Xianzhi Xie

Phytochrome B control of total leaf area and stomatal density affects drought tolerance in rice

We report that phytochrome B (phyB) mutants exhibit improved drought tolerance compared to wild type (WT) rice (Oryza sativa L. cv. Nipponbare). To understand the underlying mechanism by which phyB regulates drought tolerance, we analyzed root growth and water loss from the leaves of phyB mutants. The root system showed no significant difference between the phyB mutants and WT, suggesting that improved drought tolerance has little relation to root growth. However, phyB mutants exhibited reduced total leaf area per plant, which was probably due to a reduction in the total number of cells per leaf caused by enhanced expression of Orysa;KRP1 and Orysa;KRP4 (encoding inhibitors of cyclin-dependent kinase complex activity) in the phyB mutants. In addition, the developed leaves of phyB mutants displayed larger epidermal cells than WT leaves, resulting in reduced stomatal density. phyB deficiency promoted the expression of both putative ERECTA family genes and EXPANSIN family genes involved in cell expansion in leaves, thus causing greater epidermal cell expansion in the phyB mutants. Reduced stomatal density resulted in reduced transpiration per unit leaf area in the phyB mutants. Considering all these findings, we propose that phyB deficiency causes both reduced total leaf area and reduced transpiration per unit leaf area, which explains the reduced water loss and improved drought tolerance of phyB mutants.

Positive Regulation of Phytochrome B on Chlorophyll Biosynthesis and Chloroplast Development in Rice

Phytochromes in rice are encoded by a gene family composed of three members, PHYA, PHYB, and PHYC. Through characterizing the phytochrome mutants and wild type (WT) in terms of photomorphogenesis, roles of individual phytochromes have been preliminarily explored in regulating rice de-etiolation, flowering time and fertility. However, little information has been reported about whether or how phytochromes affect chlorophyll biosynthesis and chloroplast development in rice. In this study, we compared the chlorophyll contents of wild type and the phyA, phyB and phyAphyB mutants grown under either white light (WL) or red light (R). The results suggest that phyB perceives R to positively regulate chlorophyll biosynthesis, while the role of phyA can be detected only in the phyB-deficient mutant. Analyses of the expression levels of genes involved in chlorophyll biosynthesis revealed that phytochromes affected the chlorophyll biosynthesis by regulating protochlorophyll oxidoreductase A (PORA) expression. The role of phyB in chloroplast development was also analyzed, and the results suggest that phyB perceives R to regulate chloroplast development by affecting the numbers of chloroplasts and grana, as well as the chloroplast membrane system.

OsBBX14 delays heading date by repressing florigen gene expression under long and short-day conditions in rice

B-box (BBX) proteins are zinc finger proteins containing B-box domains, which have roles in Arabidopsis growth and development. However, little is known concerning rice BBXs. Herein, we identified a rice BBX protein, Oryza sativa BBX14 (OsBBX14). OsBBX14 is highly expressed in flag leaf blades. OsBBX14 expression shows a diurnal rhythm under photoperiodic conditions and subsequent continuous white light. OsBBX14 is located in the nucleus and has transcriptional activation potential. OsBBX14-overexpression (OsBBX14-OX) lines exhibited delayed heading date under long-day (LD) and short-day (SD) conditions, whereas RNAi lines of OsBBX14 lines had similar heading dates to the WT. The florigen genes, Hd3a and RFT1, were downregulated in the OsBBX14-OX lines under LD and SD conditions. Under LD conditions, Hd1 was expressed higher in the OsBBX14-OX lines than in the wild type (WT), and the rhythmic expression of circadian clock genes, OsLHY and OsPRR1, was changed in OsBBX14-OX lines. Thus, OsBBX14 acts as a floral repressor by promoting Hd1 expression under LD conditions, probably because of crosstalk with the circadian clock. Under SD conditions, Ehd1 expression was reduced in OsBBX14-OX lines, but Hd1 and circadian clock gene expressions were unaffected, indicating that OsBBX14 acts as a repressor of Ehd1. Our findings suggested that OsBBX14 regulates heading date differently under LD and SD conditions.

Overexpression of an S-like ribonuclease gene, OsRNS4, confers enhanced tolerance to high salinity and hyposensitivity to phytochrome-mediated light signals in rice

