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 overexpression in Arabidopsis thaliana of a Trichoderma harzianum gene that modulates glucosidase activity, and enhances tolerance to salt and osmotic stresses

Using the TrichoEST database, generated in a previous functional genomics project from the beneficial filamentous fungus Trichoderma harzianum, a gene named Thkel1, which codes for a putative kelch-repeat protein, was isolated and characterized. Silencing of this gene in T. harzianum leads to a reduction of glucosidase activity and mycelial growth under abiotic stress conditions. Expression of this gene in Arabidopsis enhances plant tolerance to salt and osmotic stresses, accompanied by an increase in glucosidase activity and a reduction of abscisic acid levels compared to those observed in wild-type plants. Data presented throughout this article suggest the high value of T. harzianum as a source of genes able to facilitate the achievement of producing plants resistant to abiotic stresses without alteration of their phenotype.     

The nucleotidase/phosphatase SAL1 is a negative regulator of drought tolerance in Arabidopsis

An Arabidopsis thaliana drought-tolerant mutant, altered expression of APX2 ( alx8 ), has constitutively increased abscisic acid (ABA) content, increased expression of genes responsive to high light stress and is reported to be drought tolerant. We have identified alx8 as a mutation in SAL1, an enzyme that can dephosphorylate dinucleotide phosphates or inositol phosphates. Previously identified mutations in SAL1, including fiery ( fry1-1 ), were reported as being more sensitive to drought imposed by detachment of rosettes. Here we demonstrate that alx8 , fry1-1 and a T-DNA insertional knockout allele all have markedly increased resistance to drought when water is withheld from soil-grown intact plants. Microarray analysis revealed constitutively altered expression of more than 1800 genes in both alx8 and fry1-1. The up-regulated genes included some characterized stress response genes, but few are inducible by ABA. Metabolomic analysis revealed that both mutants exhibit a similar, dramatic reprogramming of metabolism, including increased levels of the polyamine putrescine implicated in stress tolerance, and the accumulation of a number of unknown, potential osmoprotectant carbohydrate derivatives. Under well-watered conditions, there was no substantial difference between alx8 and Col-0 in biomass at maturity; plant water use efficiency (WUE) as measured by carbon isotope discrimination; or stomatal index, morphology or aperture. Thus, SAL1 acts as a negative regulator of predominantly ABA-independent and also ABA-dependent stress response pathways, such that its inactivation results in altered osmoprotectants, higher leaf relative water content and maintenance of viable tissues during prolonged water stress.     

Endosidin20-1 is more potent than endosidin20 in inhibiting plant cellulose biosynthesis and molecular docking analysis of cellulose biosynthesis inhibitors on modeled cellulose synthase structure

Endosidin20 (ES20) is a recently identified cellulose biosynthesis inhibitor (CBI) that targets the catalytic site of plant cellulose synthase (CESA). Here, we screened over 600 ES20 analogs and identified nine active analogs named ES20-1 to ES20-9. Among these, endosidin20-1 (ES20-1) had stronger inhibitory effects on plant growth and cellulose biosynthesis than ES20. At the biochemical level, we demonstrated that ES20-1, like ES20, directly interacts with CESA6. At the cellular level, this molecule, like ES20, induced the accumulation of cellulose synthase complexes at the Golgi apparatus and inhibited their secretion to the plasma membrane. Like ES20, ES20-1 likely targets the catalytic site of CESA. However, through molecular docking analysis using a modeled structure of full-length CESA6, we found that both ES20 and ES20-1 might have another target site at the transmembrane regions of CESA6. Besides ES20, other CBIs such as isoxaben, C17, and flupoxam are widely used tools to dissect the mechanism of cellulose biosynthesis and are also valuable resources for the development of herbicides. Here, based on mutant genetic analysis and molecular docking analysis, we have identified the potential target sites of these CBIs on a modeled CESA structure. Some bacteria also produce cellulose, and both ES20 and ES20-1 inhibited bacterial cellulose biosynthesis. Therefore, we conclude that ES20-1 is a more potent analog of ES20 that inhibits intrinsic cellulose biosynthesis in plants, and both ES20 and ES20-1 show an inhibitory effect on bacterial growth and cellulose synthesis, making them excellent tools for exploring the mechanisms of cellulose biosynthesis across kingdoms.     

Overexpression of the soybean GmWNK1 altered the sensitivity to salt and osmotic stress in Arabidopsis

The WNK (With No Lysine K) serine-threonine kinases have been shown to be osmosensitive regulators and are critical for cell volume homeostasis in humans. We previously identified a soybean root-specific WNK homolog, GmWNK1, which is important for normal late root development by fine-tuning regulation of ABA levels. However, the functions of WNKs in plant osmotic stress response remains uncertain. In this study, we generated transgenic Arabidopsis plants with constitutive expression of GmWNK1. We found that these transgenic plants had increased endogenous ABA levels and altered expression of ABA-responsive genes, and exhibited a significantly enhanced tolerance to NaCl and osmotic stresses during seed germination and seedling development. These findings suggest that, in addition to regulating root development, GmWNK1 also regulates ABA-responsive gene expression and/or interacts with other stress related signals, thereby modulating osmotic stress responses. Thus, these results suggest that WNKs are members of an evolutionarily conserved kinase family that modulates cellular response to osmotic stresses in both animal and plants.     

