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The coordinated utilization of nitrogen (N) and phosphorus (P) is vital for plants to maintain nutrient balance and achieve optimal growth. Previously, we revealed a mechanism by which nitrate induces genes for phosphate utilization; this mechanism depends on NRT1.1B-facilitated degradation of cytoplasmic SPX4, which in turn promotes cytoplasmic-nuclear shuttling of PHR2, the central transcription factor of phosphate signaling, and triggers the nitrate-induced phosphate response (NIPR) and N-P coordinated utilization in rice. In this study, we unveiled a fine-tuning mechanism of NIPR in the nucleus regulated by Highly Induced by Nitrate Gene 1 (HINGE1, also known as RLI1), a MYB-transcription factor closely related to PHR2. RLI1/HINGE1, which is transcriptionally activated by PHR2 under nitrate induction, can directly activate the expression of phosphate starvation-induced genes. More importantly, RLI1/HINGE1 competes with PHR2 for binding to its repressor proteins in the nucleus (SPX proteins), and consequently releases PHR2 to further enhance phosphate response. Therefore, RLI1/HINGE1 amplifies the phosphate response in the nucleus downstream of the cytoplasmic SPX4-PHR2 cascade, thereby enabling fine-tuning of N-P balance when nitrate supply is sufficient.

The intensive application of inorganic nitrogen underlies marked increases in crop production, but imposes detrimental effects on ecosystems1,2: it is therefore crucial for future sustainable agriculture to improve the nitrogen-use efficiency of crop plants. Here we report the genetic basis of nitrogen-use efficiency associated with adaptation to local soils in rice (Oryza sativa L.). Using a panel of diverse rice germplasm collected from different ecogeographical regions, we performed a genome-wide association study on the tillering response to nitrogen-the trait that is most closely correlated with nitrogen-use efficiency in rice-and identified OsTCP19 as a modulator of this tillering response through its transcriptional response to nitrogen and its targeting to the tiller-promoting gene DWARF AND LOW-TILLERING (DLT)3,4. A 29-bp insertion and/or deletion in the OsTCP19 promoter confers a differential transcriptional response and variation in the tillering response to nitrogen among rice varieties. The allele of OsTCP19 associated with a high tillering response to nitrogen is prevalent in wild rice populations, but has largely been lost in modern cultivars: this loss correlates with increased local soil nitrogen content, which suggests that it might have contributed to geographical adaptation in rice. Introgression of the allele associated with a high tillering response into modern rice cultivars boosts grain yield and nitrogen-use efficiency under low or moderate levels of nitrogen, which demonstrates substantial potential for rice breeding and the amelioration of negative environment effects by reducing the application of nitrogen to crops.

Nitrogen-use efficiency of indica varieties of rice is superior to that of japonica varieties. We apply 16S ribosomal RNA gene profiling to characterize root microbiota of 68 indica and 27 japonica varieties grown in the field. We find that indica and japonica recruit distinct root microbiota. Notably, indica-enriched bacterial taxa are more diverse, and contain more genera with nitrogen metabolism functions, than japonica-enriched taxa. Using genetic approaches, we provide evidence that NRT1.1B, a rice nitrate transporter and sensor, is associated with the recruitment of a large proportion of indica-enriched bacteria. Metagenomic sequencing reveals that the ammonification process is less abundant in the root microbiome of the nrt1.1b mutant. We isolated 1,079 pure bacterial isolates from indica and japonica roots and derived synthetic communities (SynComs). Inoculation of IR24, an indica variety, with an indica-enriched SynCom improved rice growth in organic nitrogen conditions compared with a japonica-enriched SynCom. The links between plant genotype and root microbiota membership established in this study will inform breeding strategies to improve nitrogen use in crops.

