Abiotic Stress-responsive Genes Research Articles

Abiotic Stress-Responsive miRNA and Transcription Factor-Mediated Gene Regulatory Network in Oryza sativa: Construction and Structural Measure Study.

Climate changes and environmental stresses have a consequential association with crop plant growth and yield, meaning it is necessary to cultivate crops that have tolerance toward the changing climate and environmental disturbances such as water stress, temperature fluctuation, and salt toxicity. Recent studies have shown that trans-acting regulatory elements, including microRNAs (miRNAs) and transcription factors (TFs), are emerging as promising tools for engineering naive improved crop varieties with tolerance for multiple environmental stresses and enhanced quality as well as yield. However, the interwoven complex regulatory function of TFs and miRNAs at transcriptional and post-transcriptional levels is unexplored in Oryza sativa. To this end, we have constructed a multiple abiotic stress responsive TF-miRNA-gene regulatory network for O. sativa using a transcriptome and degradome sequencing data meta-analysis approach. The theoretical network approach has shown the networks to be dense, scale-free, and small-world, which makes the network stable. They are also invariant to scale change where an efficient, quick transmission of biological signals occurs within the network on extrinsic hindrance. The analysis also deciphered the existence of communities (cluster of TF, miRNA, and genes) working together to help plants in acclimatizing to multiple stresses. It highlighted that genes, TFs, and miRNAs shared by multiple stress conditions that work as hubs or bottlenecks for signal propagation, for example, during the interaction between stress-responsive genes (TFs/miRNAs/other genes) and genes involved in floral development pathways under multiple environmental stresses. This study further highlights how the fine-tuning feedback mechanism works for balancing stress tolerance and how timely flowering enable crops to survive in adverse conditions. This study developed the abiotic stress-responsive regulatory network, APRegNet database (http://lms.snu.edu.in/APRegNet), which may help researchers studying the roles of miRNAs and TFs. Furthermore, it advances current understanding of multiple abiotic stress tolerance mechanisms.

Transcriptome profiling reveals the effects of drought tolerance in Giant Juncao

BackgroundGiant Juncao is often used as feed for livestock because of its huge biomass. However, drought stress reduces forage production by affecting the normal growth and development of plants. Therefore, investigating the molecular mechanisms of drought tolerance will provide important information for the improvement of drought tolerance in this grass.ResultsA total of 144.96 Gb of clean data was generated and assembled into 144,806 transcripts and 93,907 unigenes. After 7 and 14 days of drought stress, a total of 16,726 and 46,492 differentially expressed genes (DEGs) were observed, respectively. Compared with normal irrigation, 16,247, 23,503, and 11,598 DEGs were observed in 1, 5, and 9 days following rehydration, respectively. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed abiotic stress-responsive genes and pathways related to catalytic activity, methyltransferase activity, transferase activity, and superoxide metabolic process. We also identified transcription factors belonging to several families, including basic helix-loop-helix (bHLH), WRKY, NAM (no apical meristem), ATAF1/2 and CUC2 (cup-shaped cotyledon) (NAC), fatty acyl-CoA reductase (FAR1), B3, myeloblastosis (MYB)-related, and basic leucine zipper (bZIP) families, which are important drought-rehydration-responsive proteins. Weighted gene co-expression network analysis was also used to analyze the RNA-seq data to predict the interrelationship between genes. Twenty modules were obtained, and four of these modules may be involved in photosynthesis and plant hormone signal transduction that respond to drought and rehydration conditions.ConclusionsOur research is the first to provide a more comprehensive understanding of DEGs involved in drought stress at the transcriptome level in Giant Juncao with different drought and recovery conditions. These results may reveal insights into the molecular mechanisms of drought tolerance in Giant Juncao and provide diverse genetic resources involved in drought tolerance research.

Assessing the Levels of Polymorphism and Differentiation in Iris pumila L. Populations Using Three Types of PCR Markers

The genetic polymorphism of Iris pumila L., a rare ornamental species involved in hybridization, was studied by PCR analysis using three types of primers: those based on microsatellite sequences (ISSR), MGE sequences (IRAP and iPBS), and abiotic stress response genes (LP-PCR). High levels of intraspecific and intrapopulation genetic polymorphism were revealed, which were comparable to those in other species of the Iris genus. The main indicators of genetic polymorphism of five I. pumila populations from the territory of Ukraine were the proportion of polymorphic loci (P) of 26.5–68.5%, the Shannon index (S) of 0.105–0.285, and genetic diversity (He) of 0.069–0.190. The dependence of the variability level on the population size was direct in the ISSR analysis and inverse according to the other two markers. The direct relationship between genetic and geographic distances between populations was found only using ISSR markers. The highest level of genetic polymorphism was detected by LP-PCR markers, while the population identification of all individual plants was possible only using ISSR markers. The tested system of PCR markers can be used to monitor the state of the gene pool and investigate the genetic structure of populations and migration processes.