Heterologous expression of the AtDREB1A gene in chrysanthemum increases drought and salt stress tolerance

DNA cassette containing an AtDREB1A cDNA and a nos terminator, driven by a cauliflower mosaic 35S promoter, or a stress-inducible rd29A promoter, was transformed into the ground cover chrysanthemum (Dendranthema grandiflorum) ‘Fall Color’ genome. Compared with wild type plants, severe growth retardation was observed in 35S:DREB1A plants, but not in rd29A:DREB1A plants. RT-PCR analysis revealed that, under stress conditions, the DREB1A gene was over-expressed constitutively in 35S:DREB1A plants, but was over-expressed inductively in rd29A:DREB1A plants. The transgenic plants exhibited tolerance to drought and salt stress, and the tolerance was significantly stronger in rd29A:DREB1A plants than in 35S:DREB1A plants. Proline content and SOD activity were increased inductively in rd29A:DREB1A plants than in 35S:DREB1A plants under stress conditions. These results indicate that heterologous AtDREB1A can confer drought and salt tolerance in transgenic chrysanthemum, and improvement of the stress tolerance may be related to enhancement of proline content and SOD activity.     

Overexpression of an Arabidopsis thaliana galactinol synthase gene improves drought tolerance in transgenic rice and increased grain yield in the field

Drought stress has often caused significant decreases in crop production which could be associated with global warming. Enhancing drought tolerance without a grain yield penalty has been a great challenge in crop improvement. Here, we report the Arabidopsis thaliana galactinol synthase 2 gene (AtGolS2) was able to confer drought tolerance and increase grain yield in two different rice (Oryza sativa) genotypes under dry field conditions. The developed transgenic lines expressing AtGolS2 under the control of the constitutive maize ubiquitin promoter (Ubi:AtGolS2) also had higher levels of galactinol than the non‐transgenic control. The increased grain yield of the transgenic rice under drought conditions was related to a higher number of panicles, grain fertility and biomass. Extensive confined field trials using Ubi:AtGolS2 transgenic lines in Curinga, tropical japonica and NERICA4, interspecific hybrid across two different seasons and environments revealed the verified lines have the proven field drought tolerance of the Ubi:AtGolS2 transgenic rice. The amended drought tolerance was associated with higher relative water content of leaves, higher photosynthesis activity, lesser reduction in plant growth and faster recovering ability. Collectively, our results provide strong evidence that AtGolS2 is a useful biotechnological tool to reduce grain yield losses in rice beyond genetic differences under field drought stress.     

Expression of Arabidopsis CBF1 regulated by an ABA/stress inducible promoter in transgenic tomato confers stress tolerance without affecting yield

Modern‐day plants are subjected to various biotic and abiotic stresses thereby limiting plant productivity and quality. It has previously been reported that the use of a strong constitutive 35S cauliflower mosaic virus (CaMV) promoter to drive the expression of Arabidopsis CBF1 in tomato improved tolerance to cold, drought and salt loading, at the expense of growth and yield under normal growth conditions. Hence in the present study, the suitability of expressing the Arabidopsis CBF1 driven by three copies of an ABA‐responsive complex (ABRC1) from the barley HAV22 gene in order to improve the agronomic performance of the transgenic tomato plants was investigated. Northern blot analysis indicated that CBF1 gene expression was induced by chilling, water‐deficit and salt treatment in the transgenic tomato plants. Under these tested stress conditions, transgenic tomato plants exhibited enhanced tolerance to chilling, water‐deficit, and salt stress in comparison with untransformed plants. Under normal growing conditions the ABRC1‐CBF1 tomato plants maintained normal growth and yield similar to the untransformed plants. The results demonstrate the promise of using ABRC1‐CBF1 tomato plants in highly stressed conditions which will in turn benefit agriculture.     

Heterologous expression of the AtDREB1A gene in chrysanthemum increases drought and salt stress tolerance

DNA cassette containing an AtDREB1A cDNA and a nos terminator, driven by a cauliflower mosaic 35S promoter, or a stress-inducible rd29A promoter, was transformed into the ground cover chrysanthemum (Dendranthema grandiflorum) ‘Fall Color’ genome. Compared with wild type plants, severe growth retardation was observed in 35S:DREB1A plants, but not in rd29A:DREB1A plants. RT-PCR analysis revealed that, under stress conditions, the DREB1A gene was over-expressed constitutively in 35S:DREB1A plants, but was over-expressed inductively in rd29A:DREB1A plants. The transgenic plants exhibited tolerance to drought and salt stress, and the tolerance was significantly stronger in rd29A:DREB1A plants than in 35S:DREB1A plants. Proline content and SOD activity were increased inductively in rd29A:DREB1A plants than in 35S:DREB1A plants under stress conditions. These results indicate that heterologous AtDREB1A can confer drought and salt tolerance in transgenic chrysanthemum, and improvement of the stress tolerance may be related to enhancement of proline content and SOD activity.     

