Metabolite-mediated adaptation of crops to drought and the acquisition of tolerance.

Drought is one of the major and growing threats to agriculture productivity and food security. Metabolites are involved in the regulation of plant responses to various environmental stresses, including drought stress. The complex drought tolerance can be ascribed to several simple metabolic traits. These traits could then be used for detecting the genetic architecture of drought tolerance. Plant metabolomes show dynamic differences when drought occurs during different developmental stages or upon different levels of drought stress. Here, we reviewed the major and most recent findings regarding the metabolite-mediated plant drought response. Recent progress in the development of drought-tolerant agents is also discussed. We provide an updated schematic overview of metabolome-driven solutions for increasing crop drought tolerance and thereby addressing an impending agricultural challenge.

Cotton GhNAC4 promotes drought tolerance by regulating secondary cell wall biosynthesis and ribosomal protein homeostasis.

Drought has a severe impact on the quality and yield of cotton. Deciphering the key genes related to drought tolerance is important for understanding the regulation mechanism of drought stress and breeding drought-tolerant cotton cultivars. Several studies have demonstrated that NAC transcription factors are crucial in the regulation of drought stress, however, the related functional mechanisms are still largely unexplored. Here, we identified that NAC transcription factor GhNAC4 positively regulated drought stress tolerance in cotton. The expression of GhNAC4 was significantly induced by abiotic stress and plant hormones. Silencing of GhNAC4 distinctly impaired the resistance to drought stress and overexpressing GhNAC4 in cotton significantly enhanced the stress tolerance. RNA-seq analysis revealed that overexpression of GhNAC4 enriched the expression of genes associated with the biosynthesis of secondary cell walls and ribosomal proteins. We confirmed that GhNAC4 positively activated the expressions of GhNST1, a master regulator reported previously in secondary cell wall formation, and two ribosomal protein-encoding genes GhRPL12 and GhRPL18p, by directly binding to their promoter regions. Overexpression of GhNAC4 promoted the expression of downstream genes associated with the secondary wall biosynthesis, resulting in enhancing secondary wall deposition in the roots, and silencing of GhRPL12 and GhRPL18p significantly impaired the resistance to drought stress. Taken together, our study reveals a novel pathway mediated by GhNAC4 that promotes secondary cell wall biosynthesis to strengthen secondary wall development and regulates the expression of ribosomal protein-encoding genes to maintain translation stability, which ultimately enhances drought tolerance in cotton.

MdMYB44-like positively regulates salt and drought tolerance via the MdPYL8-MdPP2CA module in apple.

Abscisic acid (ABA) is involved in salt and drought stress responses, but the underlying molecular mechanism remains unclear. Here, we demonstrated that the overexpression of MdMYB44-like, an R2R3-MYB transcription factor, significantly increases the salt and drought tolerance of transgenic apples and Arabidopsis. MdMYB44-like inhibits the transcription of MdPP2CA, which encodes a type 2C protein phosphatase that acts as a negative regulator in the ABA response, thereby enhancing ABA signaling-mediated salt and drought tolerance. Furthermore, we found that MdMYB44-like and MdPYL8, an ABA receptor, form a protein complex that further enhances the transcriptional inhibition of the MdPP2CA promoter by MdMYB44-like. Significantly, we discovered that MdPP2CA can interfere with the physical association between MdMYB44-like and MdPYL8 in the presence of ABA, partially blocking the inhibitory effect of the MdMYB44-like-MdPYL8 complex on the MdPP2CA promoter. Thus, MdMYB44-like, MdPYL8, and MdPP2CA form a regulatory loop that tightly modulates ABA signaling homeostasis under salt and drought stress. Our data reveal that MdMYB44-like precisely modulates ABA-mediated salt and drought tolerance in apples through the MdPYL8-MdPP2CA module.

The ubiquitin E3 ligase RZFP1 affects drought tolerance in poplar by mediating the degradation of the protein phosphatase PP2C-9.

