Dehydration Stress Conditions Research Articles

Rice microRNA171f/SCL6 module enhances drought tolerance by regulation of flavonoid biosynthesis genes.

Plants have evolved sophisticated defense systems to enhance drought tolerance. These include the microRNA (miRNA) group of small noncoding RNAs that act as post‐transcriptional regulators; however, details of the mechanisms by which they confer drought tolerance are not well understood. Here, we show that osa‐MIR171f, a member of osa‐MIR171 gene family, is mainly expressed in response to drought stress and regulates the transcript levels of SCARECROW‐LIKE6‐I (SCL6‐I) and SCL6‐II in rice ( Oryza sativa ). The SCL6 genes are known to be involved in shoot branching and flag leaf morphology. Osa‐MIR171f‐overexpressing (osa‐MIR171f‐OE) transgenic plants showed reduced drought symptoms compared with non‐transgenic (NT) control plants under both field drought and polyethylene glycol (PEG)‐mediated dehydration stress conditions. Transcriptome analysis of osa‐MIR171f‐OE plants and osa‐mir171f‐knockout (K/O) lines generated by clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) revealed that osa‐mature‐miR171a‐f (osa‐miR171) regulates the expression of flavonoid biosynthesis genes, consequently leading to drought tolerance. This upregulation in the osa‐MIR171f‐OE plants, which did not occur in NT control plants, was observed under both normal and drought conditions. Our findings indicate that osa‐miR171 plays a role in drought tolerance by regulating SCL6‐I and SCL6‐II transcript levels.

Pepper ubiquitin-specific protease, CaUBP12, positively modulates dehydration resistance by enhancing CaSnRK2.6 stability.

Abscisic acid (ABA) is a plant hormone that activates adaptive mechanisms to environmental stress conditions. Plant adaptive mechanisms are complex and highly modulated processes induced by stress-responsive proteins; however, the precise mechanisms by which these processes function under adverse conditions remain unclear. Here, we isolated CaUBP12 (Capsicum annuum ubiquitin-specific protease 12) from pepper (C. annuum) leaves. We show that CaUBP12 expression is significantly induced after exposure to abiotic stress treatments. We conducted loss-of-function and gain-of-function genetic studies to elucidate the biological functions of CaUBP12 in response to ABA and dehydration stress. CaUBP12-silenced pepper plants and CaUBP12-overexpressing Arabidopsis plants displayed dehydration-sensitive and dehydration-tolerant phenotypes, respectively; these phenotypes were characterized by regulation of transpirational water loss and stomatal aperture. Under dehydration stress conditions, CaUBP12-silenced pepper plants and CaUBP12-overexpressing Arabidopsis plants exhibited lower and higher expression levels of stress-related genes, respectively, than the control plants. We isolated a CaUBP12 interaction protein, CaSnRK2.6, which is a homolog of Arabidopsis OST1; degradation of this protein was partially inhibited by CaUBP12. Similar to CaUBP12-silenced pepper plants and CaUBP12-overexpressing Arabidopsis plants, CaSnRK2.6-silenced pepper plants and CaSnRK2.6-overexpressing Arabidopsis displayed dehydration-sensitive and dehydration-tolerant phenotypes, respectively. Our findings suggest that CaUBP12 positively modulates the dehydration stress response by suppressing CaSnRK2.6 protein degradation.

Comparative proteomic analysis of parasitic loranthus seeds exposed to dehydration stress

