is epistatic to ssadh. Moreover, we isolated EMS-generated mutants suppressing the phenotype of ssadh revealing two new pop2 alleles. By measuring thermoluminescence at high temperature, the peroxide contents of ssadh and pop2 mutants were evaluated, showing that only ssadh plants accumulate peroxides. In addition, pop2 ssadh seedlings are more sensitive to exogenous SSA or GHB relative to wild type, because GHB and/or SSA accumulate in these plants.

Significance

We conclude that the lack of supply of succinate and NADH to the TCA cycle is not responsible for the oxidative stress and growth retardations of ssadh mutants. Rather, we suggest that the accumulation of SSA, GHB, or both, produced downstream of the GABA-T transamination step, is toxic to the plants, resulting in high ROI levels and impaired development.     

Important roles of drought- and cold-inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana

Raffinose family oligosaccharides (RFO) accumulating during seed development are thought to play a role in the desiccation tolerance of seeds. However, the functions of RFO in desiccation tolerance have not been elucidated. Here we examine the functions of RFO in Arabidopsis thaliana plants under drought- and cold-stress conditions, based on the analyses of function and expression of genes involved in RFO biosynthesis. Sugar analysis showed that drought-, high salinity- and cold-treated Arabidopsis plants accumulate a large amount of raffinose and galactinol, but not stachyose. Raffinose and galactinol were not detected in unstressed plants. This suggests that raffinose and galactinol are involved in tolerance to drought, high salinity and cold stresses. Galactinol synthase (GolS) catalyses the first step in the biosynthesis of RFO from UDP-galactose. We identified three stress-responsive GolS genes (AtGolS1, 2 and 3) among seven Arabidopsis GolS genes. AtGolS1 and 2 were induced by drought and high-salinity stresses, but not by cold stress. By contrast, AtGolS3 was induced by cold stress but not by drought or salt stress. All the GST fusion proteins of GST-AtGolS1, 2 and 3 expressed in Escherichia coli had galactinol synthase activities. Overexpression of AtGolS2 in transgenic Arabidopsis caused an increase in endogenous galactinol and raffinose, and showed reduced transpiration from leaves to improve drought tolerance. These results show that stress-inducible galactinol synthase plays a key role in the accumulation of galactinol and raffinose under abiotic stress conditions, and that galactinol and raffinose may function as osmoprotectants in drought-stress tolerance of plants.     

Overexpression of a nascent polypeptide associated complex gene (SaβNAC) of Spartina alterniflora improves tolerance to salinity and drought in transgenic Arabidopsis

Salinity and drought are the most important environmental constraints limiting crop growth and productivity. Here, we have characterized a gene 'SaβNAC' encoding the β subunit of nascent polypeptide associated complex from a halophyte Spartina alterniflora and investigated its role toward abiotic stress regulation. Expression of SaβNAC was differentially regulated by abiotic stresses, including salinity, drought, cold, and ABA in leaves and roots of S. alterniflora. Constitutive over-expression of SaβNAC in Arabidopsis exhibited normal growth under non-stress conditions but enhanced tolerance to salt and drought stresses. Transgenic SaβNAC Arabidopsis retained more chlorophyll, proline, and showed improved ionic homeostasis with less damage under stress conditions compared to WT plants. This is a first report to demonstrate the involvement of βNAC in imparting abiotic stress tolerance which might be due to protection of the newly synthesized polypeptides involved in various stress tolerance mechanisms from abiotic stress induced damage and inhibition of cell death in plant.     

The pepper extracellular peroxidase CaPO2 is required for salt, drought and oxidative stress tolerance as well as resistance to fungal pathogens

In plants, biotic and abiotic stresses regulate the expression and activity of various peroxidase isoforms. Capsicum annuum EXTRACELLULAR PEROXIDASE 2 (CaPO2) was previously shown to play a role in local and systemic reactive oxygen species bursts and disease resistance during bacterial pathogen infection. Here, we report CaPO2 expression patterns and functions during conditions of biotic and abiotic stress. In pepper plants, CaPO2 expression was strongly induced by abscisic acid, but not by defense-related plant hormones such as salicylic acid, ethylene and jasmonic acid. CaPO2 was also strongly induced by abiotic and biotic stress treatments, including drought, cold, high salinity and infection by the hemibiotrophic fungal pathogen Colletotrichum coccodes. Loss-of-function of CaPO2 in virus-induced gene silenced pepper plants led to increased susceptibility to salt- and osmotic-induced stress. In contrast, CaPO2 overexpression in transgenic Arabidopsis thaliana plants conferred enhanced tolerance to high salt, drought, and oxidative stress, while also enhancing resistance to infection by the necrotrophic fungal pathogen Alternaria brassicicola. Taken together, these results provide evidence for the involvement of pepper extracellular peroxidase CaPO2 in plant defense responses to various abiotic stresses and plant fungal pathogens.     