Abscisic acid signaling has been implicated in plant responses to water deficit-induced osmotic stress. However, the underlying molecular mechanism remains unelucidated. This study identified the RING-type E3 ubiquitin ligase RING ZINC FINGER PROTEIN1 (PtrRZFP1) in poplar (Populus trichocarpa), a woody model plant. PtrRZFP1 encodes a ubiquitin E3 ligase that participates in protein ubiquitination. PtrRZFP1 mainly functions in the nucleus and endoplasmic reticulum and is activated by drought and abscisic acid. PtrRZFP1-overexpressing transgenic poplars (35S:PtrRZFP1) showed greater tolerance to drought, whereas PtrRZFP1-knockdown lines (KD-PtrRZFP1) showed greater sensitivity to drought. Under treatment with polyethylene glycol and abscisic acid, PtrRZFP1 promoted the production of NO and H2O2 in stomatal guard cells, ultimately enhancing stomatal closure and improving drought tolerance. Additionally, PtrRZFP1 physically interacted with the clade A Protein Phosphatase 2C protein PtrPP2C-9, a core regulator of abscisic acid signaling, and mediated its ubiquitination and eventual degradation through the ubiquitination-26S proteasome system, indicating that PtrRZFP1 positively regulates the abscisic acid signaling pathway. Furthermore, the PtrPP2C-9-overexpression line was insensitive to abscisic acid and more sensitive to drought than the wild-type plants, whereas the opposite phenotype was observed in 35S:PtrRZFP1 plants. In general, PtrRZFP1 negatively regulates the stability of PtrPP2C-9 to mediate poplar drought tolerance. The results of this study provide a theoretical framework for the targeted breeding of drought-tolerant traits in perennial woody plants.

Parallel adaptation to lower altitudes is associated with enhanced plasticity in Heliosperma pusillum (Caryophyllaceae).

High levels of phenotypic plasticity are thought to be inherently costly in stable or extreme environments, but enhanced plasticity may evolve as a response to new environments and foster novel phenotypes. Heliosperma pusillum forms glabrous alpine and pubescent montane ecotypes that diverged recurrently and polytopically (parallel evolution) and can serve as evolutionary replicates. The specific alpine and montane localities are characterized by distinct temperature conditions, available moisture, and light. Noteworthy, the ecotypes show a home-site fitness advantage in reciprocal transplantations. To disentangle the relative contribution of constitutive versus plastic gene expression to altitudinal divergence, we analyze the transcriptomic profiles of two parallely evolved ecotype pairs, grown in reciprocal transplantations at native altitudinal sites. In this incipient stage of divergence, only a minor proportion of genes appear constitutively differentially expressed between the ecotypes in both pairs, regardless of the growing environment. Both derived, montane populations bear comparatively higher plasticity of gene expression than the alpine populations. Genes that change expression plastically or constitutively underlie similar ecologically relevant pathways, related to response to drought and trichome formation. Other relevant processes, such as photosynthesis, rely mainly on plastic changes. The enhanced plasticity consistently observed in the montane ecotype likely evolved as a response to the newly colonized, drier, and warmer niche. We report a striking parallelism of directional changes in gene expression plasticity. Thus, plasticity appears to be a key mechanism shaping the initial stages of phenotypic evolution, likely fostering adaptation to novel environments.

Modulation of the wheat transcriptome by TaZFP13D under well-watered and drought conditions.