Plant hormone cytokinin signals through histidine-aspartic acid (H-D) phosphorelay to regulate plant growth and development. While it is well known that the phosphorelay involves histidine kinases, histidine phosphotransfer proteins (HPs), and response regulators (RRs), how this process is regulated by external components remains unknown. Here we demonstrate that protein phosphatase with kelch-like domains (PPKL1), known as a signaling component of steroid hormone brassinosteroid, is actually a cryptic inhibitor of cytokinin phosphorelay in rice (Oryza sativa). Mutation at a specific amino acid D364 of PPKL1 activates cytokinin response and thus enlarges grain size in a semi-dominant mutant named s48. Overexpression of PPKL1 containing D364, either with the deletion of the phosphatase domain or not, rescues the s48 mutant phenotype. PPKL1 interacts with OsAHP2, one of authentic HPs, and D364 resides in a region resembling the receiver domain of RRs. Accordingly, PPKL1 can utilize D364 to suppress OsAHP2-to-RR phosphorelay, whereas mutation of D364 abolishes the effect. This function of PPKL1 is independent of the phosphatase domain that is required for brassinosteroid signaling. Importantly, editing of the D364-residential region produces a diversity of semi-dominant mutations associated with variously increased grain sizes. Further screening of the edited plants enables the identification of two genotypes that confer significantly improved grain yield. Collectively, our study uncovers a noncanonical cytokinin signaling suppressor and provides a robust tool for seed rational design.

The intensive application of inorganic nitrogen underlies marked increases in crop production, but imposes detrimental effects on ecosystems

Glycosyltransferase OsUGT90A1 helps to protect cellular membranes during chilling and salt stress by enhancing antioxidant enzyme activity to reduce ROS levels, and to balance the stress response with growth., Due to its subtropical origins, rice (Oryza sativa) is sensitive to low-temperature stress. In this study, we identify LOC_Os04g24110, annotated to encode the UDP-glycosyltransferase enzyme UGT90A1, as a gene associated with the low-temperature seedling survivability (LTSS) quantitative trait locus qLTSS4-1. Differences between haplotypes in the control region of OsUGT90A1 correlate with chilling tolerance phenotypes, and reflect differential expression between tolerant and sensitive accessions rather than differences in protein sequences. Expression of OsUGT90A1 is initially enhanced by low temperature, and its overexpression helps to maintain membrane integrity during cold stress and promotes leaf growth during stress recovery, which are correlated with reduced levels of reactive oxygen species due to increased activities of antioxidant enzymes. In addition, overexpression of OsUGT90A1 in Arabidopsis improves freezing survival and tolerance to salt stress, again correlated with enhanced activities of antioxidant enzymes. Overexpression of OsUGT90A1 in rice decreases root lengths in 3-week-old seedlings while gene-knockout increases the length, indicating that its differential expression may affect phytohormone activities. We conclude that higher OsUGT90A1 expression in chilling-tolerant accessions helps to maintain cell membrane integrity as an abiotic stress-tolerance mechanism that prepares plants for the resumption of growth and development during subsequent stress recovery.

In plants, microRNA (miRNA) functions in the post-transcriptional repression of target mRNAs have been well explored. However, the mechanisms regulating the accumulation of miRNAs remain poorly understood. Here, we report that distinct mechanisms regulate accumulation of a monocot-specific miRNA, rice (Oryza sativa) miR528. At the transcriptional level, miR528 accumulated to higher levels in older plants than in young seedlings and exhibited aging-modulated gradual accumulation and diurnal rhythms in leaves; at the post-transcriptional level, aging also modulated miR528 levels by enhancing pri-miR528 alternative splicing. We found that miR528 promotes rice flowering under long-day conditions by targeting RED AND FAR-RED INSENSITIVE 2 (OsRFI2). Moreover, natural variations in the MIR528 promoter region caused differences in miR528 expression among rice varieties, which are correlated with their different binding affinities with the transcription factor OsSPL9 that activates the expression of miR528. Taken together, our findings reveal rice plants have evolved sophisticated modes fine-tuning miR528 levels and provide insight into the mechanisms that regulate MIRNA expression in plants.

To ensure high crop yields in a sustainable manner, a comprehensive understanding of the control of nutrient acquisition is required. In particular, the signalling networks controlling the coordinated utilization of the two most highly demanded mineral nutrients, nitrogen and phosphorus, are of utmost importance. Here, we reveal a mechanism by which nitrate activates both phosphate and nitrate utilization in rice (Oryza sativa L.). We show that the nitrate sensor NRT1.1B interacts with a phosphate signalling repressor SPX4. Nitrate perception strengthens the NRT1.1B-SPX4 interaction and promotes the ubiquitination and degradation of SPX4 by recruiting NRT1.1B interacting protein 1 (NBIP1), an E3 ubiquitin ligase. This in turn allows the key transcription factor of phosphate signalling, PHR2, to translocate to the nucleus and initiate the transcription of phosphorus utilization genes. Interestingly, the central transcription factor of nitrate signalling, NLP3, is also under the control of SPX4. Thus, nitrate-triggered degradation of SPX4 activates both phosphate- and nitrate-responsive genes, implementing the coordinated utilization of nitrogen and phosphorus.