Parasitic loranthus [Taxillus chinensis (DC.) Danser] is an important medicinal plant that produces recalcitrant seeds that are sensitive to dehydration. Desiccation tolerance is critical for the survival of recalcitrant seeds in low-moisture environments. Clarifying how these seeds respond to desiccation is important for long-term conservation. Thus, the viability, germination, microstructure, and antioxidant enzyme activities of dehydrated parasitic loranthus seeds were investigated. Diverse organelles were degraded or deformed during dehydration treatments. Additionally, superoxide dismutase and catalase activities gradually decreased in response to desiccation stress. A proteomic analysis involving TMT-labeling and LC–MS/MS were performed. A total of 1479 proteins were identified, of which 141 were differentially expressed proteins (DEPs) at 16 and 36 h after initiating the dehydration treatments. A functional annotation based on gene ontology revealed that the DEPs were mainly localized in chloroplasts and were related to energy metabolism, responses to stimuli, and the regulation of biological processes. A KEGG pathway enrichment analysis determined that several of the identified proteins were associated with signal transductions, photosynthesis, and glycolysis/gluconeogenesis. The results suggest that the efficient removal of excessive ROS amounts may be crucial for promoting parasitic loranthus seed germination under dehydration stress conditions. A series of candidate dehydration stress-related proteins were identified and may be relevant for enhancing the dehydration tolerance of the recalcitrant seeds. To the best of our knowledge, this is the first study to elucidate the possible molecular mechanisms underlying the sensitivity of recalcitrant parasitic loranthus seeds to dehydration via a proteomic analysis involving TMT-labeling.

Functional Characterization of a Putative RNA Demethylase ALKBH6 in Arabidopsis Growth and Abiotic Stress Responses

RNA methylation and demethylation, which is mediated by RNA methyltransferases (referred to as “writers”) and demethylases (referred to as “erasers”), respectively, are emerging as a key regulatory process in plant development and stress responses. Although several studies have shown that AlkB homolog (ALKBH) proteins are potential RNA demethylases, the function of most ALKBHs is yet to be determined. The Arabidopsis thaliana genome contains thirteen genes encoding ALKBH proteins, the functions of which are largely unknown. In this study, we characterized the function of a potential eraser protein, ALKBH6 (At4g20350), during seed germination and seedling growth in Arabidopsis under abiotic stresses. The seeds of T-DNA insertion alkbh6 knockdown mutants germinated faster than the wild-type seeds under cold, salt, or abscisic acid (ABA) treatment conditions but not under dehydration stress conditions. Although no differences in seedling and root growth were observed between the alkbh6 mutant and wild-type under normal conditions, the alkbh6 mutant showed a much lower survival rate than the wild-type under salt, drought, or heat stress. Cotyledon greening of the alkbh6 mutants was much higher than that of the wild-type upon ABA application. Moreover, the transcript levels of ABA signaling-related genes, including ABI3 and ABI4, were down-regulated in the alkbh6 mutant compared to wild-type plants. Importantly, the ALKBH6 protein had an ability to bind to both m6A-labeled and m5C-labeled RNAs. Collectively, these results indicate that the potential eraser ALKBH6 plays important roles in seed germination, seedling growth, and survival of Arabidopsis under abiotic stresses.

Genomic dissection and expression analysis of stress-responsive genes in C4 panicoid models, Setaria italica and Setaria viridis

The study reports the identification and expression profiling of five major classes of C4 pathway-specific genes, namely, carbonic anhydrase (CaH), phosphoenolpyruvate carboxylase (PEPC), pyruvate orthophosphate dikinase (PPDK), NADP-dependent malate dehydrogenase (MDH) and NADP-dependent malic enzyme (NADP-ME), in the model species, Setaria italica and Setaria viridis. A total of 42 and 41 genes were identified in S. italica and S. viridis, respectively. Further analysis revealed that segmental and tandem duplications have contributed to the expansion of these gene families. RNA-Seq derived expression profiles of the gene family members showed their differential expression pattern in tissues and dehydration stress. Comparative genome mapping and Ks dating provided insights into their duplication and divergence in the course of evolution. Expression profiling of candidate genes in contrasting S. italica cultivars subjected to abiotic stresses and hormone treatments showed distinct stress-specific upregulation of SiαCaH1, SiβCaH5, SiPEPC2, SiPPDK2, SiMDH8, and SiNADP-ME5 in the tolerant cultivar. Overexpression of SiNADP-ME5 in heterologous yeast system enabled the transgenic cells to survive and grow in dehydration stress conditions, which highlights the putative role of SiNADP-ME5 in conferring tolerance to dehydration stress. Altogether, the study highlights key genes that could be potential candidates for elucidating their functional roles in abiotic stress response.