Arbuscular mycorrhizal symbiosis and abiotic stress in plants: A review

Abiotic stresses (such as salinity, drought, cold, heat, mineral deficiency and metals/metalloids) have become major threats to the global agricultural production. These stresses in isolation and/or combination control plant growth, development and productivity by causing physiological disorders, ion toxicity, and hormonal and nutritional imbalances. Some soil microorganisms like arbuscular mycorhizal fungi (AMF) inhabit the rhizosphere and develop a symbiotic relationship with the roots of most plant species. AMF can significantly improve resistance of host plants to varied biotic and abiotic stresses. Taking into account recent literature, this paper: (a) overviews major abiotic stresses and introduces the arbuscular mycorrhizae symbiosis (b) appraises the role and underlying major mechanisms of AMF in plant tolerance to major abiotic stresses including salinity, drought, temperature regimes (cold and heat), nutrient-deficiency, and metal/metalloids; (c) discusses major molecular mechanisms potentially involved in AMF-mediated plant-abiotic stress tolerance; and finally (d) highlights major aspects for future work in the current direction.     

Photosynthetic response of transgenic soybean plants, containing an Arabidopsis P5CR gene, during heat and drought stress

The biochemical basis of heat/drought tolerance was investigated by comparing the response of antisense and sense transgenic soybean plants (containing the L-delta1-pyrroline-5-carboxylate reductase gene) with non-transgenic wild-type plants. The plants were subjected to a simultaneous drought and heat stress of 2 days, whereafter they were rewatered at 25 degrees C. During this time the sense plants only showed mild symptoms of stress compared to the antisense plants which were severely stressed. Upon stress, nicotinamide adenine dinucleotide phosphate (NADP+) levels decreased in antisense while it increased in sense plants. Recovery with respect to NADP+ levels was best in sense plants. Sense plants had the highest ability to accumulate proline during stress and to metabolise proline after rewatering. Analyses of the fast phase chlorophyll-a fluorescence transients showed dissociation of the oxygen-evolving complex (OEC) upon stress in all plants tested. In the sense plants, which best resisted the stress, OEC dissociation was bypassed by proline feeding electrons into photosystem 2 (PSII), maintaining an acceptable nicotinamide adenine dinucleotide hydrogen phosphate (NADPH) level, preventing further damage. Upon recovery, NADPH is consumed during oxidation of accumulated proline providing high Levels of NADP+ to act as electron acceptor to PSII, which indirectly may ameliorate the inhibition and/or the effect of uncoupling of the OEC.     

Arabidopsis SAP5 functions as a positive regulator of stress responses and exhibits E3 ubiquitin ligase activity

AtSAP5, one of approximately 14 members of the Stress Associated Protein gene family in Arabidopsis, was identified by its expression in response to salinity, osmotic, drought and cold stress. AtSAP5 shows strong homology to OSISAP1, an A20/AN1-type zinc finger protein implicated in stress tolerance in rice. To evaluate the function of AtSAP5 in the regulation of abiotic stress responses, transgenic Arabidopsis plants that over-express AtSAP5 (35S::AtSAP5) were characterized, along with wild-type and T-DNA knock-down plants. Plants that over-express AtSAP5 showed increased tolerance to environmental challenges including salt stress, osmotic stress and water deficit. Comparison of gene expression patterns between 35S::AtSAP5 transgenic plants and wild-type plants under normal conditions and water deficit stress indicated that over-expression of AtSAP5 correlates with up-regulation of drought stress responsive gene expression. Analysis of transgenic plants that express GFP-AtSAP5 showed that it is localized primarily in nuclei of root cells and recombinant AtSAP5 has E3 ubiquitin ligase activity in vitro. These results indicate that AtSAP5 has E3 ligase activity and acts as a positive regulator of stress responses in Arabidopsis.     

Functional study and elite haplotype identification of soybean phosphate starvation response transcription factors GmPHR14 and GmPHR32

Molecular Breeding - Global climate change leads to the concurrence of a number of abiotic stresses including moisture stress (drought, waterlogging), temperature stress (heat, cold), and salinity...