Protective functions of alternative splicing transcripts (CdDHN4-L and CdDHN4-S) of CdDHN4 from bermudagrass under multiple abiotic stresses

Growing evidence indicates that calcium-dependent protein kinases (CDPKs) are involved in many aspects of plant’s growth, development and responses to biotic and abiotic stresses. Previous researchers reported AtCDPK1 gene used as a positive regulator in salt and drought stress signaling. This study aimed to increase transgenic potatoes’ (Solanum tuberosum L. cv. Favorita) tolerance to the drought and osmotic stresses. Transgenic potato plants expressing AtCDPK1 gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter (referred to as T-plants) were successfully generated through Agrobacterium-mediated transformation. Two representative transgenic T6 and T25 lines were evaluated for enhanced tolerance to drought and osmotic stresses. The survival rates of transgenic lines T6 and T25 (67 and 54%, respectively) were higher than that of wild type (WT) plants (25%) after the drought stress. After 15 days of PEG stress, the survival rate of T6 was 45.68%, whereas that of the WT line was approximately 20%. The survival rate of transgenic lines was very higher than that of WT after 15 days of PEG stress. The content of proline and malondialdehyde (MDA) were clear difference between control and transgenic lines after the drought and osmotic stresses. After the 24 h osmotic treatment, content of proline in transgenic plants was very higher than that of the control, enhanced 26%, reached an extremely significant level (P < 0.05). MDA content in transgenic plants was lower than the control in the same period, while the biggest difference appeared at 24 h (P < 0.05), and MDA content of transgenic plants reduced 28% than the control. qRT-PCR proves that relative expressions of StP5CS and StProDH genes in the transgenic lines both increased in early period, and then decreased after PEG 6000 stress. These results suggested that enhanced AtCDPK1 expression affected the transcription of stress-associated genes and the content of proline and MDA in transgenic plants, which leads to improved tolerance ability in transgenic potatoes.

To investigate the relationship between nuclear factor Y (NF-Y) and stress tolerance in garlic, we cloned a NF-Y family gene AsNF-YC8 from garlic, which was largely upregulated at dehydrate stage. Expression pattern analyses in garlic revealed that AsNF-YC8 is induced through abscisic acid (ABA) and abiotic stresses, such as NaCl and PEG. Compared with wild-type plants, the overexpressing-AsNF-YC8 transgenic tobacco plants showed higher seed germination rates, longer root length and better plant growth under salt and drought stresses. Under drought stress, the transgenic plants maintained higher relative water content (RWC), net photosynthesis, lower levels of malondialdehyde (MDA), and less ion leakage (IL) than wild-type control plants. These results indicate the high tolerance of the transgenic plants to drought stress compared to the WT. The transgenic tobacco lines accumulated less reactive oxygen species (ROS) and exhibited higher antioxidative enzyme activities compared with wild-type (WT) plants under drought stress, which suggested that the overexpression of AsNF-YC8 improves the antioxidant defense system by regulating the activities of these antioxidant enzymes, which in turn protect transgenic lines against drought stress. These results suggest that AsNF-YC8 plays an important role in tolerance to drought and salt stresses.

Oxidative stress caused by reactive oxygen species (ROS) is one of the major abiotic stresses in plants. Under adverse growth conditions, the incoordination of various metabolic processes in plant cells can result in increased hydrogen peroxide (H2O2), thus causing a variety of threats and injuries to plant cells. Ascorbate peroxidase (APX) is an important enzyme to remove H2O2 in plants. In Arabidopsis thaliana, there are eight APX gene family members, including APX1?APX6, sAPX and tAPX. In this study, we analyzed the expression patterns of the eight APX genes in the wild-type and apx mutant plants at different developmental stages and under different abiotic stress conditions. Meanwhile, the tolerance of each apx mutant to salt, drought and heat stresses was studied. qRT-PCR analysis showed that during development (from 4 to 8 weeks old), APX1 and APX2 exhibited the highest and lowest expression levels, respectively. In addition, the expression levels of APX4, sAPX and tAPX decreased during development, while the expression of APX6 increased with the maturity of the plants. Moreover, under different abiotic stress conditions, APX1, APX2 and APX6 were significantly induced by heat stress, sAPX actively responded to salt stress, and APX3 and APX5 exhibited obvious responses to salt, drought and heat stresses. Further tolerance analysis showed that the resistance of all apx mutants to salt and drought stresses was lower than that of the wild-type plant at both germination and maturity stages. At germination stage, all apx mutants were more sensitive to drought stress than to salt stress. At maturity stage, the apx1 and apx6 mutants were more sensitive to salt and drought stresses than the wild-type and other apx mutant plants. The physiological indexes indicated that the H2O2 content in all mutants, especially in the apx1, sapx and tapx, was significantly higher than that in the wild type 10 days after drought stress treatment, the malondialdehyde (MDA) content in all mutants was significantly higher than that in the wild type 5 days after salt stress treatment, while heat stress treatment for 2 h resulted in a significant increase in the contents of H2O2 and MDA in apx1, apx2 and apx6, especially in apx2. Taken together, our study revealed that all eight APX members of Arabidopsis participate in the growth and developmental processes and the abiotic stress responses, with some specific APXs playing a major role in a certain process.