Maintaining global food security in the context of climate changes will be an important challenge in the next century. Improving abiotic stress tolerance of major crops such as wheat can contribute to this goal. This can be achieved by the identification of the genes involved and their use to develop tools for breeding programs aiming to generate better adapted cultivars. Recently, we identified the wheat TaZFP13D gene encoding Zinc Finger Protein 13D as a new gene improving water-stress tolerance. The current work analyzes the TaZFP13D-dependent transcriptome modifications that occur in well-watered and dehydration conditions to better understand its function during normal growth and during drought. Plants that overexpress TaZFP13D have a higher biomass under well-watered conditions, indicating a positive effect of the protein on growth. Survival rate and stress recovery after a severe drought stress are improved compared to wild-type plants. The latter is likely due the higher activity of key antioxidant enzymes and concomitant reduction of drought-induced oxidative damage. Conversely, down-regulation of TaZFP13D decreases drought tolerance and protection against drought-induced oxidative damage. RNA-Seq transcriptome analysis identified many genes regulated by TaZFP13D that are known to improve drought tolerance. The analysis also revealed several genes involved in the photosynthetic electron transfer chain known to improve photosynthetic efficiency and chloroplast protection against drought-induced ROS damage. This study highlights the important role of TaZFP13D in wheat drought tolerance, contributes to unravel the complex regulation governed by TaZFPs, and suggests that it could be a promising marker to select wheat cultivars with higher drought tolerance.

Physiological traits, crop growth, and grain quality of quinoa in response to deficit irrigation and planting methods.

Climate change has become a concern, emphasizing the need for the development of crops tolerant to drought. Therefore, this study is designed to explore the physiological characteristics of quinoa that enable it to thrive under drought and other extreme stress conditions by investigating the combined effects of irrigation water levels (100%, 75%, and 50% of quinoa's water requirements, WR as I1, I2 and I3) and different planting methods (basin, on-ridge, and in-furrow as P1, P2 and P3) on quinoa's physiological traits and gas exchange. Results showed that quinoa's yield is lowest with on-ridge planting and highest in the in-furrow planting method. Notably, the seed protein concentrations in I2 and I3 did not significantly differ but they were 25% higher than those obtained in I1, which highlighted the possibility of using a more effective irrigation method without compromising the seed quality. On the other hand, protein yield (PY) was lowest in P2 (mean of I1 and I2 as 257 kg ha-1) and highest in P3 (mean of I1 and I2 as 394 kg ha-1, 53% higher). Interestingly, PY values were not significantly different in I1 and I2, but they were lower significantly in I3 by 28%, 27% and 20% in P1, P2, and P3, respectively. Essential plant characteristics including plant height, stem diameter, and panicle number were 6.1-16.7%, 6.4-24.5%, and 18.4-36.5% lower, respectively, in I2 and I3 than those in I1. The highest Leaf Area Index (LAI) value (5.34) was recorded in the in-furrow planting and I1, while the lowest value was observed in the on-ridge planting method and I3 (3.47). In I3, leaf temperature increased by an average of 2.5-3 oC, particularly during the anthesis stage. The results also showed that at a similar leaf water potential (LWP) higher yield and dry matter were obtained in the in-furrow planting compared to those obtained in the basin and on-ridge planting methods. The highest stomatal conductance (gs) value was observed within the in-furrow planting method and full irrigation (I1P3), while the lowest values were obtained in the on-ridge and 50%WR (I3P2). Finally, photosynthesis rate (An) reduction with diminishing LWP was mild, providing insights into quinoa's adaptability to drought. In conclusion, considering the thorough evaluation of all the measured parameters, the study suggests using the in-furrow planting method with a 75%WR as the best approach for growing quinoa in arid and semi-arid regions to enhance production and resource efficiency.

Evaluation of drought-tolerant varieties based on root system architecture in cotton (Gossypium hirsutum L.).

