The rice NUCLEAR FACTOR-YA5 and MICRORNA169a module promotes nitrogen utilization during nitrogen deficiency.

Nitrogen (N) is essential for plant growth and development. Therefore, understanding its utilization is essential for improving crop productivity. However, much remains to be learned about plant N sensing and signaling. Here, rice (Oryza sativa) NUCLEAR FACTOR-YA5 (OsNF-YA5) expression was tightly regulated by N status and induced under N-deficient conditions. Overexpression (OE) of OsNF-YA5 in rice resulted in increased chlorophyll levels and delayed senescence compared to control plants under normal N conditions. Agronomic traits were significantly improved in OE plants and impaired in knockout mutants under N-deficient conditions. Using a dexamethasone-inducible system, we identified the putative targets of OsNF-YA5 that include amino acid, nitrate/peptide transporters, and NITRATE TRANSPORTER 1.1A (OsNRT1.1A), which functions as a key transporter in rice. OsNF-YA5 directly enhanced OsNRT1.1A expression and N uptake rate under N-deficient conditions. Besides, overexpression of OsNF-YA5 also enhanced the expression of GLUTAMINE SYNTHETASE 1/2 (GS1/2) and GLUTAMINE OXOGLUTARATE AMINOTRANSFERASE 1/2 (GOGAT1/2), increasing free amino acid contents under N-deficient conditions. Osa-miR169a expression showed an opposite pattern with OsNF-YA5 depending on N status. Further analysis revealed that osa-miR169a negatively regulates OsNF-YA5 expression and N utilization, demonstrating that an OsNF-YA5/osa-miR169a module tightly regulates rice N utilization for adaptation to N status.

DROUGHT-INDUCED BRANCHED-CHAIN AMINO ACID AMINOTRANSFERASE enhances drought tolerance in rice.

Plants accumulate several metabolites in response to drought stress, including branched-chain amino acids (BCAAs). However, the roles of BCAAs in plant drought responses and the underlying molecular mechanisms for BCAA accumulation remain elusive. Here, we demonstrate that rice (Oryza sativa) DROUGHT-INDUCED BRANCHED-CHAIN AMINO ACID AMINOTRANSFERASE (OsDIAT) mediates the accumulation of BCAAs in rice in response to drought stress. An in vitro enzyme activity assay indicated that OsDIAT is a branched-chain amino acid aminotransferase, and subcellular localization analysis revealed that OsDIAT localizes to the cytoplasm. The expression of OsDIAT was induced in plants upon exposure to abiotic stress. OsDIAT-overexpressing (OsDIATOX) plants were more tolerant to drought stress, whereas osdiat plants were more susceptible to drought stress compared with nontransgenic (NT) plants. Amino acid analysis revealed that BCAA levels were higher in OsDIATOX but lower in osdiat compared with in NT plants. Finally, the exogenous application of BCAAs improved plant tolerance to osmotic stress compared with that in control plants. Collectively, these findings suggest that OsDIAT mediates drought tolerance by promoting the accumulation of BCAAs.

Rice NAC17 transcription factor enhances drought tolerance by modulating lignin accumulation.

Land plants have developed a comprehensive system to cope with the drought stress, and it is operated by intricate signaling networks, including transcriptional regulation. Herein, we identified the function of OsNAC17, a member of NAC (NAM, ATAF, and CUC2) transcription factor family, in drought tolerance. OsNAC17 is localized to the nucleus, and its expression was significantly induced under drought conditions. A transactivation assay in yeast revealed that the OsNAC17 is a transcriptional activator, harboring an activation domain in the C-terminal region. Overexpressing (OsNAC17OX) transgenic plants showed drought-tolerant, and knock-out (OsNAC17KO) plants exhibited drought susceptible phenotype compared to non-transgenic plants. Further investigation revealed that OsNAC17 positively regulates several lignin biosynthetic genes and promotes lignin accumulation in leaves and roots. Together, our results show that OsNAC17 contributes to drought tolerance through lignin biosynthesis in rice.

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.

