Gene Expression Analysis Of New Sprouts In Cunninghamia Lanceolata Grown Under Light And Dark Conditions

The key event in the greening process of plants is the biosynthesis of chlorophyll in plastids.The reduction of protochlorophyllide to chlorophyllide is the key rate-limiting step in chlorophyll biosynthesis,which is catalyzed by dark-operative protochlorophyllide reductase(DPOR)and light-dependent protochlorophyllide reductase(LPOR).Angiosperms only contain LPOR,so they cannot synthesize chlorophyll under dark conditions.Chinese fir(Cunninghamia lanceolata L.)cotyledons are green in darkness due to the coexistence of functional DPOR and LPOR.However,the sprouts of Chinese fir are etiolated in darkness,which is completely different from cotyledons.In this study,Chinese fir sprouts were selected as the experimental material.The transcriptome sequencing and molecular biology methods were used to analyze the gene expression of Chinese fir sprouts under light and dark conditions.The main results are as follows:(1)The expression level of POR gene for LPOR and chlB,chlL and chlN genes for three DPOR subunits were strongly down-regulated in dark-grown new sprouts in which chlorophyll biosynthesis was inhibited.(2)The total RNA from etiolated(dark-grown)and green(light-grown)sprouts was extracted,sequenced and annotated.A total of 70,499 unigenes were found,of which 47,410 were annotated.Among them,3263 differentially expressed genes were identified between dark-and light-grown samples.There were 1555 up-regulated and 2068 down-regulated genes in dark-grown samples.(3)The enrichment analysis was performed to explain the biological function of identified unigenes.It was found that the most affected pathways in GO terms were related to cellular components and catalytic.According to the KEGG databases,the five most affected metabolic pathways were flavonoid biosynthesis,photosynthesis-antenna proteins,photosynthesis,cutin,suberine and wax biosynthesis and stilbenoid,diarylheptanoid and gingerol biosynthesis.(4)In the process of photosynthetic electron transport and photophosphorylation,10 chloroplast genome-encoded genes and 28 nuclear genome-encoded genes were identified as differentially expressed genes.Compared with light-grown sprouts,the expression level of these genes was down-regulated in dark-grown sprouts.(5)Based on the acquired cDNA sequences,applied quantitative PCR was ued to further analyze the relative expression levels of 18 nuclear genes and 11 chloroplast genes related to photophosphorylation.Both transcriptomic and quantitative PCR results indicated that dark treatment can significantly block the expression of all of the identified genes related to the protein complexes of photosynthetic electron flow in new sprouts.In summary,dark treatment of Chinese fir sprouts caused etiolation via blockage of the expression of DPOR-and LPOR-encoding genes.Both nuclear and plastid genomic genes related to photosynthesis were significantly down-regulated in etiolated sprouts.The results provide new clues to understanding the different mechanisms of chloroplast development in different photosynthetic organs.