Identification of Allelic Variation in Drought Responsive Dehydrin Gene Based on Sequence Similarity in Chickpea (Cicer arietinum L.).

Chickpea (Cicer arietinum L.) is an economically important food legume grown in arid and semi-arid regions of the world. Chickpea is cultivated mainly in the rainfed, residual moisture, and restricted irrigation condition. The crop is always prone to drought stress which is resulting in flower drop, unfilled pods, and is a major yield reducer in many parts of the world. The present study elucidates the association between candidate gene and morpho-physiological traits for the screening of drought tolerance in chickpea. Abiotic stress-responsive gene Dehydrin (DHN) was identified in some of the chickpea genotypes based on the sequence similarity approach to play a major role in drought tolerance. Analysis of variance revealed a significant effect of drought on relative water content, membrane stability index, plant height, and yield traits. The genotypes Pusa1103, Pusa362, and ICC4958 were found most promising genotypes for drought tolerance as they maintained the higher value of osmotic regulations and yield characters. The results were further supported by a sequence similarity approach for the dehydrin gene when analyzed for the presence of single nucleotide polymorphisms (SNPs) and indels. Homozygous indels and single nucleotide polymorphisms were found after the sequencing in some of the selected genotypes.

Genome-wide identification of R2R3-MYB family in wheat and functional characteristics of the abiotic stress responsive gene TaMYB344

BackgroundMYB superfamily is one of the most abundant families in plants, which plays important roles in plant growth, development, and productivity. However, to date, researches on MYBs in wheat (Triticum aestivum L.) are scattered mostly, not comprehensive.ResultsIn this study, a total of 393 R2R3-MYBs and 12 R1R2R3-MYBs were identified and analyzed including gene structure, chromosomal distribution, synteny relationship, and evolutionary relationship. Then, 29 clusters tandem duplication and 8 clusters segmental duplication genes were discovered. The expression profile of the identified genes under abiotic and biotic stress was analyzed using RNA-seq data. Based on expression patterns analysis, we screened many candidate genes involved in plant response to abiotic and biotic stress. Among them, the functional characteristics of TaMYB344 were further studied. TaMYB344 was localized in the nucleus and functioned as a weak transcriptional activator. We demonstrated that TaMYB344-overexpressing transgenic tobacco plants had enhanced tolerance to drought, heat, and high salt stress.ConclusionsIn this study, 393 R2R3-MYBs and 12 R1R2R3-MYBs in wheat were systemically identified and analyzed. Differential expression analysis indicated that many R2R3-MYBs were involved in abiotic and biotic stress response. We identified a potential candidate gene TaMYB344, overexpression of which in tobacco plants enhanced drought, heat, and salt stress tolerance. These results will provide abundant molecular data for breeding new varieties of wheat in the future.

Overexpression of a Novel Arabidopsis Gene SUPA Leads to Various Morphological and Abiotic Stress Tolerance Alternations in Arabidopsis and Poplar.

With the development of sequencing technology, the availability of genome data is rapidly increasing, while functional annotation of genes largely lags behind. In Arabidopsis, the functions of nearly half of the proteins are unknown and this remains one of the main challenges in current biological research. In an attempt to identify novel and rapid abiotic stress responsive genes, a number of salt-up (SUP) regulated genes were isolated by analyzing the public transcriptomic data, and one of them, SUPA, was characterized in this study. The expression of SUPA transcripts was rapidly up-regulated by various abiotic stress factors (<15 min), and SUPA protein is mainly localized in the peroxisome. Overexpression of SUPA in Arabidopsis leads to the elevated accumulation of reactive oxygen species (ROS), strong morphological changes and alternations in abiotic stress tolerance. The transcriptome analysis showed changes in expression of genes involved in stress response and plant development. Interestingly, ectopic overexpression of SUPA in poplar leads to a dwarf phenotype with severely curved leaves and changes in the plant tolerance of abiotic stresses. Our study reinforces the potential roles of SUPA in normal plant growth and the abiotic stress response.

