The miR164b-SiNAC015 Module Regulates Drought Tolerance by Scavenging Reactive Oxygen Species in Foxtail Millet.

Foxtail millet (Setaria italica), a vital drought-resistant crop, plays a significant role in ensuring food and nutritional security. However, its drought resistance mechanism is not fully understood. N6 -methyladenosine (m6 A) modification of RNA, a prevalent epi-transcriptomic modification in eukaryotes, provides a binding site for m6 A readers and affects plant growth and stress responses by regulating RNA metabolism. In this study, we unveiled that the YT521-B homology (YTH) family gene SiYTH1 positively regulated the drought tolerance of foxtail millet. Notably, the siyth1 mutant exhibited reduced stomatal closure and augmented accumulation of excessive H2 O2 under drought stress. Further investigations demonstrated that SiYTH1 positively regulated the transcripts harboring m6 A modification related to stomatal closure and reactive oxygen species (ROS) scavenging under drought stress. SiYTH1 was uniformly distributed in the cytoplasm of SiYTH1-GFP transgenic foxtail millet. It formed dynamic liquid-like SiYTH1 cytosol condensates in response to drought stress. Moreover, the cytoplasmic protein SiYTH1 was identified as a distinct m6 A reader, facilitating the stabilization of its directly bound SiARDP and ROS scavenging-related transcripts under drought stress. Furthermore, natural variation analysis revealed SiYTH1AGTG as the dominant allele responsible for drought tolerance in foxtail millet. Collectively, this study provides novel insights into the intricate mechanism of m6 A reader-mediated drought tolerance and presents a valuable genetic resource for improving drought tolerance in foxtail millet breeding.

Anthropogenic activities in the past and present eras have created global warming and consequently a storm of drought stress, affecting both plants and animals. Being sessile, plants are more vulnerable to drought stress and consequently reduce plant growth and yield. To mitigate the effects of drought stress on plants, it is very crucial to determine the plant response mechanisms against drought stress. Drought response mechanism includes morph-physiological, biochemical, cellular and molecular processes takes place in plants underlying drought stress. These processes include improvement in root system, leaf structure, osmotic adjustment, relative water content and stomata regulation. In addition, calcium and phytohormone (Abscisic acid, Jasmonic acid, Salicylic acid, Auxins, Gibberellins, Ethylene etc.) signaling pathways and scavenging of reactive oxygen species are the key mechanisms to cope with drought stress. Moreover, microorganisms such as bacteria and fungi also have an important role in drought tolerance enhancement. To further elucidate and improve drought tolerance in plants, quantitative trait loci, transgenic approach and application of exogenous substances (nitric oxide, 24-epibrassinoide, glycine betaine and proline) are very crucial. Hereby, the present study integrates various mechanisms of drought tolerance in plants.

Dwarfism and drought tolerance are 2 valuable traits in breeding of many crops. In this study, we report the novel physiological roles of cholesterol in regulation of plant growth and drought tolerance. Compared with the wild type, sterol-C24-methyltransferase 1 (SMT1) gene transcript was greatly reduced in a bermudagrass mutant with dwarfism and enhanced drought tolerance, accompanied with cholesterol accumulation, elevated transcript levels of a small group of genes including SAMDC, and increased concentrations of putrescine (Put), spermidine (Spd), and spermine (Spm). Knock-down of OsSMT1 expression by RNA interference resulted in similar phenotypic changes in transgenic rice. Moreover, exogenously applied cholesterol also led to elevated transcripts of a similar set of genes, higher levels of Put, Spd, and Spm, improved drought tolerance, and reduced plant height in both bermudagrass and rice. We revealed that it is Spm, but not Spd, that is responsible for the height reduction in bermudagrass and rice. In conclusion, we suggest that cholesterol induces expression of SAMDC and leads to dwarfism and elevated drought tolerance in plants as a result of the promoted Spd and Spm synthesis.

