Crassulacean Acid Metabolism Pathway And Its Key Genes’ Co-expression Networks In Dendrobium Catenatum

It is a survival strategy that approximately 7%of vascular plants recruit Crassulacean Acid Metabolism(CAM)pathway to withstand periodic drought stress in habitats or ecological niches.Although the core pathway of CAM has been described well,the key genes,regulatory genes,and regulatory mechanism in CAM related biological processes,including circadian rhythm,stomatal movement,and dicarboxylate transport,are still poorly understood.Dendrobium catenatum is a facultative CAM plant that can perform the pathway in varied degrees accroding to available water in matrix.Firstly,I would explore when and where CAM pathway were recruited in D.catenatum at its developmental stages or in volumetric water contents and these related physiological characteristics.Secondly,according to physiological characteristics,mature leaf samples were collected in four CAM phases and their RNAs were subjected to transcriptome sequencing.Then,transcriptome analysis,including gene function enrichment and gene set enrichment,were performed to differentially expressed genes(DEGs)between two adjacent phases.The analysis aimed to find the key genes in processes circadian rhythm,stomatal movement,and dicarboxylate transport.At last,a gene co-expression network was constructed using large scale transcriptome data of leaf samples.The regulatory mechanism and key genes’transcript factors in CAM pathway and its related biological process were analyzed and identified,respectively,based on the network.The main results are as follows:(1)Total organic acids contents(TOAc)in D.catenatum photosynthetic tissues showed that TOAc in the stages from protocorm likebodies to no pseudobulb seedlings was low(<0.05mol kg-1 FW)and not obviously fluctuated in a diel;that of leaves in the growth stages with a pseudobulb was high at night and low at day showing a significant fluctuation;but no obvious changes were found for TOAc of pseudobulbs at 24 hours.These results indicated that the photosynthetic pathways of the orchid changed from C3 to CAM with development stages and CAM were performed at the leaves of seedling with a pseudobulb.(2)Diurnal curves of CO2 uptake were measured for the third and fourth mature leaves(from the apex)of plants under drought stress(matrix volume water contents 0%)and control(matrix volume water contents 25 to 35%).Both the two type leaves were identified with CO2exchange characteristics of CAM pathway.But only the control ones had a whole four CAM phases that occur in obligate CAM plants.TOAc of mature leaves from plants with matrix volume water contents(free water)ranged from 0 to 45%accumulated at night and earlier stage of the following day,declined at the middle and later stages of the light period,and showed a diurnal fluctuation.A linear regression analysis for the mature leaves’TOAc and matrix volume water contents at each sample times in a diel showed slope-0.0002–0.0018 and R~20.0002–0.4430,which indicated that no linear relation between the two variables and suggested that CAM pathway were obligate in the mature leaves of D.catenatum.(3)High quality transcriptomic data of the mature leaf samples that obtained from four CAM phases have been analyzed.Firstly,Gene ontology(GO)analysis for DEGs between all adjacent periods showed that terms comprised of circadian rhythm,response to light stimulus,response to temperature stimulus,and response to endogenous hormone were enriched,which suggested that CAM pathway is a physiological equilibrium resulted from the interaction of internal and external factors.Then,I had identifed 29 candidates for key genes including circadian transcription factors,transporters or channel proteins in stomatal movements and dicarboxylic acid transport according to the physiological characteristic of CAM phases.Moreover,trend analyses and gene set enrichments for all DEGs showed that genes set of circadian rhythm,transcriptional regulation,cation transport,and response to hormone have a similar expression pattern.Gene sets of photosynthesis were upgraded at CAM phase III,which meant that the stomatal closure of D.catenatum was not midday depression like C3plants.(4)A total of 75 transcriptome data from leaf samples treated with drought stress,cold stress,and daytime courses were collected.I used these data to construct a gene co-expression network based on rank of weight Pearson correlation coefficient.Based on 86 gene modules related to key genes of CAM pathway,transcription factors’and transcription coactivators’candidates for genes PEPC,PPCK,NADP-ME,NADP-MDH,SLAC1,t DT,Ti D1,PUMP5,and RP1 were identified respectively.These CAM key genes’potential regulators and regulatory networks showed strong relationships to circadian rhythm,light/temperature stimulus,ABA hormone signal pathway.Moreover,promoter regions of these genes enriched with redundant photo-responsive cis-elements and a few circadian-,hormone-responsive-,and temperature-cis-elements.These results suggested CAM key genes were regulated by circadian rhythm,ABA,light,and temperature collectively.(5)In contrast to C3 plant Arabidopsis thaliana,the core clock oscillator LHY of D.catenatum show lowed weight Pearson correlation coefficient(-0.65–0.43)with most of the genes that are responsible for ABA synthesis and ABA signaling pathway,which means that LHY lost transcriptional regulation to these latter genes.And most genes of ABA signaling pathway and negative regulation of abscisic acid-activated signaling pathway were up-and down-regulated,respectively,in the CAM phase II and III,which showed a coupling relationship between ABA and stomatal movements.These collective evidences suggested that the uncoupling regualtion among LHY and genes of ABA synthesis and signaling pathway might be a reason for the reversal of stomatal movement in CAM plants to C3 partners.In conclusion,this study described initialization of CAM pathways in the developmental stages and CAM physiological characteristic of mature leaves;identified key candidate genes for CAM related process including circadian rhythm,stomatal movement,and dicarboxylate transport;also identified the regulatory genes and networks for CAM key genes;put forward a viewpoint that CAM key genes of the orchid were regulated by circadian rhythm,ABA,light,and temperature collectively;and suggested that uncoupling among LHY and ABA genes of ABA synthesis and signaling pathway might be a reason for distinct stomatal movement in CAM plants.This thesis provides new profiles for expounding complex regulatory processes of CAM pathway and new gene candidates for CAM genetic engineering.