Overexpression of OsERF83, a Vascular Tissue-Specific Transcription Factor Gene, Confers Drought Tolerance in Rice.

Abiotic stresses severely affect plant growth and productivity. To cope with abiotic stresses, plants have evolved tolerance mechanisms that are tightly regulated by reprogramming transcription factors (TFs). APETALA2/ethylene-responsive factor (AP2/ERF) transcription factors are known to play an important role in various abiotic stresses. However, our understanding of the molecular mechanisms remains incomplete. In this study, we identified the role of OsERF83, a member of the AP2/ERF transcription factor family, in response to drought stress. OsERF83 is a transcription factor localized to the nucleus and induced in response to various abiotic stresses, such as drought and abscisic acid (ABA). Overexpression of OsERF83 in transgenic plants (OsERF83OX) significantly increased drought tolerance, with higher photochemical efficiency in rice. OsERF83OX was also associated with growth retardation, with reduced grain yields under normal growth conditions. OsERF83 is predominantly expressed in the vascular tissue of all organs. Transcriptome analysis revealed that OsERF83 regulates drought response genes, which are related to the transporter (OsNPF8.10, OsNPF8.17, OsLH1), lignin biosynthesis (OsLAC17, OsLAC10, CAD8D), terpenoid synthesis (OsTPS33, OsTPS14, OsTPS3), cytochrome P450 family (Oscyp71Z4, CYP76M10), and abiotic stress-related genes (OsSAP, OsLEA14, PCC13-62). OsERF83 also up-regulates biotic stress-associated genes, including PATHOGENESIS-RELATED PROTEIN (PR), WALL-ASSOCIATED KINASE (WAK), CELLULOSE SYNTHASE-LIKE PROTEIN E1 (CslE1), and LYSM RECEPTOR-LIKE KINASE (RLK) genes. Our results provide new insight into the multiple roles of OsERF83 in the cross-talk between abiotic and biotic stress signaling pathways.

Application of Upstream Open Reading Frames (uORFs) Editing for the Development of Stress-Tolerant Crops.

Global population growth and climate change are posing increasing challenges to the production of a stable crop supply using current agricultural practices. The generation of genetically modified (GM) crops has contributed to improving crop stress tolerance and productivity; however, many regulations are still in place that limit their commercialization. Recently, alternative biotechnology-based strategies, such as gene-edited (GE) crops, have been in the spotlight. Gene-editing technology, based on the clustered regularly interspaced short palindromic repeats (CRISPR) platform, has emerged as a revolutionary tool for targeted gene mutation, and has received attention as a game changer in the global biotechnology market. Here, we briefly introduce the concept of upstream open reading frames (uORFs) editing, which allows for control of the translation of downstream ORFs, and outline the potential for enhancing target gene expression by mutating uORFs. We discuss the current status of developing stress-tolerant crops, and discuss uORF targets associated with salt stress-responsive genes in rice that have already been verified by transgenic research. Finally, we overview the strategy for developing GE crops using uORF editing via the CRISPR-Cas9 system. A case is therefore made that the mutation of uORFs represents an efficient method for developing GE crops and an expansion of the scope of application of genome editing technology.

The Rice GLYCINE-RICH PROTEIN 3 Confers Drought Tolerance by Regulating mRNA Stability of ROS Scavenging-Related Genes.