The widely distributed heat shock protein DnaJ is renowned for its pivotal role in enhancing thermal tolerance in plants; however, its involvement in drought tolerance remains elusive. In this study, genes encoding DnaJ1 were cloned from drought-resistant wild tomato (Solanum pennellii) and drought-sensitive cultivated tomato (Solanum lycopersicum). SpDnaJ1 and SlDnaJ1 from both tomato species were localized in the chloroplast, and their gene expression was induced by various abiotic stresses. SpDnaJ1 was found to be a more potent regulator than SlDnaJ1 in oxidative stress tolerance when expressed in yeast cells. Overexpression of SpDnaJ1 was demonstrated to confer drought tolerance in transgenic plants of cultivated tomato. These transgenic plants exhibited reduced relative conductivity, leaf water loss rate, and malondialdehyde content as compared to the wild-type plants following drought treatment. RNA-seq analysis revealed that overexpression of SpDnaJ1 primarily affects the expression of genes associated with antioxidants, protease inhibitors, and MAPK signaling in response to drought stress. Screening of a tomato cDNA library in the yeast two-hybrid system identified a flavanone 3-hydroxylase-like protein (F3HL) as an interacting protein of DnaJ1. Subsequent findings revealed that F3HL enhances drought tolerance in tomato by increasing the activity of antioxidant enzymes and scavenging reactive oxygen species. These findings demonstrate a pivotal role of DnaJ1-F3HL interaction in enhancing drought tolerance, unveiling a novel molecular mechanism in drought tolerance in plants.

Foxtail millet SiMYB52 overexpression improves sulfur assimilation and antioxidant defense for enhanced drought tolerance in Arabidopsis.

Functional characterization of VrNAC15 for drought resistance in mung beans

Nitric oxide (NO), a key signaling molecule, can be induced by polyamines (PAs), which play an important role in improving drought tolerance in plants. This study was to further investigate the role of NO in spermidine (Spd)-induced drought tolerance associated with antioxidant defense in leaves of white clover (Trifolium repens) under drought stress induced by -0.3MPa polyethylene glycol (PEG-6000) solution. A hydroponic growth method was used for cultivating plants in a controlled growth chamber for 30-33days until the second leaves were fully expanded. Two relative independent experiments were carried out in our study. One is that exogenous application of Spd or an NO donor (sodium nitroprusside (SNP)) significantly improved drought tolerance in whole plants, as demonstrated by better phenotypic appearance, increased relative water content (RWC), and decreased electrolyte leakage (EL) and malondialdehyde (MDA) content in leaves as compared to untreated plants. For another detached leaf experiment, PEG induced an increase in the generation of NO in cells and significantly improved activities of nitrate reductase (NR) and nitric oxide synthase (NOS). These responses could be blocked by pre-treatment with a Spd biosynthetic inhibitor, dicyclohexyl amine (DCHA), and then reversed by application of exogenous Spd. Meanwhile, PEG induced up-regulation of activities and gene transcript levels of corresponding antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX) to varying degrees, while these effects were partially blocked by pre-treatment with DCHA, the scavenger of NO, the inhibitors of NR or NOS. In addition, Spd-induced antioxidant enzyme activities and gene expression also could be effectively inhibited by an NO scavenger as well as inhibitors of NR and NOS. These findings suggest that both Spd and NO can enhance drought tolerance. Spd was involved in drought stress-activated NR and NOS pathways associated with NO release, which mediated antioxidant defense and thus contributed to drought tolerance in white clover.

Antioxidants play a key role in maintaining cell activity in plants and animals by scavenging reactive oxygen species. Hence, it is very important to understand genes associated with antioxidant activity for improving the varieties. In this study, we compared structural and functional aspects of antioxidant genes viz., APX, DHAR, MDHAR, GR, and SOD of two contrasting genotypes viz. GP-1 (low Ca2+) and GP-45 (high Ca2+) of finger millet with other cereal crops such as rice, sorghum, and foxtail millet. The structural analysis shows that all genes are conserved and shares almost the same domains such as ascorbate peroxidase, glutathione dehydrogenase, glutathione reductase, Fe, and Cu–Zn superoxide dismutase domains which play a significant role in antioxidant activity and drought tolerance. These genes were mainly localized in chloroplast and cytoplasm which prove that both are the major ROS-scavenging sites. Furthermore, several putative cis-acting regulatory elements such as AuxRE, DRE, GARE, G-box, GATA-box, MBS, MYBR, and W-box are also studied and found that these genes are involved in defense mechanisms which allow responses against drought stress. Antioxidant activity of these genes was compared using expression analysis in terms of FPKM values and found that the genes of low Ca2+ genotype are highly expressed compared to the genes of high Ca2+ genotype and the genes of rice, sorghum, and foxtail millet. These results revealed that a low Ca2+ genotype of finger millet has higher antioxidant activity in comparison to high Ca2+ genotype and other cereal crops. Based on the results, we hypothesize that these candidate genes could be a target to develop highly antioxidative potential and drought tolerant genotypes of other cereal crops through appropriate breeding approaches.