Jasmonates induce growth and modulation in pigments and vitamins of Brassica oleracea var. capitata, italica and botrytis edible heads (foliage/inflorescence)

Jasmonates (JAs) are a rising class of lipid-derived signalling molecules with varied functions ranging from induction of abiotic and biotic stress-responsive genes to regulation of plant growth and development under natural condition. The rationale of present study was to investigate the nutritional importance of JAs in inducing and triggering accumulation of carbohydrates, sugars, nutritional pigments and vitamins in three varieties of Brassica oleracea L. (variety botrytis, capitata, italica) edible heads. Exogenous application of Jasmonic acid (JA) and methyl ester of Jasmonic acid (Me–JA), made edible heads of var. botrytis, capitata and italica nutritionally more accrete as compared to control untreated edible heads. Results showed that the treatment of JA and Me-JA enhanced growth of edible heads in terms of head diameter, head length, fresh weight, dry weight, moisture content, total protein, total starch, total carbohydrates, total soluble sugar, non-reducing sugar, vitamins C, A, E and B2 in var. botrytis but at the same time dramatic decline in reducing sugar, beta-carotene and lycopene was seen. In var. capitata certain reduction in vitamin E accumulation was recorded by the exogenous application of JA and Me–JA while overall growth of edible heads was accelerated which could also be supported by enhanced nutritional and productivity level. In variety italica, JAs reduced total starch and vitamin C, but overall growth was surged along with productivity. Both JA and Me-JA could be attributed to induce growth and enhance the nutritional potential of edible heads in three varieties of Brassica oleracea as compared to their untreated control edible heads. From the present results, it has been suggested that these two eco-friendly oxylipins can be further explored for enhancing the growth and nutritional value of these vegetables by exogenous application of JAs in μM to pM concentration.

Overexpression of wheat transcription factor (TaHsfA6b) provides thermotolerance in barley.

Overexpressing a heat shock factor gene (TaHsfA6bT) from wheat provides thermotolerance in barley by constitutive expression of heat and other abiotic stress-response genes. Temperature is one of the most crucial abiotic factors defining the yield potential of temperate cereal crops, such as barley. The regulators of heat shock response (HSR), heat stress transcription factors (Hsfs), modulate the transcription level of heat-responsive genes to protect the plants from heat stress. In this study, an Hsf from wheat (TaHsfA6b) is overexpressed in barley for providing thermotolerance. Transgenic barley lines overexpressing TaHsfA6b showed improvement in thermotolerance. The constitutive overexpression of a TaHsfA6b gene upregulated the expression of major heat shock proteins and other abiotic stress-responsive genes. RNA-seq and qRT-PCR analysis confirmed the upregulation of Hsps, chaperonins, DNAJ, LEA protein genes, and genes related to anti-oxidative enzymes in transgenic lines. Excessive generation and accumulation of reactive oxygen species (ROS) occurred in wild-type (WT) plants during heat stress; however, the transgenic lines reflected improved ROS homeostasis mechanisms, showing lesser ROS accumulation under high temperature. No negative phenotypic changes were observed in overexpression lines. These results suggest that TaHsfA6b is a regulator of HSR and its overexpression altered the expression patterns of some main stress-related genes and enhanced the thermotolerance of this cereal crop.

Soybean nuclear factor YA10 positively regulates drought resistance in transgenic Arabidopsis thaliana