BACKGROUND: Root system architecture (RSA) exhibits significant genetic variability and is closely associated with drought tolerance. However, the evaluation of drought-tolerant cotton cultivars based on RSA in the field conditions is still underexplored. RESULTS: So, this study conducted a comprehensive analysis of drought tolerance based on physiological and morphological traits (i.e., aboveground and RSA, and yield) within a rain-out shelter, with two water treatments: well-watered (75 ± 5% soil relative water content) and drought stress (50 ± 5% soil relative water content). The results showed that principal component analysis identified six principal components, including highlighting the importance of root traits and canopy parameters in influencing drought tolerance. Moreover, the systematic cluster analysis was used to classify 80 cultivars into 5 categories, including drought-tolerant cultivars, relatively drought-tolerant cultivars, intermediate cultivars, relatively drought-sensitive cultivars, and drought-sensitive cultivars. Further validation of the drought tolerance index showed that the yield drought tolerance index and biomass drought tolerance index of the drought-tolerant cultivars were 8.97 and 5.05 times higher than those of the drought-sensitive cultivars, respectively. CONCLUSIONS: The RSA of drought-tolerant cultivars was characterised by a significant increase in average length-all lateral roots, a significant decrease in average lateral root emergence angle and a moderate root/shoot ratio. In contrast, the drought-sensitive cultivars showed a significant decrease in average length-all lateral roots and a significant increase in both average lateral root emergence angle and root/shoot ratio. It is therefore more comprehensive and accurate to assess field crop drought tolerance by considering root performance.

Overexpression of MtIPT gene enhanced drought tolerance and delayed leaf senescence of creeping bentgrass (Agrostis stolonifera L.).

BACKGROUND: Isopentenyltransferases (IPT) serve as crucial rate-limiting enzyme in cytokinin synthesis, playing a vital role in plant growth, development, and resistance to abiotic stress. RESULTS: Compared to the wild type, transgenic creeping bentgrass exhibited a slower growth rate, heightened drought tolerance, and improved shade tolerance attributed to delayed leaf senescence. Additionally, transgenic plants showed significant increases in antioxidant enzyme levels, chlorophyll content, and soluble sugars. Importantly, this study uncovered that overexpression of the MtIPT gene not only significantly enhanced cytokinin and auxin content but also influenced brassinosteroid level. RNA-seq analysis revealed that differentially expressed genes (DEGs) between transgenic and wild type plants were closely associated with plant hormone signal transduction, steroid biosynthesis, photosynthesis, flavonoid biosynthesis, carotenoid biosynthesis, anthocyanin biosynthesis, oxidation-reduction process, cytokinin metabolism, and wax biosynthesis. And numerous DEGs related to growth, development, and stress tolerance were identified, including cytokinin signal transduction genes (CRE1, B-ARR), antioxidase-related genes (APX2, PEX11, PER1), Photosynthesis-related genes (ATPF1A, PSBQ, PETF), flavonoid synthesis genes (F3H, C12RT1, DFR), wax synthesis gene (MAH1), senescence-associated gene (SAG20), among others. CONCLUSION: These findings suggest that the MtIPT gene acts as a negative regulator of plant growth and development, while also playing a crucial role in the plant's response to abiotic stress.

RhMED15a-like, a subunit of the Mediator complex, is involved in the drought stress response in Rosa hybrida.

BACKGROUND: Rose (Rosa hybrida) is a globally recognized ornamental plant whose growth and distribution are strongly limited by drought stress. The role of Mediator, a multiprotein complex crucial for RNA polymerase II-driven transcription, has been elucidated in drought stress responses in plants. However, its physiological function and regulatory mechanism in horticultural crop species remain elusive. RESULTS: In this study, we identified a Tail module subunit of Mediator, RhMED15a-like, in rose. Drought stress, as well as treatment with methyl jasmonate (MeJA) and abscisic acid (ABA), significantly suppressed the transcript level of RhMED15a-like. Overexpressing RhMED15a-like markedly bolstered the osmotic stress tolerance of Arabidopsis, as evidenced by increased germination rate, root length, and fresh weight. In contrast, the silencing of RhMED15a-like through virus induced gene silencing in rose resulted in elevated malondialdehyde accumulation, exacerbated leaf wilting, reduced survival rate, and downregulated expression of drought-responsive genes during drought stress. Additionally, using RNA-seq, we identified 972 differentially expressed genes (DEGs) between tobacco rattle virus (TRV)-RhMED15a-like plants and TRV controls. Gene Ontology (GO) analysis revealed that some DEGs were predominantly associated with terms related to the oxidative stress response, such as 'response to reactive oxygen species' and 'peroxisome'. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment highlighted pathways related to 'plant hormone signal transduction', in which the majority of DEGs in the jasmonate (JA) and ABA signalling pathways were induced in TRV-RhMED15a-like plants. CONCLUSION: Our findings underscore the pivotal role of the Mediator subunit RhMED15a-like in the ability of rose to withstand drought stress, probably by controlling the transcript levels of drought-responsive genes and signalling pathway elements of stress-related hormones, providing a solid foundation for future research into the molecular mechanisms underlying drought tolerance in rose.