WRKY transcription factors are transcriptional regulators of signaling pathways involved in biotic and abiotic stress responses. In this study, we report that ectopic expression of the GhWRKY6-like gene significantly improved salt tolerance in Arabidopsis thaliana while silencing the GhWRKY6-like increase the sensitivity to abiotic stresses in cotton. GhWRKY6-like was localized to the nucleus. Expression of GhWRKY6-like was remarkably induced by salt, polyethylene glycol (PEG) and abscisic acid (ABA) treatments. For further characterization, the GhWRKY6-like gene was cloned and transformed into Arabidopsis. Our findings showed that the germination rate and root length were significantly improved in plants overexpressing GhWRKY6-like vs wild type (WT) under salt, mannitol and ABA treatments. Additionally, the overexpressing lines showed greater salt tolerance than WT plants in soil. In addition, overexpressing plants accumulated less H2 O2 and malondialdehyde (MDA), while higher proline content, superoxide dismutase (SOD) and peroxidase (POD) activities were detected under salt and osmotic stresses. In contrast, virus-induced gene silencing (VIGS) of GhWRKY6-like in cotton showed enhanced sensitivity compared to WT plants during salt and drought stresses. Additionally, expression analysis of stress-responsive genes in GhWRKY6-like Arabidopsis revealed that there was increased expression of genes involved in the ABA signaling pathway (AtABF4, AtABI5 and AtMYC2) and osmotic stress (AtSOS2, AtRD29a and AtRD29b). Our results revealed that GhWRKY6-like enhanced salt tolerance in Arabidopsis by scavenging reactive oxygen species and regulating the ABA signaling pathway. We suggest that overexpression of the GhWRKY6-like gene in cotton will enhance tolerance against salt, drought and osmotic stresses.

Protein ubiquitination is an important regulatory mechanism for biological growth and development against environmental influences, and can affect several biological processes, including the growth, development, and stress responses of plants. However, the function of potato-related ubiquitin-conjugating enzymes in abiotic stress tolerance is poorly understood. In this study, a StUBC13 with a UBC conserved structural domain was identified in potato and its function was investigated under osmotic stress and salt stress conditions. The observation of plant phenotypes under stress conditions revealed that overexpressed plants grew better than wild-type plants. In line with the above results, the determination of stress-related physiological indices revealed that the overexpression transgenic plants had better stress tolerance and stronger adaptation to environmental stress, and the transgenic plants were found to tolerate better drought and salt stress by decreasing their malondialdehyde (MDA) content and increasing their superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) contents under stress conditions. Based on these results, StUBC13 has an important regulatory role in the response of plants to abiotic stresses (osmotic stress and salt stress), and overexpression of this gene can improve the tolerance of potatoes to osmotic and salt stresses.

