Cloning And Biochemical Analysis Of A Key Gene OsCOL13 In Regulation Of Flowering Time In Rice

Rice(Oryza sativa)as one of the most important crops provides energy for billions of people in the world.Rice is a short day(SD)plant that shows strong photoperiod sensitivity in flowering.Flowering plays an important role in the whole life cycle of plants,which determines the adaptability of plants to different external environments,and affected by both internal signals and external environment stimuli.Among all the flowering regulatory factors,the photoperiod is the most important and widely studied impact factor.Currently,a number of genes that participate in the photoperiod pathway have been characterized in long-day plants such as Arabidopsis,as well as in short-day plants such as Oryza sativa,and a signal network of Os GI/GI-Hd1/CO-Hd3a/FT has been built up.Of those,CONSTANS(CO)as a floral integrator promotes flowering in Arabidopsis under both long and short day conditions.In rice,Heading date1(Hd1),a homologue of CO,functions in an opposite way,which inhibits flowering under long day conditions and induces flowering under short day conditions.In this study,we reported a rice CONSTANS-like(COL)gene,OsCOL13,which participated in the rice photoperiod regulation of flowering.To know the roles of OsCOL13 in the photoperiodic flowering,we analyzed the function of OsCOL13 by genetics,molecular biology,biochemistry and cytology.The main results are as follows:(1)OsCOL13 inhibited rice flowering.We found that the OsCOL13-OE plants always showed 3-week late-flowering compared with wild type(Kitaake)under both SD conditions(14 h dark h /10 light)and LD conditions(10 h dark h /14 light).At the same time,the flowering time of OsCOL13-OE plants in two other photoperiod sensitive japonica rice cultivars Zhonghua 11(ZH11)and W030 was also significantly delayed.Using CRISPR-Cas9 system,we obtained three independent OsCOL13 mutants,but there was no significant change in flowering time compared with WT,suggesting that OsCOL13 may be functionally redundant with other Os COL gene(s).(2)OsCOL13 was mainly expressed in leaf,sheath,stem and panicle with low expression in root,and the expression level in leaf was stable during diff erent developmental stages.Histochemical assays showed that the GUS activity were detected in all tissues,suggesting that OsCOL13 is a constitutively expressed gene.RNA in situ hybridization showed that OsCOL13 was highly expressed in the mesophyll cells.(3)OsCOL13 is a rhythmic expression gene,which is modulated by light and the circadian clock.The transcription level of OsCOL13 began to accumulate slowly from the dusk and reached a peak at dawn,and then sharply decreased until the dusk under both SD and LD.The expression of OsCOL13 basically remained unchanged along vegetative development but was higher under LD than under SD,and the expression of OsCOL13 also exhibited rhythm characteristic and had no change in the amplitude under continuous light(LL),but the amplitude decreased sharply under continuous darkness(DD).(4)By observing the fluorescence signal of OsCOL13-GFP fusion protein,we found that OsCOL13 was strictly located in the nucleus,which was overlapped with the nuclear maker(D53-m Cherry)in rice protoplast.OsCOL13-BD fusion protein displayed stronger transcriptional activation activity than the BD control in yeast strain AH109,and the activation domain was in the middle region,between the conserved B-box and CCT domains,similar to that reported for CO and DTH2.These results indicate that OsCOL13 may act as a transcriptional activator to regulate the expression of downstream genes.(5)OsCOL13 physically interacted with itself and functions as homodimers in plants.When we detected the OsCOL13-Flag protein from the overexpressing transgenic plants with anti-Flag antibody,we found a major protein band with the size of 70 k Da,which is twice of the predicted OsCOL13-Flag protein.However,with increasing concentration of DTT added in the extraction buffer,an extra protein band with a molecular mass of 35 k Da appeared.Therefore,we speculated that OsCOL13 might form a dimer in its native state.In addition,in vitro pull-down assay and Bi FC assay further confirmed that OsCOL13 can form a dimer.(6)In rice flowering regulatory network,OsCOL13 is located downstream of Osphy B and upstream of Ehd1.OsCOL13 functions as a constitutive negative regulator to delay floral transition by down-regulating the expression of Ehd1,independent from other Ehd1 regulators,and consequently decreasing the expression level of Hd3 a and RFT1.The transcription level of OsCOL13 was also measured in rice flowering related near isogenic lines(NILs)and mutants.We found that the expression of OsCOL13 was significantly down-regulated in osphyb mutant,suggesting that Osphy B is a positive regulator of OsCOL13.(7)OsCOL13 directly regulates the expression of OsLBD38.By transcriptome analysis,we found that a gene called OsLBD38 may be directly regulated by OsCOL13.Further,using chromatin immunoprecipitation(Ch IP)analysis and firefly luciferase assay,we confirmed that OsCOL13 could bind to the promoter of OsLBD38 and regulate the expression of OsLBD38.But the OsLBD38-OE plants have no change in the flowering time,suggesting that OsLBD38 is directly regulated by OsCOL13,and plays other unknown functions.