It has long been established that premature leaf senescence negatively impacts the yield stability of rice, but the underlying molecular mechanism driving this relationship remains largely unknown. Here, we identified a dominant premature leaf senescence mutant, prematurely senile 1 (ps1-D). PS1 encodes a plantspecific NAC (no apical meristem, Arabidopsis ATAF1/2, and cupshaped cotyledon2) transcriptional activator, Oryza sativa NAC-like, activated by apetala3/pistillata (OsNAP). Overexpression of OsNAP significantly promoted senescence, whereas knockdown of OsNAP produced a marked delay of senescence, confirming the role of this gene in the development of rice senescence. OsNAP expression was tightly linked with the onset of leaf senescence in an age-dependent manner. Similarly, ChIP-PCR and yeast onehybrid assays demonstrated that OsNAP positively regulates leaf senescence by directly targeting genes related to chlorophyll degradation and nutrient transport and other genes associated with senescence, suggesting that OsNAP is an ideal marker of senescence onset in rice. Further analysis determined that OsNAP is induced specifically by abscisic acid (ABA), whereas its expression is repressed in both aba1 and aba2, two ABA biosynthetic mutants. Moreover, ABA content is reduced significantly in ps1-D mutants, indicating a feedback repression of OsNAP on ABA biosynthesis. Our data suggest that OsNAP serves as an important link between ABA and leaf senescence. Additionally, reduced OsNAP expression leads to delayed leaf senescence and an extended grain-filling period, resulting in a 6.3% and 10.3% increase in the grain yield of two independent representative RNAi lines, respectively. Thus, fine-tuning OsNAP expression should be a useful strategy for improving rice yield in the future.hormones | nutrition remobilization | programmed cell death L eaf senescence is an integral part of the final stages of plant development and is controlled by a fine-tuned, complex regulatory network (1). During senescence, leaf cells undergo dramatic changes in cellular metabolism, structure, and gene expression (2, 3). The most striking feature of these changes is the yellowing of the leaves caused by the breakdown of chlorophyll during chloroplast degeneration, followed by the hydrolysis of macromolecules such as lipids, proteins, and nucleic acids, which, in turn, results in mitochondria and nuclei dissociation and cell death (4, 5). This process facilitates both hydrolysis and the recycling of nutrients from source to sink tissues to increase reproductive success (6). Thus, senescence is not a passive process but rather is a developmentally programmed procedure that has a strong adaptive advantage (7,8). Although leaf senescence is controlled primarily by developmental age, the onset and progression of this process also is influenced by a number of endogenous and external factors (1, 9, 10). For example, abscisic acid (ABA) is thought to be one of the phytohormones that promote leaf senescence (11, 12). Specifically, both an upregu...

Asian cultivated rice (Oryza sativa L.) consists of two main subspecies, indica and japonica. Indica has higher nitrate-absorption activity than japonica, but the molecular mechanisms underlying that activity remain elusive. Here we show that variation in a nitrate-transporter gene, NRT1.1B (OsNPF6.5), may contribute to this divergence in nitrate use. Phylogenetic analysis revealed that NRT1.1B diverges between indica and japonica. NRT1.1B-indica variation was associated with enhanced nitrate uptake and root-to-shoot transport and upregulated expression of nitrate-responsive genes. The selection signature of NRT1.1B-indica suggests that nitrate-use divergence occurred during rice domestication. Notably, field tests with near-isogenic and transgenic lines confirmed that the japonica variety carrying the NRT1.1B-indica allele had significantly improved grain yield and nitrogen-use efficiency (NUE) compared to the variety without that allele. Our results show that variation in NRT1.1B largely explains nitrate-use divergence between indica and japonica and that NRT1.1B-indica can potentially improve the NUE of japonica.

Nitric oxide (NO) is a key redox-active, small molecule involved in various aspects of plant growth and development. Here, we report the identification of an NO accumulation mutant, nitric oxide excess1 (noe1), in rice (Oryza sativa), the isolation of the corresponding gene, and the analysis of its role in NO-mediated leaf cell death. Map-based cloning revealed that NOE1 encoded a rice catalase, OsCATC. Furthermore, noe1 resulted in an increase of hydrogen peroxide (H 2 O 2 ) in the leaves, which consequently promoted NO production via the activation of nitrate reductase. The removal of excess NO reduced cell death in both leaves and suspension cultures derived from noe1 plants, implicating NO as an important endogenous mediator of H 2 O 2 -induced leaf cell death. Reduction of intracellular S-nitrosothiol (SNO) levels, generated by overexpression of rice S-nitrosoglutathione reductase gene (GSNOR1), which regulates global levels of protein S-nitrosylation, alleviated leaf cell death in noe1 plants. Thus, S-nitrosylation was also involved in light-dependent leaf cell death in noe1. Utilizing the biotin-switch assay, nanoliquid chromatography, and tandem mass spectrometry, S-nitrosylated proteins were identified in both wild-type and noe1 plants. NO targets identified only in noe1 plants included glyceraldehyde 3-phosphate dehydrogenase and thioredoxin, which have been reported to be involved in S-nitrosylation-regulated cell death in animals. Collectively, our data suggest that both NO and SNOs are important mediators in the process of H 2 O 2 -induced leaf cell death in rice.

One of the most common challenges for both conventional and modern crop improvement is that the appearance of one desirable trait in a new crop variety is always balanced by the impairment of one or more other beneficial characteristics. The best way to overcome this problem is the flexible utilization of regulatory genes, especially genes that provide more efficient and precise regulation in a targeted manner. MicroRNAs (miRNAs), a type of short non-coding RNA, are promising candidates in this area due to their role as master modulators of gene expression at the post-transcriptional level, targeting messenger RNAs for cleavage or directing translational inhibition in eukaryotes. We herein highlight the current understanding of the biological role of miRNAs in orchestrating distinct agriculturally important traits by summarizing recent functional analyses of 65 miRNAs in 9 major crops worldwide. The integration of current miRNA knowledge with conventional and modern crop improvement strategies is also discussed.