BACKGROUND: Plant glycine-rich proteins are categorized into several classes based on their protein structures. The glycine-rich RNA binding proteins (GRPs) are members of class IV subfamily possessing N-terminus RNA-recognition motifs (RRMs) and proposed to be involved in post-transcriptional regulation of its target transcripts. GRPs are involved in developmental process and cellular stress responses, but the molecular mechanisms underlying these regulations are still elusive. RESULTS: Here, we report the functional characterization of rice GLYCINE-RICH PROTEIN 3 (OsGRP3) and its physiological roles in drought stress response. Both drought stress and ABA induce the expression of OsGRP3. Transgenic plants overexpressing OsGRP3 (OsGRP3OE) exhibited tolerance while knock-down plants (OsGRP3KD) were susceptible to drought compared to the non-transgenic control. In vivo, subcellular localization analysis revealed that OsGRP3-GFP was transported from cytoplasm/nucleus into cytoplasmic foci following exposure to ABA and mannitol treatments. Comparative transcriptomic analysis between OsGRP3OE and OsGRP3KD plants suggests that OsGRP3 is involved in the regulation of the ROS related genes. RNA-immunoprecipitation analysis revealed the associations of OsGRP3 with PATHOGENESIS RELATED GENE 5 (PR5), METALLOTHIONEIN 1d (MT1d), 4,5-DOPA-DIOXYGENASE (DOPA), and LIPOXYGENASE (LOX) transcripts. The half-life analysis showed that PR5 transcripts decayed slower in OsGRP3OE but faster in OsGRP3KD, while MT1d and LOX transcripts decayed faster in OsGRP3OE but slower in OsGRP3KD plants. H2O2 accumulation was reduced in OsGRP3OE and increased in OsGRP3KD plants compared to non-transgenic plants (NT) under drought stress. CONCLUSION: OsGRP3 plays a positive regulator in rice drought tolerance and modulates the transcript level and mRNA stability of stress-responsive genes, including ROS-related genes. Moreover, OsGRP3 contributes to the reduction of ROS accumulation during drought stress. Our results suggested that OsGRP3 alleviates ROS accumulation by regulating ROS-related genes' mRNA stability under drought stress, which confers drought tolerance.

OsCRP1, a Ribonucleoprotein Gene, Regulates Chloroplast mRNA Stability That Confers Drought and Cold Tolerance.

Chloroplast ribonucleoproteins (cpRNPs) are nuclear-encoded and highly abundant proteins that are proposed to function in chloroplast RNA metabolism. However, the molecular mechanisms underlying the regulation of chloroplast RNAs involved in stress tolerance are poorly understood. Here, we demonstrate that CHLOROPLAST RNA-BINDING PROTEIN 1 (OsCRP1), a rice (Oryza sativa) cpRNP gene, is essential for stabilization of RNAs from the NAD(P)H dehydrogenase (NDH) complex, which in turn enhances drought and cold stress tolerance. An RNA-immunoprecipitation assay revealed that OsCRP1 is associated with a set of chloroplast RNAs. Transcript profiling indicated that the mRNA levels of genes from the NDH complex significantly increased in the OsCRP1 overexpressing compared to non-transgenic plants, whereas the pattern in OsCRP1 RNAi plants were opposite. Importantly, the OsCRP1 overexpressing plants showed a higher cyclic electron transport (CET) activity, which is essential for elevated levels of ATP for photosynthesis. Additionally, overexpression of OsCRP1 resulted in significantly enhanced drought and cold stress tolerance with higher ATP levels compared to wild type. Thus, our findings suggest that overexpression of OsCRP1 stabilizes a set of mRNAs from genes of the NDH complex involved in increasing CET activity and production of ATP, which consequently confers enhanced drought and cold tolerance.

Overexpression of OsTF1L, a rice HD-Zip transcription factor, promotes lignin biosynthesis and stomatal closure that improves drought tolerance.

Drought stress seriously impacts on plant development and productivity. Improvement of drought tolerance without yield penalty is a great challenge in crop biotechnology. Here, we report that the rice (Oryza sativa) homeodomain-leucine zipper transcription factor gene, OsTF1L (Oryza sativa transcription factor 1-like), is a key regulator of drought tolerance mechanisms. Overexpression of the OsTF1L in rice significantly increased drought tolerance at the vegetative stages of growth and promoted both effective photosynthesis and a reduction in the water loss rate under drought conditions. Importantly, the OsTF1L overexpressing plants showed a higher drought tolerance at the reproductive stage of growth with a higher grain yield than nontransgenic controls under field-drought conditions. Genomewide analysis of OsTF1L overexpression plants revealed up-regulation of drought-inducible, stomatal movement and lignin biosynthetic genes. Overexpression of OsTF1L promoted accumulation of lignin in shoots, whereas the RNAi lines showed opposite patterns of lignin accumulation. OsTF1L is mainly expressed in outer cell layers including the epidermis, and the vasculature of the shoots, which coincides with areas of lignification. In addition, OsTF1L overexpression enhances stomatal closure under drought conditions resulted in drought tolerance. More importantly, OsTF1L directly bound to the promoters of lignin biosynthesis and drought-related genes involving poxN/PRX38, Nodulin protein, DHHC4, CASPL5B1 and AAA-type ATPase. Collectively, our results provide a new insight into the role of OsTF1L in enhancing drought tolerance through lignin biosynthesis and stomatal closure in rice.