Glutathione S-transferases (GSTs) have been well-documented to be involved in oxidative stress metabolism. However, reports on GST gene participating in regulatory function are limited. Previously our lab had found that glutathione S-transferase U17 (AtGSTU17) knock-out plants were more tolerant to drought and salt stresses than wild-type plants. The mechanism causing these phenotypes of atgstu17 can be explained mostly by the combined effect of elevated contents of both glutathione and abscisic acid. Thus, AtGSTU17 plays a negative role in regulating abiotic stress tolerance. Three homozygous knockout mutants, atgstu7, atgstu8, atgstu18, were chosen for further study with their function in response to abiotic stress. Preliminary stress experiments indicated that the mentioned above atgstu mutants showed different phenotypes from the atgstu17 mutant plants in terms of salt and drought tolerance. However, the germination rates of the three mutants were higher than wild-type plants when they were treated with abscisic acid, salt, and osmotic stress. We suggested that these three genes might play a negative regulatory role during seed imbibitions and seedling development under stress conditions. Genetic analysis of F2 segergating population indicated that wild-type (AtGSTU25/AtGSTU25), hemizygous (AtGSTU25/atgstu25), and homozygous mutant (atgstu25/atgstu25) plants were in a ratio of 1:2:0, indicating that the homozygous mutant allele is lethal.

A wheat salinity-induced WRKY transcription factor TaWRKY93 confers multiple abiotic stress tolerance in Arabidopsis thaliana

Drought and salt stress are two important environmental factors that significantly restrict plant growth and reproduction. Malate dehydrogenase is essential to life as it is engaged in numerous physiological processes in cells, particularly those related to abiotic stress reactions. However, a complete understanding of MDH family members in kenaf is not clear yet. In this study, subcellular localization analysis and a yeast transcriptional activation assay revealed that HcMDH1 was localized in chloroplasts but had no transcriptional activation activity. When exposed to salt or drought stress, yeast cells expressing the HcMDH1 gene exhibit an increased survival rate. Overexpression of HcMDH1 in Arabidopsis increased seed germination rate and root growth when transgenic lines were exposed to varying concentrations of mannitol and NaCl. Subsequent physiological studies revealed that transgenic lines had higher concentrations of soluble carbohydrates, proline, and chlorophyll and lower concentrations of malondialdehyde (MDA) and reactive oxygen species (ROS). Furthermore, inhibiting HcMDH1 in kenaf using virus-induced gene silencing (VIGS) decreased salt and drought tolerance due to elevated ROS and MDA levels. In these silenced lines, the expression of six essential genes engaged in stress-resistance and photosynthesis, namely HcGAPDH, HcGLYK, HcFBA, HcFBPase, HcPGA, and HcLSD, is significantly altered under salt and drought stress. In summary, HcMDH1 is a complex and positive regulatory gene that plays a key role in regulating chlorophyll content, antioxidant enzyme activity and osmotic regulation under salt and drought stress, which may have implications for kenaf transgenic breeding.Graphical

Salinity and drought conditions commonly result in osmotic and oxidative stresses, while salinity additionally causes ionic stress. In this study, we identified specific genes regulated by osmotic and ionic stresses in five Arabidopsis ecotypes. Shahdara (SHA) and C24 ecotypes were more tolerant to salt and drought stresses at the seedling growth stage, as evidenced by lower water loss rate, lower electrolyte leakage, and higher survival rate when compared to the other three ecotypes under drought and salinity conditions. Transcriptomic analysis revealed that 3700 and 2242 genes were differentially regulated by salt and osmotic stresses, respectively. Totally 78.1% of upregulated and 62.0% of downregulated genes by osmotic stress were also commonly regulated by salt stress. Gene ontology term enrichment analysis showed that auxin indole-3-acetic acid (IAA), abscisic acid, cytokinin, and gibberellic acid pathways were regulated by the osmotic stress, while IAA, jasmonic acid, and ethylene pathways were changed by the ionic stress. The nutrient and water uptake pathways were regulated by both the osmotic and ionic stresses, whereas ion transportation and kinase pathways were modulated by the ionic stress. Additionally, we characterized bHLH61 as a negative regulator in response to salt and drought stresses. This study provided new clues of plant responses to salt and drought stresses.

