Cell-type-specific calcium responses to drought, salt and cold in the Arabidopsis root

Little is known about the signalling processes involved in the response of roots to abiotic stresses. The Arabidopsis root is a model system of root anatomy with a simple architecture and is amenable to genetic manipulation. Although it is known that the root responds to cold, drought and salt stress with increases in cytoplasmic free calcium, there is currently no information about the role(s) of the functionally diverse cell types that comprise the root. Transgenic Arabidopsis with enhancer-trapped GAL4 expression in specific cell types was used to target the calcium reporting protein, aequorin, fused to a modified yellow fluorescent protein (YFP). The luminescence output of targeted aequorin enabled in vivo measurement of changes in cytosolic free calcium concentrations ([Ca2+]cyt) in specific cell types during acute cold, osmotic and salt stresses. In response to an acute cold stress, all cell types tested as well as plants constitutively expressing aequorin displayed rapid [Ca2+]cyt peaks. However, there were significant quantitative differences between different cell types in terms of their response to cold stress, osmotic stress (440 mM mannitol) and salt stress (220 mM NaCl), implying specific roles for certain cell types in the detection and/or response to these stimuli. In response to osmotic and salt stress, the endodermis and pericycle displayed prolonged oscillations in cytosolic calcium that were distinct from the responses of the other cell types tested. Targeted expression of aequorin circumvented the technical difficulties involved in fluorescent dye injection as well as the lack of cell specificity of constitutively expressed aequorin, and revealed a new level of complexity in root calcium signalling.     

Constitutive overexpression of the calcium sensor CBL5 confers osmotic or drought stress tolerance in Arabidopsis

Calcium serves as a critical messenger in many adaptation and developmental processes. Cellular calcium signals are detected and transmitted by sensor molecules such as calcium-binding proteins. In plants, the calcineurin B-like protein (CBL) family represents a unique group of calcium sensors and plays a key role in decoding calcium transients by specifically interacting with and regulating a family of CBL-interacting protein kinases (CIPKs). In this study, we report the role of Arabidopsis CBL5 gene in high salt or drought tolerance. CBL5 gene is expressed significantly in green tissues, but not in roots. CBL5 was not induced by abiotic stress conditions such as high salt, drought or low temperature. To determine whether the CBL5 gene plays a role in stress response pathways, we ectopically expressed the CBL5 protein in transgenic Arabidopsis plants (35S-CBL5) and examined plant responses to abiotic stresses. CBL5-overexpressing plants displayed enhanced tolerance to high salt or drought stress. CBL5 overexpression also rendered plants more resistant to high salt or hyperosmotic stress during early development (i.e., seed germination) but did not alter their response to abiscisic acid (ABA). Furthermore, overexpression of CBL5 alters the gene expression of stress gene markers, such as RD29A, RD29B and Kin1 etc. These results suggest that CBL5 may function as a positive regulator of salt or drought responses in plants.     

CIPK3, a calcium sensor-associated protein kinase that regulates abscisic acid and cold signal transduction in Arabidopsis

Plants respond to environmental stress by activating "stress genes." The plant hormone abscisic acid (ABA) plays an important role in stress-responsive gene expression. Although Ca(2+) serves as a common second messenger in signaling stress and ABA, little is known about the molecular basis of Ca(2+) action in these pathways. Here, we show that CIPK3, a Ser/Thr protein kinase that associates with a calcineurin B-like calcium sensor, regulates ABA response during seed germination and ABA- and stress-induced gene expression in Arabidopsis. The expression of the CIPK3 gene itself is responsive to ABA and stress conditions, including cold, high salt, wounding, and drought. Disruption of CIPK3 altered the expression pattern of a number of stress gene markers in response to ABA, cold, and high salt. However, drought-induced gene expression was not altered in the cipk3 mutant plants, suggesting that CIPK3 regulates select pathways in response to abiotic stress and ABA. These results identify CIPK3 as a molecular link between stress- and ABA-induced calcium signal and gene expression in plant cells. Because the cold signaling pathway is largely independent of endogenous ABA production, CIPK3 represents a cross-talk "node" between the ABA-dependent and ABA-independent pathways in stress responses.     