NGS sequencing reveals that many of the genetic variations in transgenic rice plants match the variations found in natural rice population.

BACKGROUND: As the transformation process can induce mutations in host plants, molecular characterization of the associated genomic changes is important not only for practical food safety but also for understanding the fundamental theories of genome evolution. OBJECTIVES: To investigate a population-scale comparative study of the genome-wide spectrum of sequence variants in the transgenic genome with the variations present in 3000 rice varieties. RESULTS: On average, we identified 19,273 SNPs (including Indels) per transgenic line in which 10,729 SNPs were at the identical locations in the three transgenic rice plants. We found that these variations were predominantly present in specific regions in chromosomes 8 and 10. Majority (88%) of the identified variations were detected at the same genomic locations as those in natural rice population, implying that the transgenic induced mutations had a tendency to be common alleles. CONCLUSION: Genomic variations in transgenic rice plants frequently occurred at the same sites as the major alleles found in the natural rice population, which implies that the sequence variations occur within the limits of a biological system to ensure survival.

Allantoin accumulation through overexpression of ureide permease1 improves rice growth under limited nitrogen conditions.

In legumes, nitrogen (N) can be stored as ureide allantoin and transported by ureide permease (UPS) from nodules to leaves where it is catabolized to release ammonium and assimilation to amino acids. In non-leguminous plants especially rice, information on its roles in N metabolism is scarce. Here, we show that OsUPS1 is localized in plasma membranes and are highly expressed in vascular tissues of rice. We further evaluated an activation tagging rice overexpressing OsUPS1 (OsUPS1OX ) under several N regimes. Under normal field conditions, panicles from OsUPS1OX plants (14 days after flowering (DAF)) showed significant allantoin accumulation. Under hydroponic system at the vegetative stage, plants were exposed to N-starvation and measured the ammonium in roots after resupplying with ammonium sulphate. OsUPS1OX plants displayed higher ammonium uptake in roots compared to wild type (WT). When grown under low-N soil supplemented with different N-concentrations, OsUPS1OX exhibited better growth at 50% N showing higher chlorophyll, tiller number and at least 20% increase in shoot and root biomass relative to WT. To further confirm the effects of regulating the expression of OsUPS1, we evaluated whole-body-overexpressing plants driven by the GOS2 promoter (OsUPS1GOS2 ) as well as silencing plants (OsUPS1RNAi ). We found significant accumulation of allantoin in leaves, stems and roots of OsUPS1GOS2 while in OsUPS1RNAi allantoin was significantly accumulated in roots. We propose that OsUPS1 is responsible for allantoin partitioning in rice and its overexpression can support plant growth through accumulation of allantoin in sink tissues which can be utilized when N is limiting.

A Nitrogen Molecular Sensing System, Comprised of the ALLANTOINASE and UREIDE PERMEASE 1 Genes, Can Be Used to Monitor N Status in Rice.

