Studies On The Regulatory Mechanism Of PIF4 On Anthocyanin Accumulation In Arabidopsis And Respiration Metabolism On Cross-tolerance In Highland Barley

We firstly investigated the mechanism of PIF4 on Arabidopsis anthocyanin accumulation induced by several factors under normal light conditions using related mutants and transgenic plants.In addition,the effects of respiratory metabolism pathway-mediated ROS signalling on UV-B absorption compound,photosynthesis,stomata movement,and UV-B tolerance in highland barley were also studied.The results were summarized as follows:1.PIF4 negatively regulates anthocyanin accumulation in Arabidopsis.PIF4,a key transcription factor in light signaling,can regulate hypocotyl growth,anthocyanin accumulation,and some other physiological processes in Arabidopsis.Our results showed that when Arabidopsis plants were treated with 50?M Me JA,10?M 6-BA,or other factors under normal light conditions,the anthocyanin was significantly accumulated in PIF single mutants,pif1,pif3,pif4,and pif5,as well as transgenic lines 35S::PIF3 and 35S::PIF5,but not in35S::PIF4 over-expression plants.Further studies found that Me JA and 6-BA also significantly up-regulated the expression of PIF1,PIF3 and PIF5 but down-regulated the expression of PIF4.Data also showed that the transcripts of biosynthetic and regulatory genes of anthocyanin production,including CHI,CHS,DFR,PAP1,PAP2,and TTG1,were markedly induced in pif4mutant and pifq but inhibited in 35S::PIF4 over-expression line.We also carried out a yeast two-hybrid(Y2H)screen with PIF4 as the bait,and found that PIF4 can interact with PAP1 and PAP2 in yeast and this result was further validated by Bi FC.Moreover,Y2H results showed that the N terminus(contains the R2R3 domain)of PAP protein and the APB domain of PIF4 were required for their interaction.The co-location of PIF4 and PAP1 in the nucleus was also observed in transfected Arabidopsis protoplasts and transformed N.benthamiana.PIF4 can inhibit the promoter activity of PAP1 and PAP2 through LUC analysis in N.benthamiana,and PIF4 also competed with the b HLH protein TT8 for binding to PAP1 and PAP2 using the yeast three-hybrid system.Over-expression of PAP1 in 35S::PIF4 failed to accumulate any anthocyanin not only under normal condition but also under Me JA or 6-BA treatments;while the opposite results were obtained when over-expression of PAP1 in Col-0.Similar results were found by crossing35S::PIF4 with pap1-D.Taken together,these results demonstrated that PIF4 negatively regulates the accumulation of anthocyanin induced by several factors under normal conditions,which is mediated through inhibiting transcriptional activities of PAP1 and PAP2 as well as the integrity of the MBW complex.2.Respiration metabolism mediated the cross-tolerance of highland barley.200 m M Na Cl pretreatment for 24 h markedly improved the growth of highland barley under UV-B radiation evidenced by reduction of the number of wilting leaves MDA content but increase in the relative water content compared to UV-B treatment alone.At the same time,the antioxidant enzyme activities,the G6PDH activity,and the alternative pathway capacity were also significantly up-regulated by salt pretreatment.Under UV-B stress,the excessive NADPH in chloroplasts can be transported via shuttle machineries,such as the malate-oxaloacetate shuttle into mitochondria,where excessive NADPH is consumed by alternative pathway(AP)respiration.Thus,the NADPH/NADP⁺ratio was obviously increased when the AP was inhibited.At the same time,the NADPH/NADP⁺ratio was decreased when G6PDH was inhibited.G6PDH is another major source of NAPDH production in addition to chloroplasts.So,the interaction between AP and G6PDH was involved in the cross-tolerance of UV-B and salt in highland barley.NADPH is the reducing power for GSH-As A and NADPH oxidase.When the NAPDH content is insufficient,the content of As A and GSH as well as the activity of NADPH oxidase also decreased.Furthermore,the content of H₂O₂ was very high under UV-B,UV-B+Na Cl+SHAM,or UV-B+Na Cl+Glucm treatments.These treatments also posed some negative effects,including the damage of photosynthesis system,the decrease of photosynthesis-related parameters,the closed stomata and the abnormal cell morphology as well as very high content of UV-B absorption compound.Together,these results demonstrated that salt pretreatment up-regulates AP and G6PDH,which coordinately maintain the normal NADPH content.NADPH,on one hand,feedback regulates the activity of G6PDH and the capacity of AP;on the other hand,controls the cycle of As A-GSH and NADPH oxidase activity.Both pathways and other pathways together maintain the balance of ROS homeostasis and alleviate some negative effects resulted from ROS,including photosynthesis system,stomatal movement,UV-B absorption compound,and some other physiological processes and finally enhance the tolerance of highland barley to UV-B radiation.