A tomato glutaredoxin gene SlGRX1 regulates plant responses to oxidative, drought and salt stresses

Glutaredoxins (Grxs) are ubiquitous small heat-stable disulfide oxidoreductases that play a crucial role in plant development and response to oxidative stress. Here, a novel cDNA fragment (SlGRX1) from tomato encoding a protein containing the consensus Grx family domain with a CGFS active site was isolated and characterized. Southern blot analysis indicated that SlGRX1 gene had a single copy in tomato genome. Quantitative real-time RT-PCR analysis revealed that SlGRX1 was expressed ubiquitously in tomato including leaf, root, stem and flower, and its expression could be induced by oxidative, drought, and salt stresses. Virus-induced gene silencing mediated silencing of SlGRX1 in tomato led to increased sensitivity to oxidative and salt stresses with decreased relative chlorophyll content, and reduced tolerance to drought stress with decreased relative water content. In contrast, over-expression of SlGRX1 in Arabidopsis plants significantly increased resistance of plants to oxidative, drought, and salt stresses. Furthermore, expression levels of oxidative, drought and salt stress related genes Apx2, Apx6, and RD22 were up-regulated in SlGRX1-overexpressed Arabidopsis plants when analyzed by quantitative real-time PCR. Our results suggest that the Grx gene SlGRX1 plays an important role in regulating abiotic tolerance against oxidative, drought, and salt stresses.     

SCABP8/CBL10, a putative calcium sensor, interacts with the protein kinase SOS2 to protect Arabidopsis shoots from salt stress

The SOS (for Salt Overly Sensitive) pathway plays essential roles in conferring salt tolerance in Arabidopsis thaliana. Under salt stress, the calcium sensor SOS3 activates the kinase SOS2 that positively regulates SOS1, a plasma membrane sodium/proton antiporter. We show that SOS3 acts primarily in roots under salt stress. By contrast, the SOS3 homolog SOS3-LIKE CALCIUM BINDING PROTEIN8 (SCABP8)/CALCINEURIN B-LIKE10 functions mainly in the shoot response to salt toxicity. While root growth is reduced in sos3 mutants in the presence of NaCl, the salt sensitivity of scabp8 is more prominent in shoot tissues. SCABP8 is further shown to bind calcium, interact with SOS2 both in vitro and in vivo, recruit SOS2 to the plasma membrane, enhance SOS2 activity in a calcium-dependent manner, and activate SOS1 in yeast. In addition, sos3 scabp8 and sos2 scabp8 display a phenotype similar to sos2, which is more sensitive to salt than either sos3 or scabp8 alone. Overexpression of SCABP8 in sos3 partially rescues the sos3 salt-sensitive phenotype. However, overexpression of SOS3 fails to complement scabp8. These results suggest that SCABP8 and SOS3 are only partially redundant in their function, and each plays additional and unique roles in the plant salt stress response.     

Otoferlin is a calcium sensor that directly regulates SNARE-mediated membrane fusion

Otoferlin is a large multi-C2 domain protein proposed to act as a calcium sensor that regulates synaptic vesicle exocytosis in cochlear hair cells. Although mutations in otoferlin have been associated with deafness, its contribution to neurotransmitter release is unresolved. Using recombinant proteins, we demonstrate that five of the six C2 domains of otoferlin sense calcium with apparent dissociation constants that ranged from 13-25 μM; in the presence of membranes, these apparent affinities increase by up to sevenfold. Using a reconstituted membrane fusion assay, we found that five of the six C2 domains of otoferlin stimulate membrane fusion in a calcium-dependent manner. We also demonstrate that a calcium binding-deficient form of the C2C domain is incapable of stimulating membrane fusion, further underscoring the importance of calcium for the protein's function. These results demonstrate for the first time that otoferlin is a calcium sensor that can directly regulate soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor-mediated membrane fusion reactions.     

AtSIA1, an ABC1-like kinase, regulates salt response in Arabidopsis

Atypical kinase family, ABC1 (the activity of bc1 complex) genes play essential roles in plant growth, development and responses to be environmental stresses. Here, a novel gene, AtSIA1 (A. thaliana Salt-Induced ABC1 kinase 1) was cloned and characterized in Arabidopsis. Semi-quantitative RT-PCR analysis revealed that it constitutively expressed and mainly induced by salt treatment. AtSIA1-GFP fusion analysis showed that AtSIA1 is localized in chloroplasts. Transgenic plants with overexpression of AtSIA1 show higher tolerance to salt stress than that of the Col-0 and the knock-out mutant sia1 during seedling growth. Taken together, our results suggested that AtSIA1 may be involved in salt stress tolerance in Arabidopsis.     