Expression Analyses of Soybean VOZ Transcription Factors and the Role of GmVOZ1G in Drought and Salt Stress Tolerance.

Vascular plant one-zinc-finger (VOZ) transcription factor, a plant specific one-zinc-finger-type transcriptional activator, is involved in regulating numerous biological processes such as floral induction and development, defense against pathogens, and response to multiple types of abiotic stress. Six VOZ transcription factor-encoding genes (GmVOZs) have been reported to exist in the soybean (Glycine max) genome. In spite of this, little information is currently available regarding GmVOZs. In this study, GmVOZs were cloned and characterized. GmVOZ genes encode proteins possessing transcriptional activation activity in yeast cells. GmVOZ1E, GmVOZ2B, and GmVOZ2D gene products were widely dispersed in the cytosol, while GmVOZ1G was primarily located in the nucleus. GmVOZs displayed a differential expression profile under dehydration, salt, and salicylic acid (SA) stress conditions. Among them, GmVOZ1G showed a significantly induced expression in response to all stress treatments. Overexpression of GmVOZ1G in soybean hairy roots resulted in a greater tolerance to drought and salt stress. In contrast, RNA interference (RNAi) soybean hairy roots suppressing GmVOZ1G were more sensitive to both of these stresses. Under drought treatment, soybean composite plants with an overexpression of hairy roots had higher relative water content (RWC). In response to drought and salt stress, lower malondialdehyde (MDA) accumulation and higher peroxidase (POD) and superoxide dismutase (SOD) activities were observed in soybean composite seedlings with an overexpression of hairy roots. The opposite results for each physiological parameter were obtained in RNAi lines. In conclusion, GmVOZ1G positively regulates drought and salt stress tolerance in soybean hairy roots. Our results will be valuable for the functional characterization of soybean VOZ transcription factors under abiotic stress.

The pepper AP2 domain-containing transcription factor CaDRAT1 plays a negative role in response to dehydration stress

APETALA2 (AP2) domain-containing transcription factors play critical regulatory roles in plant growth, development, and response to abiotic stresses, including dehydration stress. In Capsicum annuum (pepper), data are lacking regarding AP2 domain-containing transcription factors and their precise functions. Here, we identified six putative AP2 domain-containing transcription factors in pepper plants; the expression levels of these transcription factors were induced or repressed by dehydration stress. We named the gene having the highest fold change as Capsicum annuumDehydration Responsive AP2 domain-containing Transcription factor 1 (CaDRAT1). CaDRAT1 belongs to the ERF subfamily B-4, but the roles of these ERF proteins in dehydration responses are not well known. CaDRAT1 transcript levels were significantly induced after exposure to abscisic acid (ABA), mannitol, low temperature, and H2O2. The acidic domain in the CaDRAT1 C-terminal region had a transactivation effect on the reporter gene in the yeast GAL4 system. CaDRAT1-silenced pepper plants displayed a dehydration-resistant phenotype characterized by enhanced stomatal closure and low levels of transpirational water loss. Moreover, under dehydration stress conditions, expression levels of stress-related genes including CaNCED3 were higher in CaDRAT1-silenced pepper plants than in control plants and ABA content was also higher. Taken together, our findings suggest that CaDRAT1 functions as a negative regulator of dehydration resistance through modulation of ABA biosynthesis and signaling.

NF-YB2 and NF-YB3 Have Functionally Diverged and Differentially Induce Drought and Heat Stress-Specific Genes.