Gene expression analysis of drought tolerance and cuticular wax biosynthesis in diploid and tetraploid induced wallflowers.

Whole-genome doubling leads to cell reprogramming, upregulation of stress genes, and establishment of new pathways of drought stress responses in plants. This study investigated the molecular mechanisms of drought tolerance and cuticular wax characteristics in diploid and tetraploid-induced Erysimum cheiri. According to real-time PCR analysis, tetraploid induced wallflowers exhibited increased expression of several genes encoding transcription factors (TFs), including AREB1 and AREB3; the stress response genes RD29A and ERD1 under drought stress conditions. Furthermore, two cuticular wax biosynthetic pathway genes, CER1 and SHN1, were upregulated in tetraploid plants under drought conditions. Leaf morphological studies revealed that tetraploid leaves were covered with unique cuticular wax crystalloids, which produced a white fluffy appearance, while the diploid leaves were green and smooth. The greater content of epicuticular wax in tetraploid leaves than in diploid leaves can explain the decrease in cuticle permeability as well as the decrease in water loss and improvement in drought tolerance in wallflowers. GC‒MS analysis revealed that the wax components included alkanes, alcohols, aldehydes, and fatty acids. The most abundant wax compound in this plant was alkanes (50%), the most predominant of which was C29. The relative abundance of these compounds increased significantly in tetraploid plants under drought stress conditions. These findings revealed that tetraploid-induced wallflowers presented upregulation of multiple drought-related and wax biosynthesis genes; therefore, polyploidization has proved useful for improving plant drought tolerance.

Characterization of DREB family genes in Lotus japonicus and LjDREB2B overexpression increased drought tolerance in transgenic Arabidopsis.

BACKGROUND: Drought stress affects plant growth and development. DREB proteins play important roles in modulating plant growth, development, and stress responses, particularly under drought stress. To study the function of DREB transcription factors (TFs), we screened key DREB-regulating TFs for drought in Lotus japonicus. RESULTS: Forty-two DREB TFs were identified, and phylogenetic analysis of proteins from L. japonicus classified them into five subfamilies (A1, A2, A4, A5, A6). The gene motif composition of the proteins is conserved within the same subfamily. Based on the cis-acting regulatory element analysis, we identified many growth-, hormone-, and stress-responsive elements within the promoter regions of DREB. We further analyzed the expression pattern of four genes in the A2 subfamily in response to drought stress. We found that the expression of most of the LjDREB A2 subfamily genes, especially LjDREB2B, was induced by drought stress. We further generated LjDREB2B overexpression transgenic Arabidopsis plants. Under drought stress, the growth of wild-type (WT) and overexpressing LjDREB2B (OE) Arabidopsis lines was inhibited; however, OE plants showed better growth. The malondialdehyde content of LjDREB2B overexpressing lines was lower than that of the WT plants, whereas the proline content and antioxidant enzyme activities in the OE lines were significantly higher than those in the WT plants. Furthermore, after drought stress, the expression levels of AtP5CS1, AtP5CS2, AtRD29A, and AtRD29B in the OE lines were significantly higher than those in the WT plants. CONCLUSIONS: Our results facilitate further functional analysis of L. japonicus DREB. LjDREB2B overexpression improves drought tolerance in transgenic Arabidopsis. These results indicate that DREB holds great potential for the genetic improvement of drought tolerance in L. japonicus.

Overexpression of OsNAR2.1 by OsNAR2.1 promoter increases drought resistance by increasing the expression of OsPLDα1 in rice.