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MicroRNAs (miRNAs) play key regulatory roles in numerous developmental and physiological processes in animals and plants. The elaborate mechanism of miRNA biogenesis involves transcription and multiple processing steps. Here, we report the identification of a pair of evolutionarily conserved NOT2_3_5 domain–containing-proteins, NOT2a and NOT2b (previously known as At-Negative on TATA less2 [NOT2] and VIRE2-INTERACTING PROTEIN2, respectively), as components involved in Arabidopsis thaliana miRNA biogenesis. NOT2 was identified by its interaction with the Piwi/Ago/Zwille domain of DICER-LIKE1 (DCL1), an interaction that is conserved between rice (Oryza sativa) and Arabidopsis thaliana. Inactivation of both NOT2 genes in Arabidopsis caused severe defects in male gametophytes, and weak lines show pleiotropic defects reminiscent of miRNA pathway mutants. Impairment of NOT2s decreases the accumulation of primary miRNAs and mature miRNAs and affects DCL1 but not HYPONASTIC LEAVES1 (HYL1) localization in vivo. In addition, NOT2b protein interacts with polymerase II and other miRNA processing factors, including two cap binding proteins, CBP80/ABH1, CBP20, and SERRATE (SE). Finally, we found that the mRNA levels of some protein coding genes were also affected. Therefore, these results suggest that NOT2 proteins act as general factors to promote the transcription of protein coding as well as miRNA genes and facilitate efficient DCL1 recruitment in miRNA biogenesis.

Although the role of H3K9 methylation in rice (Oryza sativa) is unclear, in Arabidopsis thaliana the loss of histone H3K9 methylation by mutation of Kryptonite [also known as SU(VAR)3-9 homolog] reduces genome-wide DNA methylation and increases the transcription of transposable elements. Here, we report that rice SDG714 (for SET Domain Group Protein714) encodes a histone H3K9-specific methyltransferase. The C terminus of SDG714 confers enzymatic activity and substrate specificity, whereas the N terminus localizes it in the nucleus. Loss-of-function mutants of SDG714 (SDG714IR transformants) generated by RNA interference display a mostly glabrous phenotype as a result of the lack of macro trichomes in glumes, leaves, and culms compared with control plants. These mutants also show decreased levels of CpG and CNG cytosine methylation as well as H3K9 methylation at the Tos17 locus, a copia-like retrotransposon widely used for the generation of rice mutants. Most interestingly, loss of function of SDG714 can enhance transcription and cause the transposition of Tos17. Together, these results suggest that histone H3K9 methylation mediated by SDG714 is involved in DNA methylation, the transposition of transposable elements, and genome stability in rice.

Elongation of rice internodes is one of the most important agronomic traits, which determines the plant height and underlies the grain yield. It has been shown that the elongation of internodes is under genetic control, and various factors are implicated in the process. Here, we report a detailed characterization of an elongated uppermost internode1 (eui1) mutant, which has been used in hybrid rice breeding. In the eui1-2 mutant, the cell lengths in the uppermost internodes are significantly longer than that of wild type and thus give rise to the elongated uppermost internode. It was found that the level of active gibberellin was elevated in the mutant, whereas its growth in response to gibberellin is similar to that of the wild type, suggesting that the higher level accumulation of gibberellin in the eui1 mutant causes the abnormal elongation of the uppermost internode. Consistently, the expression levels of several genes which encode gibberellin biosynthesis enzymes were altered. We cloned the EUI1 gene, which encodes a putative cytochrome P450 monooxygenase, by map-based cloning and found that EUI1 was weakly expressed in most tissues, but preferentially in young panicles. To confirm its function, transgenic experiments with different constructs of EUI1 were conducted. Overexpression of EUI1 gave rise to the gibberellin-deficient-like phenotypes, which could be partially reversed by supplementation with gibberellin. Furthermore, apart from the alteration of expression levels of the gibberellin biosynthesis genes, accumulation of SLR1 protein was found in the overexpressing transgenic plants, indicating that the expression level of EUI1 is implicated in both gibberellin-mediated SLR1 destruction and a feedback regulation in gibberellin biosynthesis. Therefore, we proposed that EUI1 plays a negative role in gibberellin-mediated regulation of cell elongation in the uppermost internode of rice.