Nuclear factor (NF)-Y is composed of three subunits, NF-YA, NF-YB and NF-YC, and plays important roles in the regulation of plant abiotic stress response gene expression. However, the function of individual soybean NF-YA proteins in response to drought stress is unclear. In this study, the soybean NF-YA subunit gene GmNF-YA10 was cloned, and its function in response to drought stress was identified. GmNF-YA10 is broadly expressed in soybean tissues and is specifically located in the nucleus. In soybean roots, the transcription level of the GmNF-YA10 gene was induced by polyethylene glycol (PEG)-simulated drought stress and abscisic acid (ABA) treatment. Under mannitol stress, the germination rate of transgenic Arabidopsis overexpressing GmNF-YA10 was higher than that of wild-type (WT) Arabidopsis. After drought stress, the survival rate, relative water content, ABA content, proline content and chlorophyll content of transgenic Arabidopsis overexpressing GmNF-YA10 were higher than those of WT Arabidopsis while the rate of water loss and the contents of malondialdehyde and sucrose were lower than those of WT Arabidopsis, indicating that transgenic Arabidopsis overexpressing GmNF-YA10 has better drought resistance than WT Arabidopsis. Under the ABA treatment, the root length of transgenic Arabidopsis overexpressing GmNF-YA10 was shorter than that of WT Arabidopsis and the degree of stomatal closure was greater than that of WT Arabidopsis, indicating that transgenic Arabidopsis overexpressing GmNF-YA10 was more sensitive to ABA at the post-germination stage. Transcriptome sequencing and real-time quantitative PCR analysis showed that GmNF-YA10 affected the expression of both ABA-dependent and ABA-independent signalling pathway genes. These results indicate that GmNF-YA10 significantly improves the drought resistance of transgenic Arabidopsis through ABA-dependent and ABA-independent signal transduction pathways.

Genomic survey and gene expression analysis of the MYB-related transcription factor superfamily in potato (Solanum tuberosum L.)

The MYB transcription factors (TFs) comprise a major TF family in the plant kingdom. Studies increasingly show that MYB-related genes drive physiological functions in plants. However, little is known regarding their regulatory networks and downstream pathways in potato. We conducted a genome-wide analysis of MYB TFs and related proteins in potato (Solanum tuberosum, abbreviated as St), and identified 138 StMYB-related TFs that were phylogenetically classified into three distinct subgroups based on highly conserved gene structures, consensus motifs and protein domain architecture. Segmental duplication events were detected in the StMYB-related gene family by collinearity analysis, which likely contributed to the expansion of this family. Synteny analysis indicated that 41 StMYB-related genes were orthologous to Arabidopsis and 24 to wheat. In addition, RNA-seq analysis identified several tissue-specific and abiotic stress-responsive StMYB-related genes. To determine a potential role of these genes in anthocyanin biosynthesis and drought response, we analyzed the transcriptomes of the white, pigmented, drought-sensitive (‘Atlantic’) and drought-resistant (‘Qingshu No.9’) tetraploid potato cultivars from three flowering stages: early, peak (full blooms) and late (foliage falling). The interaction networks of StMYB-related proteins that were differentially expressed between pigmented versus white, as well as the drought-tolerant versus sensitive cultivars were also predicted. Our findings lay the foundation for prospective functional studies of potato StMYB-related TFs.

Ectopic expression of BrALDH7B2 gene encoding an antiquitin from Brassica rapa confers tolerance to abiotic stresses and improves photosynthetic performance under salt stress in tobacco

Improved plant performance under salt and drought stresses has been a challenging task to the plant due to the complexity and multitude of genes that govern them. We describe here, isolation and characterization of an aldehyde dehydrogenase gene (BrALDH7B2) that encodes an antiquitin isolated from Brassica rapa. Overexpression of the antiquitin gene in tobacco conferred tolerance to salt and drought stresses at the seedling stage as reflected by fresh weight, root length, and chlorophyll and carotenoid contents. The improved physiological characteristics observed in the transgenics may be ascribed to better PIabs, Pn, gs, WUE and E leading to better endurance under salt stress. The overexpression of BrALDH7B2 improved photosynthetic performance accompanied by reduced ROS formation followed by repressed cell-death in roots as determined by intracellular accumulation of H2DCF-DA and PI during salt stress in transgenic plants. Under salt stress, ALDH expressing tobacco transgenic plants showed activation of abiotic stress-responsive genes and vigorous ROS-scavenging system at the molecular level. Altogether, BrALDH7B2 represents a good candidate gene for orchestrating stress responses and with potential for engineering stress tolerance in different crops. To the best of our knowledge, this is the first report on functional study of BrALDH gene in the heterologous system tobacco with enhanced photosynthetic performance under salt stress.

