Molecular Regulated Mechanism During Adventitious Root Development In IBA-induced Tetraploid Black Locust Cuttings

Tetraploid black locust, with the characteristics of fast growing, drought and barren resistance and saline-alkaline tolerance, is the prior choice of tree species for windbreak and sand fixation as well as soil and water conservation in Loess Plateau Region. Tetraploid black locust has difficulties in rooting so it is hard to be extensively planted. However, auxin from phytohormone is able to promote adventitious root formation of cuttings. Indole-3-butyric acid(IBA) is widely used in inducing adventitious root in cutting propagation in forest tree and can effectively promote the formation of adventitious root. IBA can promote rooting of tetraploid black locust cuttings. However, the molecular mechanism of induction of adventitious root formation by IBA remains unclear. Therefore, it is necessary to study the molecular mechanisms in regulation of cuttings rooting and in regulation of cuttings rooting by exogenous hormone IBA in tetraploid black locust.This study was based on differential proteins SAMS and MTN, which were selected by two-dimensional electrophoresis in previous studies, in various rooting stages of softwood cuttings under the induction of IBA. With semi-lignified softwood cuttings of tetraploid black locust from the same year as the material, full-length cloning, bioinformatics analysis and subcellular localization were performed on SAMS and MTN genes using homology-based cloning. Quantitative PCR was then used to analyze their expression profile in cutting base in different rooting stages and their expression profile in root, stem, leaf and phloem tissues in adventitious roots formed stage. Variation of corresponding enzyme activities were analyzed simultaneously. Further research was done on spatiotemporal expression pattern of genes related to metabolic cycle of methionine containing these 2 genes, relative enzyme activities and variation of metabolite contents. Transgenic Arabidopsis of SAMS and MTN genes was then prepared by transgenic technology to investigate the biological effect of the two genes on Arabidopsis rooting. At last, the differences in transcriptome between IBA-treated group and control group in hardwood cuttings adventitious roots growing stage were studied by high-throughput sequencing techniques. The dynamic changes of transcriptome in cuttings adventitious root growing stage were described in detail. The main results are summarized below.1. The clone and subcellular localization of SAMS and MTN genesDegenerate primers were designed according to conservative regions of homologous genes and were used to clone SAMS and MTN genes. Full length of c DNA was obtained by RACE technology. SAMS was 1498 bp long with an open reading frame of 1179 bp, encoding 392 amino acids and it shared 66-87% homology with Glycine max, Lupinus luteus, Lotus japonicas and Cicer arirtinum. MTN was 1158 bp long with an open reading frame of 810 bp, encoding 269 amino acids and it shared 94-96% homology with Glycine max, Lupinus luteus, Lotus japonicas and Cicer arirtinum. SAMS and MTN proteins were mainly located in cell membranes and cytoplasm based on analysis by subcellular localization.2. The expression profile of SAMS and MTN genes and their enzyme activityExpression profile of genes were analyzed by q PCR technique. The results showed that SAMS and MTN genes both had the highest expression in primordia formed stage in IBA group. In primordia formed and adventitious roots formed stages, IBA-treated group had greater expression levels of SAMS and MTN genes than CK control group. In IBA group, during adventitious roots formed stage both of SAMS and MTN genes had highest expression in stem tissues, followed by leaves, buds and roots. In various rooting stage, MTN enzyme activity increased firstly, decreased subsequently and then increased again. In addition, MTN enzyme activity was higher in IBA-treated group than in CK control group in primordia formed and adventitious roots formed stages. SAMS enzyme activity increased firstly and decreased subsequently and was higher in IBA-treated group than in CK control group in primordia formed and adventitious roots formed stages.3. The biological effect of MTN-transgenic and SAMS-transgenic Arabidopsis thalianaArabidopsis was transfected by constructing overexpression vectors of SAMS and MTN genes. The results showed that SAMS-transgenic Arabidopsis of T3 generation had longer adventitious root