Functional diversification of transcription factors allows the precise regulation of transcriptomic changes under different environmental conditions. The NUCLEAR FACTOR Y (NF-Y) transcription factor comprises three subunits, NF-YA, NF-YB, and NF-YC, and is broadly diversified in plant species, whereas Humans (Homo sapiens) have one protein for each subunit. However, there remains much to be learned about the diversified functions of each subunit in plants. Here, we found that NF-YB2 and NF-YB3, which have the greatest sequence similarity to each other among NF-YB family proteins in Arabidopsis (Arabidopsis thaliana), are functionally diversified and specifically activate dehydration-inducible and heat-inducible genes, according to environmental conditions. Overexpression of NF-YB2 and NF-YB3 specifically enhanced drought and heat stress tolerance, respectively, and each single knockout mutant showed adverse stress-sensitive phenotypes. Transcriptomic analyses confirmed that overexpression of NF-YB2 and NF-YB3 largely affected the transcriptomic changes under dehydration and heat stress conditions, respectively. The DNA-binding profiles of each protein in planta also suggested that dehydration and heat stress increased the DNA-binding activity of NF-YB2 and NF-YB3 to dehydration-inducible and heat stress-inducible target genes, respectively. Moreover, phylogenetic analysis suggested that the NF-YB proteins of angiosperm plants belong to divergent NF-YB2 and NF-YB3 subgroups. These results demonstrate the functional diversification of NF-Y through evolutionary processes and how plants adapt to various abiotic stresses under fluctuating environments.

Evolution of the modular, disordered stress proteins known as dehydrins.

Dehydrins, plant proteins that are upregulated during dehydration stress conditions, have modular sequences that can contain three conserved motifs (the Y-, S-, and K-segments). The presence and order of these motifs are used to classify dehydrins into one of five architectures: Kn, SKn, KnS, YnKn, and YnSKn, where the subscript n describes the number of copies of that motif. In this study, an architectural and phylogenetic analysis was performed on 426 dehydrin sequences that were identified in 53 angiosperm and 3 gymnosperm genomes. It was found that angiosperms contained all five architectures, while gymnosperms only contained Kn and SKn dehydrins. This suggests that the ancestral dehydrin in spermatophytes was either Kn or SKn, and the Y-segment containing dehydrins first arose in angiosperms. A high-level split between the YnSKn dehydrins from either the Kn or SKn dehydrins could not be confidently identified, however, two lower level architectural divisions appear to have occurred after different duplication events. The first likely occurred after a whole genome duplication, resulting in the duplication of a Y3SK2 dehydrin; the duplicate subsequently lost an S- and K- segment to become a Y3K1 dehydrin. The second split occurred after a tandem duplication of a Y1SK2 dehydrin, where the duplicate lost both the Y- and S- segment and gained four K-segments, resulting in a K6 dehydrin. We suggest that the newly arisen Y3K1 dehydrin is possibly on its way to pseudogenization, while the newly arisen K6 dehydrin developed a novel function in cold protection.

Xerophyta viscosa Aldose Reductase, XvAld1, Enhances Drought Tolerance in Transgenic Sweetpotato.

Sweetpotato is a significant crop which is widely cultivated particularly in the developing countries with high and stable yield. However, drought stress is a major limiting factor that antagonistically influences the crop's productivity. Dehydration stress caused by drought causes aggregation of reactive oxygen species (ROS) in plants, and aldose reductases are first-line safeguards against ROS caused by oxidative stress. In the present study, we generated transgenic sweetpotato plants expressing aldose reductase, XvAld1 isolated from Xerophyta viscosa under the control of a stress-inducible promoter via Agrobacterium-mediated transformation. Our results demonstrated that the transgenic sweetpotato lines displayed significant enhanced tolerance to simulated drought stress and enhanced recuperation after rehydration contrasted with wild-type plants. In addition, the transgenic plants exhibited improved photosynthetic efficiency, higher water content and more proline accumulation under dehydration stress conditions compared with wild-type plants. These results demonstrate that exploiting the XvAld1 gene is not only a compelling and attainable way to improve sweetpotato tolerance to drought stresses without causing any phenotypic imperfections but also a promising gene candidate for more extensive crop improvement.