The IBI1 Receptor of β-Aminobutyric Acid Interacts with VOZ Transcription Factors to Regulate Abscisic Acid Signaling and Callose-Associated Defense

External and internal signals can prime the plant immune system for a faster and/or stronger response to pathogen attack. β-aminobutyric acid (BABA) is an endogenous stress metabolite that induces broad-spectrum disease resistance in plants. BABA perception in Arabidopsis is mediated by the aspartyl tRNA synthetase IBI1, which activates priming of multiple immune responses, including callose-associated cell wall defenses that are under control by abscisic acid (ABA). However, the immediate signaling components after BABA perception by IBI1, as well as the regulatory role of ABA therein, remain unknown. Here, we have studied the early signaling events controlling IBI1-dependent BABA-induced resistance (BABA-IR), using untargeted transcriptome and protein interaction analyses. Transcriptome analysis revealed that IBI1-dependent expression of BABA-IR against the biotrophic oomycete Hyaloperonospora arabidopsidis is associated with suppression of ABA-inducible abiotic stress genes. Protein interaction studies identified the VOZ1 and VOZ2 transcription factors (TFs) as IBI1-interacting partners, which are transcriptionally induced by ABA but suppress pathogen-induced expression of ABA-dependent genes. Furthermore, we show that VOZ TFs require nuclear localization for their contribution to BABA-IR by mediating augmented expression of callose-associated defense. Collectively, our study indicates that the IBI1-VOZ signaling module channels pathogen-induced ABA signaling toward cell wall defense while simultaneously suppressing abiotic stress-responsive genes.

Expression of Zinc Finger Protein Zat12 from Arabidopsis thaliana in Escherichia coli

The C2H2 zinc finger protein ZAT12 has been classified as a plant core abiotic stress response gene in the early response to multiple stresses. ZAT12 links the iron deficiency and oxidative stress responses through the direct interaction with/and negative regulation of a central regulator - FIT. For further research on the regulation of the ZAT12 protein in planta, a huge quantity of ZAT12 proteins is required to inject into mice for the generation of ZAT12 antiserum. In this study, the gene encoding the ZAT12 protein from Arabidopsis thaliana was cloned into the expression vector - pETBlue-2 and then overexpressed in E. coli T7. A high expression level was indicated by SDS-PAGE. Immunoblot demonstrated successful expression using a bacterial expression system.

Genome-Wide Identification of the Physic Nut WUSCHEL-Related Homeobox Gene Family and Functional Analysis of the Abiotic Stress Responsive Gene JcWOX5.

Plant-specific WOX transcription factors have important regulatory functions in plant development and response to abiotic stress. However, the identification and functional analysis of members of the WOX family have rarely been reported in the physic nut plant until now. Our research identified 12 WOX genes (JcWOXs) in physic nut, and these genes were divided into three groups corresponding to the ancient clade, WUS clade, and intermediate clade. Expression analysis based on RNA-seq and qRT-PCR showed that most of the JcWOX genes were expressed in at least one of the tissues tested, whereas five genes were identified as being highly responsive to drought and salt stresses. Subcellular localization analysis in Arabidopsis protoplast cells showed that JcWOX5 encoded a nuclear-localized protein. JcWOX5-overexpression plants increased sensitivity to drought stress, and transgenic plants suggested a lower proline content and CAT activity, higher relative electrolyte leakage, higher MDA content, and higher rate of water loss under drought conditions. Expression of some stress-related genes was obviously lower in the transformed rice lines as compared to their expression in wild-type rice lines under drought stress. Further data on JcWOX5-overexpressing plants reducing drought tolerance verified the potential role of JcWOX genes in responsive to abiotic stress. Collectively, the study provides a foundation for further functional analysis of JcWOX genes and the improvement of physic nut crops.

Genome-wide analysis and transcript profiling identify several abiotic and biotic stress-responsive Glutathione S-transferase genes in soybean

Abiotic and biotic stresses stimulate the generation of free radicals and reactive oxygen species, which results in oxidative stress. Glutathione-S-transferase (GST) is a large class of multifunctional enzymes that exists widely in plants. The addition of reduced glutathione by GSTs protects the cell from various toxic molecules and thus provide tolerance against adverse conditions. In this study, we have performed a genome-wide identification, characterization and expression analysis of GST gene family in soybean (Glycine max) to know their function. We have identified the presence of at least 126 putative GST genes in soybean. Among them, presence of a total of 33 pairs of duplicated genes might contribute towards the expansion of GST family members in soybean. Based on the phylogenetic analysis, GmGSTs could be further subdivided into 12 classes. GmGST tau class possessed the maximum number of 68 members, followed by phi (14 members) and lambda (7 members) classes. Expression of all 126 GmGST transcripts was analyzed in 26 anatomical tissues and 7 developmental stages. Moreover, expression profiling was expanded in response to various abiotic and biotic stresses. Four genes (GmGSTU63, GmGSTU73, GmGSTF2, and GmGSTT5) were found to be highly upregulated in response to four abiotic stresses- dehydration, drought, temperature and ozone; and two biotic stress conditions. This comprehensive genome-wide analysis and expression profiling will help to understand the function of GST in adverse environmental circumstances as well as enhancing the genetic modification of soybean.

