Mechanism Of AbGLK1 And Ksu On Regulating Green-white Chimeric Leaf Formation In Ananas Comosus Var.bracteatus Cv. ’chiyan’

Leaf color mutants are ubiquitous in nature,which are important sources for germplasm resource innovation.Ananas comosus var.bracteatus is an important tropical ornamental plant.Ananas comosus var.bracteatus cv.‘chiyan’ is a leaf color mutant selected from A.bracteatus,and its leaves present typical green-white chimeric phenotype with albino margin and green middle.In this study,integrative application of histocytology,physiology,reverse genetics and modified omics were conducted.And the aim of this study is to reveal the mechanism of green-white chimeric leaf color formation in Ananas comosus var.bracteatus cv.‘chiyan’.The main findings of the study are as follows:(1)Ultrastructural observation showed that chloroplasts of albino tissues were vesiclelike and had no thylakoid,indicating that albino tissues lack thylakoid membrane structure.Chlorophyll fluorescence analysis showed PS II complex assembly in albino tissues was disordered.Because albino tissues had less PS II active reaction center.Meanwhile,electron transfer process from QA to QB was blocked in albino tissues,and oxygen-evolving complex was seriously damaged in albino tissues.Besides,the distribution of sugars and endogenous hormones in albino tissues and green tissues is uneven.(2)In order to analysis expression characteristics of genes under different spatiotemporal conditions and hormone treated,stable reference genes were screened under specific experiments.We first selected ten candidate reference genes from transcriptome and common housekeeping genes.And then five common statistics tools(ge Norm,Norm Finder,Best Keeper,ΔCt method,Ref Finder)were used to evaluate ten candidate reference genes.The results showed that Unigene.16454 and Unigene.16459 were the optimal reference genes for different tissues,Unigene.16454 and ZRANB2 for green-white chimeric leaf at different developmental stages,IDH and SDP for seedlings under different hormone treatments.(3)AbGLK1 expression characteristic was as following: 1)AbGLK1 was mainly expressed in leaves,and expression level in albino tissues was significantly lower than that in green tissue;2)The expression level of AbGLK1 in green tissues was upregulated following chimeric leaves development,while the expression level of AbGLK1 in albino tissues had no significant change;3)GA3 and IAA could induce expression of AbGLK1.(4)AbGLK1 protein had a GOLDEN2 C-terminal box and a GAPR DNA-binding domain at the N terminal,belonged to GLK transcription factor family,which was located in nucleus and closely related with pineapple,rice and corn.And transactivation analysis showed that AbGLK1 has transcriptional activation activity.Besides,42 AbGLK1 interacting proteins were obtained by yeast two-hybrid.Among them,NADP-ME,MDH and ALDO are key enzymes in carbon fixation.This showed that AbGLK1 may be involved in regulating photosynthesis in green-white chimeric leaves.(5)BSMV-AbGLK1 silenced plant had less chlorophyll and abnormal thylakoids stacking,resulting in etiolation phenotype.Chlorophyll fluorescence analysis showed PS II complex assembly in BSMV-AbGLK1 silenced plant was disordered.Because BSMVAbGLK1 silenced plant had less PS II active reaction center.Meanwhile,electron transfer process from QA to QB was blocked in BSMV-AbGLK1 silenced plant,and oxygenevolving complex was seriously damaged in BSMV-AbGLK1 silenced plant.RT-q PCR results showed that the expression level of AbGLK1 in BSMV-AbGLK1 silenced plant was significantly downregulated,which could lead to downregulation of key genes of chlorophyll synthesis(Chl M,Chl27 and Por B),as well as genes related to photosystem assembly(Lhca1,Lhca2,Lhcb1.4,Lhcb5,and Psa H).(6)In total,855 Ksu sites in 335 proteins were identified and 593 Ksu sites in 237 proteins were quantified in green-white chimeric leaves.Compared to green tissues,232 Ksu sites in 128 proteins were quantified as upregulated targets,and 148 Ksu sites in 70 proteins were quantified as downregulated targets in albino tissues.Three conserved motifs were identified from 855 succinylated sites,and Ksu(X9)K only exist in Ananas comosus var.bracteatus cv.‘chiyan’.(7)Many differentially succinylated proteins were identified in carbohydrate metabolism,photoreaction,CAM pathway photosynthesis and photorespiration,which may play roles in green-chimeric leaf color formation.The severe damage of the oxygen release complex in albino tissues may be related to differentially succinylation on Psb O and Psb P.On the other hand,the change in the carbohydrate content of the albino tissues may be related to differentially succinylation on AbGLK1 interacting proteins(NADP-ME,MDH and ALDO),which are key enzyme in carbon fixation.