Nitrogen (N) is an essential nutrient for plant growth and development, but its concentration in the soil is often insufficient for optimal crop production. Consequently, improving N utilization in crops is considered as a major target in agricultural biotechnology. However, much remains to be learnt about crop N metabolism for application. In this study, we have developed a molecular sensor system to monitor the N status in rice (Oryza sativa). We first examined the role of the ureide, allantoin, which is catabolized into allantoin-derived metabolites and used as an N source under low N conditions. The expression levels of two genes involved in ureide metabolism, ALLANTOINASE (OsALN) and UREIDE PERMEASE 1 (OsUPS1), were highly responsive to the N status. OsALN was rapidly up-regulated under low N conditions, whereas OsUPS1 was up-regulated under high N conditions. Taking advantage of the responses of these two genes to N status, we generated transgenic rice plants harboring the molecular N sensors, proALN::ALN-LUC2 and proUPS1::UPS1-LUC2, comprising the gene promoters driving expression of the luciferase reporter. We observed that expression of the transgenes mimicked transcriptional regulation of the endogenous OsALN and OsUPS1 genes in response to exogenous N status. Importantly, the molecular N sensors showed similar levels of specificity to nitrate and ammonium, from which we infer their sensing abilities. Transgenic rice plants expressing the proUPS1::UPS1-LUC2 sensor showed strong luminescence under high exogenous N conditions (>1 mM), whereas transgenic plants expressing the proALN::ALN-LUC2 sensor showed strong luminescence under low exogenous N conditions (<0.1 mM). High exogenous N (>1 mM) substantially increased internal ammonium and nitrate levels, whereas low exogenous N (<0.1 mM) had no effect on internal ammonium and nitrate levels, indicating the luminescence signals of molecular sensors reflect internal N status in rice. Thus, proALN::ALN-LUC2 and proUPS1::UPS1-LUC2 represent N molecular sensors that operate over a physiological and developmental range in rice.

Overexpression of OsNAC14 Improves Drought Tolerance in Rice.

Plants have evolved to have sophisticated adaptation mechanisms to cope with drought stress by reprograming transcriptional networks through drought responsive transcription factors. NAM, ATAF1-2, and CUC2 (NAC) transcription factors are known to be associated with various developmental processes and stress tolerance. In this study, we functionally characterized the rice drought responsive transcription factor OsNAC14. OsNAC14 was predominantly expressed at meiosis stage but is induced by drought, high salinity, ABA, and low temperature in leaves. Overexpression of OsNAC14 resulted in drought tolerance at the vegetative stage of growth. Field drought tests demonstrated that OsNAC14 overexpressing transgenic rice lines exhibited higher number of panicle and filling rate compared to non-transgenic plants under drought conditions. RNA-sequencing analysis revealed that OsNAC14 overexpression elevated the expression of genes for stress response, DNA damage repair, defense related, and strigolactone biosynthesis. In addition, chromatin immunoprecipitation analysis confirmed the direct interaction of OsNAC14 with the promoter of OsRAD51A1, a key component in homologous recombination in DNA repair system. Collectively, these results indicate that OsNAC14 mediates drought tolerance by recruiting factors involved in DNA damage repair and defense response resulting in improved tolerance to drought.

A bioinformatics approach for identifying transgene insertion sites using whole genome sequencing data.

BACKGROUND: Genetically modified crops (GM crops) have been developed to improve the agricultural traits of modern crop cultivars. Safety assessments of GM crops are of paramount importance in research at developmental stages and before releasing transgenic plants into the marketplace. Sequencing technology is developing rapidly, with higher output and labor efficiencies, and will eventually replace existing methods for the molecular characterization of genetically modified organisms. METHODS: To detect the transgenic insertion locations in the three GM rice gnomes, Illumina sequencing reads are mapped and classified to the rice genome and plasmid sequence. The both mapped reads are classified to characterize the junction site between plant and transgene sequence by sequence alignment. RESULTS: Herein, we present a next generation sequencing (NGS)-based molecular characterization method, using transgenic rice plants SNU-Bt9-5, SNU-Bt9-30, and SNU-Bt9-109. Specifically, using bioinformatics tools, we detected the precise insertion locations and copy numbers of transfer DNA, genetic rearrangements, and the absence of backbone sequences, which were equivalent to results obtained from Southern blot analyses. CONCLUSION: NGS methods have been suggested as an effective means of characterizing and detecting transgenic insertion locations in genomes. Our results demonstrate the use of a combination of NGS technology and bioinformatics approaches that offers cost- and time-effective methods for assessing the safety of transgenic plants.

