A cellulose synthase‐like protein is required for osmotic stress tolerance in Arabidopsis

Osmotic stress imposed by soil salinity and drought stress significantly affects plant growth and development, but osmotic stress sensing and tolerance mechanisms are not well understood. Forward genetic screens using a root‐bending assay have previously identified salt overly sensitive (sos) mutants of Arabidopsis that fall into five loci, SOS1 to SOS5. These loci are required for the regulation of ion homeostasis or cell expansion under salt stress, but do not play a major role in plant tolerance to the osmotic stress component of soil salinity or drought. Here we report an additional sos mutant, sos6‐1, which defines a locus essential for osmotic stress tolerance. sos6‐1 plants are hypersensitive to salt stress and osmotic stress imposed by mannitol or polyethylene glycol in culture media or by water deficit in the soil. SOS6 encodes a cellulose synthase‐like protein, AtCSLD5. Only modest differences in cell wall chemical composition could be detected, but we found that sos6‐1 mutant plants accumulate high levels of reactive oxygen species (ROS) under osmotic stress and are hypersensitive to the oxidative stress reagent methyl viologen. The results suggest that SOS6/AtCSLD5 is not required for normal plant growth and development but has a critical role in osmotic stress tolerance and this function likely involves its regulation of ROS under stress.     

The microfilament cytoskeleton plays a vital role in salt and osmotic stress tolerance in Arabidopsis

Although recent studies have suggested that the microfilament (MF) cytoskeleton of plant cells participates in the response to salt stress, it remains unclear as to whether the MF cytoskeleton actually plays an active role in a plant's ability to withstand salt stress. In the present study, we report for the first time the role of MFs in salt tolerance of Arabidopsis thaliana . Our experiments revealed that Arabidopsis seedlings treated with 150 m m NaCl maintained MF assembly and bundle formation, whereas treatment with 250 m m NaCl initially induced MF assembly but subsequently caused MF disassembly. A corresponding change in the fluorescence intensity of MFs was also observed; that is, a sustained rise in fluorescence intensity in seedlings exposed to 150 m m NaCl and an initial rise and subsequent fall in seedlings exposed to 250 m m NaCl. These results suggest that MF assembly and bundles are induced early after salt stress treatment, while MF polymerization disappears after high salt stress. Facilitation of MF assembly with phalloidin rescued wild-type seedlings from death, whereas blocking MFs assembly with latrunculin A and cytochalasin D resulted in few survivors under salt stress. Pre-treatment of seedlings with phalloidin also clearly increased plant ability to withstand salt stress. MF assembly increased survival of Arabidopsis salt-sensitive sos2 mutants under salt stress and rescued defective sos2 mutants. Polymerization of MFs and its role in promoting survival was also found in plants exposed to osmotic stress. These findings suggest that the MF cytoskeleton participates and plays a vital role in responses to salt and osmotic stress in Arabidopsis .     

Abscisic acid and osmotic stress or slow drying independently induce desiccation tolerance in mutant seeds of Arabidopsis thaliana

Action of endogenous abscisic acid (ABA) is absent in the ABA-deficient and -insensitive double mutant ( aba-1abi3–1 ) seeds of Arabidopsis thaliana . Thus, responses to osmotic stress and dehydration can be studied without interference of endogenous ABA. Seeds of this double mutunt are viable hut desiceation-intolerant. However, desiccation tolerance can he induced by either (1) slow dehydration of immature seeds; (2) treatment of immature seeds with osmotica or; (31 due to the leakiness of the ABA-insensitivty mutation, by application of exogenous ABA. Consequently it is concluded that either ABA or osmotic- or dehydration-stress and related gene expression meets the minimal requirements for acquisition of desiccation tolerance in seeds of Arabidopsis thalianna .     

