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Summary: 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. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Plant stress responses and mechanisms determining tolerance are controlled by diverse sets of genes. Transcription factors (TFs) have been implicated in conferring drought tolerance under drought stress conditions, and the identification of their target genes can elucidate molecular regulatory networks that orchestrate tolerance mechanisms. Results: We generated transgenic rice plants overexpressing the 4 rice TFs, OsNAC5, 6, 9, and 10, under the control of the root-specific RCc3 promoter. We showed that they were tolerant to drought stress with reduced loss of grain yield under drought conditions compared with wild type plants. To understand the molecular mechanisms underlying this tolerance, we here performed chromatin immunoprecipitation (ChIP)-Seq and RNA-Seq analyses to identify the direct target genes of the OsNAC proteins using the RCc3:6MYC-OsNAC expressing roots. A total of 475 binding loci for the 4 OsNAC proteins were identified by cross-referencing their binding to promoter regions and the expression levels of the corresponding genes. The binding loci were distributed among the promoter regions of 391 target genes that were directly up-regulated by one of the OsNAC proteins in four RCc3:6MYC-OsNAC transgenic lines. Based on gene ontology (GO) analysis, the direct target genes were related to transmembrane/transporter activity, vesicle, plant hormones, carbohydrate metabolism, and TFs. The direct targets of each OsNAC range from 4.0-8.7% of the total number of up-regulated genes found in the RNA-Seq data sets. Thus, each OsNAC up-regulates a set of direct target genes that alter root system architecture in the RCc3:OsNAC plants to confer drought tolerance. Our results provide a valuable resource for functional dissection of the molecular mechanisms of drought tolerance. Conclusions: Many of the target genes, including transmembrane/transporter, vesicle related, auxin/hormone related, carbohydrate metabolic processes, and transcription factor genes, that are up-regulated by OsNACs act as the cellular components which would alter the root architectures of RCc3:OsNACs for drought tolerance. [ABSTRACT FROM AUTHOR]

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 Os ERF48, 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 Os ERF48 transcriptional activity. When Os ERF48 was overexpressed in an either a root-specific ( ROX O s ERF 48) or whole-body ( OX O s ERF 48) 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, ROX O s ERF 48 plants showed a more vigorous root growth than OX O s ERF 48 and NT plants. In addition, the ROX O s ERF 48 plants exhibited higher grain yield than OX O s ERF 48 and NT plants under field-drought conditions. We constructed a putative Os ERF48 regulatory network by cross-referencing ROX O s ERF 48 root-specific RNA-seq data with a co-expression network database, from which we inferred the involvement of 20 drought-related genes in Os ERF48-mediated responses. These included genes annotated as being involved in stress signalling, carbohydrate metabolism, cell-wall proteins and drought responses. They included, Os CML16, a key gene in calcium signalling during abiotic stress, which was shown to be a direct target of Os ERF48 by chromatin immunoprecipitation- qPCR analysis and a transient protoplast expression assay. Our results demonstrated that Os ERF48 regulates Os CML16, a calmodulin-like protein gene that enhances root growth and drought tolerance. [ABSTRACT FROM AUTHOR]

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 Os NAC6-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 Os NAC6 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 Os NAC6 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 Os NAC6, promoted the accumulation of the metal chelator NA and, consequently, drought tolerance. Collectively, Os NAC6 orchestrates novel molecular drought tolerance mechanisms and has potential for the biotechnological development of high-yielding crops under water-limiting conditions. [ABSTRACT FROM AUTHOR]

Auxin signaling is a fundamental part of many plant growth processes and stress responses and operates through Aux/IAA protein degradation and the transmission of the signal via auxin response factors (ARFs). A total of 31 Aux/IAA genes have been identified in rice ( Oryza sativa ), some of which are induced by drought stress. However, the mechanistic link between Aux/IAA expression and drought responses is not well understood. In this study we found that the rice Aux/IAA gene OsIAA6 is highly induced by drought stress and that its overexpression in transgenic rice improved drought tolerance, likely via the regulation of auxin biosynthesis genes. We observed that OsIAA6 was specifically expressed in the axillary meristem of the basal stem, which is the tissue that gives rise to tillers. A knock-down mutant of OsIAA6 showed abnormal tiller outgrowth, apparently due to the regulation of the auxin transporter OsPIN1 and the rice tillering inhibitor OsTB1. Our results confirm that the OsIAA6 gene is involved in drought stress responses and the control of tiller outgrowth. [ABSTRACT FROM AUTHOR]