Heterologous expression of the AtDREB1A gene in chrysanthemum increases drought and salt stress tolerance

DNA cassette containing an AtDREB1A cDNA and a nos terminator,driven by a cauli- flower mosaic 35S promoter,or a stress-inducible rd29A promoter,was transformed into the ground cover chrysanthemum(Dendranthema grandiflorum)'Fall Color'genome.Compared with wild type plants,severe growth retardation was observed in 35S:DREB1A plants,but not in rd29A:DREB1A plants.RT-PCR analysis revealed that,under stress conditions,the DREB1A gene was over-expressed constitutively in 35S:DREB1A plants,but was over-expressed inductively in rd29A:DREB1A plants.The transgenic plants exhibited tolerance to drought and salt stress,and the tolerance was significantly stronger in rd29A:DREB1A plants than in 35S:DREB1A plants.Proline content and SOD activity were increased inductively in rd29A:DREB1A plants than in 35S:DREB1A plants under stress conditions.These results indicate that heterologous AtDREB1A can confer drought and salt tolerance in transgenic chrysanthemum,and improvement of the stress tolerance may be related to enhancement of proline content and SOD activity.     

Expression of an Arabidopsis molybdenum cofactor sulphurase gene in soybean enhances drought tolerance and increases yield under field conditions

LOS5/ABA3 gene encoding molybdenum cofactor sulphurase is involved in aldehyde oxidase (AO) activity in Arabidopsis, which indirectly regulates ABA biosynthesis and increased stress tolerance. Here, we used a constitutive super promoter to drive LOS5/ABA3 overexpression in soybean (Glycine max L.) to enhance drought tolerance in growth chamber and field conditions. Expression of LOS5/ABA3 was up‐regulated by drought stress, which led to increasing AO activity and then a notable increase in ABA accumulation. Transgenic soybean under drought stress had reduced water loss by decreased stomatal aperture size and transpiration rate, which alleviated leaf wilting and maintained higher relative water content. Exposed to drought stress, transgenic soybean exhibited reduced cell membrane damage by reducing electrolyte leakage and production of malondialdehyde and promoting proline accumulation and antioxidant enzyme activities. Also, overexpression of LOS5/ABA3 enhanced expression of stress‐up‐regulated genes. Furthermore, the seed yield of transgenic plants is at least 21% higher than that of wide‐type plants under drought stress conditions in the field. These data suggest that overexpression of LOS5/ABA3 could improve drought tolerance in transgenic soybean via enhanced ABA accumulation, which could activate expression of stress‐up‐regulated genes and cause a series of physiological and biochemical resistant responses.     

The Arabidopsis GIBBERELLIN METHYL TRANSFERASE 1 suppresses gibberellin activity,reduces whole‐plant transpiration and promotes drought tolerance in transgenic tomato

Previous studies have shown that reduced gibberellin (GA) level or signal promotes plant tolerance to environmental stresses, including drought, but the underlying mechanism is not yet clear. Here we studied the effects of reduced levels of active GAs on tomato (Solanum lycopersicum) plant tolerance to drought as well as the mechanism responsible for these effects. To reduce the levels of active GAs, we generated transgenic tomato overexpressing the Arabidopsis thaliana GA METHYL TRANSFERASE 1 (AtGAMT1) gene. AtGAMT1 encodes an enzyme that catalyses the methylation of active GAs to generate inactive GA methyl esters. Tomato plants overexpressing AtGAMT1 exhibited typical GA‐deficiency phenotypes and increased tolerance to drought stress. GA application to the transgenic plants restored normal growth and sensitivity to drought. The transgenic plants maintained high leaf water status under drought conditions, because of reduced whole‐plant transpiration. The reduced transpiration can be attributed to reduced stomatal conductance. GAMT1 overexpression inhibited the expansion of leaf‐epidermal cells, leading to the formation of smaller stomata with reduced stomatal pores. It is possible that under drought conditions, plants with reduced GA activity and therefore, reduced transpiration, will suffer less from leaf desiccation, thereby maintaining higher capabilities and recovery rates.     

Arabidopsis thaliana KORRIGAN1 protein: N-glycan modification,localization, and function in cellulose biosynthesis and osmotic stress responses

Plant cellulose biosynthesis is a complex process involving cellulose-synthase complexes (CSCs) and various auxiliary factors essential for proper orientation and crystallinity of cellulose microfibrils in the apoplast. Among them is KORRIGAN1 (KOR1), a type-II membrane protein with multiple N-glycans within its C-terminal cellulase domain. N-glycosylation of the cellulase domain was important for KOR1 targeting to and retention within the trans-Golgi network (TGN), and prevented accumulation of KOR1 at tonoplasts. The degree of successful TGN localization of KOR1 agreed well with in vivo-complementation efficacy of the rsw2–1 mutant, suggesting non-catalytic functions in the TGN. A dynamic interaction network involving microtubules, CSCs, KOR1, and currently unidentified glycoprotein component(s) likely determines stress-triggered re-organization of cellulose biosynthesis and resumption of cell-wall growth under stress.