Salt and drought stresses are major environmental conditions that severely limit grape growth and productivity, while exogenous melatonin can alleviate the drought and salt damage to grapevines. N-acetylserotonin methyltransferase (ASMT) is the key enzyme in melatonin synthesis, which plays a critical role in regulating stress responses. However, the roles of ASMTs from grapevine under drought and salt stresses responses remain largely unclear. In this study, the VvASMT1 gene was isolated from grapevine, and its physiological functions in salt and mimic drought stress tolerance were investigated. Expression pattern analysis revealed that VvASMT1 was significantly induced by different salt and osmotic stresses. Ectopic expression of VvASMT1 in Nicotiana benthamiana significantly enhanced melatonin production in transgenic plants. Compared with wild-type plants, the transgenic lines exhibited a higher germination ratio, longer root length, lower degree of leaf wilting and relative water content (RWC) under salt and osmotic stresses. In addition, under salt and osmotic stresses, overexpression of VvASMT1 improved proline and malondialdehyde (MDA) contents, increased the activity of antioxidant enzymes and decreased the accumulation of reactive oxygen species (ROS). Taken together, our results demonstrate the explicit role of VvASMT1 in salt and osmotic stress responses, which provides a theoretical foundation for the genetic engineering of grapevine.

GhMAPK3, a mitogen-activated protein kinase, enhance salt and drought tolerance in cotton (Gossypium hirsutum)

Biochemical and biomolecular response of Arabidopsis seedlings to osmotic and salt stress: Mitigation by biostimulant formulation enriched in betalain degradation products

Drought and salinity are serious environmental factors limiting the growth and productivity of plants worldwide. Therefore, it is necessary to develop ways to improve drought and salinity stress tolerance in plants. In this study, a drought-responsive nuclear factor Y subunit A gene, ZmNF-YA12, was cloned from maize. qPCR revealed ZmNF-YA12 transcript in all vegetative and reproductive tissues, with higher levels in young roots. Expression analyses of maize revealed that ZmNF-YA12 was induced by abscisic acid (ABA), jasmonic acid (JA), and abiotic stresses, including dehydration, high salinity, cold, and polyethylene glycol (PEG) treatment. The heterologous expression of ZmNF-YA12 in Arabidopsis plants resulted in increased root length and better plant growth than in wild-type (WT) plants under conditions of mannitol, salt, and JA stress on 1/2 MS medium. Transgenic Arabidopsis showed improved tolerance to drought and salt stresses in soil, and higher proline content and lower malondialdehyde (MDA) content than WT controls. The transgenic plants also maintained higher peroxidase (POD) activities than WT plants under conditions of NaCl stress. A yeast two-hybrid experiment demonstrated that ZmNF-YA12 interacted with ZmNF-YC1 and ZmNF-YC15. Moreover, the transcript levels of stress-responsive genes (RD29A, RD29B, RAB18, and RD22) were markedly increased in transgenic lines under conditions of drought and salt stress. These observations suggested that the ZmNF-YA12 gene may confers drought and salt stress tolerance by regulating stress-related genes or interacting with ZmNF-YC1 and ZmNF-YC15, and has potential applications in molecular breeding with maintenance of production under conditions of stress.

Overexpression of alfalfa Orange gene in tobacco enhances carotenoid accumulation and tolerance to multiple abiotic stresses

Cotton (Gossypium hirsutum) as one of the most important economic crop in the world is often suffering from biotic and abiotic stresses during its growth seasons. Mitogen-activated protein kinase (MAPK) cascades participate in signal transduction of extracellular stimuli and regulate multiple biotic and abiotic stress responses in plants. In this study, a MAPK gene (GhMPK4) belonging to the group B of MAPK family was identified in cotton. Quantitative RT-PCR analysis showed that expression of GhMPK4 was induced by high salinity and osmotic stresses. Overexpression of GhMPK4 in Arabidopsis significantly enhanced the transgenic plants’ sensitivity to salt and osmotic stresses and exogenous abscisic acid (ABA). Under NaCl, mannitol and ABA treatments, the rates of seed germination and cotyledon expansion/greening of the GhMPK4 overexpression transgenic lines were remarkably declined compared with those of wild type, and roots of the GhMPK4 overexpression transgenic seedlings were shorter than those of wild type. Chlorophyll and proline contents in leaves of the GhMPK4 transgenic lines were obviously lower than those in wild type. Growth status of the GhMPK4 transgenic plants was worse than that of wild type when being subjected to drought and salinity stresses. Furthermore, the expression levels of the stress-related genes were altered in the transgenic plants under NaCl, mannitol and ABA treatments, compared with those in wild type. Taken the data together, it is suggested that GhMPK4 as a negative regulator may participate in response to salt and osmotic stresses and ABA signaling through affecting the expression of the stress-related genes in plants.