Drought conditions are among the most serious challenges to crop production worldwide. Here, we report the results of field evaluations of transgenic rice plants overexpressing OsNAC5, under the control of either the root-specific ( RCc3) or constitutive ( GOS2) promoters. Field evaluations over three growing seasons revealed that the grain yield of the RCc3:OsNAC5 and GOS2:OsNAC5 plants were increased by 9%-23% and 9%-26% under normal conditions, respectively. Under drought conditions, however, RCc3:OsNAC5 plants showed a significantly higher grain yield of 22%-63%, whilst the GOS2:OsNAC5 plants showed a reduced or similar yield to the nontransgenic ( NT) controls. Both the RCc3:OsNAC5 and GOS2:OsNAC5 plants were found to have larger roots due to an enlarged stele and aerenchyma at flowering stage. Cell numbers per cortex layer and stele of developing roots were higher in both transgenic plants than NT controls, contributing to the increase in root diameter. The root diameter was enlarged to a greater extent in the RCc3:OsNAC5, suggesting the importance of this phenotype for enhanced drought tolerance. Microarray experiments identified 25 up-regulated genes by more than three-fold ( P < 0.01) in the roots of both transgenic lines. Also identified were 19 and 18 up-regulated genes that are specific to the RCc3:OsNAC5 and GOS2:OsNAC5 roots, respectively. Of the genes specifically up-regulated in the RCc3:OsNAC5 roots, GLP, PDX, MERI5 and O-methyltransferase were implicated in root growth and development. Our present findings demonstrate that the root-specific overexpression of OsNAC5 enlarges roots significantly and thereby enhances drought tolerance and grain yield under field conditions. [ABSTRACT FROM AUTHOR]

Drought conditions limit agricultural production by preventing crops from reaching their genetically predetermined maximum yields. Here, we present the results of field evaluations of rice overexpressing OsNAC9, a member of the rice NAC domain family. Root-specific ( RCc3) and constitutive ( GOS2) promoters were used to overexpress OsNAC9 and produced the transgenic RCc3:OsNAC9 and GOS2:OsNAC9 plants. Field evaluations over two cultivating seasons showed that grain yields of the RCc3:OsNAC9 and the GOS2:OsNAC9 plants were increased by 13%-18% and 13%-32% under normal conditions, respectively. Under drought conditions, RCc3:OsNAC9 plants showed an increased grain yield of 28%-72%, whilst the GOS2:OsNAC9 plants remained unchanged. Both transgenic lines exhibited altered root architecture involving an enlarged stele and aerenchyma. The aerenchyma of RCc3:OsNAC9 roots was enlarged to a greater extent than those of GOS2:OsNAC9 and non-transgenic (NT) roots, suggesting the importance of this phenotype for enhanced drought resistance. Microarray experiments identified 40 up-regulated genes by more than threefold ( P < 0.01) in the roots of both transgenic lines. These included 9-cis-epoxycarotenoid dioxygenase, an ABA biosynthesis gene, calcium-transporting ATPase, a component of the Ca2+ signalling pathway involved in cortical cell death and aerenchyma formation, cinnamoyl CoA reductase 1, a gene involved in lignin biosynthesis, and wall-associated kinases¸ genes involved in cell elongation and morphogenesis. Interestingly, O-methyltransferase, a gene necessary for barrier formation, was specifically up-regulated only in the RCc3:OsNAC9 roots. Such up-regulated genes that are commonly and specifically up-regulated in OsNAC9 transgenic roots may account for the altered root architecture conferring increased drought resistance phenotype. [ABSTRACT FROM AUTHOR]

Coordination of multiple transgenes is essential for metabolic engineering of biosynthetic pathways. Here, we report the utilization of two bicistronic systems involving the 2A sequence from the foot-and-mouth disease virus and the internal ribosome entry site (IRES) sequence from the crucifer-infecting tobamovirus to the biosynthesis of carotenoids in rice endosperm. Two carotenoid biosynthetic genes, phytoene synthase ( Psy) from Capsicum and carotene desaturase ( CrtI) from Pantoea, were linked via either the synthetic 2A sequence that was optimized for rice codons or the IRES sequence under control of the rice globulin promoter, generating PAC ( sy-2 - rtI) and PIC ( sy- RES- rtI) constructs, respectively. The transgenic endosperm of PAC rice had a more intense golden color than did PIC rice, demonstrating that 2A was more efficient than IRES in coordinating gene expression. The 2A and IRES constructs were equally effective in driving transgene transcription. However, immunoblot analysis of CRTI, a protein encoded by the downstream open reading frame of the bicistronic constructs, revealed that 2A was ninefold more effective than IRES in driving translation. The PAC endosperms accumulated an average of 1.3 μg/g of total carotenoids, which was ninefold higher than was observed for PIC endosperms. In particular, accumulation of β-carotene was much higher in PAC endosperms than in PIC endosperms. Collectively, these results demonstrate that both 2A and IRES systems can coordinate the expression of two biosynthetic genes, with the 2A system exhibiting greater efficiency. Thus, the 2A expression system described herein is an effective new tool for multigene stacking in crop biotechnology. [ABSTRACT FROM AUTHOR]

