Jiuyou Tang

Nitric Oxide and Protein S-Nitrosylation Are Integral to Hydrogen Peroxide-Induced Leaf Cell Death in Rice

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 (H2O2) 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 H2O2-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 H2O2-induced leaf cell death in rice.

OsNAP connects abscisic acid and leaf senescence by fine-tuning abscisic acid biosynthesis and directly targeting senescence-associated genes in rice

Significance Premature leaf senescence is known to decrease rice yield severely, but the molecular mechanism underlying this relationship remains largely unknown. Similarly, although abscisic acid (ABA)-induced leaf senescence has long been observed, the mechanism of this pathway has yet to be determined. In this study we identified and characterized a dominant premature leaf senescence mutant, prematurely senile 1 (ps1-D). The data demonstrated both that PS1/Oryza sativa NAC (no apical meristem, Arabidopsis ATAF1/2, and cup-shaped cotyledon2)-like, activated by apetala3/pistillata (OsNAP) is an ideal marker of natural senescence onset and that it functions as an important link between ABA and leaf senescence in rice. Furthermore, reduced OsNAP expression led to extended grain filling and an improved seed-setting rate, which significantly enhanced the grain yield. Thus, fine-tuning OsNAP expression should be a means of improving rice yield. 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 plant-specific NAC (no apical meristem, Arabidopsis ATAF1/2, and cup-shaped 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 one-hybrid 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.

LEAF TIP NECROSIS1 plays a pivotal role in the regulation of multiple phosphate starvation responses 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::β-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.

Mutations of genes in synthesis of the carotenoid precursors of ABA lead to pre-harvest sprouting and photo-oxidation in rice.

Pre-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), ζ-carotene desaturase (OsZDS), carotenoid isomerase (OsCRTISO) and lycopene β-cyclase (β-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/phs3-1 mutant 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.

Variation in NRT1.1B contributes to nitrate-use divergence between rice subspecies

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.

Rice DENSE AND ERECT PANICLE 2 is essential for determining panicle outgrowth and elongation

The architecture of the panicle, including grain size and panicle morphology, directly determines grain yield. Panicle erectness, which is selected for achieving ideal plant architecture in the northern part of China, has drawn increasing attention of rice breeders. Here, dense and erect panicle 2 (dep2) mutant, which shows a dense and erect panicle phenotype, was identified. DEP2 encodes a plant-specific protein without any known functional domain. Expression profiling of DEP2 revealed that it is highly expressed in young tissues, with most abundance in young panicles. Morphological and expression analysis indicated that mutation in DEP2 mainly affects the rapid elongation of rachis and primary and secondary branches, but does not impair the initiation or formation of panicle primordia. Further analysis suggests that decrease of panicle length in dep2 is caused by a defect in cell proliferation during the exponential elongation of panicle. Despite a more compact plant type in the dep2 mutant, no significant alteration in grain production was found between wild type and dep2 mutant. Therefore, the study of DEP2 not only strengthens our understanding of the molecular genetic basis of panicle architecture but also has important implications for rice breeding.

The Histone Methyltransferase SDG724 Mediates H3K36me2/3 Deposition at MADS50 and RFT1 and Promotes Flowering in Rice

In rice, the two florigens Hd3a and RFT1 are separated by only 11.5 kb in the genome. This study reveals that rice SDG724 specifically affects the histone H3 lysine 36 me2/3 level of RFT1 but not its close paralog Hd3a. Therefore, RFT1 and Hd3a have functionally diverged to control flowering time under long-day and short-day conditions partly via a fine-tuned epigenetic mechanism. Chromatin modifications affect flowering time in the long-day plant Arabidopsis thaliana, but the role of histone methylation in flowering time regulation of rice (Oryza sativa), a short-day plant, remains to be elucidated. We identified a late-flowering long vegetative phase1 (lvp1) mutant in rice and used map-based cloning to reveal that lvp1 affects the SET domain group protein 724 (SDG724). SDG724 functions as a histone methyltransferase in vitro and contributes to a major fraction of global histone H3 lysine 36 (H3K36) methylation in vivo. Expression analyses of flowering time genes in wild-type and lvp1 mutants revealed that Early heading date1, but not Heading date1, are misregulated in lvp1 mutants. In addition, the double mutant of lvp1 with photoperiod sensitivity5 (se5) flowered later than the se5 single mutant, indicating that lvp1 delays flowering time irrespective of photoperiod. Chromatin immunoprecipitation assays showed that lvp1 had reduced levels of H3K36me2/3 at MADS50 and RFT1. This suggests that the divergent functions of paralogs RFT1 and Hd3a, and of MADS50 and MADS51, are in part due to differential H3K36me2/3 deposition, which also correlates with higher expression levels of MADS50 and RFT1 in flowering promotion in rice.