Expression patterns of members of the ethylene signaling-related gene families in response to dehydration stresses in cassava.

Drought is the one of the most important environment stresses that restricts crop yield worldwide. Cassava (Manihot esculenta Crantz) is an important food and energy crop that has many desirable traits such as drought, heat and low nutrients tolerance. However, the mechanisms underlying drought tolerance in cassava are unclear. Ethylene signaling pathway, from the upstream receptors to the downstream transcription factors, plays important roles in environmental stress responses during plant growth and development. In this study, we used bioinformatics approaches to identify and characterize candidate Manihot esculenta ethylene receptor genes and transcription factor genes. Using computational methods, we localized these genes on cassava chromosomes, constructed phylogenetic trees and identified stress-responsive cis-elements within their 5’ upstream regions. Additionally, we measured the trehalose and proline contents in cassava fresh leaves after drought, osmotic, and salt stress treatments, and then it was found that the regulation patterns of contents of proline and trehalose in response to various dehydration stresses were differential, or even the opposite, which shows that plant may take different coping strategies to deal with different stresses, when stresses come. Furthermore, expression profiles of these genes in different organs and tissues under non-stress and abiotic stress were investigated through quantitative real-time PCR (qRT-PCR) analyses in cassava. Expression profiles exhibited clear differences among different tissues under non-stress and various dehydration stress conditions. We found that the leaf and tuberous root tissues had the greatest and least responses, respectively, to drought stress through the ethylene signaling pathway in cassava. Moreover, tuber and root tissues had the greatest and least reponses to osmotic and salt stresses through ethylene signaling in cassava, respectively. These results show that these plant tissues had differential expression levels of genes involved in ethylene signaling in response to the stresses tested. Moreover, after several gene duplication events, the spatiotemporally differential expression pattern of homologous genes in response to abiotic and biotic stresses may imply their functional diversity as a mechanism for adapting to the environment. Our data provide a framework for further research on the molecular mechanisms of cassava resistance to drought stress and provide a foundation for breeding drought-resistant new cultivars.

Three zinc-finger RNA-binding proteins in cabbage (Brassica rapa) play diverse roles in seed germination and plant growth under normal and abiotic stress conditions.

Despite the increasing understanding of the stress-responsive roles of zinc-finger RNA-binding proteins (RZs) in several plant species, such as Arabidopsis thaliana, wheat (Triticum aestivum) and rice (Oryza sativa), the functions of RZs in cabbage (Brassica rapa) have not yet been elucidated. In this study, the functional roles of the three RZ family members present in the cabbage genome, designated as BrRZ1, BrRZ2 and BrRZ3, were investigated in transgenic Arabidopsis under normal and environmental stress conditions. Subcellular localization analysis revealed that all BrRZ proteins were exclusively localized in the nucleus. The expression levels of each BrRZ were markedly increased by cold, drought or salt stress and by abscisic acid (ABA) treatment. Expression of BrRZ3 in Arabidopsis retarded seed germination and stem growth and reduced seed yield of Arabidopsis plants under normal growth conditions. Germination of BrRZ2- or BrRZ3-expressing Arabidopsis seeds was delayed compared with that of wild-type seeds under dehydration or salt stress conditions and cold stress conditions, respectively. Seedling growth of BrRZ3-expressing transgenic Arabidopsis plants was significantly inhibited under salt, dehydration or cold stress conditions. Notably, seedling growth of all three BrRZ-expressing transgenic Arabidopsis plants was inhibited upon ABA treatment. Importantly, all BrRZs possessed RNA chaperone activity. Taken together, these results indicate that the three cabbage BrRZs harboring RNA chaperone activity play diverse roles in seed germination and seedling growth of plants under abiotic stress conditions as well as in the presence of ABA.