Drought is one of the major environmental stresses adversely affecting crop productivity worldwide. Precise characterization of genes involved in drought response is necessary to develop new crop varieties with enhanced drought tolerance. Previously, we identified 66 drought-induced miRNAs in rice plants. For the further functional investigation of the miRNAs, we applied recombinant codon-optimized Cas9 (rCas9) for rice with single-guide RNAs specifically targeting mature miRNA sequences or sites required for the biogenesis of mature miRNA. A total of 458 T0 transgenic plants were analyzed to determine the frequency and type of mutations induced by CRISPR/rCas9 on 13 independent target miRNAs. The average mutation frequency for 13 genes targeted by single guide RNAs (sgRNAs) in T0 generation was 59.4%, including mono-allelic (8.54%), bi-allelic (11.1%), and hetero-allelic combination (39.7%) mutations. The mutation frequency showed a positive correlation with Tm temperature of sgRNAs. For base insertion, one base insertion (99%) was predominantly detected in transgenic plants. Similarly, one base deletion accounted for the highest percentage, but there was also a significant percentage of cases in which more than one base was deleted. The deletion of more than two bases in OsmiR171f and OsmiR818b significantly reduced the level of corresponding mature miRNAs. Further functional analysis using CRISPR/Cas9-mediated mutagenesis confirmed that OsmiR818b is involved in drought response in rice plants. Overall, this study suggests that the CRISPR/rCas9 system is a powerful tool for loss-of-function analysis of miRNA in rice. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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., (© 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Background: Plant transcriptome profiling has provided a tool for understanding the mechanisms by which plants respond to stress conditions. Analysis of genome-wide transcriptome will provides a useful dataset of drought responsive noncoding RNAs and their candidate target genes that may be involved in drought stress responses., Results: Here RNA-seq analyses of leaves from drought stressed rice plants was performed, producing differential expression profiles of noncoding RNAs. We found that the transcript levels of 66 miRNAs changed significantly in response to drought conditions and that they were negatively correlated with putative target genes during the treatments. The negative correlations were further validated by qRT-PCR using total RNAs from both drought-treated leaves and various tissues at different developmental stages. The drought responsive miRNA/target pairs were confirmed by the presence of decay intermediates generated by miRNA-guided cleavages in Parallel Analysis of RNA Ends (PARE) libraries. We observed that the precursor miR171f produced two different mature miRNAs, miR171f-5p and miR171f-3p with 4 candidate target genes, the former of which was responsive to drought conditions. We found that the expression levels of the miR171f precursor negatively correlated with those of one candidate target gene, but not with the others, suggesting that miR171f-5p was drought-responsive, with Os03g0828701-00 being a likely target. Pre-miRNA expression profiling indicated that miR171f is involved in the progression of rice root development and growth, as well as the response to drought stress. Ninety-eight lncRNAs were also identified, together with their corresponding antisense transcripts, some of which were responsive to drought conditions., Conclusions: We identified rice noncoding RNAs (66 miRNAs and 98 lncRNAs), whose expression was highly regulated by drought stress conditions, and whose transcript levels negatively correlated with putative target genes.

CORONATINE INSENSITIVE 1 (COI1) encodes an E3 ubiquitin ligase complex component that interacts with JAZ proteins and targets them for degradation in response to JA signaling. The Arabidopsis genome has a single copy of COI1, but the Oryza sativa genome has three closely related COI homologs. To examine the functions of the three OsCOIs, we used yeast two-hybrid assays to examine their interactions with JAZ proteins and found that OsCOIs interacted with OsJAZs and with JAZs, in a coronatine dependent manner. We also tested whether OsCOI1a and OsCOI1b could complement Arabidopsis coi1-1 mutants and found that overexpression of either gene in the coi1-1 mutant resulted in restoration of JA signal transduction and production of seeds, indicating successful complementation. Although OsCOI2 interacted with a few OsJAZs, we were not able to successfully complement the coi1-1 mutant with OsCOI2. Molecular modeling revealed that the three OsCOIs adopt 3D structures similar to COI1. Structural differences resulting from amino acid variations, especially among amino acid residues involved in the interaction with coronatine and JAZ proteins, were tested by mutation analysis. When His-391 in OsCOI2 was substituted with Tyr-391, OsCOI2 interacted with a wider range of JAZ proteins, including OsJAZ1, 2, 5∼9 and 11, and complemented coi1-1 mutants at a higher frequency than the other OsCOIs and COI1. These results indicate that the three OsCOIs are orthologues of COI1 and play key roles in JA signaling.