Analysis On Vascular Endogenous Hormone Contents, Ultra-Structure And Gene Differential Expression In Sugarcane Under Lxx-infection

Sugarcane is the largest sugar crop, originated in tropical and subtropical regions, and is a thermophilic crop. Ratoon stunting disease (RSD) of sugarcane (Sacchrum spp. Hyb.), caused by bacterium Leifsonia xyli subsp. xyli (Lxx), is one of the most important economically damaging diseases worldwide. RSD has become a major limiting factor for sugarcane production. The current research is mainly focusing on the detection methods and pathogen of RSD, and less on the interaction between the pathogen and host. The present study investigated the effects of RSD on cane yield components, sucrose content, and endogenous hormones content and ultrastructure of stalk and leaf in sugarcane. SSH library was established for Lxx-infected sugarcane stalk, gene differential expressions related to response to Lxx-inoculation were detected by real-time PCR technique, and five important genes responded to RSD pathogen-induction were cloned and their expressions were analyzed. The results showed as follows:1. Two commercial cane varieties (i.e. sugar cane variety of ROC22and chewing cane variety of Badila) were selected as the plant materials. The emergence rate of seedcane, plant height, stalk diameter, internode length, single stalk weight and sucrose content in ROC22and Badila were decreased by2.94%and8.50%,16.7%and19.4%,0.28cm and0.60cm,3.50cm and3.00cm,0.36kg and1.10kg,0.9%and1.1%(absolute value), respectively, in the RSD infection treatment than in the control. The plant height in the RSD infected seedcane treatment was significantly lower than those in the control and thermal treatment; The contents of GA3and IAA were obviously lower in the RSD infected seedcane treatment than those in the control and thermal treatment, in contrast to the content of ABA which was higher in the infected seedcane treatment as compared with the control and thermal treatment. Two cane varieties showed the same pattern and there was no significant difference between the control and thermal treatment in plant height and endogenous hormone levels. The thermal therapy is efficient to control RSD in sugarcane. The ultras tructure of stalks and leaves under transmission electron microscopy showed apparent difference between healthy and infected plants in chloroplast, mitochondria and nucleus of mesophyll cells, bundle sheath cells and stalk cells. The chloroplasts in diseased leaves were deformed, most chloroplast grana were dissolved, and the inner and outer membrane of chloroplast delaminated. The abnormal mitochondria expanded with indistinct cristae. In serious abnormal mitochondria, cristae disappeared and vacuolation of mitochondria with a few remains were found. The abnormal nucleuses were found and envelop of nucleus was split. The chromatin showed asymmetrical and degraded. There was a large amount of electron-dense substances accumulated in the infected stalk cells that might be associated with bacterial infection, and xylem cell walls were degraded and broken in different degrees. The results indicate that sugarcane quality and yield reduced due to RSD infection might associate to the disorder of water and nutrition transportation and photosynthetic efficiency reduce in the plants.2. ROC22was selected as the experimental material for establishing a SSH library for Lxx-infected sugarcane stalks, and195differentially expressed genes responsed to RSD were sceened out. Through the BLASTN functional comparison, it was found that there were41genes having a high degree of similarity with the genes related to disease and defence, accounted for21%of all, and this is the largest number. In addition,14genes were for protein synthesis and degradation, accounting for7%;17for metabolism, accounting for9%;12for ribosome biogenesis, accounting for6%;10for cell structure, accounting for5%;9translation related, accounting for5%;18transcription related, accounting for9%;12for signal transduction, accounting for6%;8for membrane trafficking protein, accounting for4%; and54for hypothetical protein and showed no significant similarity to the found genes, accounted for28%. the gene sequences were acquired and classified based on the GO classification standard. The results indicated that the genes involving in biological processes most were response to stress, metabolic processes, and transcription and translation, which were25.1%,17.7%and16.6%, respectively. The genes for methylation and glycolysis were the least, accounting for only0.06%. For molecular function, the mosts were for protein binding and structural constituent of ribosome, accounting for15%and11%, respectively, the least for ubiquitin-protein ligase activity and calciumion binding, accounting for only0.6%. For cellular components, the mosts were for composing cytoplasm, accounting for 36%; followed by nucleus and mitochondrion,15%and13%. But the least were for endoplasmic reticulum membrane and transcriptional repressor complex, accounting for0.6%.3. The expressions of14differential genes in response to Lxx-induction were analyzed using real-time PCR technology, and found that they showed upregulated expression under Lxx-infection.4. Five genes responded to RSD pathogen induction, remorin membrane protein (REMORIN), glycogen synthase kinase3(GSK3), ras-related protein (RASP), nascent peptide complexes transcription factor3(BTF3), splicing regulatory protein (SRP1) were cloned. Their full-length cDNAs were778bp,936bp,935bp,901bp,1119bp, respectively, which showed upregulated expression under Lxx-infection, and their expression quantity varied under PEG, NaCl, H2O2and4℃stresses.