Reprogramming of the Developmental Program of Rhus javanica During Initial Stage of Gall Induction by Schlechtendalia chinensis.

Insect galls are unique organs that provide shelter and nutrients to the gall-inducing insects. Although insect galls are fascinating structures for their unique shapes and functions, the process by which gall-inducing insects induce such complex structures is not well understood. Here, we performed RNA-sequencing-based comparative transcriptomic analysis of the early developmental stage of horned gall to elucidate the early gall-inducing process carried out by the aphid, Schlechtendalia chinensis, in the Chinese sumac, Rhus javanica. There was no clear similarity in the global gene expression profiles between the gall tissue and other tissues, and the expression profiles of various biological categories such as phytohormone metabolism and signaling, stress-response pathways, secondary metabolic pathways, photosynthetic reaction, and floral organ development were dramatically altered. Particularly, master transcription factors that regulate meristem, flower, and fruit development, and biotic and abiotic stress-responsive genes were highly upregulated, whereas the expression of genes related to photosynthesis strongly decreased in the early stage of the gall development. In addition, we found that the expression of class-1 KNOX genes, whose ectopic overexpression is known to lead to the formation of de novo meristematic structures in leaf, was increased in the early development stage of gall tissue. These results strengthen the hypothesis that gall-inducing insects convert source tissues into fruit-like sink tissues by regulating the gene expression of host plants and demonstrate that such manipulation begins from the initial process of gall induction.

Pre-harvest climate and post-harvest acclimation to cold prevent from superficial scald development in Granny Smith apples

Superficial scald is one of the most serious postharvest physiological disorders that can affect apples after a prolonged cold storage period. This study investigated the impact of pre- and post-harvest climatic variations on superficial scald in a susceptible apple cultivar. Fruit batches with contrasting phenotypes for superficial scald incidence were identified among several years of “Granny Smith” fruit production. The “low scald” year pre-harvest climate was characterised by a warm period followed by a sudden decrease in temperature, playing the part of an in vivo acclimation to cold storage. This was associated with many abiotic stress responsive genes which were induced in fruit peel. In particular 48 Heat Shock Proteins (HSPs) and 5 Heat Shock transcription Factors (HSFs) were strongly induced at harvest when scald incidence was low. For “high scald” year, a post-harvest acclimation of 1 week was efficient in reducing scald incidence. Expression profiles of stress related genes were affected by the acclimation treatment and indicate fruit physiological adaptations to cold storage. The identified stress-responsive genes, and in particular HSPs, could be useful indicators of the fruit physiological status to predict the risk of scald occurrence as early as harvest.

Cloning, Genetic Transformation and Cellular Localization of Abiotic Stress Responsive Universal Stress Protein Gene (GUSP1) in Gossypium hirsutum.

Background: Drought stress seriously affects the cotton fiber development. Universal stress protein gene isolated from native species Gossypium arboreum has the promising tolerance role against these stresses.Objectives: This study aimed to clone, characterize, and genetically transform the GUSP1 gene in local cotton and to observe its expression in transgenic plants under drought stress.Materials and Methods: Universal Stress Protein (GUSP1) gene from Gossypium arboreum was cloned in pCEMBIA (-) 1301plant expression vector by replacing Hygromycin and GUS exon with GUSP1-GFP fusion fragment. The construct was transformed into Agrobacterium tumefaciens and transient expression assay was confirmed by agro-infiltration of Nicotiana benthamiana leaves and green fluorescence under a confocal microscope. Gene integration and expression in transgenic plants was observed through Southern blot and real-time PCR analyses. Cellular localization was observed through a confocal microscope and the copy number of the transgene was observed in progeny plants. Results: Transformation efficiency was 1.9%. Developmental and spatial expression of GUSP1 was observed through Real-time PCR in stem, root, leaf, inflorescence, and seeds of transgenic plants at the vegetative and flowering stage. Integration of GUSP1 revealed a fragment of approximately 500 bp in Southern Blot analyses. Localization of GUSP1 was detected in the intact leaf of transgenic plants through GFP fluorescence in midrib, guard cells of stomata, and trichomes. Single gene copy was detected in the chromosome of transgenic seeds.Conclusion: GUSP1 has cloned from native species of local cotton and its integration and expression in transgenic plants confirmed that the role of GUSP1 will provide direction to breed economically important cotton varieties.

