It is well known that abscisic acid (ABA)-induced leaf senescence and premature leaf senescence negatively affect the yield of rice (Oryza sativa). However, the molecular mechanism underlying this relationship, especially the upstream transcriptional network that modulates ABA level during leaf senescence, remains largely unknown. Here, we demonstrate a rice NAC transcription factor, OsNAC2, that participates in ABA-induced leaf senescence. Overexpression of OsNAC2 dramatically accelerated leaf senescence, whereas its knockdown lines showed a delay in leaf senescence. Chromatin immunoprecipitation-quantitative PCR, dual-luciferase, and yeast one-hybrid assays demonstrated that OsNAC2 directly activates expression of chlorophyll degradation genes, OsSGR and OsNYC3. Moreover, ectopic expression of OsNAC2 leads to an increase in ABA levels via directly up-regulating expression of ABA biosynthetic genes (OsNCED3 and OsZEP1) as well as down-regulating the ABA catabolic gene (OsABA8ox1). Interestingly, OsNAC2 is upregulated by a lower level of ABA but downregulated by a higher level of ABA, indicating a feedback repression of OsNAC2 by ABA. Additionally, reduced OsNAC2 expression leads to about 10% increase in the grain yield of RNAi lines. The novel ABA-NAC-SAGs regulatory module might provide a new insight into the molecular action of ABA to enhance leaf senescence and elucidates the transcriptional network of ABA production during leaf senescence in rice.Senescence is the last stage of leaf development. During this period, various changes occur at the physiological, biochemical, and molecular levels. For example, macromolecules including lipids, proteins, and nucleic acids are hydrolyzed, which leads to disassembly of mitochondria and nuclei, and to cell death (Buchanan-Wollaston et al., 2005;Ulker et al., 2007). Although senescence is an active process to salvage nutrients from old tissues, precocious senescence will shorten the growth stage of crops and be unfavorable to agronomic production (Woo et al., 2013).The most distinguishing feature in leaf senescence is the yellowing phenotype, which is a visible marker of the degradation of macromolecules (Kim et al., 2006). The chlorophyll degradation pathway is one of the most characterized ones for macromolecule degradation in plants (Hörtensteiner, 2006). Overexpressing NON-YELLOW COLORING1 (NYC1) or NYC1-like genes in rice (Oryza sativa) can induce degradation of chlorophyll . A pph (encoding pheophytinase) mutant is abnormal in chlorophyll degradation during senescence and therefore exhibits a stay-green phenotype (Schelbert et al., 2009). Mutation of the PAO (Pheophorbide a oxygenase) gene leads to retention of chlorophyll in leaves during dark-induced senescence in Arabidopsis (Arabidopsis thaliana; Pruzinská et al., 2005). Recently, the highly conserved STAY-GREEN (SGR) in higher plants has been identified to be chloroplast-localized dechelatase (Shimoda et al., 2016).The senescence process is highly regulated by a range of important factors. It has been demon...

Plants respond to environmental stresses by altering gene expression, and several genes have been found to mediate stress-induced expression, but many additional factors are yet to be identified. OsNAP is a member of the NAC transcription factor family; it is localized in the nucleus, and shows transcriptional activator activity in yeast. Analysis of the OsNAP transcript levels in rice showed that this gene was significantly induced by ABA and abiotic stresses, including high salinity, drought and low temperature. Rice plants overexpressing OsNAP did not show growth retardation, but showed a significantly reduced rate of water loss, enhanced tolerance to high salinity, drought and low temperature at the vegetative stage, and improved yield under drought stress at the flowering stage. Microarray analysis of transgenic plants overexpressing OsNAP revealed that many stress-related genes were up-regulated, including OsPP2C06/OsABI2, OsPP2C09, OsPP2C68 and OsSalT, and some genes coding for stress-related transcription factors (OsDREB1A, OsMYB2, OsAP37 and OsAP59). Our data suggest that OsNAP functions as a transcriptional activator that plays a role in mediating abiotic stress responses in rice.

SummaryUV-B radiation damage in leaves is prevented by epidermal UV-screening compounds that can be modulated throughout ontogeny. In epiphytic orchids, roots need to be protected against UV-B because they photosynthesize, sometimes even replacing the leaves. How orchid roots, which are covered by a dead tissue called velamen, avoid UV-B radiation is currently unknown.We tested for a UV-B protective function of the velamen using gene expression analyses, mass spectrometry, histochemistry, and chlorophyll fluorescence in Phalaenopsis 9 hybrida roots. We also investigated its evolution using comparative phylogenetic methods.Our data show that two paralogues of the chalcone synthase (CHS) gene family are UV-B-induced in orchid root tips, triggering the accumulation of two UV-B-absorbing flavonoids and resulting in effective protection of the photosynthetic root cortex. Phylogenetic and dating analyses imply that the two CHS lineages duplicated c. 100 million yr before the rise of epiphytic orchids.These findings indicate an additional role for the epiphytic orchid velamen previously thought to function solely in absorbing water and nutrients. This new function, which fundamentally differs from the mechanism of UV-B avoidance in leaves, arose following an ancient duplication of CHS, and has probably contributed to the family's expansion into the canopy during the Cenozoic.

