| Light provides the energy resource for all the organisms on earth,and is essential to plant growth and development.In the adaptation with the diurnal changed environmental light signal,the organism has established an evolutionary conserved regulatory mechanism to regulate energy storage and metabolism.In the daytime,sugar produced by photosynthesis in leaves provides the energy for all kinds of life activities,and the residual sugar was storage in starch form for the short-term storage of energy.In the night,the transient starch in plant leaves was degraded to sugar thus to satisfy plant growth and metabolism.Under energy starvation state,a series of carbon-starvation related gene expression is induced,the metabolism process is altered and even caused the autophagy to restrain the plant growth and development.Previous studies have provided that the starch synthesis and degradation in plants is regulated by the diurnal changed of light signals,endogenous sugar content and biological rhythms,but the underlying molecular mechanism is still unclear.Light is not only providing the energy source for photosynthesis,but is also an important environment signal to regulate plant growth and development.In order to reveal the molecular mechanism of how light signal protein regulate plant energy storage and metabolism,we investigated the effect of light signal protein FHY3 and FAR1 on starch synthesis in the light and energy starvation response in the night in Arabidopsis thaliana.FHY3 and FAR1 are transposase derived transcription factors,and involved in multiple signal transduction pathway.They bind to the FBS cis-elements to regulate the expression of its downstream target genes in the homodimer or heterodimer manner.In this study,based on the phenotype of reduced starch accumulation and abnormal plant cell death after extend darkness on the mutants of fhy3 and fhy3 far1,the researchers investigated the molecular mechanism of how FHY3 mediated the starch synthesis and energy metabolism.The main results are as follows:(1)The iodine staining showed that the starch of fhy3 and fhy3 far1 mutants are much lower than wild type at the end of light,which indicate that FHY3 and FAR1 are the positive regulators during starch synthesis.Based on the quantitative determination of starch,WSP and the morphology of starch granules,we found that the content of starch is decreased,the WSP is increased,and the structure of starch granules are abnormal in fhy3 and fhy3 far1 mutants.All these results indicated that FHY3 and FAR1 regulate the synthesis of starch positively.(2)According to the ChIP-seq of FHY3,and the altered expression of starch metabolism related genes in the microarray results of fhy3 and fhy3 far1 mutants,the starch debranching enzyme ISA2 was supposed to be the candidate gene of FHY3 involved in starch metabolism.Further,yeast one-hybrid,ChIP-qPCR and RT-qPCR showed that FHY3 directly binds to the promotor of ISA2 and regulate its gene expression positively.In addition,over-expression of ISA2 in fhy3 far1 rescued the content of starch and WSP and the abnormal starch granules.Therefore,FHY3 directly activates the expression of ISA2 thus to promote the synthesis of starch.(3)RT-qPCR analyses showed that the expression of ISA2 is induced by exogenous sugar obviously,while the sugar induced expression of ISA2 is decreased in glucose sensor HXK1 mutant gin2 and fhy3 far1 double mutant.Therefore,light signal protein FHY3 mediated both light signal and sugar signal regulated the expression of ISA2 simultaneously and thus promote the synthesis of starch.(4)After extended darkness treatment then transferred to light conditions,fhy3 and fhy3 far1 mutants show serious abnormal cell death compared to wild type,which indicated that FHY3 is essential during the process of dark-light transition.Furthermore,the highly accumulation of singlet oxygen,and the altered protein level of photosynthesis related proteins probably are the direct reasons cause to cell death in fhy3 far1 mutants during the process of dark-light transition in the diurnal conditions.(5)The ultrastructure of chloroplast membrane and thylakoid was structural abnormality in fhy3 and fhy3 far1 mutants through the TEM observations in darkness conditions,which maybe the intrinsic reasons of plant oxidation damage during the dark-light transition.Meantime,autophagy related genes expression significantly increased in fhy3 and fhy3 far1 mutants,and disrupt the expression of the autophagy key factors ATG5 and FHY3 in double mutant of atg5 fhy3 showed the premature senescence indicated that FHY3 negatively regulate cell autophagy in darkness.(6)Under the extended darkness conditions,the expression of energy starvation related genes DIN1 and DIN6 are significantly increased in fhy3 far1 mutant,and supplied with exogenous sugar rescued the phenotype of abnormal cell death and photo-oxidative damage significantly.It proved that the energy deficiency resulted from carbon starvation is the important reason to cause cell death in fhy3 far1 mutant during the dark-light transition,and FHY3 is prevent energy starvation stress in plant.(7)Further,in order to explore the relationship between FHY3 and AKIN10,the ?-subunit of the SnRK1 kinase during the energy starvation response,RNA-seq and yeast two-hybrid assay were performed and found that the physiological function of FHY3 and AKIN10 is antagonistic,while they interact directly with each other.Multiple AKIN10 induced target genes are increased in fhy3 and fhy3 far1 mutants,whereas the expression of FHY3 direct activates the target gene CCA1 and ELF4 are decreased in akin10 mutant.Protein sequence analysis revealed that predicted AKIN10 phosphorylation site is existed in the protein sequence of FHY3.So AKIN10 may interact with FHY3 directly thus to coordinately regulate the energy starvation response.Our results indicated that light signal protein FHY3 and FAR1 directly bind,and activate the gene expression of ISA2,thus involved in the starch synthesis mediated by light signal and sugar signal in the daytime.Meanwhile,FHY3 is the important negative regulator prevent carbon starvation under darkness conditions,and interact with AKIN10 to regulate the response of energy metabolism thus keep the plant adapting with the diurnal changed light-dark cycle. |
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