S-like ribonucleases (S-like RNases) are homologous to S-ribonucleases (S-RNases), but are not involved in self-incompatibility. In dicotyledonous plants, S-like RNases play an important role in phosphate recycling during senescence and are induced by inorganic phosphate-starvation and in response to defense and mechanical wounding. However, little information about the functions of the S-like RNase in monocots has been reported. Here, we investigated the expression patterns and roles of an S-like RNase gene, OsRNS4, in abscisic acid (ABA)-mediated responses and phytochrome-mediated light responses as well as salinity tolerance in rice. The OsRNS4 gene was expressed at relatively high levels in leaves although its transcripts were detected in various organs. OsRNS4 expression was regulated by salt, PEG and ABA. The seedlings overexpressing OsRNS4 had longer coleoptiles and first leaves than wild-type seedlings under red light (R) and far-red light (FR), suggesting negative regulation of OsRNS4 in photomorphogenesis in rice seedlings. Moreover, ABA-induced growth inhibition of rice seedlings was significantly increased in the OsRNS4-overexpression (OsRNS4-OX) lines compared with that in WT, suggesting that OsRNS4 probably acts as a positive regulator in ABA responses in rice seedlings. In addition, our results demonstrate that OsRNS4-OX lines have enhanced tolerance to high salinity compared to WT. Our findings supply new evidence on the functions of monocot S-like RNase in regulating photosensitivity and abiotic stress responses.

Overexpression of OsPIL15, a phytochrome-interacting factor-like protein gene, represses etiolated seedling growth in rice

Phytochrome-interacting factors (PIFs) regulate an array of developmental responses ranging from seed germination to vegetational architecture in Arabidopsis. However, information regarding the functions of the PIF family in monocots has not been widely reported. Here, we investigate the roles of OsPIL15, a member of the rice (Oryza sativa L. cv. Nipponbare) PIF family, in regulating seedling growth. OsPIL15 encodes a basic helix-loop-helix factor localized in the nucleus. OsPIL15-OX seedlings exhibit an exaggerated shorter aboveground part and undeveloped root system relative to wild-type seedlings, suggesting that OsPIL15 represses seedling growth in the dark. Microarray analysis combined with gene ontology analysis revealed that OsPIL15 represses a set of genes involved in auxin pathways and cell wall organization or biogenesis. Given the important roles of the auxin pathway and cell wall properties in controlling plant growth, we speculate that OsPIL15 represses seedling growth likely by regulating the auxin pathway and suppressing cell wall organization in etiolated rice seedlings. Additionally, exposure to red light or far-red light relieved growth retardation and promoted seedling elongation in the OsPIL15-OX lines, despite higher levels of OsPIL15 transcripts under red light and far-red light than in the dark. These results suggest that light regulation of OsPIL15 expression is probably involved in photomorphogenesis in rice.

Overexpression of a phytochrome-regulated tandem zinc finger protein gene, OsTZF1, confers hypersensitivity to ABA and hyposensitivity to red light and far-red light in rice seedlings

AbstractTandem zinc finger proteins (TZFs) in plants are involved in gene regulation, developmental responses, and hormone-mediated environmental responses in Arabidopsis. However, little information about the functions of the TZF family in monocots has been reported. Here, we investigated a cytoplasmic TZF protein, OsTZF1, which is involved in photomorphogenesis and ABA responses in rice seedlings. The OsTZF1 gene was expressed at relatively high levels in leaves and shoots, although its transcripts were detected in various organs. Red light (R)- and far-red light (FR)-mediated repression of OsTZF1 gene expression was attributed to phytochrome B (phyB) and phytochrome C (phyC), respectively. In addition, OsTZF1 expression was regulated by salt, PEG, and ABA. Overexpression of OsTZF1 caused a long leaf sheath relative to wild type (WT) under R and FR, suggesting that OsTZF1 probably acts as a negative regulator of photomorphogenesis in rice seedlings. Moreover, ABA-induced growth inhibition of rice seedlings was marked in the OsTZF1-overexpression lines compared with WT, suggesting the positive regulation of OsTZF1 to ABA responses. Genome-wide expression analysis further revealed that OsTZF1 also functions in other hormone or stress responses. Our findings supply new evidence on the functions of monocot TZF proteins in phytochrome-mediated light and hormone responses. Key message OsTZF1 encodes a cytoplasm-localized tandem zinc finger protein and is regulated by both ABA and phytochrome-mediated light signaling. OsTZF1 functions in phytochrome-mediated light and ABA responses in rice.

Phytochrome B Negatively Affects Cold Tolerance by Regulating OsDREB1 Gene Expression through Phytochrome Interacting Factor-Like Protein OsPIL16 in Rice

Cross talk between light signaling and cold signaling has been elucidated in the model plant Arabidopsis and tomato, but little is known about their relationship in rice. Here, we report that phytochrome B (phyB) mutants exhibit improved cold tolerance compared with wild type (WT) rice (Oryza sativa L. cv. Nipponbare). The phyB mutants had a lower electrolyte leakage index and malondialdehyde concentration than the WT, suggesting that they had greater cell membrane integrity and less lipid peroxidation. Real-time PCR analysis revealed that the expression levels of dehydration-responsive element binding protein 1 (OsDREB1) family genes, which functions in the cold stress response in rice, were increased in the phyB mutant under normal and cold stress conditions. PIFs are central players in phytochrome-mediated light signaling networks. To explore the relationship between rice PIFs and OsDREB1 gene expression, we produced overexpression lines of rice PIF genes. OsDREB1 family genes were up-regulated in OsPIL16-overexpression lines, which had improved cold tolerance relative to the WT. Chromatin immunoprecipitation (ChIP)-qPCR assay revealed that OsPIL16 can bind to the N-box region of OsDREB1B promoter. Expression pattern analyses revealed that OsPIL16 transcripts were induced by cold stress and was significantly higher in the phyB mutant than in the WT. Moreover, yeast two-hybrid assay showed that OsPIL16 can bind to rice PHYB. Based on these results, we propose that phyB deficiency positively regulates OsDREB1 expression through OsPIL16 to enhance cell membrane integrity and to reduce the malondialdehyde concentration, resulting in the improved cold tolerance of the phyB mutants.