BACKGROUND: pOsNAR2.1:OsNAR2.1 expression could significantly increase nitrogen uptake efficiency and grain yield of rice. RESULT: This study reported the effects of overexpression of OsNAR2.1 by OsNAR2.1 promoter on physiological and agronomic traits associated with drought tolerance. In comparison to the wild-type (WT), the pOsNAR2.1:OsNAR2.1 transgenic lines exhibited a significant improvement in survival rate when subjected to drought stress and then irrigation. Under limited water supply conditions, compared with WT, the photosynthesis and water use efficiency (WUE) of transgenic lines were increased by 39.2% and 28.8%, respectively. Finally, the transgenic lines had 25.5% and 66.4% higher grain yield than the WT under full watering and limited water supply conditions, respectively. Compared with the WT, the agronomic nitrogen use efficiency (NUE) of transgenic lines increased by 25.5% and 66.4% under full watering and limited water supply conditions, and the N recovery efficiency of transgenic lines increased by 29.3% and 50.2%, respectively. The interaction between OsNAR2.1 protein and OsPLDα1 protein was verified by yeast hybrids. After drought treatment, PLDα activity on the plasma membrane of the transgenic line increased 85.0% compared with WT. CONCLUSION: These results indicated that pOsNAR2.1:OsNAR2.1 expression could improve the drought resistance of rice by increasing nitrogen uptake and regulating the expression of OsPLDα1.

Genome-wide identification and expression analysis of the BZR gene family in Zanthoxylum armatum DC and functional analysis of ZaBZR1 in drought tolerance.

MAIN CONCLUSION: In this study, six ZaBZRs were identified in Zanthoxylum armatum DC, and all the ZaBZRs were upregulated by abscisic acid (ABA) and drought. Overexpression of ZaBZR1 enhanced the drought tolerance of transgenic Nicotiana benthamian. Brassinosteroids (BRs) are a pivotal class of sterol hormones in plants that play a crucial role in plant growth and development. BZR (brassinazole resistant) is a crucial transcription factor in the signal transduction pathway of BRs. However, the BZR gene family members have not yet been identified in Zanthoxylum armatum DC. In this study, six members of the ZaBZR family were identified by bioinformatic methods. All six ZaBZRs exhibited multiple phosphorylation sites. Phylogenetic and collinearity analyses revealed a closest relationship between ZaBZRs and ZbBZRs located on the B subgenomes. Expression analysis revealed tissue-specific expression patterns of ZaBZRs in Z. armatum, and their promoter regions contained cis-acting elements associated with hormone response and stress induction. Additionally, all six ZaBZRs showed upregulation upon treatment after abscisic acid (ABA) and polyethylene glycol (PEG), indicating their participation in drought response. Subsequently, we conducted an extensive investigation of ZaBZR1. ZaBZR1 showed the highest expression in the root, followed by the stem and terminal bud. Subcellular localization analysis revealed that ZaBZR1 is present in the cytoplasm and nucleus. Overexpression of ZaBZR1 in transgenic Nicotiana benthamiana improved seed germination rate and root growth under drought conditions, reducing water loss rates compared to wild-type plants. Furthermore, ZaBZR1 increased proline content (PRO) and decreased malondialdehyde content (MDA), indicating improved tolerance to drought-induced oxidative stress. The transgenic plants also showed a reduced accumulation of reactive oxygen species. Importantly, ZaBZR1 up-regulated the expression of drought-related genes such as NbP5CS1, NbDREB2A, and NbWRKY44. These findings highlight the potential of ZaBZR1 as a candidate gene for enhancing drought resistance in transgenic N. benthamiana and provide insight into the function of ZaBZRs in Z. armatum.

Mutation in Arabidopsis mitochondrial Pentatricopeptide repeat 40 gene affects tolerance to water deficit.