These authors contributed equally to the work. SUMMARYPlant height and flowering time are key agronomic traits affecting yield in rice (Oryza sativa). In this study, we investigated the functions in rice growth and development of OsNAC2, encoding a NAC transcription factor in rice. Transgenic plants that constitutively expressed OsNAC2 had shorter internodes, shorter spikelets, and were more insensitive to gibberellic acid (GA 3 ). In addition, the levels of GAs decreased in OsNAC2 overexpression plants, compared with the wild-type. Moreover, flowering was delayed for approximately 5 days in transgenic lines. The transcription of Hd3a, a flowering-time related gene, was suppressed in transgenic lines. In addition, transgenic Arabidopsis plants expressing OsNAC2 were also more insensitive to GA 3 . The expression levels of GA biosynthetic genes OsKO2 and OsKAO were repressed. The expression of OsSLRL, encoding a repressor in the GA signal pathway, and OsEATB, which encodes a repressor of GA biosynthesis, were both enhanced. Western blotting indicated that DELLA also accumulated at the protein level. Dual-luciferase reporter analyses, yeast one-hybrid assays and ChIP-qPCR suggested that OsNAC2 directly interacted with the promoter of OsEATB and OsKO2. Taken together, we proposed that OsNAC2 is a negative regulator of the plant height and flowering time, which acts by directly regulating key genes of the GA pathway in rice.

Development of genetic varieties with improved nitrogenuse efficiency (NUE) is essential for sustainable agriculture.In this study, we developed a growth system for rice wherein N was the growth-limiting factor, and identified Nresponsive genes by a whole genome transcriptional profiling approach. Some genes were selected to test their functionality in NUE by a transgenic approach. One such example with positive effects on NUE is an early nodulin gene OsENOD93-1. This OsENOD93-1 gene responded significantly to both N induction and N reduction. Transgenic rice plants over-expressing the OsENOD93-1 gene had increased shoot dry biomass and seed yield. This OsENOD93-1 gene was expressed at high levels in roots of wild-type (WT) plants, and its protein product was localized in mitochondria. Transgenic plants accumulated higher concentrations of total amino acids and total N in roots. A higher concentration of amino acids in xylem sap was detected in transgenic plants, especially under N stress. In situ hybridization revealed that OsENOD93-1 is expressed in vascular bundles, as well as in epidermis and endodermis. This work demonstrates that transcriptional profiling, coupled with a transgenic validation approach, is an effective strategy for gene discovery. The knowledge gained from this study could be applied to other important crops.

Among the heat shock proteins (HSPs) of higher plants, those belonging to the small HSP (sHSP) family remain the least characterized in functional terms. To improve our understanding of sHSPs, we have characterized RcHSP17.8 from Rosa chinensis. Sequence alignments and phylogenetic analysis reveal this to be a cytosolic class I sHSP. RcHSP17.8 expression in R. chinensis was induced by heat, cold, salt, drought, osmotic and oxidative stresses. Recombinant RcHSP17.8 was overexpressed in Escherichia coli and yeast to study its possible function under stress conditions. The recombinant E. coli and yeast cells that accumulated RcHSP17.8 showed improved viability under thermal, salt and oxidative stress conditions compared with control cultures. We also produced transgenic Arabidopsis thaliana that constitutively expressed RcHSP17.8. These plants exhibited increased tolerance to heat, salt, osmotic and drought stresses. These results suggest that R. chinensis cytosolic class I sHSP (RcHSP17.8) has the ability to confer stress resistance not only to E. coli and yeast but also to plants grown under a wide variety of unfavorable environmental conditions.

SummaryThe rice root system is important for growth. The crosstalk between auxin and cytokinin mediates root initiation and elongation. However, it remains unclear how the transcriptional network upstream of the auxin and cytokinin signalling pathways determines root development. Here, we observed that the knockdown of OsNAC2, which encodes a NAC transcription factor, increased the primary root length and the number of crown roots. OsNAC2 predominantly expressed in primary root tips, crown roots and lateral root primordia, implying it influences root development. Molecular analyses revealed that the expressions of auxin‐ and cytokinin‐responsive genes were affected in OsNAC2‐overexpressing (OsNAC2‐OX; ON7 and ON11), RNA interference (OsNAC2‐RNAi; RNAi25 and RNAi31) and CRISPR/Cas9 plants. Additionally, OsNAC2 can directly bind to the promoters of IAA inactivation‐related genes (GH3.6 and GH3.8), an IAA signalling‐related gene (OsARF25), and a cytokinin oxidase gene (OsCKX4). Furthermore, genetic analysis of ON11/osgh3.6 and RNAi31/osckx4 homozygote confirmed that OsCKX4 and OsGH3.6 functioned downstream of OsNAC2. The mRNA levels of CROWN ROOTLESS (CRL) genes and cyclin‐dependent protein kinase (CDK) genes increased in OsNAC2‐RNAi and OsNAC2‐cas9 lines while reduced in OsNAC2‐OX lines. Thus, we describe that OsNAC2 functions as an upstream integrator of auxin and cytokinin signals that affect CRL and CDK production to regulate cell division during root development. This novel auxin‐OsNAC2‐cytokinin model should provide a new insight into the understanding of NAC TFs and crosstalk of auxin and cytokinin pathway, and can be potentially applied in agriculture to enhance rice yields by genetic approaches.

Plants can perceive environmental changes and respond to external stressors. Here, we show that OsNAC2, a member of the NAC transcription factor family, was strongly induced by ABA and osmotic stressors such as drought and high salt. With reduced yields under drought conditions at the flowering stage, OsNAC2 overexpression lines had lower resistance to high salt and drought conditions. RNAi plants showed enhanced tolerance to high salinity and drought stress at both the vegetative and flowering stages. Furthermore, RNAi plants had improved yields after drought stress. A microarray assay indicated that many ABA-dependent stress-related genes were down-regulated in OsNAC2 overexpression lines. We further confirmed that OsNAC2 directly binds the promoters of LATE EMBRYOGENESIS ABUNDANT 3 (OsLEA3) and Stress-Activated Protein Kinases 1 (OsSAPK1), two marker genes in the abiotic stress and ABA response pathways, respectively. Our results suggest that in rice OsNAC2 regulates both abiotic stress responses and ABA-mediated responses, and acts at the junction between the ABA and abiotic stress pathways.