Signal transduction during cold, salt, and drought stresses in plants

Abiotic stresses, especially cold, salinity and drought, are the primary causes of crop loss worldwide. Plant adaptation to environmental stresses is dependent upon the activation of cascades of molecular networks involved in stress perception, signal transduction, and the expression of specific stress-related genes and metabolites. Plants have stress-specific adaptive responses as well as responses which protect the plants from more than one environmental stress. There are multiple stress perception and signaling pathways, some of which are specific, but others may cross-talk at various steps. In this review article, we first expound the general stress signal transduction pathways, and then highlight various aspects of biotic stresses signal transduction networks. On the genetic analysis, many cold induced pathways are activated to protect plants from deleterious effects of cold stress, but till date, most studied pathway is ICE-CBF-COR signaling pathway. The Salt-Overly-Sensitive (SOS) pathway, identified through isolation and study of the sos1, sos2, and sos3 mutants, is essential for maintaining favorable ion ratios in the cytoplasm and for tolerance of salt stress. Both ABA-dependent and -independent signaling pathways appear to be involved in osmotic stress tolerance. ROS play a dual role in the response of plants to abiotic stresses functioning as toxic by-products of stress metabolism, as well as important signal transduction molecules and the ROS signaling networks can control growth, development, and stress response. Finally, we talk about the common regulatory system and cross-talk among biotic stresses, with particular emphasis on the MAPK cascades and the cross-talk between ABA signaling and biotic signaling.     

FKF1, a clock-controlled gene that regulates the transition to flowering in Arabidopsis

Plant reproduction requires precise control of flowering in response to environmental cues. We isolated a late-flowering Arabidopsis mutant, fkf1, that is rescued by vemalization or gibberellin treatment. We positionally cloned FKF1, which encodes a novel protein with a PAS domain similar to the flavin-binding region of certain photoreceptors, an F box characteristic of proteins that direct ubiquitin-mediated degradation, and six kelch repeats predicted to fold into a beta propeller. FKF1 mRNA levels oscillate with a circadian rhythm, and deletion of FKF1 alters the waveform of rhythmic expression of two clock-controlled genes, implicating FKF1 in modulating the Arabidopsis circadian clock.     

GhTZF1 regulates drought stress responses and delays leaf senescence by inhibiting reactive oxygen species accumulation in transgenic Arabidopsis

Redox homeostasis is important for plants to be able to maintain cellular metabolism, and disrupting cellular redox homeostasis will cause oxidative damage to cells and adversely affect plant growth. In this study, a cotton CCCH-type tandem zinc finger gene defined as GhTZF1, which was isolated from a cotton cell wall regeneration SSH library in our previous research, was characterized. GhTZF1 was predominantly expressed during early cell wall regeneration, and it was expressed in various vegetative and reproductive tissues. The expression of GhTZF1 was substantially up-regulated by a variety of abiotic stresses, such as PEG and salt. GhTZF1 also responds to methyl jasmonate (MeJA) and H₂O₂ treatment. Overexpression of GhTZF1 enhanced drought tolerance and delayed drought-induced leaf senescence in transgenic Arabidopsis. Subsequent experiments indicated that dark- and MeJA-induced leaf senescence was also attenuated in transgenic plants. The amount of H₂O₂ in transgenic plants was attenuated under both drought conditions and with MeJA-treatment. The activity of superoxide dismutase and peroxidase was higher in transgenic plants than in wild type plants under drought conditions. Quantitative real-time PCR analysis revealed that overexpression of GhTZF1 reduced the expression of oxidative-related senescence-associated genes (SAGs) under drought conditions. Overexpression of GhTZF1 also enhanced oxidative stress tolerance, which was determined by measuring the expression of a set of antioxidant genes and SAGs that were altered in transgenic plants during H₂O₂ treatment. Hence, we conclude that GhTZF1 may serve as a regulator in mediating drought stress tolerance and subsequent leaf senescence by modulating the reactive oxygen species homeostasis.     

Two cysteine proteinase inhibitors from Arabidopsis thaliana, AtCYSa and AtCYSb, increasing the salt, drought, oxidation and cold tolerance

Two cysteine proteinase inhibitors (cystatins) from Arabidopsis thaliana, designated AtCYSa and AtCYSb, were characterized. Recombinant GST-AtCYSa and GST-AtCYSb were expressed in Escherichia coli and purified. They inhibit the catalytic activity of papain, which is generally taken as evidence for cysteine proteinase inhibitor function. Northern blot analyses showed that the expressions of AtCYSa and AtCYSb gene in Arabidopsis cells and seedlings were strongly induced by multiple abiotic stresses from high salt, drought, oxidant, and cold. Interestingly, the promoter region of AtCYSa gene contains a dehydration-responsive element (DRE) and an abscisic acid (ABA)-responsive element (ABRE), which identifies it as a DREB1A and AREB target gene. Under normal conditions, AtCYSa was expressed in 35S: DREB1A and 35S: AREB1 plants at a higher level than in WT plants, while AtCYSa gene was expressed in 35S: DREB2A plants at the same level as in WT plants. Under stress conditions (salt, drought and cold), AtCYSa was expressed more in all three transgenic plants than in WT plants. Over-expression of AtCYSa and AtCYSb in transgenic yeast and Arabidopsis plants increased the resistance to high salt, drought, oxidative, and cold stresses. Taken together, these data raise the possibility of using AtCYSa and AtCYSb to genetically improve environmental stresses tolerance in plants.     