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are two types of noncoding RNAs involved in developmental regulation, genome maintenance, and defense in eukaryotes. The activity of Dicer or Dicer-like (DCL) proteins is required for the maturation of miRNAs and siRNAs. In this study, we cloned and sequenced 66 candidate rice (Oryza sativa) miRNAs out of 1,650 small RNA sequences (19 to approximately 25 nt), and they could be further grouped into 21 families, 12 of which are newly identified and three of which, OsmiR528, OsmiR529, and OsmiR530, have been confirmed by northern blot. To study the function of rice DCL proteins (OsDCLs) in the biogenesis of miRNAs and siRNAs, we searched genome databases and identified four OsDCLs. An RNA interference approach was applied to knock down two OsDCLs, OsDCL1 and OsDCL4, respectively. Strong loss of function of OsDCL1IR transformants that expressed inverted repeats of OsDCL1 resulted in developmental arrest at the seedling stage, and weak loss of function of OsDCL1IR transformants caused pleiotropic developmental defects. Moreover, all miRNAs tested were greatly reduced in OsDCL1IR but not OsDCL4IR transformants, indicating that OsDCL1 plays a critical role in miRNA processing in rice. In contrast, the production of siRNA from transgenic inverted repeats and endogenous CentO regions were not affected in either OsDCL1IR or OsDCL4IR transformants, suggesting that the production of miRNAs and siRNAs is via distinct OsDCLs.

A rice mutant with reduced heading date (designated rhd1) found in a transgenic line of cultivar Teqing 2 (Oryza sativa L. ssp. indica) was used to identify the genes related to rice heading and thereby to study its molecular mechanism. Genetic analysis showed that rhd1 was a dominant mutation and did not result from T-DNA insertion. By using the differential display polymerase chain reaction (DD-PCR) technique, differential gene expression between rhd1 and Teqing 2 was compared and a rhd1-down-regulated c DNA fragment was identified. Sequence analysis showed that this fragment shared 99% similarity to the OsGRF1 (O. sativa growth-regulating factor 1) gene. The OsGRF1 gene encodes a putative transcription factor, which contains two conserved regions: the QLQ (Gln, Leu, Gln) and WRC (Trp, Arg, Cys) domains. Southern analysis indicates that OsGRF1 may be encoded by single copy gene in the rice genome. RNA interference results revealed that transgenic lines with reduced OsGRF1 transcript displayed delayed growth and development, developed small leaves, and had delayed heading. The extent of the phenotypes developed was well-correlated with the OsGRF1 gene transcript. Our results clearly demonstrate that the OsGRF1 gene is not only involved in regulating growth at the juvenile stage, but that it may also be involved in the regulation of heading in rice. (Managing editor: Li-Hui ZHAO)

ThegdcsP promoter isolated from C3-C4 intermediate plantFlaveria anomala was fused to the β-glucuronidase (GUS) gene. The chimeric gene was inserted into the binary vector pBin19 and introduced into the rice (Oryza sativa L.) cv. 8706 byAgrobacteriummediated gene transfer. GUS activity can be detected in leaf, leaf sheath, stem and root tissues via fluorometric GUS assay. However, no GUS activity was found in mature endosperm. Histochemical localization revealed that GUS expression was exclusively restricted to vascular tissues in transgenic plants. This promoter also showed spatial-temporal expression patterns that GUS expression declined significantly with the maturity of plants. These expression patterns make thegdcsP promoter extremely valuable in the applied biotechnology that needs target gene expression restricted to vascular tissues.

A cloned gene, Xa21 was transferred into five widely-used Chinese rice varieties through an Agrobacterium- mediated system, and over 110 independent transgenic lines were obtained. PCR and Southern analysis of transgenic plants revealed the integration of the whole Xa21 gene into the host genomes. The integrated Xa21 gene was stably inherited, and segregated in a 3: 1 ratio in the selfed T(1) generation when one copy of the gene was integrated in the transformants. Inoculation tests displayed that transgenic T(0) plants and Xa21 PCR-positive T(1) plants were highly resistant to bacterial blight disease. The selected Xa21 homozygous resistant transgenic lines with desirable qualities may be propagated as new varieties or utilized in hybrid rice breeding.

