The Functional Analysis Of DHD1 In Regulating Heading Date And Characterization Of OsCHLI In Controlling Leaf Color In Rice

Rice(Oryza sativa L.)is an important food crop,the yield of rice is close related to the security of world food.Heading date of rice has a decisive influence on the agronomic traits related to rice yield.Therefore,the cultivation of suitable heading varieties which adapt to different areas of planting conditions has been the breeders' pursuit.Heading date of rice is affected by many factors,the most important factor is photoperiod,which is the most reported flowering pathway in heading date of rice.In recent years,there are some reports involved drought,temperature,gibberellin and other factors on the impact of rice heading date.Chlorophyll is the main component of photosynthesis and is essential for the growth and development of plants.It is important to study the relevant processes of chlorophyll synthesis and to elucidate that the relevant regulatory pathways are important for improving the photosynthetic efficiency and yield of crop plants.In this study,we mainly analyzed Delayed Heading Date 1(DHD1)from the GRAS family,which overexpressed delayed the heading date of rice and increased the yield-related agronomic traits.DHD1 can also interact with OsHAP5C and OsHAP5D to form complex to delay the heading date by inhibiting Ehdl expression.In addition,the analysis of a rice leaf colour mutant ell showed that OsCHLI played an essential role in chlorophyll synthesis of rice.A single base mutation of the conserved domain of OsCHLI makes it completely in capable of function,leading to complete failure to synthesize chlorophyll in rice and then die during seedling.The main results of these two studies are as follows:1.Overexpression of DHD1 significantly delayed the heading date of rice.Overexpression of DHD1 with the Ubi promoter of maize can significantly delay heading date in a variety Kitaake which with a short growth period.Under natural long day conditions,the heading date was delayed about one month,and the yield-related agronomic traits were greatly improved.The flowering time of the overexpression lines was 3 weeks longer than that of the wild type under long day conditions(10h dark/14h light)and short day conditions(14h dark/1 Oh light)in the controlled incubator.There is no difference in the leaf emergence rate before flowering,indicating that the delayed flowering time is not due to retarded growth.2.Reduction of DHD1 expression by RNA interference(RNAi)technique or knock-out of DHD1 by CRISPR-Cas9 technique in Nipponbare did not make the heading date earlier.DHD1L,which is highly similar to DHD1 sequence,was found in the rice genome,and the double mutant of dhdl dhdll was constructed by CRIPSR-Cas9 technique.There was no difference in the heading date of the double mutant,but the plant height and some agronomic traits were reduced.It is speculated that there are other redundant genes in rice that regulate the flowering time in the same way.3.DHD1 was constitutively expressed in rice,but mainly expressed in leaves,leaf sheaths and spikes,and the expression level in roots and stems was relatively low.DHD1 is a rhythm expression gene has the highest expression in the dawn and land to a minimum after sunset under both LD and SD conditions.4.DHD1 is a typical transcription factor.By fusion of GFP protein and rice protoplast transformation experiments,DHD1 was found to be located in the nucleus and overlapped with the nuclear marker protein D53-mCherry fusion protein.BD-DHD1 vector was constructed and transformed into yeast strain AH 109.DHD1 was found to have no transcriptional activity in yeast by using empty BD and BD-Ehd4 as negative and positive controls,respectively.DHD1 may be associated with other transcription factors to regulate the transcription of downstream genes.5.The expression of flowering-related gens was detected in overexpression lines of DHD1 and the expression levels of Ehd1.Hd3a and RFT1 were significantly decreased in overexpression lines.While the expression of other genes did not change significantly,indicating that DHD1 is through the inhibition of Ehd1 expression to regulate the downstream flowering genes expression and delay the heading date.In addition,we also detected the expression level of DHD1 in the collected mutant or near-isogenic lines(NIL),and there was no difference.The upstream gene of DHD1 was still unknown.6.Through the yeast two-hybrid screening library,we screened a series of proteins that might interact with DHD1.After further validation,OsHAP5C and OsHAP5D could interact with DHD1.DHD1 and OsHAP5C/D have physical interactions in vitro by using the Pull-down experiment.Subsequently,we performed two-molecule fluorescence complementary(BiFC)experiments and co-immunoprecipitation(Co-IP)experiments in tobacco and rice protoplasts,respectively,to prove their interactions in plants also exist.7.In order to further verify the function of OsHAP5C1D,we also constructed OsHAP5C/D overexpressing plants with Kitaake as the receptor.It was found that the heading date of OsHAP5C/D overexpression lines were delayed compare to wild type Kitaake under natural long day conditions,control long day conditions and control short day conditions,and the expression of Ehdl in overexpression plants was also inhibited.These results suggest that OsHAP5C1D may co-regulate the flowering time of rice with DHD1 in the same pathway.8.Considering DHD1 is homologous to the important flowering regulatory factor DELLA protein in Arabidopsis,it is speculated whether DHD1 has similar properties to DELLA protein.We used abscisic acid(ABA),gibberellin(GA)and brassinosteroid(BR)to treat rice protoplasts that transiently expressed DHD1-Flag protein,and found that ABA could increase the accumulation of DHD1-Flag protein,while GA could reduce the accumulation of the protein.This result suggests that DHD1 may be involved in stress-related or GA-related flowering pathways.9.OsCHLI encodes the smallest subunit of the three subunits of the magnesium chelatase.ell mutant has a single base substitution mutation in the key conserved domain of OsCHLI,resulting in OsCHLI completely losing its function.Thus,the mutant completely lost the ability to synthesize chlorophyll,and died in the seedling stage after the consumption of endosperm nutrition.Further studies have shown that this critical mutation site affects the interaction between OsCHLI and OsCHLD,which makes the synthesis of magnesium protoporphyrinIX blocked.In addition,OsCHLI also interacts with thioredoxin(TRX),and a novel thioredoxin TRXZ may be involved in the redox regulation of OsCHLI.