Expression Patterns of OsPIL11, a Phytochrome-Interacting Factor in Rice, and Preliminary Analysis of Its Roles in Light Signal Transduction

The expression patterns of OsPIL11, one of six putative phytochrome-interacting factors, were analyzed in different organs of transgenic tobacco (Nicotiana tabacum). The expression of OsPIL11 was organ-specific and was regulated by leaf development, abscisic acid (ABA), jasmonic acid (JA) and salicylic acid (SA). To further explore the role of OsPIL11 in plant light signal transduction, a plant expression vector of OsPIL11 was constructed and introduced into tobacco. When grown under continuous red light, OsPIL11-overexpressed transgenic tobacco exhibited shorter hypocotyls and larger cotyledons and leaves compared to wild-type seedlings. When grown under continuous far-red light, however, transgenic and wild-type seedlings showed similar phenotypes. These results indicate that OsPIL11 is involved in red light induced de-etiolation, but not in far-red light induced de-etiolation in transgenic tobacco, which lays the foundation for dissecting the function of OsPIL11 in phytochrome-mediated light signal transduction in rice.

Light signals mediated by phytochrome B affect abscisic acid pathway in rice

Several evidences revealed the interaction between phytochrome-mediated light signals and plant hormones abscisic acid (ABA) pathway in Arabidopsis . However, interaction between ABA signaling and phytochrome-mediated light signaling in mediating rice growth and development remain unclear. In the present study, we analyzed effects of PhyB -mediated light signals on ABA metabolism and ABA responses using rice wild type (WT) and the phyB mutant. It was observed that transcript levels of ABA biosynthetic genes (including OsNCED1 , OsNCED2 , OsNCED3 and OsNCED4 ) were higher in the phyB mutant than those in WT, whereas transcript level of ABA deactivating gene OsABA8OX1 was lower in the phyB mutant than that in WT, which probably contributed to the relatively high ABA content in the phyB mutant. ABA treatment inhibited germination of rice seeds grown either in the dark or under light. However, inhibitory effects of ABA treatment on seed germination were more obvious in phyB mutants relative to that in WT when seeds were grown under light conditions, suggesting that PhyB -mediated light signals attenuated the inhibitory effects triggered by ABA. Meantime, we compared the expression patterns of genes related to seed germination in WT and the phyB mutant grown in the medium with or without ABA. It was deduced that these genes is unlikely to contribute for the promotive effects of phyB-mediated light signals on seed germination. In addition, ABA treatment inhibited growth of both above-ground part and seminal root in rice seedlings. PhyB -mediated light signals did not affect the ABA-induced inhibition of above-ground part growth, but negatively regulate the inhibition of root growth. Taken together, our results suggest that PhyB -mediated light signals negatively regulate ABA accumulation and ABA responses in rice. This work reveals the influence of PhyB -mediated light signals on ABA pathway, which lays the foundation for dissecting the molecular mechanism of coordinated regulation of rice development by light and ABA in rice.

The Rice Phytochrome Genes, PHYA and PHYB , Have Synergistic Effects on Anther Development and Pollen Viability

Phytochromes are the main plant photoreceptors regulating multiple developmental processes. However, the regulatory network of phytochrome-mediated plant reproduction has remained largely unexplored. There are three phytochromes in rice, phyA, phyB and phyC. No changes in fertility are observed in the single mutants, whereas the seed-setting rate of the phyA phyB double mutant is significantly reduced. Histological and cytological analyses showed that the reduced fertility of the phyA phyB mutant was due to defects in both anther and pollen development. The four anther lobes in the phyA phyB mutant were developed at different stages with fewer pollen grains, most of which were aborted. At the mature stage, more than one lobe in the double mutant was just consisted of several cell layers. To identify genes involved in phytochrome-mediated anther development, anther transcriptomes of phyA, phyB and phyA phyB mutants were compared to that of wild-type rice respectively. Analysis of 2,241 double-mutant-specific differentially expressed transcripts revealed that the metabolic profiles, especially carbohydrate metabolism, were altered greatly, and heat-shock responses were activated in the double mutant. This study firstly provides valuable insight into the complex regulatory networks underlying phytochrome-mediated anther and pollen development in plants, and offers novel clues for hybrid rice breeding.