Studies On Physiologycial And Molecular Foundation Of Sugarcane-Endophytic Diazotroph DX120E Interactions

Sugarcane is the most important sugar and energy crop in the world. Guangxi is the most important sugarcane growing region and its annual cane sugar production takes up almost 70% of the total in China. However, sugarcane production in Guangxi needs to apply of a large amount of nitrogen and N fertilizers are expensive and proved to be ecologically unsafe, which prompted the search for alternatives. Among them utilization of biological nitrogen fixation is the best choice. A lot of nirogen fixing bateria has been isolated and identified from the main sugarcane cultivars in Guangxi. Some of them have been found to be beneficial to plants after inoculation to sugarcane. However, report about systematic research on the interaction between associative nitrogen-fixing bacteria and sugarcane has not been found so far. This study carried out the whole genome sequencing of nitrogen-fixing bacterium strain DX120E isolated from the main sugarcane cultivar ROC22 grown in Guangxi.Experiments also were carried out to study the effects on growth and physiological characteristics of tissue cultured sugarcane seedlings after inoculated to two different sugarcane cultivars. Meanwhile we used the strain DX120E to compare the nitrogen-fixing capability with the model of nitrogen-fixing bacterium strain PAL5. Proteomics of the interaction between sugarcane and nitrogen fixing bacteria also was studied. The aim is to elucidate themechanism ofplant growth promotion and nitrogen fixationby the strain DX120E on physiological and molecular levels. The main results are as follows.1. The strain DX120E propagated in root, leaf sheath and leaf of two sugarcane cultivars B8 and GT21, and the bacteria quantities in different parts were root> leaf sheath> leaf. The bacteria could invade root through the surface cracks, generating sites of main root, lateral root, and root fracture, intensively propagate in intercellular spaces and cells of the root, and also transfer to mesophyll cells and vascular bundle sheath cells in leaf.There were no differences in the maximum propagation quantity under various inoculating concentrations, with the optimum concentration of 1×102 CFU mL-2. The DX120E inoculation could effectively promote plant growth and nutrient uptake, significantly improve nitrate reductase (NR) activities, and increase the glutamine synthetase (GS) activities and nitrate concentration in certain degree in the leaves, compared with the uninoculated seedlings. The DX120E inoculation also significantly improved the net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr) in leaves of two sugarcane culitivars. Comparing thenitrogen-fixing capability between the strains DX120E and PAL5 after inoculation, both sugarcane culitivars fixed nitrogen, and strain DX120E showed higher nitrogen fixation efficiency than strain PAL5.3. According to phylogenetic analyses of 16S rRNA, rpoB and gyrA sequences, strain DX120E is affiliated toKlebsiella variicola species. The result of complete genome sequencing showed that the whole genome contains one circular chromosome and two plasmids, and contains 5,718,434 nucleotides with 57.1% GC content,5,172 protein-coding genes,25 rRNA genes,87 tRNA genes, 7 ncRNA genes,25 pseudo genes and 2 CRISPR repeats. COGs functional analyses showed that the highest percentage was for carbohydrate transport and metabolism, amino acid transport and metabolism, and transcription, accounting for 11.3%,10.4% and 9.5%of the total predicted proteins, respectively.4. It was found that 22 differentially expressed proteins of strain DX120E after 48 hours of co-culture with tissue culturedsugarcane seedlings in 1/10 MS liquid medium by two-dimensional gel electrophoresis. Eighteen differentially expressed proteins were identified with mass spectrometry, including 8 up-regulated proteins and 10 down-regulated proteins.The differentially expressed proteins were classified into 4 categories as follows:energy accounting for 38.9%, metabolism (amino acid metabolism and carbohydrate metabolism) accounting for 27.8%, defense response accounting for 22.2%, and transporters accounting for 11.1%. We successfully identified 246 differentially expressed proteinsby ITRAQ combined with mass spectrometry proteomics technique from the the aerial part of the tissue cultured sugarcane plant after 48 h of inoculation, including 240 up-regulated proteins (109 known)and 6 down-regulated proteins(4 known). Based on their molecular functions, the known proteins were classified into 11 categories as follows:stress and defense accounting for 23.9%, metabolism accounting for 16.5%, protein turnover (including protein synthesis, folding and proteolysis) accounting for 12.8%, energy and photosynthesis equally accounting for 10.1% respectively, transporters and intracellular traffic accounting for 6.4%, transcription accounting for 5.5%, signal transduction accounting for 4.6% and cell structure and unclassified proteins equally accounting for 3.7%, respectively. Those for cell growth and division are the least, accounting for 2.8%.