Stress-responsive expression patterns and functional characterization of cold shock domain proteins in cabbage (Brassica rapa) under abiotic stress conditions

Although the functional roles of cold shock domain proteins (CSDPs) have been demonstrated during the growth, development, and stress adaptation of Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and wheat (Triticum aestivum), the functions of CSDPs in other plants species, including cabbage (Brassica rapa), are largely unknown. To gain insight into the roles of CSDPs in cabbage under stress conditions, the genes encoding CSDPs in cabbage were isolated, and the functional roles of CSDPs in response to environmental stresses were analyzed. Real-time RT-PCR analysis revealed that the levels of BrCSDP transcripts increased during cold, salt, or drought stress, as well as upon ABA treatment. Among the five BrCSDP genes found in the cabbage genome, one CSDP (BRU12051), named BrCSDP3, was unique in that it is localized to the chloroplast as well as to the nucleus. Ectopic expression of BrCSDP3 in Arabidopsis resulted in accelerated seed germination and better seedling growth compared to the wild-type plants under high salt or dehydration stress conditions, and in response to ABA treatment. BrCSDP3 did not affect the splicing of intron-containing genes and processing of rRNAs in the chloroplast. BrCSDP3 had the ability to complement RNA chaperone-deficient Escherichia coli mutant cells under low temperatures as well as DNA- and RNA-melting abilities, suggesting that it possesses RNA chaperone activity. Taken together, these results suggest that BrCSDP3, harboring RNA chaperone activity, plays a role as a positive regulator in seed germination and seedling growth under stress conditions.

Pyramiding of AtEDT1/HDG11 and Cry2Aa2 into pepper (Capsicum annuum L.) enhances drought tolerance and insect resistance without yield decrease

Improvement of biotic and abiotic stress tolerance without yield decrease has been a great challenge in vegetable breeding. Functional stacking of multiple resistant genes could potentially be an effective strategy against more different stress. Here, we report that constitutive co-expression of Arabidopsis thaliana homodomain-leucine zipper transcription factor Enhanced Drought Tolerance/HOMEODOMAIN GLABROUS11 (AtEDT1/HDG11) and Bacillus thuringiensis Cry2Aa2 was able to increase pepper drought tolerance and insect resistance. More importantly, fruit yield was improved under both normal and drought stress conditions. The improved tolerance to drought stress was associated with improved root system and reduced stomatal density. Compared to the non-transgenic control, the transgenic pepper plants also had higher levels of chlorophyll content, chlorophyll fluorescence (Fm/Fv), proline, soluble sugar, abscisic acid, and ROS scavenging capacity under dehydration stress conditions. The increased flower number, elevated fruit weight, and improved fruit number all contributed to the increased yield of transgenic pepper. In addition, bioassays showed that the transgenic plants were effective to control the Prodenia litura, producing a mortality rate of 81.3 % after 5 days of infection. Therefore, our study indicates that stacking of AtEDT1/HDG11 and Cry2Aa2 in pepper plants can improve drought tolerance and insect resistance, demonstrating application of AtEDT1/HDG11 and Cry2Aa2 has the potential to enhance drought tolerance and insect resistance.

Over-expression of a glutamate dehydrogenase gene, MgGDH, from Magnaporthe grisea confers tolerance to dehydration stress in transgenic rice.

Heterologous expression of a fungal NADP(H)-GDH gene ( MgGDH ) from Magnaporthe grisea can improve dehydration stress tolerance in rice by preventing toxic accumulation of ammonium. Glutamate dehydrogenase (GDH; EC 1.4.1.2 and EC 1.4.1.4) may act as a stress-responsive enzyme in detoxification of high intracellular ammonia and production of glutamate for proline synthesis under stress conditions. In present study, a fungal NADP(H)-GDH gene (MgGDH) from Magnaporthe grisea was over-expressed in rice (Oryza sativa L. cv. 'kitaake'), and the transgenic plants showed the improvement of tolerance to dehydration stress. The kinetic analysis showed that His-TF-MgGDH preferentially utilizes ammonium to produce L-glutamate. Moreover, the affinity of His-TF-MgGDH for ammonium was dramatically higher than that of His-TF-OsGDH for ammonium. Over-expressing MgGDH transgenic rice plants showed lower water-loss rate and higher completely close stomata than the wild-type plants under dehydration stress conditions. In transgenic plants, the NADP(H)-GDH activities were markedly higher than those in wild-type plants and the amination activity was significantly higher than the deamination activity. Compared with wild-type plants, the transgenic plants accumulated much less NH4 (+) but higher amounts of glutamate, proline and soluble sugar under dehydration stress conditions. These results indicate that heterologous expression of MgGDH can prevent toxic accumulation of ammonium and in return improve dehydration stress tolerance in rice.