Oryza sativa drought-, heat-, and salt-induced RING finger protein 1 (OsDHSRP1) negatively regulates abiotic stress-responsive gene expression.

Plants are sessile and unable to avoid environmental stresses, such as drought, high temperature, and high salinity, which often limit the overall plant growth. Plants have evolved many complex mechanisms to survive these abiotic stresses via post-translational modifications. Recent evidence suggests that ubiquitination plays a crucial role in regulating abiotic stress responses in plants by regulating their substrate proteins. Here, we reported the molecular function of a RING finger E3 ligase, Oryza sativa Drought, Heat and Salt-induced RING finger protein 1 (OsDHSRP1), involved in regulating plant abiotic stress tolerance via the Ub/26S proteasome system. The OsDHSRP1 gene transcripts were highly expressed under various abiotic stresses such as NaCl, drought, and heat and the phytohormone abscisic acid (ABA). In addition, in vitro ubiquitination assays demonstrated that the OsDHSRP1 protein possesses a RING-H2 type domain that confers ligase functionality. The results of yeast two-hybrid (Y2H), in vitro pull-down, and bimolecular fluorescence complementation assays support that OsDHSRP1 is able to regulate two substrates, O. sativa glyoxalase (OsGLYI-11.2) and O. sativa abiotic stress-induced cysteine proteinase 1 (OsACP1). We further confirmed that these two substrate proteins were ubiquitinated by OsDHSRP1 E3 ligase and caused protein degradation via the Ub/26S proteasome system. The Arabidopsis plants overexpressing OsDHSRP1 exhibited hypersensitivity to drought, heat, and NaCl stress and a decrease in their germination rates and root lengths compared to the control plants because the degradation of the OsGLYI-11.2 protein maintained lower glyoxalase levels, which increased the methylglyoxal amount in transgenic Arabidopsis plants. However, the OsDHSRP1-overexpressing plants showed no significant difference when treated with ABA. Our finding supports the hypothesis that the OsDHSRP1 E3 ligase acts as a negative regulator, and the degradation of its substrate proteins via ubiquitination plays important roles in regulating various abiotic stress responses via an ABA-independent pathway.

Excess sterols disrupt plant cellular activity by inducing stress-responsive gene expression.

Sterols are important lipid constituents of cellular membranes in plants and other organisms. Sterol homeostasis is under strict regulation in plants because excess sterols negatively impact plant growth. HIGH STEROL ESTER 1 (HISE1) functions as a negative regulator of sterol accumulation. If sterol production exceeds a certain threshold, excess sterols are detoxified via conversion to sterol esters by PHOSPHOLIPID STEROL ACYL TRANSFERASE 1 (PSAT1). We previously reported that the Arabidopsis thaliana double mutant hise1-3 psat1-2 shows 1.5-fold higher sterol content than the wild type and consequently a severe growth defect. However, the specific defects caused by excess sterol accumulation in plants remain unknown. In this study, we investigated the effects of excess sterols on plants by analyzing the phenotypes and transcriptomes of the hise1-3 psat1-2 double mutant. Transcriptomic analysis revealed that 435 genes were up-regulated in hise1-3 psat1-2 leaves compared with wild-type leaves. Gene ontology (GO) enrichment analysis revealed that abiotic and biotic stress-responsive genes including RESPONSIVE TO DESICCATION 29B/LOW-TEMPERATURE-INDUCED 65 (RD29B/LTI65) and COLD-REGULATED 15A (COR15A) were up-regulated in hise1-3 psat1-2 leaves compared with wild-type leaves. Expression levels of senescence-related genes were also much higher in hise1-3 psat1-2 leaves than in wild-type leaves. hise1-3 psat1-2 leaves showed early senescence, suggesting that excess sterols induce senescence of leaves. In the absence of sucrose, hise1-3 psat1-2 exhibited defects in seedling growth and root elongation. Together, our data suggest that excess sterol accumulation disrupts cellular activities of vegetative organs including leaves and roots, resulting in multiple damages to plants.