Overexpression of OsERF48 causes regulation of OsCML16, a calmodulin-like protein gene that enhances root growth and drought tolerance.

The AP2/ERF family is a plant-specific transcription factor family whose members have been associated with various developmental processes and stress tolerance. Here, we functionally characterized the drought-inducible OsERF48, a group Ib member of the rice ERF family with four conserved motifs, CMI-1, -2, -3 and -4. A transactivation assay in yeast revealed that the C-terminal CMI-1 motif was essential for OsERF48 transcriptional activity. When OsERF48 was overexpressed in an either a root-specific (ROXOsERF48 ) or whole-body (OXOsERF48 ) manner, transgenic plants showed a longer and denser root phenotype compared to the nontransgenic (NT) controls. When plants were grown on a 40% polyethylene glycol-infused medium under in vitro drought conditions, ROXOsERF48 plants showed a more vigorous root growth than OXOsERF48 and NT plants. In addition, the ROXOsERF48 plants exhibited higher grain yield than OXOsERF48 and NT plants under field-drought conditions. We constructed a putative OsERF48 regulatory network by cross-referencing ROXOsERF48 root-specific RNA-seq data with a co-expression network database, from which we inferred the involvement of 20 drought-related genes in OsERF48-mediated responses. These included genes annotated as being involved in stress signalling, carbohydrate metabolism, cell-wall proteins and drought responses. They included, OsCML16, a key gene in calcium signalling during abiotic stress, which was shown to be a direct target of OsERF48 by chromatin immunoprecipitation-qPCR analysis and a transient protoplast expression assay. Our results demonstrated that OsERF48 regulates OsCML16, a calmodulin-like protein gene that enhances root growth and drought tolerance.

Rice OsERF71-mediated root modification affects shoot drought tolerance.

Drought is the most serious problem that impedes crop development and productivity worldwide. Although several studies have documented the root architecture adaption for drought tolerance, little is known about the underlying molecular mechanisms. Our latest study demonstrated that overexpression of the OsERF71 in rice roots under drought conditions modifies root structure including larger aerenchyma and radial root growth, and thereby, protects the rice plants from drought stresses. The OsERF71-mediated root modifications are caused by combinatory overexpression of general stress-inducible, cell wall-associated and lignin biosynthesis genes that contribute to drought tolerance. Here we addressed that the OsERF71-mediated root modifications alter physiological capacity in shoots without evidence of developmental changes for drought tolerance. Thus, the OsERF71-mediated root modifications provide novel molecular insights into drought tolerance mechanisms.

The rice OsNAC6 transcription factor orchestrates multiple molecular mechanisms involving root structural adaptions and nicotianamine biosynthesis for drought tolerance.

Drought has a serious impact on agriculture worldwide. A plant's ability to adapt to rhizosphere drought stress requires reprogramming of root growth and development. Although physiological studies have documented the root adaption for tolerance to the drought stress, underlying molecular mechanisms is still incomplete, which is essential for crop engineering. Here, we identified OsNAC6-mediated root structural adaptations, including increased root number and root diameter, which enhanced drought tolerance. Multiyear drought field tests demonstrated that the grain yield of OsNAC6 root-specific overexpressing transgenic rice lines was less affected by drought stress than were nontransgenic controls. Genome-wide analyses of loss- and gain-of-function mutants revealed that OsNAC6 up-regulates the expression of direct target genes involved in membrane modification, nicotianamine (NA) biosynthesis, glutathione relocation, 3'-phophoadenosine 5'-phosphosulphate accumulation and glycosylation, which represent multiple drought tolerance pathways. Moreover, overexpression of NICOTIANAMINE SYNTHASE genes, direct targets of OsNAC6, promoted the accumulation of the metal chelator NA and, consequently, drought tolerance. Collectively, OsNAC6 orchestrates novel molecular drought tolerance mechanisms and has potential for the biotechnological development of high-yielding crops under water-limiting conditions.

Overexpression of the OsERF71 Transcription Factor Alters Rice Root Structure and Drought Resistance.

