Functions Of Phytochrome-interacting Factor-like OsPIL16 In The Regulation Of Rice Growth And Development

Phytochrome-interacting factors(PIFs)is a kind of basic helix-loop-helix(b HLH)transcription factors,belonging to the 15 th subfamily of b HLH family.PIFs can interact with photochromes(PHYs)to participate in red/far-red signal transduction and regulate the whole life process of plants from seed germination to flowering and ripening.Although six PIFs genes(OsPIL11-OsPIL16)have been identified in rice,the molecular functions of these genes in the regulation of photosignaling and growth and development in rice is not as well studied as that in Arabidopsis thaliana.In this study,we analyzed the structural characteristics of OsPIL16 protein and the expression patterns in rice tissues.In order to study the molecular functions of OsPIL16,we constructed OsPIL16-overexpressed(OsPIL16-OX)and loss-of-function(Ospil16)lines in rice.We compared their phenotypes with wild type(WT)in the regulation of photomorphogenesis and agronomic traits.The main results are as followed:(1)Based on bioinformatics analysis,we predicted that OsPIL16 protein contained one b HLH domain,one nuclear localization signal(NLS)motif and one active phytochrome binding(APB)domain.Subcellular localization assay showed that OsPIL16 was a nuclear protein;LCI assay showed that OsPIL16 could physically interact with PHYA and PHYB;Yeast two hybrid experiments showed that the APB domain was required for the interaction between OsPIL16 and PHYB.Transcriptional activation analysis showed that OsPIL16 had potential transcriptional activation,and the active site was located at the N-terminal.(2)The expression level of OsPIL16 was higher in female gamete,flower,seedling,leaf blade,panicle and stem based on the data from PPRD(Plant Public RNA-seq Database);q RT-PCR was conducted to analyze the expression level of OsPIL16 in three-leaf-seedlings of WT under LD condition,and the results showed that OsPIL16 gene was a rhythmic gene,but not completely regulated by circadian clock.Comparison of OsPIL16 expression levels in WT under the dark(D),red light(R)and far-red light(FR)conditions revealed that R and FR had no significant effect on the transcription level of OsPIL16.(3)Under the dark conditions,OsPIL16-OX lines demonstrated skotomorphogenesis,and OsPIL16-SRDX lines showed photomorphogenesis.RNA-seq was conducted to compare the gene expression profile of WT and OsPIL16-SRDX seedlings which were grown in red light for 6 days for the former and grown in darkness for 6 days for the latter respectively.The results showed that the gene expression profile of OsPIL16-SRDX seedlings grown in darkness was similar to that of wild-type seedlings grown in red light;KEGG enrichment analysis showed that the expression levels of photosynthesis-related genes were upregulated and the expression levels of plant hormones-related genes,such as auxin,were downregulated in darkness in OsPIL16-SRDX transgenic lines.It is speculated that OsPIL16-SRDX may inhibit skotomorphogenesis of rice by promoting photosynthesis process and inhibiting auxin pathway in darkness.(4)The heading date is delayed under LD conditions in OsPIL16-OX lines.q RT-PCR analysis showed that the expression levels of clock-related genes were not affected in OsPIL16-OX and Ospil16 lines,suggesting that the effects of OsPIL16 on heading date may be independent of circadian clock.Then,we analyzed the transcriptional levels of florigen genes(Hd3a and RFT1)and upstream genes in WT,OsPIL16-OX and Ospil16 seedling lines growing for 60 days after sowing in natural conditions.The results showed that the expressions of RFT1,Hd3 a and Ehd1 were inhibited in OsPIL16-OX.Further analysis revealed that the expression levels of flowering suppressors COL4 and DTH8,upstream of Ehd1,were up-regulated in OsPIL16-OX and downXregulated in Ospil16,respectively.Ch IP-q PCR and LCI experiments confirmed that OsPIL16 could directly bind to the promoter of COL4 and DTH8 to regulate their expression levels.Based on these results,we speculated the model of delaying rice heading date by OsPIL16: OsPIL16 binds to the promoter of COL4 and DTH8 to upregulate their transcription levels,and then downregulates the expression levels of Ehd1 and inhibits the expression of florigen genes,leading to delayed heading date of rice.(5)OsPIL16-OX exhibited more number of rice tillers,and longer mesocotyl under darkness,and Ospil16 had the opposite phenotype,which is consistent with the function of strigolactone in inhibiting mesocotyl elongation and tillering in rice.Exogenous rac-GR24 treatments could inhibit the mesocotyl elongation of OsPIL16-OX lines,suggesting that the mesocotyl elongation regulated by OsPIL16 is related to the regulation of SL.Thus,the content of SL component 2′-epi-5DS was determined in the roots of WT,OsPIL16-OX and Ospil16.The results showed that the content of SL in OsPIL16-OX line was significantly lower than that of WT,while the content of SL in Ospil16 line was significantly higher than that of WT,which implied that OsPIL16 negatively regulated SL synthesis.Furthermore,the RNA-seq,q RT-PCR and Ch IP-q PCR were conducted to confirm that OsPIL16 might directly bind to the E-box of the SLB1 promoter to regulate the expression levels of SLB1 gene,and thus inhibit the synthesis of SL and positively regulate rice tillering.(6)The chlorophyll content of OsPIL16-OX lines was significantly higher than that of wild type at tillering stage and heading stage,while the chlorophyll content of Ospil16 lines was significantly lower than that of wild type at heading stage,suggesting that OsPIL16 might affect the uptake and utilization of nitrogen in rice.Analysis of the response of OsPIL16-OX and Ospil16 to low nitrogen treatment showed that OsPIL16-OX exhibited higher resistance to nitrogen starvation and its nitrogen absorption capacity might be enhanced.Further analysis through Ch IPSeq showed that OsPIL16 protein could bind to the promoter of nitrogen metabolism and nitrate transport-related gene NR1.1 and GDH3,which affecting the nitrogen-use efficiency in rice.This study preliminarily revealed the regulatory role of OsPIL16 in rice growth and development,and our comprehensive findings provide supportive insights into the exploration of the potential regulatory role of PIFs in crop improvement.