Higher plants have evolved multiple RNA-dependent RNA polymerases (RDRs), which work with Dicer-like (DCL) proteins to produce different classes of small RNAs with specialized molecular functions. Here we report that OsRDR6, the rice (Oryza sativa L.) homolog of Arabidopsis RDR6, acts in the biogenesis of various types and sizes of small RNAs. We isolated a rice osrdr6-1 mutant, which was temperature sensitive and showed spikelet defects. This mutant displays reduced accumulation of tasiR-ARFs, the conserved trans-acting siRNAs (tasiRNAs) derived from the TAS3 locus, and ectopic expression of tasiR-ARF target genes, the Auxin Response Factors (including ARF2 and ARF3/ETTIN). The loss of tasiR-mediated repression of ARFs in osrdr6-1 can explain its morphological defects, as expression of two non-targeted ARF3 gene constructs (ARF3muts) in a wild-type background mimics the osrdr6 and osdcl4-1 mutant phenotypes. Small RNA high-throughput sequencing also reveals that besides tasiRNAs, 21-nucleotide (nt) phased small RNAs are also largely dependent on OsRDR6. Unexpectedly, we found that osrdr6-1 has a strong impact on the accumulation of 24-nt phased small RNAs, but not on unphased ones. Our work uncovers the key roles of OsRDR6 in small RNA biogenesis and directly illustrates the crucial functions of tasiR-ARFs in rice development.

Higher plants have evolved multiple proteins in the RNase III family to produce and regulate different classes of small RNAs with specialized molecular functions. In rice (Oryza sativa), numerous genomic clusters are targeted by one of two microRNAs (miRNAs), miR2118 and miR2275, to produce secondary small interfering RNAs (siRNAs) of either 21 or 24 nucleotides in a phased manner. The biogenesis requirements or the functions of the phased small RNAs are completely unknown. Here we examine the rice Dicer-Like (DCL) family, including OsDCL1, -3a, -3b and -4. By deep sequencing of small RNAs from different tissues of the wild type and osdcl4-1, we revealed that the processing of 21-nucleotide siRNAs, including trans-acting siRNAs (tasiRNA) and over 1000 phased small RNA loci, was largely dependent on OsDCL4. Surprisingly, the processing of 24-nucleotide phased small RNA requires the DCL3 homolog OsDCL3b rather than OsDCL3a, suggesting functional divergence within DCL3 family. RNA ligase-mediated 5' rapid amplification of cDNA ends and parallel analysis of RNA ends (PARE)/degradome analysis confirmed that most of the 21- and 24-nucleotide phased small RNA clusters were initiated from the target sites of miR2118 and miR2275, respectively. Furthermore, the accumulation of the two triggering miRNAs requires OsDCL1 activity. Finally, we show that phased small RNAs are preferentially produced in the male reproductive organs and are likely to be conserved in monocots. Our results revealed significant roles of OsDCL4, OsDCL3b and OsDCL1 in the 21- and 24-nucleotide phased small RNA biogenesis pathway in rice.

About 25,000 rice T-DNA insertional mutant lines were generated using the vector pCAS04 which has both promoter-trapping and activation-tagging function. Southern blot analysis revealed that about 40% of these mutants were single copy integration and the average T-DNA insertion number was 2.28. By extensive phenotyping in the field, quite a number of agronomically important mutants were obtained. Histochemical GUS assay with 4,310 primary mutants revealed that the GUS-staining frequency was higher than that of the previous reports in various tissues and especially high in flowers. The T-DNA flanking sequences of some mutants were isolated and the T-DNA insertion sites were mapped to the rice genome. The flanking sequence analysis demonstrated the different integration pattern of the right border and left border into rice genome. Compared with Arabidopsis and poplar, it is much varied in the T-DNA border junctions in rice.

Betaine as one of osmolytes plays an important role in osmoregulation of most high plants. Betaine aldehyde dehydrogenase (BADH) is the second enzyme involved in betaine biosynthesis. The BADH gene from a halophite,Atriplex hortensis, was transformed into rice cultivars by bombarment method. Totally 192 transgenic rice plants were obtained and most of them had higher salt tolerance than controls. Among transgenic plants transplanted in the saline pool containing 0.5% NaCl in a greenhouse, 22 survived. 13 of which set seeds, and the frequency of seed setting was very low, only 10%. But the controls could not grow under the same condition. The results of BADH activity assay and Northern blot showed that the BADH gene was integrated into chromosomes of transgenic plants and expressed.