While involved in a functional genomics program, we found that the overexpression of potato (Solanum tuberosum) glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene in yeast improves its water-deficit stress (drought) tolerance. But, the effect of altered (under and over) expression of GAPDH on water-deficit stress tolerance of higher plants is not yet studied. In this study, we used a versatile reverse genetics tool called virus-induced gene silencing and down-regulated the expression of GAPDH gene in tobacco (Nicotiana benthamiana) to examine the relevance (effect of underexpression) of GAPDH on drought tolerance of higher plants. Leaf discs made from silenced and nonsilenced tobacco plants were subjected to water-deficit stress. Measure of cell viability and the content of chlorophyll in stressed and nonstressed leaf discs were determined to quantify the effect of stress. Leaf discs made from the gene-silenced plants were found to be more severely affected by the stress than the leaf discs made from nonsilenced plants, implying the importance of GAPDH gene in drought tolerance of plants. Furthermore, to reiterate the involvement of GAPDH in drought tolerance of plants, potato transgenic plants constitutively overexpressing the GAPDH gene were generated and their performance under drought condition was analyzed. Transgenic potato plants showed improved drought tolerance when compared to wild-type potato. On the whole, our results confirm that the GAPDH gene plays an important role in drought tolerance of higher plants, and its constitutive overexpression by genetic engineering can be used to improve drought tolerance of crop plants like potato.

NAC (NAM, ATAF1/2, CUC2) transcription factors (TFs) constitute a plant-specific gene family. It is reported that NAC TFs play important roles in plant growth and developmental processes and in response to biotic/abiotic stresses. Nevertheless, little information is known about the functional and evolutionary characteristics of NAC TFs in mangrove plants, a group of species adapting coastal intertidal habitats. Thus, we conducted a comprehensive investigation for NAC TFs in Avicennia marina, one pioneer species of mangrove plants. We totally identified 142 NAC TFs from the genome of A. marina. Combined with NAC proteins having been functionally characterized in other organisms, we built a phylogenetic tree to infer the function of NAC TFs in A. marina. Gene structure and motif sequence analyses suggest the sequence conservation and transcription regulatory regions-mediated functional diversity. Whole-genome duplication serves as the driver force to the evolution of NAC gene family. Moreover, two pairs of NAC genes were identified as positively selected genes of which AmNAC010/040 may be imposed on less constraint toward neofunctionalization. Quite a few stress/hormone-related responsive elements were found in promoter regions indicating potential response to various external factors. Transcriptome data revealed some NAC TFs were involved in pneumatophore and leaf salt gland development and response to salt, flooding and Cd stresses. Gene co-expression analysis found a few NAC TFs participates in the special biological processes concerned with adaptation to intertidal environment. In summary, this study provides detailed functional and evolutionary information about NAC gene family in mangrove plant A. marina and new perspective for adaptation to intertidal habitats.

