Functional Studies Of High Light Responsive Transcription Factor GATAX In Chlamydomonas

Photosynthetic organisms often experience extreme light conditions,and excessive light absorption can damage the photosynthetic complexes.As one of the ancient photosynthetic organisms,Chlamydomonas reinhardtii has developed an elaborate mechanism to resist high light during the long evolutionary process.One of them involves the transcription regulatory network,that helps cells adapt to high light and maintain normal growth by activating or suppressing the gene expressions.In this study,GATAX,a transcription factor responsive to high light,was characterized in Chlamydomonas reinhardtii.By analyzing the transcriptome results,we identified NY was downstream target gene of GATAX.NY-like proteins possess NYX1 type zinc finger structure and are involved in Target of Rapamycin(TOR)signaling pathway in yeast cells.In eukaryotes,Target of Rapamycin(TOR)is one of the key regulators integrating nutrition,hormone and various stress signals during various biological processes,such as translation,cell metabolism,autophagy,and stress response.Compared to other species,the TOR signaling network in algae is currently poorly understood.In particular,it is not well known about the molecular mechanism by which TOR promotes the growth of photosynthetic cells.The main results were as follows:(1)We performed transcriptome sequencing of wild type Chlamydomonas treated with low light,high light for 15 minutes and high light for 30 minutes,and finally identified the transcription factors GATAX,A,B,C,D,which were significantly upregulated after short high light treatment.Then we identified mutants gatax,a,b,c-1,c-2 and d from DNA level,transcription level and phenotype.The results showed that all were knockout mutants except a and c-2.High-light phenotype characterization showed that a was not hypersensitive to high light compared to the wild type,and the rest of mutants had a high light lethal phenotype.Besides the significant high-light sensitive phenotype,gatax also forms palmelloid structure under normal growth condition,which is accompanied by the secretion of extracellular polysaccharides and the increased starch content.So,we focused on the functional study on GATAX.(2)We first constructed a complement vector with GATAX promoter,and transformed the recombinant plasmid into gatax mutants by electrical transformation.After around ten-day growth,we screened 5 positive transgenic strains with wild-type level GATAX transcript accumulation and high light resistance.(3)Transcriptome sequencing was performed on gr-,gatax and gatax C to search for genes differentially expressed between mutant and wild type,and partially or completely recovered in complement strain.Among those differently expressed 2029 genes,the down-regulated genes in gatax were mainly involved in protein synthesis,ribosomal biogenesis and chloroplast transport,and the up-regulated genes were involved in autophagy and some metabolic pathways indicated by GO and KEGG enrichment analyses.The one with most significant difference in transcript accumulation was NY,which has not been studied in Chlamydomonas.Some other potential target genes of GATAX were also verified by Semi-q PCR and Real-time q PCR,which could confirm the results from RNA-seq.(4)The GATAX protein was revealed to bind at GATA motif of NY promoter by Electrophoretic Mobility Shift Assay(EMSA).GATAX and NY have similar expression patterns,and both proteins were located in the nucleus.Yeast two-hybrid experiments demonstrated that GATAX interacted with NY,and NY also interacted with the immunophilin protein FKB12,a key component in the TOR signaling pathway.(5)Further phenotypic characterization of gatax mutant and analyses of RNA-seq results concluded GATAX is involved in the TOR signaling pathway by regulating NY expression.(6)We searched the homologous genes of GATAX and NY in Arabidopsis,and ordered homozygous mutants atgatam,atgatan,atnym and atnyn.Phenotypic observation showed that the single mutant atgatam,atgatan,atnym and atnyn had similar growth phenotype with wild-type.We then crossed and obtained the double mutants atgatamatgatan with genotype aabb,which both provide material basis for studying the function of GATAX and NY homologues in Arabidopsis thaliana.In conclusion,this study identified transcription factor GATAX in Chlamydomonas reinhardtii,and found its downstream target gene NY.By exploring the function of GATAX,we propose that GATAX is involved in the regulation of TOR signaling by affecting the expression of NY.