Functional characterization of a chloroplast-targeted RNA-binding protein CRP1 in Arabidopsis thaliana under abiotic stress conditions

Although recent analysis of proteomes in specific cellular organelles of plants has demonstrated that more than 2,000 nuclear-encoded proteins are targeted to the chloroplast, the physiological functions of a majority of the proteins in chloroplasts are largely unknown. Here, we investigated the functional role of a chloroplast-targeted RNA-binding protein in Arabidopsis thaliana (At4g20030), designated CRP1 (for chloroplast-targeted ṞNA-binding protein1), during seed germination and seedling growth under different light environments and various stress conditions. Confocal analysis of subcellular localization of CRP1-GFP fusion protein revealed that CRP1 protein is localized to the chloroplast. The T-DNA insertion loss-offunction crp1 mutant displayed poorer seedling growth than the wild-type plants under UV and high temperature stress conditions. Germination of crp1 mutant seeds was delayed compared with that of the wild-type seeds under cold or salt stress but not under dehydration stress conditions. The transgenic Arabidopsis plants that overexpress CRP1 showed better root and hypocotyl growth than wild type under heat stress conditions. Taken together, these results suggest that the chloroplast-targeted CRP1 plays a role in seed germination and seedling growth under UV, heat, cold, or salt stress but not under dehydration stress conditions.

A chloroplast-localized DEAD-box RNA helicaseAtRH3 is essential for intron splicing and plays an important role in the growth and stress response in Arabidopsis thaliana

Although many DEAD-box RNA helicases (RHs) are targeted to chloroplasts, the functional roles of the majority of RHs are still unknown. Recently, the chloroplast-localized Arabidopsis thaliana AtRH3 has been demonstrated to play important roles in intron splicing, ribosome biogenesis, and seedling growth. To further understand the functional role of AtRH3 in intron splicing and growth and the stress response in Arabidopsis, the newly-generated artificial microRNA-mediated knockdown plants as well as the previously characterized T-DNA tagged rh3-4 mutant were analyzed under normal and stress conditions. The rh3 mutants displayed retarded growth and pale-green phenotypes, and the growth of mutant plants was inhibited severely under salt or cold stress but marginally under dehydration stress conditions. Splicing of several intron-containing chloroplast genes was defective in the mutant plants. Importantly, splicing of ndhA and ndhB genes was severely inhibited in the mutant plants compared with the wild-type plants under salt or cold stress but not under dehydration stress conditions. Moreover, AtRH3 complemented the growth-defect phenotype of the RNA chaperone-deficient Escherichia coli mutant and had the ability to disrupt RNA and DNA base pairs, indicating that AtRH3 possesses RNA chaperone activity. Taken together, these results demonstrate that AtRH3 plays a prominent role in the growth and stress response of Arabidopsis, and suggest that proper splicing of introns governed by RNA chaperone activity of AtRH3 is crucial for chloroplast function and the growth and stress response of plants.

