Drought resistance and protein changes induced by chitosan in rice Oryza sativa L.

Abstract

This research aims to determine the appropriate chitosan types and concentrations for drought resistant induction in rice based on the hypothesized that the antioxidant system should be one of chitosan targets. Then, the proteomic approach was used to identify the other systems that resulted from chitosan treatment for drought resistant induction. Finally, the genes responsible for drought resistance induced by chitosan was identified and the mutant lines in some chitosan responsive genes were identified by TILLING method. Two rice lines, ‘Leung Pratew 123’ (‘LPT123’) (Oryza sativa L. ‘Leung Pratew123’) and ‘LPT123-TC171’ rice lines, which have the similar genotype, except the genes responsible for salt and drought resistance were used in this study. To determine of the appropriate chitosan types and concentrations for drought resistance induction, four types of chitosan molecules, P-80, O-80, P-90 and O-90 at 20 or 40 mg/L were applied during and after drought stress. O-80 chitosan at the concentration of 40 mg/L significantly increased of SFW and SDW in ‘LPT123’, while shoot water content tended to increase, but chitosan did not affect ‘LPT123-TC171’ growth under drought stress. Application of oligomeric chitosan (O-80) before drought stress could increase photosynthetic pigments in both rice lines. After drought stress for 7 days, ‘LPT123’ could maintain the pigment contents at the same level on control without chitosan application, while it showed the negative effects on ‘LPT123-TC171’. Drought induced H2O2 production was detected in both rice lines, but chitosan treatment clearly showed the reduction of H2O2 content only in ‘LPT123-TC171’ rice during drought stress. Moreover, antioxidative systems were quantified during drought stress. Ascorbic acid, GSH and GSSG contents did not seem to be involved in drought resistance, induced by chitosan in ‘LPT123’. In ‘LPT123’, more antioxidant enzyme activities are found in roots than shoots, which is opposite to what found in ‘LPT123-TC171, this supports lower H2O2 production during drought stress in ‘LPT123-TC171’ resulted in less growth and drought resistant enhancement by chitosan, when compared to ‘LPT123’. This suggested that H2O2 might be the required signal for chitosan responses in rice. Furthermore, chitosan treatment did not affect the increasing of ATPase activities in leaves and roots of both lines under drought stress. The proteomics approach using LC-MS/MS was employed to analyze protein changes in response to chitosan during drought stress. The results showed the changing of total of 168 proteins in leaves and 92 proteins in roots. Within the significant protein expression in leaves and roots of ‘LPT123’, 20 and 21 proteins were found to be down-regulated, whereas 15 and 7 proteins were up-regulated, respectively. On the other hand, in leaves and roots of ‘LPT123-TC171’, 49 and 12 proteins were found to be down-regulated, while, 4 and 8 proteins were up-regulated, respectively. The significant changes in abundance of proteins during drought stress indicated that several pathways including metabolic process, signal transduction, transcription, transport, disease resistance/defense, growth and protein degradation. These data suggest the potential for identification of the novel proteins involving in drought resistance in rice. Moreover, the up-regulated proteins during drought stress in leaves of ‘LPT123’ which were predicted to have the nucleic acid binding activity, was investigated by qPCR method. The expression of the genes; Os12g23700 and Os02g58440 involving in signal transduction and transcription, respectively, when subject to chitosan were higher than the control especially in the early phase. This suggests that both genes may play a role in chitosan response in ‘LPT123’ during drought stress. Finally, TILLING was used to identify the mutant in the 5 genes of interest for further characterization. The 66 nucleotide changes were identified in the sodium azide and methyl nitrosourea (Az-MNU) treated ‘Nipponbare’ population. The various mutations were silent mutation, non-severe mutation (NSM), possibly-severe mutation (PSM), truncation, splicing and mutation in intron.