MAIN CONCLUSION: The Arabidopsis Pentatricopeptide repeat 40 (PPR40) insertion mutants have increased tolerance to water deficit compared to wild-type plants. Tolerance is likely the consequence of ABA hypersensitivity of the mutants. Plant growth and development depend on multiple environmental factors whose alterations can disrupt plant homeostasis and trigger complex molecular and physiological responses. Water deficit is one of the factors which can seriously restrict plant growth and viability. Mitochondria play an important role in cellular metabolism, energy production, and redox homeostasis. During drought and salinity stress, mitochondrial dysfunction can lead to ROS overproduction and oxidative stress, affecting plant growth and survival. Alternative oxidases (AOXs) and stabilization of mitochondrial electron transport chain help mitigate ROS damage. The mitochondrial Pentatricopeptide repeat 40 (PPR40) protein was implicated in stress regulation as ppr40 mutants were found to be hypersensitive to ABA and high salinity during germination. This study investigated the tolerance of the knockout ppr40-1 and knockdown ppr40-2 mutants to water deprivation. Our results show that these mutants display an enhanced tolerance to water deficit. The mutants had higher relative water content, reduced level of oxidative damage, and better photosynthetic parameters in water-limited conditions compared to wild-type plants. ppr40 mutants had considerable differences in metabolic profiles and expression of a number of stress-related genes, suggesting important metabolic reprogramming. Tolerance to water deficit was also manifested in higher survival rates and alleviated growth reduction when watering was suspended. Enhanced sensitivity to ABA and fast stomata closure was suggested to lead to improved capacity for water conservation in such environment. Overall, this study highlights the importance of mitochondrial functions and in particular PPR40 in plant responses to abiotic stress, particularly drought.

TaMYB44-5A reduces drought tolerance by repressing transcription of TaRD22-3A in the abscisic acid signaling pathway.

MAIN CONCLUSION: TaMYB44-5A identified as a transcription factor negatively regulates drought tolerance in transgenic Arabidopsis. Drought can severely reduce yields throughout the wheat-growing season. Many studies have shown that R2R3-MYB transcription factors are involved in drought stress responses. In this study, the R2R3-MYB transcription factor MYB44-5A was identified in wheat (Triticum aestivum L.) and functionally analyzed. Three homologs of TaMYB44 were isolated, all of which localized to the nucleus. Overexpression of TaMYB44-5A reduced drought tolerance in Arabidopsis thaliana. Further analysis showed that TaMYB44-5A reduced the sensitivity of transgenic Arabidopsis to ABA. Genetic and transcriptional regulation analyses demonstrated that the expression levels of drought- and ABA-responsive genes were downregulated by TaMYB44-5A, and TaMYB44-5A directly bound to the MYB-binding site on the promoter to repress the transcription level of TaRD22-3A. Our results provide insights into a novel molecular pathway in which the R2R3-MYB transcription factor negatively regulates ABA signaling in response to drought stress.

Gene pyramiding for boosted plant growth and broad abiotic stress tolerance.

Abiotic stresses such as salinity, heat and drought seriously impair plant growth and development, causing a significant loss in crop yield and ornamental value. Biotechnology approaches manipulating specific genes prove to be effective strategies in crop trait modification. The Arabidopsis vacuolar pyrophosphatase gene AVP1, the rice SUMO E3 ligase gene OsSIZ1 and the cyanobacterium flavodoxin gene Fld have previously been implicated in regulating plant stress responses and conferring enhanced tolerance to different abiotic stresses when individually overexpressed in various plant species. We have explored the feasibility of combining multiple favourable traits brought by individual genes to acquire superior plant performance. To this end, we have simultaneously introduced AVP1, OsSIZ1 and Fld in creeping bentgrass. Transgenic (TG) plants overexpressing these three genes performed significantly better than wild type controls and the TGs expressing individual genes under both normal and various abiotic stress conditions, exhibited significantly enhanced plant growth and tolerance to drought, salinity and heat stresses as well as nitrogen and phosphate starvation, which were associated with altered physiological and biochemical characteristics and delicately fine-tuned expression of genes involved in plant stress responses. Our results suggest that AVP1, OsSIZ1 and Fld function synergistically to regulate plant development and plant stress response, leading to superior overall performance under both normal and adverse environments. The information obtained provides new insights into gene stacking as an effective approach for plant genetic engineering. A similar strategy can be extended for the use of other beneficial genes in various crop species for trait modifications, enhancing agricultural production.

