Investigation On The Functions Of Red Light Signal Influencing Grain Filling And Phytochrome Interacting Factor-Like Protein OsPIL15 Regulating Grain Size In Rice

Rice(Oryza sativa L.)is one of the most important cereal crops all over the world,providing staple diet for almost half of the world’s population.High and stable production of rice is of great significance to China’s food security.Rice yield is mainly formed in the whole filling duration.Of the agronomic traits determining yield potential,grain filling and grain size are the most important.It is generally known that light is one of the decisive environmental factors to the plant growth and development.Plants utilize light not only as a source of energy for photosynthesis,but also as an environmental cue to modulate a wide range of responses from germination to fruiting,a complex developmental program called photomorphogenesis.In this study,we studied the effects of red light signal on grain filling of rice,and then the differentially expressed genes(DEGs)of rice grains with red light treatment at night were identified by RNA-Seq.We predicted a possible pathway of red light signal influencing grain filling.Furthermore,we selected the phytochrome interacting factor Os PIL15,which belongs to members of the basic helix-loop-helix(b HLH)transcription factor superfamily as research subjects,and thoroughly studied the molecular functions of Os PIL15 in grain size.The results are as below:1.Rice was treated with longtime irradiation of red light at night(red light for 10 h,lasted 36d)during grain filling stage in 2011.The results showed that red light significantly decreased 1000-grain weight of superior and inferior spikelets,while the inferior spikelets were mainly affected.Rice was treated by short-time irradiation of red light at night(red light for 2h,lasted 36d)during grain filling stage in 2012 and 2013.The results showed that red light could decrease 1000-grain weight of rice,and the middle and inferior spikelets were more easily affected.Three consecutive years experiment indicated that both long-time and short-time irradiation of red light at night could affect rice grain filling.The determining factor,which resulted in these changes,was light signal,not irradiation time.Then we determined the sugar and starch content of rice grains at 36 d.The results showed that red light decreased the content of sucrose,soluble sugar and starch in grains.Finally these resulted in reduction of the 1000-grain weight and yield in rice.Photomorphogenesis of rice could be affected by red light which showed a longer panicle neck than the control,and the plant height increased slightly higher than the control,but the difference did not arrive at significant level.2.The circadian rhythms of rice could be broken up by red light irradiation during grain filling stage.RNA-Seq analysis was carried out using rice endosperm samples with red light treatment and control,and 425 DEGs were identified.Then these genes were divided into 4 groups according to their relationships.(1).Red light negatively regulates circadian rhythms related genes;(2).Red light does not regulate circadian rhythms related genes;(3).Red light positively regulates genes unrelated circadian rhythms;(4).Red light negatively regulates genes unrelated circadian rhythms.So irradiation of red light could regulate the genes involved in seed storage proteins,anabolism of sugar and starch,hormone signal transduction and concentration of Ca2+,thus affected grain filling of rice.3.Taking phytochrome interacting factor Os PIL15 for research objects.We generated knockout(KO)Os PIL15 lines in rice using CRISPR/Cas9 technology,the silencing expression of Os PIL15 led to enhanced grain size and weight.Moreover,overexpression and suppression of Os PIL15 in the rice endosperm resulted in brown rice showing grain sizes and weights that were decreased and increased respectively.Histological analysis of the spikelet hull revealed that grain size was enhanced by increased numbers of cells in the Os PIL15-KO plants.Digital gene expression analysis further revealed the role of Os PIL15 in cell development,seed storage proteins,and the hormone signal transduction pathway in rice grains.Further studies indicated that Os PIL15 binds to N1-box(CACGCG)motifs of the purine permease gene Os PUP7 promoter.Measurement of isopentenyl adenosine(i PA),a bioactive form of cytokinin(CTK),revealed increased contents in the Os PIL15-KO spikelets compared with the wild-type.Overall,our results demonstrate a possible pathway whereby Os PIL15 directly targets Os PUP7,affecting CTK transport and thereby influencing cell division and subsequent grain size.These findings provide a valuable insight into the molecular functions of Os PIL15 in rice grains,highlighting a useful genetic improvement leading to increased rice yield.