Phytochromes Antagonize OsGI In GHD7 Degradation To Regulate Heading Date In Rice(Oryza Sativa L.)

Rice is one of important food crops in the word,and it is also the staple food of China and Southeast Asian countries.Rice heading date is an important agronomic trait of rice,which determines the regional adaptability of rice varieties and determines the area where rice is planted.The flowering time of plants is affected by environmental factors,such as,temperature and photoperiod Photoperiod is an important environmental factor affecting rice heading.Rice is a typically short-day plant,which heading is promoted in short days but delayed in long days.During rice growth,long day condition is the conditions of the photoperiod in high latitudes.Due to long day suppression of rice flowering,rice heading at high latitudes is delayed.Delays in the heading of rice at high latitudes make it difficult to harvest before cold weather arrives.In order to increase the cultivation range of cultivated rice,people selected heading traits during long-term breeding selection.This selection process made the heading of rice less responsive to long-day or even no response.Therefore,understanding the molecular mechanism of rice heading response to long day is important for molecular breeding of heading rice.In contrast to rice,Arabidopsis thaliana is a typical long-day plant,which flowering time is promoted in long day,but delayed in short days.Genetics research in rice heading date showed that some rice heading-related genes have similar functions to the corresponding Arabidopsis orthologous genes.It shows that the molecular mechanism of the plant response to photoperiod is relatively conservative.Therefore,the difference in photoperiod response between two species may be related to rice unique genes compare to Arabidopsis.Previous research on the molecular mechanisms of rice heading date indicated that the photoperiodic response differences between rice and Arabidopsis may be caused by different functions of the Hd1 gene in rice and its orthologous gene CO in Arabidopsis thaliana.Hd1 inhibited rice heading,but CO promoted flowering of Arabidopsis under long days.However,further research has shown that GHD7 is the reason for the inhibition of Hd1 to flowering under long-day conditions.GHD7 has no orthologous genes in Arabidopsis and GHD7 is a long-day specific inhibitor.The study of regulation mechanism of GHD7 in long day condition helps to explain the molecular mechanism of rice photoperiod sensitity.It also helps to provide a theoretical basis for the application of GHD7 in rice heading breeding.In this study,we focused on the molecular regulation mechanism of GHD7 protein level by phytochrome under long-day conditions.The details are as follows:1.In this study,GHD7 was overexpressed in the background of Nipponbare,phyb and se5 mutants(Se5 gene encodes a key gene for the synthesis of chromophoric chromophore and se5 mutant is functionally equivalent to phyaphybphyc three mutant).It was found that overexpression of GHD7 in Nipponbare significantly delayed rice heading.Overexpression of GHD7 in phyb mutants significantly reduced the delay of heading.However,overexpression of GHD7 in se5 mutants had little effect on heading date,suggesting that Phytochrome effects GHD 7 function.Further studies revealed that the GHD7 protein level was significantly decreased in the se5 mutant,indicating that the Phytochrome can stabilize the GHD7 protein at the post-transcriptional level.2.The transgenic plants overexpressing GHD7 in the se5 mutant were treated with the 26S proteasome inhibitor MG 132 and GHD7 protein levels were significantly restored.This indicates that degradation of GHD7 in the se5 mutant may be dependent on the 26S proteasome degradation pathway.Three methods were used to demonstrate the interaction between PHYA,PHYB,and GHD7.While PHYC and GHD7 do not interact in yeast,they interact in vitro with tobacco leaves.3.Previous research showed that Se5 is an OsGI inhibitor,and OsGI orthologous gene GI in Arabidopsis thaliana mediates the degradation of flowering-related target proteins by the 26S proteasome degradation pathway.In this study we overexpressed both OsGI and GHD7 in Nipponbare.We found that overexpression of OsGI could inhibit GHD7 function of inhibiting heading.The GHD7 protein level in OsGI and GHD7 overexpressed plants was significantly lower than plants that GHD7 was overexpressed,indicating that OsGI can inhibit the stability of GHD7 protein.4.The transgenic plants overexpressing both OsGI and GHD7 were treated with MG 132 and GHD7 protein levels were significantly restored.It shows that OsGI may be involved in the degradation of GHD7,and this degradation is dependent on the 26S proteasome degradation pathway.Three methods were used to demonstrate the interaction between OsGI and GHD7 in vivo and in vitro.5.OsGI protein was divided into three sections and further studied using yeast two-hybrid method.Both the Phytochrome and GHD7 interacted with the N-terminal fragment of OsGI.This indicates that the Phytochrome may stabilize GHD7 protein by inhibiting the interaction between OsGI and GHD7.We used yeast three-hybrid and found that PHYA and PHYB can inhibit the interaction between OsGI and GHD7 but PHYC notThese results indicate that GHD7 protein level is regulated by OsGI-mediated degradation during long days.The phytochrome may inhibit the degradation process by inhibiting the interaction between OsGI and GHD7 under long-day conditions,thereby stabilizing the GHD7 protein level.Phytochromes pretect GHD7 from being degradated and GHD7 function under long-day conditions.This study allows us to understand more deeply the molecular mechanism of plant flowering response to photoperiod,and also provide theoretical support for the application of GHD7 in molecular design of rice heading breeding.