Mechanism Of Leaf Coloration In The Variety Of Ginkgo Biloba

Ginkgo biloba is one of the world famous relic species,known as a "living fossil".It not only has pharmaceutical value,edible value and timber value,but also has special landscape ornamental value because of the unique leaf shape and yellow coloration in autumn.G.biloba is widely distributed in China,and has many cultivated varieties due to the different site conditions or the effect of natural and artificial selection.Leaf color mutants are ideal materials for studying chloroplast development,photosynthesis and photomorphogenesis in plants.Previous studies mostly focused on the occurrence and genetic regulation of mutants,but few researches about the transcriptional regulation of leaf color variation were reported.In this study,we used G.biloba gold-colored mutant leaves and normal green leaves as the materials,by cytological observation and pigment content determination to clarify the physiological mechanism of yellow coloration in mutant leaves.Then,the RNA-seq of mutant yellow,leaves and normal green leaves was carried out.We further analyzed the molecular regulation mechanism of leaf color variation in G.biloba by the methods of bioinformatics and molecular biology.The main results are as follows.(1)Morphological observation showed that two types of G.biloba leaves showed similar phenotypic characteristics at the early stage of leaf development in March.Then,the mutant leaves were gradually differentiated and developed into yellow-green striped leaves from April to August,while normal leaves kept green during the same period.Finally,the mutant leaves and normal leaves gradually turned yellow and shedding from September to December.(2)Ultrastructure observation found that in the mesophyll cells of normal green leaves,chloroplasts showed typical shapes,with distinct thylakoid membranes,complete lamellar structure and few plastoglobuli.In contrast,chloroplasts in the mutant leaves revealed ruptured thylakoid membranes,indistinct or absent stromal lamellae and contained a large number of vesicles with numerous plastoglobuli.Additionally,the average number of chloroplasts per cell and mean chloroplast size in the mutant leaves were significantly lower than those of normal green leaves.(3)Physiological determination of pigment content results showed that the mutant leaves showed an 18.4%decrease in chlorophyll a content and a 59.5%decrease in chlorophyll b content compared with the normal leaves.The total chlorophyll in the mutant leaves was significantly lower than that of normal green leaves.On the other hand,the protoporphyrin IX(Proto IX),magnesium protoporphyrin(Mg-Proto)and protochlorophyllide(Pchlide)contents decreased markedly,by about 10?15%.In addition,the total carotenoid content of mutant leaves was higher than that of normal green leaves and the lutein content of the mutant was significantly higher,whereas we detected no significant differences among the other carotenoid components.The ratio of carotenoids to chlorophylls in the mutant was two-fold higher than that in green leaves.Additionally,the total flavonoid contents in the mutant were lower than those in the green leaves.(4)The transcriptome sequencing of the mutant yellow leaves and normal green leaves of G.biloba was carried out based on the Illumina HiSeqT,4000 platform.About 39.2?54.9 million clean reads were obtained through sequencing of the cDNA libraries.Q20 is above 97.93%and the content of GC is about 46.3%.Then,a total of 37.0?51.7 million reads were mapped to the G.biloba genomic database,with match ratios in the range of 94.16?94.69%.(5)A total of 5,235 differentially expressed genes(DEGs)were detected by comparing the two types of the library,including 2,933 up-regulated genes and 2,302 down-regulated genes.The functions of these DEGs were classified according to the GO database and DEGs were enriched in the GO terms 'catalytic activity' and 'carbohydrate phosphatase activity'.In addition,all DEGs were matched and assigned to 116 KEGG pathways.The main enriched pathways were'porphyrin and chlorophyll metabolism','photosynthesis','carotenoids biosynthesis' and some other pathways.(6)The chloroplast development and the chlorophyll accumulation are influenced by the expression level of their encoding genes.In the mutant,the gene GLK and Ftsz,which regulate the development and division of chloroplasts,showed down-regulated expression.Moreover,the DEGs involved in the chlorophyll biosynthesis and photosynthesis were down-regulated,while the DEGs related to chlorophyll degradation were up-regulated.Carotenoid and flavonoid can also affect the leaf coloration in plants.Comparing with the normal green leaves,the DEGs associated with carotenoid biosynthesis showed up-regulated expression,while the DEGs involved in flavonoid biosynthesis were down-regulated in the mutant leaves.(7)Transcription factors(TFs)play a key role in the regulation of plant development and secondary metabolism.In our dataset,we found that 276 DEGs were putatively identified as TFs associated with 56 TF families.The most abundant TF family was the MYB superfamily(10.51%),followed by bHLH(6.16%),AP2-EREBP(5.80%),orphans(5.07%)and WRKY(4.35%).Additionally,the DEGs encoding MYB,bHLH,AP2-EREBP,WRKY,TCP and C2H2 family TFs showed lower expression in the mutant compared to normal green leaves.(8)To confirm the changes in expression of genes,we further selected 15 DEGs related to chlorophyll metabolism,chloroplast development,photosynthesis,carotenoid and flavonoid biosynthesis,then examined by qRT-PCR.In general,the qRT-PCR results were consistent with the RNA-Seq data,suggesting the high throughput sequencing results were reliable.These DEGs profiles also provide more information on the expression level of genes at three stages of the growth and development in G.biloba.In summary,our results reveal the cytological,physiological and transcriptomic aspects of leaf color variation in the mutant of G.biloba and provide a useful reference for leaf coloration studies of other plant species.