Drought stress is one of the most significant limiting factors limiting crop productions. Foxtail millet (Setaria italica) is among the most drought-tolerant crop plants, with a high degree of collinearity with other staple cereals. The present study used a meta-analysis approach to identify genomic regions and candidate genes associated with drought tolerance and yield-related traits in foxtail millet. A meta-analysis employing all 448 collected original quantitative trait loci (QTL) identified 41 meta-QTL (MQTL) on the nine foxtail millet chromosomes. The confidence interval (CI) of the identified MQTL was determined to be 0.31–14.47 cM (5.23 cM average), which was 3.5 times narrower than the mean CI of the original QTL. Based on the available RNA-seq and microarray data, 1631 differentially expressed genes (DEGs) were detected in 41 MQTL. Furthermore, through synteny analysis, 8, 4, and 2 ortho-MQTL were recognized within co-linear regions of foxtail millet with rice (Oryza sativa), barley (Hordeum vulgare), and maize (Zea mays), respectively. To detect the most significant genome regions involved in the genetic control of drought tolerance and yield maintenance in foxtail millet, 10 MQTL with physical intervals of less than 1 Mb and seven hotspot regions with a high QTL-overview index were identified. Several candidate genes involved in foxtail millet sensing and signaling, transcription regulation, ROS inhibition, and adaptation to abiotic stress were detected by seeking drought-responsive genes in MQTL regions with a CI < 1 Mb. We hope that the achieved results would aid in developing new high-yielding drought-tolerant genotypes.

Drought is one of the most important environmental constraints limiting plant growth and agricultural productivity. To understand the underlying mechanism of drought tolerance and to identify genes for improving this important trait, we conducted a gain-of-function genetic screen for improved drought tolerance in Arabidopsis thaliana. One mutant with improved drought tolerance was isolated and designated as enhanced drought tolerance1. The mutant has a more extensive root system than the wild type, with deeper roots and more lateral roots, and shows a reduced leaf stomatal density. The mutant had higher levels of abscisic acid and Pro than the wild type and demonstrated an increased resistance to oxidative stress and high levels of superoxide dismutase. Molecular genetic analysis and recapitulation experiments showed that the enhanced drought tolerance is caused by the activated expression of a T-DNA tagged gene that encodes a putative homeodomain-START transcription factor. Moreover, overexpressing the cDNA of the transcription factor in transgenic tobacco also conferred drought tolerance associated with improved root architecture and reduced leaf stomatal density. Therefore, we have revealed functions of the homeodomain-START factor that were gained upon altering its expression pattern by activation tagging and provide a key regulator that may be used to improve drought tolerance in plants.

In order to screen candidate aquaporin genes involved in resisting osmotic stress, we analyzed the physiological responses and the expression levels of aquaporin genes in garlic under drought and salt stress with ‘Er Shuizao’ as plant material. Different physiological indicators were detected under drought and salt stress treatments. RT-qPCR was used to detect the expression levels of the candidate aquaporin genes in specific tissues. Finally, we screened AsPIP1-3 as a candidate gene and analyzed its function. The results showed that the relative water content and chlorophyll content of leaves decreased, the O2− production rate increased, and H2O2 accumulated in garlic under drought and salt stress. The activities of SOD, POD, and CAT enzymes first increased and then decreased in garlic. The content of soluble sugar and proline increased to maintain cell osmotic balance, and the content of MDA and relative conductivity continued to increase. Most aquaporin gene expression first increased and then decreased in garlic under drought and salt stress. AsPIP1-3 gene expression is up-regulated under drought and salt stress in garlic. The relative expression was the highest on the 6th day of stress, being related to antioxidant enzyme activity and osmotic regulation. The consistent changes in gene expressions and physiological responses indicated that AsPIP1-3 played a role in resisting garlic osmotic stress. AsPIP1-3 was located on the cell membrane, being consistent with the predicted results of subcellular localization. The germination rate and root length of transgenic Arabidopsis under drought stress were significantly different from the wild type. Drought stress reduced the ROS accumulation of transgenic Arabidopsis, and the antioxidant enzyme activity was significantly higher than the wild type. The relative conductivity and MDA content significantly decreased, and the proline content increased under drought stress. The expression level of the genes related to drought stress response (AtRD22, AtP5CS, AtABF3, and AtLEA) significantly increased. The overexpression of AsPIP1-3 genes improved the drought tolerance of transgenic Arabidopsis plants, showing that AsPIP1-3 proteins enhanced drought tolerance. Our study laid a foundation for exploring the regulatory mechanism of garlic to abiotic stress.

Meta-analysis reveals key features of the improved drought tolerance of plants overexpressing NAC transcription factors

Activated Expression of WRKY57 Confers Drought Tolerance in Arabidopsis