A novel kinesin-like protein with a calmodulin-binding domain

Calcium regulates diverse developmental processes in plants through the action of calmodulin. A cDNA expression library from developing anthers of tobacco was screened with ³⁵S-labeled calmodulin to isolate cDNAs encoding calmodulin-binding proteins. Among several clones isolated, a kinesin-like gene (TCK1) that encodes a calmodulin-binding kinesin-like protein was obtained. The TCK1 cDNA encodes a protein with 1265 amino acid residues. Its structural features are very similar to those of known kinesin heavy chains and kinesin-like proteins from plants and animals, with one distinct exception. Unlike other known kinesin-like proteins, TCK1 contains a calmodulin-binding domain which distinguishes it from all other known kinesin genes. Escherichia coli-expressed TCK1 binds calmodulin in a Ca²⁺-dependent manner. In addition to the presence of a calmodulin-binding domain at the carboxyl terminal, it also has a leucine zipper motif in the stalk region. The amino acid sequence at the carboxyl terminal of TCK1 has striking homology with the mechanochemical motor domain of kinesins. The motor domain has ATPase activity that is stimulated by microtubules. Southern blot analysis revealed that TCK1 is coded by a single gene. Expression studies indicated that TCK1 is expressed in all of the tissues tested. Its expression is highest in the stigma and anther, especially during the early stages of anther development. Our results suggest that Ca²⁺/calmodulin may play an important role in the function of this microtubule-associated motor protein and may be involved in the regulation of microtubule-based intracellular transport.     

Glycine-rich RNA-binding protein 7 affects abiotic stress responses by regulating stomata opening and closing in Arabidopsis thaliana

Despite the fact that glycine-rich RNA-binding proteins (GRPs) have been implicated in the responses of plants to environmental stresses, their physiological functions and mechanisms of action in stress responses remain largely unknown. Here, we assessed the functional roles of GRP7, one of the eight GRP family members in Arabidopsis thaliana , on seed germination, seedling growth, and stress tolerance under high salinity, drought, or cold stress conditions. The transgenic Arabidopsis plants overexpressing GRP7 under the control of the cauliflower mosaic virus 35S promoter displayed retarded germination and poorer seedling growth compared with the wild-type plants and T-DNA insertional mutant lines under high salinity or dehydration stress conditions. By contrast, GRP7 overexpression conferred freezing tolerance in Arabidopsis plants. GRP7 is expressed abundantly in the guard cells, and has been shown to influence the opening and closing of the stomata, in accordance with the prevailing stress conditions. GRP7 is localized to both the nucleus and the cytoplasm, and is involved in the export of mRNAs from the nucleus to the cytoplasm under cold stress conditions. Collectively, these results provide compelling evidence that GRP7 affects the growth and stress tolerance of Arabidopsis plants under high salt and dehydration stress conditions, and also confers freezing tolerance, particularly via the regulation of stomatal opening and closing in the guard cells.     

Overexpression of the rice Osmyb4 gene increases chilling and freezing tolerance of Arabidopsis thaliana plants

The expression of the gene Osmyb4, detected at low level in rice (Oryza sativa) coleoptiles grown for 3 days at 29 degrees C, is strongly induced by treatments at 4 degrees C. At sublethal temperatures of 10 and 15 degrees C, its expression in rice seedlings is already evident, but this effect cannot be vicariated by other stresses or ABA treatment. We demonstrate by transient expression that Myb4 transactivates the PAL2, ScD9 SAD and COR15a cold-inducible promoters. The Osmyb4 function in vivo is demonstrated overexpressing its cDNA in Arabidopsis thaliana plants (ecotype Wassilewskija) under the control of the constitutive CaMV 35S promoter. Myb4 overexpressing plants show a significant increased cold and freezing tolerance, measured as membrane or Photosystem II (PSII) stability and as whole plant tolerance. Finally, in Osmyb4 transgenic plants, the expression of genes participating in different cold-induced pathways is affected, suggesting that Myb4 represents a master switch in cold tolerance.     

Arabidopsis IQD1, a novel calmodulin-binding nuclear protein, stimulates glucosinolate accumulation and plant defense

Glucosinolates are a class of secondary metabolites with important roles in plant defense and human nutrition. To uncover regulatory mechanisms of glucosinolate production, we screened Arabidopsis thaliana T-DNA activation-tagged lines and identified a high-glucosinolate mutant caused by overexpression of IQD1 (At3g09710). A series of gain- and loss-of-function IQD1 alleles in different accessions correlates with increased and decreased glucosinolate levels, respectively. IQD1 encodes a novel protein that contains putative nuclear localization signals and several motifs known to mediate calmodulin binding, which are arranged in a plant-specific segment of 67 amino acids, called the IQ67 domain. We demonstrate that an IQD1-GFP fusion protein is targeted to the cell nucleus and that recombinant IQD1 binds to calmodulin in a Ca(2+)-dependent fashion. Analysis of steady-state messenger RNA levels of glucosinolate pathway genes indicates that IQD1 affects expression of multiple genes with roles in glucosinolate metabolism. Histochemical analysis of tissue-specific IQD1::GUS expression reveals IQD1 promoter activity mainly in vascular tissues of all organs, consistent with the expression patterns of several glucosinolate-related genes. Interestingly, overexpression of IQD1 reduces insect herbivory, which we demonstrated in dual-choice assays with the generalist phloem-feeding green peach aphid (Myzus persicae), and in weight-gain assays with the cabbage looper (Trichoplusia ni), a generalist-chewing lepidopteran. As IQD1 is induced by mechanical stimuli, we propose IQD1 to be novel nuclear factor that integrates intracellular Ca(2+) signals to fine-tune glucosinolate accumulation in response to biotic challenge.     

