SHB1 And CCA1 Interaction Desensitizes Light Responses And Enhances Thermomorphogenesis In Arabidopsis

Plant growth and development are regulated by many environmental factors,such as light,temperature and humidity.All of them are key determinants for normal growth and development.Plant photosynthesis provides energy for their biological activities,and light as an important environmental signal can regulate various adaptive responses of plants.In addition,temperature is also an indispensable factor for plant growth and development.Over the long evolutionary history,plants develop a complex system monitoring the temperature changes in the environment and this is beneficial to their fitness to these changes.However,excess light or drastic temperature changes may damage the normal growth and development of plants.Therefore,knowledge about how plants sense the environmental changes and how plants regulate their intrinsic signaling transduction network to shape adaptive changes and behavior is very important for improving crop quality in the future.This study was carried out in model plant Arabidopsis thaliana,and focused on the regulation of PIF4 expression by SHB1 and CCA1/LHY under red light.PIF4(PHYTOCHROME INTERACTING FACTOR 4)is involved in many aspects of plant growth and development,and is a key regulator in photomorphogenesis and thermomorphogenesis.SHB1(SHORT HYPOCOTYL UNDER BLUE 1)is involved in the blue light signal transduction,and it also influences seed size by regulating the expression of MINISEED 3(MINI3)and HAIKU 2(IKU2).CCA1(CIRCADIAN CLOCK ASSOCIATED 1)is a core player in the plant circadian clock system,and regulates plant growth and development along with its homologous gene LHY(LATE ELONGATED HYPOCOTYL).Our research discovered a desensitization step mediated by SHB1 under red light condition,and this is helpful for maintaining a balanced state of photomorphogenesis and avoiding photoinhibition.Meanwhile,as the temperature increases during the day,this can enhance plant thermomorphogenesis and make plants to better fit the temperature changes.In this study,we analyzed the mechanism by which SHB1 and CCA1/LHY regulated PIF4 expression from the molecular,biochemical and genetic perspectives,and the following results were achieved.(1)SHB1 specifically induced PIF4 expression under red light but did not change its rhythmic expression pattern.In the gain-of-function mutant shb1-D,PIF4 expression was increased dramatically under red light and was reduced in the partial loss-of-function mutant shb1.In both mutants,the rhythmic expression pattern of PIF4 was not altered.(2)The change in PIF4 protein accumulation was the same as its transcription.PIF4 expression achieved a peak at ZT6-ZT9 and it was decreased gradually thereafter.We found that PIF4 protein accumulation followed a similar pattern.After a transfer from dark to light,PIF4 was degraded rapidly.However,PIF4 was gradually accumulated with time and reached a peak at the period of ZT6-ZT9.In shb1-D,PIF4 protein was more abundant than that in wild type,and was less in shb1 than that in wild type.Both mutations did not alter the rhythmic accumulation pattern of PIF4.(3)CCA1 and LHY,the two core factors in plant circadian clock system,were involved in the regulation of PIF4 expression under red light.PIF4 expression was down-regulated in cca1,lhy and cca1 lhy double mutant.Through genetic analysis,either cca1 or lhy single mutant has a short hypocotyl compared with the wild type although CCA1 and LHY are highly homologous genes.Furthermore,cca1 lhy double mutant has a much shorter hypocotyl.In addition,shb1-D cca1 lhy triple mutant has a similar hypocotyl phenotype with cca1 lhy double mutant.The rhythmic expression pattern of PIF4 was disappeared in cca1 lhy double mutant.(4)SHB1 enhanced the thermomorphogenesis by inducing PIF4 expression.Compared with the normal temperature(20-22°C),the hypocotyl of shb1-D was elongated obviously,and PIF4 expression was also increased under the condition of high temperature(27-29°C).The hypocotyl of cca1 lhy double mutant was still elongated,but it was shorter than that of wild type.In addition,the hypocotyl of shb1-D cca1 lhy triple mutant was longer than cca1 lhy,but shorter than shb1-D.PIF4 expression in these mutants showed a similar pattern.(5)Both SHB1 and CCA1/LHY bind to PIF4 promoter under red light.SHB1 contains no recognizable DNA binding domain,but the ChIP results showed that both SHB1 and CCA1/LHY associated with the PIF4 promoter.In cca1 lhy double mutant,SHB1 failed to associate with the PIF4 promoter.The ability of SHB1 associating with its target DNA was relied on CCA1/LHY.In the dark,SHB1 and CCA1/LHY did not bind to PIF4 promoter.Preliminary data suggests that the evening complex,containing ELF3(EARLY FLOWERING 3),ELF4(EARLY FLOWERING 4)and LUX(LUXARRHYTHMO),may interfere with the binding of CCA1 or LHY to the PIF4 promoter.Furthermore,SHB1 and CCA1/LHY bound PIF4 promoter at ZT3-ZT9 in the rhythmic ChIP assays,and this coincided with the rhythmic expression pattern of PIF4 gene.(6)SHB1 interacted with CCA1/LHY in the dark and under red light.Both BiFC and co-IP results showed that SHB1 interacted with CCA1/LHY in the dark and under red light.Furthermore,the N terminus that contains the SPX domain but not the C terminus that contains the EXS domain of SHB1 interacted with CCA1/LHY.The C terminus but not the N terminus that contains the MYB domain of CCA1/LHY interacted with SHB1.(7)Activation of PIF4 expression by SHB1 depends on CCA1/LHY and this activation was red light-specific.In the protoplasts of cca1 lhy double mutant,we performed trans-activation assays and found that,when delivering CCA1 or LHY alone back,PIF4 expression had no obvious change;when delivering SHB1 alone back,PIF4 expression was also not altered;when SHB1 and CCA1 or LHY were co-delivered,PIF4 expression was greatly increased under red light.In addition,in phyB-9 mutant,the active pattern of PIF4 expression promoted by SHB1 was blocked.In conclusion,the highly expressed circadian clock protein CCA1 brought circadian signals to the regulatory region of the PIF4 signaling hub through its binding to the PIF4 promoter.SHB1 was subsequently recruited to the PIF4 promoter to specifically induce PIF4 expression.This strategy likely desensitizes the light response and maintains a balanced state of photomorphogenesis.Meanwhile,as the temperature increases during the day,this also enhances plant thermomorphogenesis.Therefore,this step is conducive for plant growth and development under the conditions of strong light or high temperature.