Semi‐dominant mutations in the CC‐NB‐LRR‐type R gene, NLS1, lead to constitutive activation of defense responses in rice

In this study, we characterized the semi-dominant mutant nls1-1D (necrotic leaf sheath 1) of rice, which displays spontaneous lesions, specifically on leaf sheaths, with a developmental pattern. nls1-1D plants also exhibited constitutively activated defense responses, including extensive cell death, excess hydrogen peroxide and salicylic acid (SA) accumulation, up-regulated expressions of pathogenesis-related genes, and enhanced resistance to bacterial pathogens. Map-based cloning revealed that NLS1 encodes a typical CC-NB-LRR-type protein in rice. The nls1-1D mutation causes a S367N substitution in the non-conserved region close to the GLPL motif of the NB domain. An adjacent S366T substitution was found in another semi-dominant mutant, nls1-2D, which exhibited the same phenotypes as nls1-1D. Combined analyses of wild-type plants transformed with the mutant NLS1 gene (nls1-1D), NLS1 RNAi and over-expression transgenic lines showed that nls1-2D is allelic to nls1-1D, and both mutations may cause constitutive auto-activation of the NLS1 R protein. Further real-time PCR analysis revealed that NLS1 is expressed constitutively in an age-dependent manner. In addition, because the morphology and constitutive defense responses of nls1-1D were not suppressed by blocking SA or NPR1 transcript accumulation, we suggest that NLS1 mediates both SA and NPR1-independent defense signaling pathways in rice.

Activation of the jasmonic acid pathway by depletion of the hydroperoxide lyase OsHPL3 reveals crosstalk between the HPL and AOS branches of the oxylipin pathway in rice.

The allene oxide synthase (AOS) and hydroperoxide lyase (HPL) branches of the oxylipin pathway, which underlie the production of jasmonates and aldehydes, respectively, function in plant responses to a range of stresses. Regulatory crosstalk has been proposed to exist between these two signaling branches; however, there is no direct evidence of this. Here, we identified and characterized a jasmonic acid (JA) overproduction mutant, cea62, by screening a rice T-DNA insertion mutant library for lineages that constitutively express the AOS gene. Map-based cloning was used to identify the underlying gene as hydroperoxide lyase OsHPL3. HPL3 expression and the enzyme activity of its product, (E)-2-hexenal, were depleted in the cea62 mutant, which resulted in the dramatic overproduction of JA, the activation of JA signaling, and the emergence of the lesion mimic phenotype. A time-course analysis of lesion formation and of the induction of defense responsive genes in the cea62 mutant revealed that the activation of JA biosynthesis and signaling in cea62 was regulated in a developmental manner, as was OsHPL3 activity in the wild-type plant. Microarray analysis showed that the JA-governed defense response was greatly activated in cea62 and this plant exhibited enhanced resistance to the T1 strain of the bacterial blight pathogen Xanthomonasoryzaepvoryzae (Xoo). The wounding response was attenuated in cea62 plants during the early stages of development, but partially recovered when JA levels were elevated during the later stages. In contrast, the wounding response was not altered during the different developmental stages of wild-type plants. These findings suggest that these two branches of the oxylipin pathway exhibit crosstalk with regards to biosynthesis and signaling and cooperate with each other to function in diverse stress responses.

RLIN1, encoding a putative coproporphyrinogen III oxidase, is involved in lesion initiation in rice

Lesion mimic is necrotic lesions on plant leaf or stem in the absence of pathogenic infection, and its exact biological mechanism is varied. By a large-scale screening of our T-DNA mutant population, we identified a mutant rice lesion initiation 1 (rlin1), which was controlled by a single nuclear recessive gene. Map-based cloning revealed that RLIN1 encoded a putative coproporphyrinogen III oxidase in tetrapyrrole biosynthesis pathway. Sequencing results showed that a G to T substitution occurred in the second exon of RLIN1 and led to a missense mutation from Asp to Tyr. Ectopic expression of RLIN1 could rescue rlin1 lesion mimic phenotype. Histochemical analysis demonstrated that lesion formation in rlin1 was light-dependent accompanied by reactive oxygen species accumulated. These results suggest that tetrapyrrole participates in lesion formation in rice.