MicroRNA169 integrates multiple factors to modulate plant growth and abiotic stress responses.

MicroRNA169 (miR169) has been implicated in multi-stress regulation in annual species such as Arabidopsis, maize and rice. However, there is a lack of experimental functional and mechanistic studies of miR169 in plants, especially in perennial species, and its impact on plant growth and development remains unexplored. Creeping bentgrass (Agrostis stolonifera L.) is a C3 cool-season perennial turfgrass of significant environmental and economic importance. In this study, we generated both miR169 overexpression and knockdown transgenic creeping bentgrass lines. We found that miR169 acts as a positive regulator in abiotic stress responses but is negatively associated with plant growth and development, playing multiple critical roles in the growth and environmental adaptation of creeping bentgrass. These roles include differentiated spatial hormone accumulation patterns associated with growth and stress accommodation, elevated antioxidant activity that alleviates oxidative damage induced by stress, ion-channelling membrane components for maintaining homeostasis under saline conditions, and potential cross-talks with stress-regulating transcription factors such as AsHsfA and AsWRKYs. Our results unravel the role of miR169 in modulating plant development and stress responses in perennial grass species. This underlines the potential of manipulating miR169 to generate crop cultivars with desirable traits to meet diverse agricultural demands.

Combined GWAS and eGWAS reveals the genetic basis underlying drought tolerance in emmer wheat (Triticum turgidum L.).

Drought is one of the major environmental constraints for wheat production world-wide. As the progenitor and genetic reservoir of common wheat, emmer wheat is considered as an invaluable gene pool for breeding drought-tolerant wheat. Combining GWAS and eGWAS analysis of 107 accessions, we identified 86 QTLs, 105 462 eQTLs as well as 68 eQTL hotspots associating with drought tolerance (DT) in emmer wheat. A complex regulatory network composed of 185 upstream regulator and 2432 downstream drought-responsive candidates was developed, of which TtOTS1 was found to play a negative effect in determining DT through affecting root development. This study sheds light on revealing the genetic basis underlying DT, which will provide the indispensable genes and germplasm resources for elite drought tolerance wheat improvement and breeding.

Rare variations within the serine/arginine-rich splicing factor PtoRSZ21 modulate stomatal size to determine drought tolerance in Populus.

Rare variants contribute significantly to the 'missing heritability' of quantitative traits. The genome-wide characteristics of rare variants and their roles in environmental adaptation of woody plants remain unexplored. Utilizing genome-wide rare variant association study (RVAS), expression quantitative trait loci (eQTL) mapping, genetic transformation, and molecular experiments, we explored the impact of rare variants on stomatal morphology and drought adaptation in Populus. Through comparative analysis of five world-wide Populus species, we observed the influence of mutational bias and adaptive selection on the distribution of rare variants. RVAS identified 75 candidate genes correlated with stomatal size (SS)/stomatal density (SD), and a rare haplotype in the promoter of serine/arginine-rich splicing factor PtoRSZ21 emerged as the foremost association signal governing SS. As a positive regulator of drought tolerance, PtoRSZ21 can recruit the core splicing factor PtoU1-70K to regulate alternative splicing (AS) of PtoATG2b (autophagy-related 2). The rare haplotype PtoRSZ21hap2 weakens binding affinity to PtoMYB61, consequently affecting PtoRSZ21 expression and SS, ultimately resulting in differential distribution of Populus accessions in arid and humid climates. This study enhances the understanding of regulatory mechanisms that underlie AS induced by rare variants and might provide targets for drought-tolerant varieties breeding in Populus.