Enhancing Arabidopsis salt and drought stress tolerance by chemical priming for its abscisic acid responses

Drought and salt stress tolerance of Arabidopsis (Arabidopsis thaliana) plants increased following treatment with the nonprotein amino acid beta-aminobutyric acid (BABA), known as an inducer of resistance against infection of plants by numerous pathogens. BABA-pretreated plants showed earlier and higher expression of the salicylic acid-dependent PR-1 and PR-5 and the abscisic acid (ABA)-dependent RAB-18 and RD-29A genes following salt and drought stress. However, non-expressor of pathogenesis-related genes 1 and constitutive expressor of pathogenesis-related genes 1 mutants as well as transgenic NahG plants, all affected in the salicylic acid signal transduction pathway, still showed increased salt and drought tolerance after BABA treatment. On the contrary, the ABA deficient 1 and ABA insensitive 4 mutants, both impaired in the ABA-signaling pathway, could not be protected by BABA application. Our data demonstrate that BABA-induced water stress tolerance is based on enhanced ABA accumulation resulting in accelerated stress gene expression and stomatal closure. Here, we show a possibility to increase plant tolerance for these abiotic stresses through effective priming of the preexisting defense pathways without resorting to genetic alterations.     

Brassinosteroid and brassinosteroid-mimic differentially modulate Arabidopsis thaliana fitness under drought

Plant Growth Regulation - Brassinosteroids (BRs) are widely used to promote plant growth/development and alleviate environmental stresses’ adverse effects. However, its low stability in the...     

MCL-1 is a stress sensor that regulates autophagy in a developmentally regulated manner

Apoptosis has an important role during development to regulate cell number. In differentiated cells, however, activation of autophagy has a critical role by enabling cells to remain functional following stress. In this study, we show that the antiapoptotic BCL-2 homologue MCL-1 has a key role in controlling both processes in a developmentally regulated manner. Specifically, MCL-1 degradation is an early event not only following induction of apoptosis, but also under nutrient deprivation conditions where MCL-1 levels regulate activation of autophagy. Furthermore, deletion of MCL-1 in cortical neurons of transgenic mice activates a robust autophagic response. This autophagic response can, however, be converted to apoptosis by either reducing the levels of the autophagy regulator Beclin-1, or by a concomitant activation of BAX. Our results define a pathway whereby MCL-1 has a key role in determining cell fate, by coordinately regulating apoptosis and autophagy.     

AtA6PR1 and AtA6PR2 encode putative aldose 6-phosphate reductases that are cytosolically localized and respond differentially to cold and salt stress in Arabidopsis thaliana

Sorbitol is synthesized in Rosaceae species, especially in source organs, in a pathway involving aldose 6-phosphate reductase (A6PR, EC 1.1.1.200). As compatible solutes, sorbitol and other sugar alcohols assist in the ability of the plant to withstand abiotic stress conditions. Here, we identify two tandemly-duplicated genes in a non-Rosaceae species (Arabidopsis thaliana L.), and show that the proteins encoded by At2g21250 (AtA6PR1) and At2g21260 (AtA6PR2) possess the molecular characteristics of A6PRs. Consistent with bioinformatic predictions, we determined that green fluorescent protein-tagged versions of AtA6PR1 and AtA6PR2 are cytosolically localized, a finding supported by immunoblotting using a specific anti-AtA6PR1 antisera after subcellular fractionation of Arabidopsis leaves. We also show that under standard growth conditions, both genes are widely-expressed, whilst AtA6PR1 protein accumulates in both source and sink organs. Under short term (24 h) cold (4 °C) or saline (150 mM NaCl) stress, both genes respond differentially and reciprocally in leaf and root tissues, suggesting a sub-functionalization of their roles. The identification of homozygous T-DNA insertional ata6pr1-mutants will facilitate the functional characterization of this gene, and help to determine its role under abiotic stress conditions.