Identification Of A Clorina Mutant In Betula Platyphylla×B.Pendula And Function Research Of BpGLK1

Leaf color variation is one of the most common mutated traits as it is easily to be discovered in higher plants.Among the leaf color mutants,yellow leaf is the most striking and brightest color in nature.Yellow leaf mutants are ideal materials for studying chlorophyll synthesis,chloroplast development and plastid to-nucleus retrograde signaling.Previously,we obtained a yellow leaf mutant yl from BpCCR1 transgenic lines in Betula platyphylla×B.pendula.In this study,we identified the insertion sites in yl genome and mutated gene responsible for yl phenotype.The research not only provided guidance for improving photosynthesis in birch,but also provided new transgenic birch varieties with different degrees of yellowing leaves for tree breeding.BpCCR1 overexpressing line(C11)and wild type birch(WT)were selected as control lines.Pigment contents,leaf anatomical structure,chloroplast development,photosynthetic characteristics,growth traits and gene expression characteristics in leaves of yl were investigated.The results showed that the content of chlorophyll a,chlorophyll b and carotenoid in yl was significantly lower than that in the two control lines,while the ratio of chlorophyll a/b was significantly higher than that of the control lines.Paraffin section result showed that the thickness of lamina,upper epidermis,lower epidermis,palisade parenchyma and spongy parenchyma thickness in yl were significantly lower than that of the two control lines.Chloroplast ultrastructure observation showed that yl mutant had a much-reduced thylakoid membrane network and grana stacking compared to control lines.The measurement of photosynthesis and seedling height showed that the net photosynthetic rate,stomatal conductance,transpiration rate and seedling height of yl were significantly lower than these of control lines.However,the effective quantum yield of PSII and maximal efficiency of PSII photochemistry showed no difference.Transcriptome sequencing result showed that the expression of many genes in leaves of yl were significantly changed.Compared with WT,a total of 930 differentially expressed genes were detected in yl,including 175 up-regulated genes and 755 down-regulated genes.Compared with C11,a total of 1163 differentially expressed genes were detected in yl,including 289 up-regulated genes and 874 down-regulated genes.KEGG enrichment analysis revealed that the differentially expressed genes of yl vs.C11 and yl vs.WT were significantly enriched in the photosynthesis-antenna protein pathway.TAIL-PCR,NGS and PacBio genome resequencing were used to analyzed T-DNA integration in yl genome.A total of 6 insertion sites,including IS1(Chr2 23466399),IS2(Chr2 26269259),IS3(Chr8 5168622),IS4(Chr8 17725909),IS5(Chr91671992)and IS6(Chr11 9184715)were detected in the yl genome.No genes were detected located within 5 kb upstream and downstream of IS1,IS2,IS4 and IS6.IS3 was located 885 bp upstream of BpPAL2 gene(Bpev01.c0990.g0002.m0001),and IS5 was located 2386 bp downstream of BpSMT2 gene(BpevO 1.c0577.g0001.m0001)and 1674 bp upstream of BpCDSP32 gene(Bpev01.c0577.g0002.m0001).However,qRT-PCR result showed no significant difference in the expression of BpPAL2,BpSMT2 and BpCDSP32 genes between yl and control lines.Further analysis of the T-DNA integration in yl revealed that three chromosomal fragments Chr2-3,Chr8-1 and Chr8-3 translocated to other chromosomes,and Chr2-2,Chr2-5 and Chr9-1 were lost after T-DNA integration.These variations lead to a homozygous deletion of a 40 kb chromosomal fragment on Chr2 and BpGLKl(Bpev01.c0167.g0013.m0001)gene was found to be located in the 40-kb deletion region.These results provided preliminary evidence that BpGLKl was responsible for the yl phenotype.PCR and RT-PCR results showed that BpGLK1 gene could not be detected at the level of both gene and transcription in yl.We imported BpGLK1 gene into yl by transformed yl using Agrobacterium or crossing yl(female parent)with WT(male parent).The functional complementation experiment showed that the leaf color were all restored in C-yl lines and yl offspring.In addition,chlorophyll a,chlorophyll b,carotenoid and chlorophyll a/b in the leaves of C-yl lines were all restored to the level of wild type.A total of six BpGLK1 overexpressing lines and 8 BpGLK1 repression lines were obtained by genetic transformation of birch.The BpGLK1 overexpressing lines showed deeper leaf color and slightly higher chlorophyll contents than the wild type.Starch granules in the chloroplasts were larger and more abundant than the wild type.The phenotypic characterization of BpGLK1 repression expression lines were similar to the yl line.They produced yellow leaves and had less chlorophyll contents,and showed defective chloroplast development,such as less and smaller starch granules and decreased grana and stroma lamellae.Transcriptome sequencing of functional leaves in BpGLKl overexpressing and repressing lines showed that 883 differently expressed genes,containing 546 up-regulated genes and 337 down-regulated genes were screened in BpGLK1 overexpressing transgenic lines.A total of 1553 differentially expressed genes,including 638 up-regulated genes and 915 down-regulated genes were screened in the BpGLK1 repression lines.GO function enrichment analysis showed differentially expressed genes were significantly enriched in photosynthesis-related process,including photosystem I light-harvesting antenna,light-harvesting antenna and photoreaction in BpGLK1 repression lines.Proteome sequencing analysis showed that the differentially expressed proteins were mainly enriched in the biological process of carbohydrate metabolism in BpGLK1 repression lines.And this was associated with the decrease of starch granules in the chloroplasts of BpGLK1 repression lines.ChIP-PCR experiments confirmed that BpGLK1 protein could bind to the promoters of eight antenna protein-related genes,five photosystem subunit-related genes and four chlorophyll biosynthesis-related genes and regulate the expression of these genes.In summary,we identified the phenotype and mutated gene of T-DNA mutant yl in birch,summarized the integration pattern of T-DNA in the yl genome,and explored the function of BpGLK1 gene.The research revealed that BpGLK1 gene was involved in the regulation of chloroplast development and leaf color formation in birch.The results obtained in this study could not only provide theoretical support for the functional study of GLK1 gene in woody plants,but also provide guidance for colored leaf tree breeding.People can obtain trees with yellow leaf trait through molecular biology techniques for landscaping.Meanwhile,the BpGLK1 repression transgenic lines obtained in this study could be resources for landscaping.