compared to wild type. Therefore, SAMS may directly involve in regulation of adventitious root growing. MTN-transgenic Arabidopsis had no remarkable variation compared to wild type, indicating that MTN may not regulate adventitious root growing.4. The enzyme activity and expression profile of gene from MTA recyclingEexpression profile of relative genes in metabolic cycle of methionine, relative enzyme activities and metabolite contents were analyzed. The results showed that SAMDC, SPDS and MTK genes all had the greatest expression levels in primordia formed stage. Their expressions in primordia formed and adventitious roots formed stages were higher in IBA-treated group than CK control group. ACS exhibited the highest expression level in adventitious roots formed stage. IBA-treated group had greater ACS enzyme activity than CK control group in primordia formed and adventitious roots formed stages. Polyamines and ethylene contents were the highest in primordia formed stage and were greater in IBA-treated group than CK control group in primordia formed and adventitious roots formed stages. During adventitious roots formed stage in IBA-treated group, SAMDC, SPDS, ACS and MTK genes all demonstrated the highest expression levels in stem tissues, followed by leaves, buds and roots. The results showed that the expression levels of relative genes in metabolic cycle of methionine and related enzyme activities increased after IBA treatment.5. The de novo transcriptome sequencing of cutting base tissuesHigh-throughput sequencing technology was used for de novo transcriptome sequencing of cutting base tissues in IBA-treated group and CK control group in 4 rooting stages(first stage, white callus stage, primordia formed stage and adventitious stage). 64876258 clean reads were obtained and assembled into 59931 unigenes with an average length of 732 bp. Among them, 38845 unigenes were annotated into 50 Gene Ontology functional categories, 17118 unigenes were classified into 25 COG functional categories and 17306 unigenes were categorized into 296 KEGG metabolic pathways. 12281 SSRs and 55800 SNPs were found in our transcriptome sequences. 45923 Inc RNAs were obtained by CPC software prediction.6. Screening, expression profiles and function of differentially expressed geneData analysis of transcriptome in 4 rooting stages after IBA treatment: 12261 genes in total were detected to be expressed in at least one stage and among them 4825 genes were expressed in all four stages. Clustering analysis was performed on data from 8 transcriptome using Cluster software and all of the 12321 genes were clustered into 40 categories. Results from clustering analysis also indicated that in cuttings of IBA-treated group 86.7% of the gene expression varied in the adventitious root formed stage. Comparing IBA-treated group and CK group, 30 categories had the same gene expression profile between the 2 groups. In the IBA-treated group, there were 8976, 1677 and 4383 differentially expressed genes in early and white callus stages, in white callus and primordia formed stages and in primordia formed and adventitious root formed stages, respectively. In addition, up-regulated gene expression outweighs the down-regulated. Comparing IBA-treated group with CK control group, there were 2570, 1685, 1512 and 10558 differentially expressed genes in early stage, white callus stage, primordia formed stage and adventitious root formed stage, respectively(p<0.05, |log2fold-change|≥1). This difference may be due to the fact that samples were obtained from different developmental stages, suggesting that some of the m RNAs were expressed in specific temporal patterns and dynamic.The 21 differentially expressed genes in different rooting stages from IBA and CK groups were verified by q PCR, the correlation coefficient between the two groups was 0.7056 indicating that the acquired gene expression profile substantially agreed with the results from high-throughput sequencing analysis. Functional enrichment analysis of Gene Ontology and KEGG was conducted on differentially expressed genes in various rooting stages in IBA-treated group. The results showed that significantly differentially expressed genes were mainly recruited to carbon and energy metabolisms and pathways including plant hormone signal transduction, cell differentiation and proliferation, metabolic cycle of methionine etc. Genes that may be related to tetraploid black locust cuttings rooting were also found, e.g. SAMS, SAMDC, ACO, SPD, POD, ZIF, AIN and ARE etc.