Plant responses to drought stress require the regulation of transcriptional networks via drought-responsive transcription factors, which mediate a range of morphological and physiological changes. AP2/ERF transcription factors are known to act as key regulators of drought resistance transcriptional networks; however, little is known about the associated molecular mechanisms that give rise to specific morphological and physiological adaptations. In this study, we functionally characterized the rice (Oryza sativa) drought-responsive AP2/ERF transcription factor OsERF71, which is expressed predominantly in the root meristem, pericycle, and endodermis. Overexpression of OsERF71, either throughout the entire plant or specifically in roots, resulted in a drought resistance phenotype at the vegetative growth stage, indicating that overexpression in roots was sufficient to confer drought resistance. The root-specific overexpression was more effective in conferring drought resistance at the reproductive stage, such that grain yield was increased by 23% to 42% over wild-type plants or whole-body overexpressing transgenic lines under drought conditions. OsERF71 overexpression in roots elevated the expression levels of genes related to cell wall loosening and lignin biosynthetic genes, which correlated with changes in root structure, the formation of enlarged aerenchyma, and high lignification levels. Furthermore, OsERF71 was found to directly bind to the promoter of OsCINNAMOYL-COENZYME A REDUCTASE1, a key gene in lignin biosynthesis. These results indicate that the OsERF71-mediated drought resistance pathway recruits factors involved in cell wall modification to enable root morphological adaptations, thereby providing a mechanism for enhancing drought resistance.

The NF-YA transcription factor OsNF-YA7 confers drought stress tolerance of rice in an abscisic acid independent manner.

The mechanisms of plant response and adaptation to drought stress require the regulation of transcriptional networks via the induction of drought-responsive transcription factors. Nuclear Factor Y (NF-Y) transcription factors have aroused interest in roles of plant drought stress responses. However, the molecular mechanism of the NF-Y-induced drought tolerance is not well understood. Here, we functionally analyzed two rice NF-YA genes, OsNF-YA7 and OsNF-YA4. Expression of OsNF-YA7 was induced by drought stress and its overexpression in transgenic rice plants improved their drought tolerance. In contrast, OsNF-YA4 expression was not increased by drought stress and its overexpression in transgenic rice plants did not affect their sensitivity to drought stress. OsNF-YA4 expression was highly induced by the stress-related hormone abscisic acid (ABA), while OsNF-YA7 was not, indicating that OsNF-YA7 mediates drought tolerance in an ABA-independent manner. Analysis of the OsNF-YA7 promoter revealed three ABA-independent DRE/CTR elements and RNA-seq analysis identified 48 genes downstream of OsNFYA7 action putatively involved in the OsNF-YA7-mediated drought tolerance pathway. Taken together, our results suggest an important role for OsNF-YA7 in rice drought stress tolerance.

The activities of four constitutively expressed promoters in single-copy transgenic rice plants for two homozygous generations.

MAIN CONCLUSION: We have characterized four novel constitutive promoters ARP1, H3F3, HSP and H2BF3 that are active in all tissues/stages of transgenic plants and stable over two homozygous generations. Gene promoters that are active and stable over several generations in transgenic plants are valuable tools for plant research and biotechnology. In this study, we characterized four putative constitutive promoters (ARP1, H3F3, HSP and H2BF3) in transgenic rice plants. Promoter regions were fused to the green fluorescence protein (GFP) reporter gene and transformed into rice. Single-copy transgenic lines were then selected and promoter activity was analyzed in various organs and tissues of two successive homozygous generations. All four promoters showed a broad expression profile in most tissues and developmental stages, and indeed the expression of the ARP1 and H3F3 promoters was even greater than that of the PGD1 promoter, a previously described constitutive promoter that has been used in transgenic rice. This observation was based on expression levels in leaves, roots, dry seeds and flowers in both the T2 and T3 generations. Each promoter exhibited comparable levels of activity over two homozygous generations with no sign of transgene silencing, which is an important characteristic of promoters to be used in crop biotechnology applications. These promoters therefore have considerable potential value for the stable and constitutive expression of transgenes in monocotyledonous crops.