SummaryPre-harvest sprouting (PHS) or vivipary in cereals is an important agronomic trait that results in significant economic loss. A considerable number of mutations that cause PHS have been identified in several species. However, relatively few viviparous mutants in rice (Oryza sativa L.) have been reported. To explore the mechanism of PHS in rice, we carried out an extensive genetic screening and identified 12 PHS mutants (phs). Based on their phenotypes, these phs mutants were classified into three groups. Here we characterize in detail one of these groups, which contains mutations in genes encoding major enzymes of the carotenoid biosynthesis pathway, including phytoene desaturase (OsPDS), f-carotene desaturase (OsZDS), carotenoid isomerase (OsCRTISO) and lycopene b-cyclase (b-OsLCY), which are essential for the biosynthesis of carotenoid precursors of ABA. As expected, the amount of ABA was reduced in all four phs mutants compared with that in the wild type. Chlorophyll fluorescence analysis revealed the occurrence of photoinhibition in the photosystem and decreased capacity for eliminating excess energy by thermal dissipation. The greatly increased activities of reactive oxygen species (ROS) scavenging enzymes, and reduced photosystem (PS) II core proteins CP43, CP47 and D1 in leaves of the Oscrtiso/phs3-1mutant and OsLCY RNAi transgenic rice indicated that photo-oxidative damage occurred in PS II, consistent with the accumulation of ROS in these plants. These results suggest that the impairment of carotenoid biosynthesis causes photo-oxidation and ABA-deficiency phenotypes, of which the latter is a major factor controlling the PHS trait in rice.

Although phosphate (Pi) starvation signaling is well studied in Arabidopsis (Arabidopsis thaliana), it is still largely unknown in rice (Oryza sativa). In this work, a rice leaf tip necrosis1 (ltn1) mutant was identified and characterized. Map-based cloning identified LTN1 as LOC_Os05g48390, the putative ortholog of Arabidopsis PHO2, which plays important roles in Pi starvation signaling. Analysis of transgenic plants harboring a LTN1 promoter::b-glucuronidase construct revealed that LTN1 was preferentially expressed in vascular tissues. The ltn1 mutant exhibited increased Pi uptake and translocation, which led to Pi overaccumulation in shoots. In association with enhanced Pi uptake and transport, some Pi transporters were up-regulated in the ltn1 mutant in the presence of sufficient Pi. Furthermore, the elongation of primary and adventitious roots was enhanced in the ltn1 mutant under Pi starvation, suggesting that LTN1 is involved in Pi-dependent root architecture alteration. Under Pi-sufficient conditions, typical Pi starvation responses such as stimulation of phosphatase and RNase activities, lipid composition alteration, nitrogen assimilation repression, and increased metal uptake were also activated in ltn1. Moreover, analysis of OsmiR399-overexpressing plants showed that LTN1 was down-regulated by OsmiR399. Our results strongly indicate that LTN1 is a crucial Pi starvation signaling component downstream of miR399 involved in the regulation of multiple Pi starvation responses in rice.Plants require large amounts of phosphate (Pi) to maintain growth and development (Raghothama, 1999). However, Pi is often a limiting factor for plants because of its low availability in the soil, which is mainly due to its low abundance and easy chelation with cations or organic compounds to form insoluble complexes (Misson et al., 2005). To cope with Pi starvation, plants have evolved multiple strategies to increase Pi availability (Raghothama, 1999). For example, changes in root architecture enable plants to increase surface contact with the soil and to acquire Pi more efficiently, and up-regulation of Pi transporter genes enhances Pi uptake and transport efficiency (Liu et al., 1998;Karthikeyan et al., 2002;Xiao et al., 2006). It is also thought that acid phosphatases are involved in Pi acquisition or recycling during Pi starvation (Duff et al., 1994;del Pozo et al., 1999), and acid phosphatase and RNase induction is a significant adaptive response to increase Pi availability (Duff et al., 1994;Trull and Deikman, 1998). In addition, Pi-starved plants can regulate multiple metabolic processes to reprioritize the utilization of internal Pi and maximize the acquisition of external Pi to adapt to low-Pi environments (Vance et al., 2003;Wasaki et al., 2003). Alteration in lipid metabolism, resulting in phospholipid degradation and galactolipid and sulfolipid synthesis, could increase intracellular Pi availability under Pi depletion (Gniazdowska et al., 1999;Misson et al., 2005). Gene chip analysis also revealed that genes relat...

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Cold stress is a major factor limiting production and geographic distribution of rice (Oryza sativa). Although the growth range of japonica subspecies has expanded northward compared to modern wild rice (O. rufipogon), the molecular basis of the adaptation remains unclear. Here we report bZIP73, a bZIP transcription factor-coding gene with only one functional polymorphism (+511 G>A) between the two subspecies japonica and indica, may have facilitated japonica adaptation to cold climates. We show the japonica version of bZIP73 (bZIP73Jap) interacts with bZIP71 and modulates ABA levels and ROS homeostasis. Evolutionary and population genetic analyses suggest bZIP73 has undergone balancing selection; the bZIP73Jap allele has firstly selected from standing variations in wild rice and likely facilitated cold climate adaptation during initial japonica domestication, while the indica allele bZIP73Ind was subsequently selected for reasons that remain unclear. Our findings reveal early selection of bZIP73Jap may have facilitated climate adaptation of primitive rice germplasms.