Isolation, promoter analysis and expression profile of Dreb2 in response to drought stress in wheat ancestors

Drought is one of the most important abiotic stresses, constraining crop production seriously. The dehydration responsive element binding proteins (DREBs) are important plant-specific transcription factors that respond to various abiotic stresses and consequently induce abiotic stress-related genes that impart stress endurance in plants. Wild species are naturally exposed to various abiotic stresses and potentially harbor suitable alleles through natural selection. In this study we isolated and characterized Dreb2 from Triticum urartu (GenBank: KF731664), Aegilops speltoides (GenBank: KF731665) and Aegilops tauschii (GenBank: KF731663), the A, B and D genome ancestors of bread wheat, respectively. Analysis of over 1.3kb upstream region of the gene revealed the presence of several conserved cis-acting regulatory elements including ABA-responsive elements, low temperature responsive elements, and several light and environmental signaling related motifs potentially vindicate Dreb2 responses to environmental signals. Moreover, the gene exhibited an alternative splicing, conserved among orthologous genes in grasses, and produced a non-functional isoform due to splicing in an exon resulted frame-shift creating an early stop codon before the functional domain. The expression analysis of Dreb2 under normal and different levels of dehydration stress conditions indicated that the two active spliced isoforms are upregulated when the plant exposed to drought stress whereas the non-functional isoform is downregulated in severe drought.

The Arabidopsis NAC Transcription Factor ANAC096 Cooperates with bZIP-Type Transcription Factors in Dehydration and Osmotic Stress Responses

Multiple transcription factors (TFs) play essential roles in plants under abiotic stress, but how these multiple TFs cooperate in abiotic stress responses remains largely unknown. In this study, we provide evidence that the NAC (for NAM, ATAF1/2, and CUC2) TF ANAC096 cooperates with the bZIP-type TFs ABRE binding factor and ABRE binding protein (ABF/AREB) to help plants survive under dehydration and osmotic stress conditions. ANAC096 directly interacts with ABF2 and ABF4, but not with ABF3, both in vitro and in vivo. ANAC096 and ABF2 synergistically activate RD29A transcription. Our genome-wide gene expression analysis revealed that a major proportion of abscisic acid (ABA)-responsive genes are under the transcriptional regulation of ANAC096. We found that the Arabidopsis thaliana anac096 mutant is hyposensitive to exogenous ABA and shows impaired ABA-induced stomatal closure and increased water loss under dehydration stress conditions. Furthermore, we found the anac096 abf2 abf4 triple mutant is much more sensitive to dehydration and osmotic stresses than the anac096 single mutant or the abf2 abf4 double mutant. Based on these results, we propose that ANAC096 is involved in a synergistic relationship with a subset of ABFs for the transcriptional activation of ABA-inducible genes in response to dehydration and osmotic stresses.

Functional characterization of a plastid-specific ribosomal protein PSRP2 in Arabidopsis thaliana under abiotic stress conditions

Plastids possess a small set of proteins unique to plastid ribosome, named plastid-specific ribosomal proteins (PSRPs). Among the six PSRPs found in Arabidopsis thaliana, PSRP2 is unique in that it harbors two RNA-recognition motifs found in diverse RNA-binding proteins. A recent report demonstrated that PSRP2 is not essential for ribosome function and plant growth under standard greenhouse conditions. Here, we investigated the functional role of PSRP2 during Arabidopsis seed germination and seedling growth under different light environments and various stress conditions, including high salinity, dehydration, and low temperature. The transgenic Arabidopsis plants overexpressing PSRP2 showed delayed germination compared with that of the wild-type plants under salt, dehydration, or low temperature stress conditions. The T-DNA insertion psrp2 mutant displayed better seedling growth but PSRP2-overexpressing transgenic plants showed poorer seedling growth than that of the wild-type plants under salt stress conditions. No noticeable differences in seedling growth were observed between the genotypes when grown under different light environments including dark, red, far-red, and blue light. Interestingly, the PSRP2 protein possessed RNA chaperone activity. Taken together, these results suggest that PSRP2 harboring RNA chaperone activity plays a role as a negative regulator in seed germination under all three abiotic stress conditions tested and in seedling growth of Arabidopsis under salt stress but not under cold or dehydration stress conditions.