Pre-conditioning the epigenetic response to high vapor pressure deficit increases the drought tolerance of Arabidopsis thaliana

Epigenetic modification of the genome via cytosine methylation is a dynamic process that responds to changes in the growing environment. This modification can also be heritable. The combination of both properties means that there is the potential for the life experiences of the parental generation to modify the methylation profiles of their offspring and so potentially to “pre-condition” them to better accommodate abiotic conditions encountered by their parents. We recently identified high vapor pressure deficit (vpd)-induced DNA methylation at 2 gene loci in the stomatal development pathway and an associated reduction in leaf stomatal frequency.¹ Here, we test whether this epigenetic modification pre-conditioned parents and their offspring to the more severe water stress of periodic drought. We found that 3 generations of high vpd-grown plants were better able to withstand periodic drought stress over 2 generations. This resistance was not directly associated with de novo methylation of the target stomata genes, but was associated with the cmt3 mutant’s inability to maintain asymmetric sequence context methylation. If our finding applies widely, it could have significant implications for evolutionary biology and breeding for stressful environments.     

A novel role for oleosins in freezing tolerance of oilseeds in Arabidopsis thaliana

Oil bodies in seeds of higher plants are surrounded with oleosins. Here we demonstrate a novel role for oleosins in protecting oilseeds against freeze/thaw-induced damage of their cells. We detected four oleosins in oil bodies isolated from seeds of Arabidopsis thaliana , and designated them OLE1, OLE2, OLE3 and OLE4 in decreasing order of abundance in the seeds. For reverse genetics, we isolated oleosin-deficient mutants ( ole1 , ole2 , ole3 and ole4 ) and generated three double mutants ( ole1 ole2 , ole1 ole3 and ole2 ole3 ). Electron microscopy showed an inverse relationship between oil body sizes and total oleosin levels. The double mutant ole1 ole2 , which had the lowest levels of oleosins, had irregular enlarged oil-containing structures throughout the seed cells. Germination rates were positively associated with oleosin levels, suggesting that defects in germination are related to the expansion of oil bodies due to oleosin deficiency. We found that freezing followed by imbibition at 4°C abolished seed germination of single mutants ( ole1 , ole2 and ole3 ), which germinated normally without freezing treatment. The treatment accelerated the fusion of oil bodies and the abnormal-positioning and deformation of nuclei in ole1 seeds, which caused seed mortality. In contrast, ole1 seeds that had undergone freezing treatment germinated normally when incubated at 22°C instead of 4°C, because degradation of oils abolished the acceleration of fusion of oil bodies during imbibition. Taken together, our findings suggest that oleosins increase the viability of over-wintering oilseeds by preventing abnormal fusion of oil bodies during imbibition in the spring.     

Costs and benefits of cold tolerance in transgenic Arabidopsis thaliana

Cold tolerance in plants is an ecologically important trait that has been under intensive study for basic and applied reasons. Determining the fitness benefits and costs of cold tolerance has previously been difficult because cold tolerance is normally an induced trait that is not expressed in warm environments. The recent creation of transgenic plants constitutively expressing cold tolerance genes enables the investigation of the fitness consequences of cold tolerance in multiple temperature environments. We studied three genes from the CBF (C-repeat/dehydration responsive element binding factor) cold tolerance pathway, CBF1, 2 and 3, in Arabidopsis thaliana to test for benefits and costs of constitutive cold tolerance. We used multiple insertion lines for each transgene and grew the lines in cold and control conditions. Costs of cold tolerance, as determined by fruit number, varied by individual transgene. CBF2 and 3 overexpressers showed costs of cold tolerance, and no fitness benefits, in both environments. CBF1 overexpressing plants showed no fitness cost of cold tolerance in the control environment and showed a marginal fitness benefit in the cold environment. These results suggest that constitutive expression of traits that are normally induced in response to environmental stress will not always lead to costs in the absence of that stress, and that the ecological risks of CBF transgene escape should be assessed prior to their use in commercial agriculture.