Molecular Mechanism Of AFC2 Regulating Thermomorphogenesis In Arabidopsis Thaliana By Regulating Alternative Splicing Of PIF4 Target Genes

As one of the most important environmental factors,temperature plays a crucial role in plant growth and development.Meanwhile,plants are sensitive to temperature changes,including day and night temperature changes,soil temperature changes and seasonal temperature changes.Plants usually display elongated hypocotyls or petioles and early flowering when grown under high ambient temperature.These adaptive changes are named thermomorphogenesis.Recently,studies on the regulatory mechanism of thermomorphogenesis mainly focus on the central transcription factor,PHYTOCHROME INTERACTING FACTOR 4(PIF4).PIF4 controls the majority of high temperature responsive transcriptome,including auxin biosynthesis related gene expressions.PIF4 can also regulate plant thermomorphogenesis through regulating alternative splicing of genes.More and more studies focus on the regulatory mechanism of alternative splicing to balance plants growth and development under high temperature.Mostly,pre-m RNA produces proteins with different functions or degraded by(nonsense-mediated decay)NMD pathway.The mechanism has some progress in monitoring flowering time.For example,the flowering time control factor FLOWERING CONTROL LOCUS A(FAC)produces functional full-length protein induced by high temperature.Transcription factor FLOWERING LOCUS M(FLM)can also balance plant growth and development by producing multiple splicing forms.FLM similar gene MADS AFFECTING FLOWERING 2(MAF2)occurs intron retention at 21? to produce nonfunctional proteins to regulate flowering time.In the past,it has been shown that mouse CDC-like kinase(CLK)regulates splicing process through directly phosphorylating SR proteins.Recent research shows that high temperature represses CLK kinase activity.Sequence alignment reveals that CLK is highly conserved.There are three homologues(AFC1/2/3)in Arabidopsis thaliana.Therefore,we attempt to find novel regulatory factor of thermomorphogenesis and explore different regulatory mechanisms.We find that afc2 mutants are hypersensitive to high temperature,suggesting that AFC2 inhibits thermomorphogenesis.Transcriptome analysis shows that temperature modulates alternative splicing events in an AFC2 dependent manner.Furthermore,AFC2 kinase activity is also impaired by high temperature,therefore inhibiting SR protein phosphorylation.It is hypothesized that AFC2 might regulate plant growth through controlling alternative splicing.PIF4 target gene COLD–CIRCADIAN RHYTHM–RNABINDING 2(CCR2)easier occurs alternative splicing in the mutant of NMD pathway factors.Lastly,we demonstrate that both PIF4 and auxin synthesis are necessary for the afc2 hypersensitivity to high temperature.Taken together,this study reveals that AFC2 is a negative regulator in thermomorphogenesis.High temperature regulates AFC2 kinase activity to modulate SR phosphorylation,inducing pre-m RNA alternative splicing or degraded by NMD pathway to regulate plant growth and development.