Molecular Mechanism Characterization Of FHY3 And FAR1 In Plant Cell Death In Arabidopsis

Light is one of the most important and basic environment cues affecting plant growth and development. Light acts not only as a source of energy for photosynthesis, but also a signal to regulate plant growth and development. Light signals are percepted by a series of photoreceptors in higher plants. When photoreceptors recieved the light changes of external environment, the protein conformation and location of photoreceptors was changed, and transmit the light signal to the downstream pathways by interaction with other signal proteins to regulate plant growth and deveiopment and acclimatize the environment exchange.FHY3 and FAR1 are two important transcription factors involved in phyA signaling, derived from a kind of Mutator-like transposase. They bind the FBS cis-elements by homogenous or heterogenous dimers to control the expression of target genes. Previous studies have proved that FHY3/FAR1 involved in the photomorphogenesis of seedlings, flowering time regulation and organelles development. Here, based on the phenotype of cell death on fhy3far 1, we parsed the molecular mechanism on FHY3 regulating cell death using molecular biology and reverse genetics method.(1) According to the different levels of cell death phenotypes under short-day condition, FHY3/FAR1 are the negative regulators of cell death. Based on the DAB and NBT staining, ROS is highly accumulated in fhy3farl double mutant and fhy3farl is more sensitive to exogenous MV treatment. we vertify that FHY3/FAR1 are important to acclimatize the oxidative stress.(2) Because of constitutive expression of S3H under fhy3farl double mutant easing the cell death under SD condition obviously, then we confirmed that the cell death of fhy3far1 is dependent on the accumulation of SA.(3) FHY3/FAR1 are the important transcription factors in phyA signaling, and they participate in controlling multiple genes to regulate plant growth and development. According to the ChIP-seq of FHY3 and gene chip on fhy3farl double mutant, we blasted 25 downstream target genes and finally because of the similar cell death phenotype between fhy3farl and mipsl, we confirmed myo-inositol 1-phosphate synthase 1(MIPS1) as candidate gene. Eventurally, we proved that FHY3 activates the expression of MIPS1 directly, and over-expression of MIPS1 rescues the inositol level of fhy3farl. So we proved that FHY3/FAR1 is essential to the de novo of myo-inositol.(4) FHY3 is impotant to the transcriptional level of MIPS1. To further investigate the importance of FHY3 to the synthesis of inositol, we detect the expression of multiple genes associated with the synthesis of inositol. For example, the expression of MIK, MIOX2, VTC4 and INT1 are changed remarkably in fhy3farl. The result indicated that FHY3 mediate the whole mechanism of inositol.(5) Inositol pathway is one of the ways to synthesize ascorbic acid (AsA) and VTC4 as a bi-functional enzyme involved in the synthesis of inositol and ascorbic acid at the same time. By RT-PCR, the expression of VTC1-5 and the level of AsA decreased in fhy3farl, and the content of AsA was improved in the complementary stain of fhy3. At last, we confirmed that FHY3 facilitated the synthesis of AsA by regulating positively the expression of VTC indirectly.(6) Inositol is essential to maintain normal growth and development of plants and resist the oxidative stress. To study the function of inositol to fhy3farl double mutant, using high light treatment we proved that MIPS1-OX1fhy3farl not only improved the level of inositol, but also released the premature senescence of fhy3farl double mutant under high light condition.All in all, our date indicated FHY3/FAR1 are negative regulators of cell death, and FHY3/FAR1 directly activate the gene expression of MIPS1/2 to promote the biosynthesis of inositol and strengthen the adaptation of oxidative stress in plants finally. Establish the basis and evidiences to further resolve the molecular mechanism of plant resistance to stress.