Conventional crop production is increasingly being challenged by various problems such as decreased soil fertility and pollution due to the use of hazardous chemical pesticides and fertilizers at a global scale. In the same time, there has been mass awareness of quality and safety of food production. This situation escalates public concerns regarding the use of eco-friendly biostimulants which contribute to not only improve plant growth and development but also produce organic greens. Chitooligosacchrides (COS) is natural linear oligosaccharide and exhibits numerous interesting physicochemical and biological properties, which make it suitable for use in many fields. In agriculture, numerous studies had reported that COS can be used to promote the plant growth. Moreover, it is reported that the bioactivity of COS is closely related to its degree of polymerization (DP). Depending on different bioactivities, the function–structure relationship between DP of COS and its bioactivities could be different. However, most of previous studies were conducted using heterogeneous COS with different DP. On the basis of those prior studies, it was hard to confirm which COS with well-defined DP played a leading role in promoting plant growth. In addition, as for the bioactivity of COS on promoting plant growth, previous studies mostly focused on the apparent effects of COS on the plant physiology and growth characteristics. There are few reports about the metabolic response mechanism of plant to COS. In order to provide a fundamental guidance for future large-scale application of chitosan in agricultural, this thesis was performed to investigate the size effect of COS on its growth-promoting effect and COS-induced metabolic regulation on plant via metabonomics and transcriptomics technologies. Results were displayed as follows:
1. After treated with nine COSs with well-defined DP, including seven single COSs (chitobiose to chitooctaose) and two COS fractions with narrow degrees of polymerization (DPs) (DP8-10, DP10-12), we determined the size effects of COSs on the shoot length, root length, dry weight, fresh weight, and parameters related to photosynthesis and chlorophyll fluorescence of wheat seedlings in all treatment groups. The results showed that the activities of COS on plant growth were closely related to their DPs, and DP > 3 was necessary to insure a significant promotion effect on the growth and photosynthesis. (GlcN)5, (GlcN)6, (GlcN)7, (GlcN)8 and DP 8-10 seemed to be more effective than other COS fragments, and (GlcN)7 exhibited the optimal activity. (GlcN)7 could significantly increase the root length, dry weight and fresh weight. The contents of soluble sugar, soluble protein and chlorophyll in (GlcN)7 treatment group was improved by 59.4%, 22.0% and 20.3%, respectively. The size effect of COS on photosynthesis was in accordance with its effect on the growth parameters of wheat seedlings. The values of net photosynthetic rate (Pn), PSⅡ potential photochemical efficiency (Fv/Fo), photochemical quenching coefficient (qP) and variable chlorophyll fluorescence decrease ratio (Rfd) in (GlcN)7 treatment group were improved up to 35.2%, 11.0%, 18.6% and 14.7%, separately, while nonphotochemical quenching coefficient (NPQ) was decreased 48.6%, which resulted in the enhancement of photosynthesis and the promotion of light utilization efficiency of wheat seedlings.
2. According to the analysis of structure-fuctional relationship between the DP of COS and its growth-promoting effect, the most effective COS fragments [(GlcN)6, (GlcN)7 and (GlcN)8] were applied in our experiments to performed an integrative analysis of metabolite profiles with GC-TOF-MS in COSs-treated wheat seedlings. Metabolite profiling revealed that three chitosan fragments all could induce significant difference of organic acids, sugars and amino acids involved in photosynthetic carbon metabolism, glycolysis, tricarboxylic acid (TCA) cycle and central nitrogen metabolism in leaves of wheat seedlings. (GlcN)6, (GlcN)7 and (GlcN)8 induced 29, 55 and 48 differentially changed metabolites, respectively, which suggested that (GlcN)7 and (GlcN)8 were much more effective than (GlcN)6 in regulating plant metabolism. According to the KEGG analysis, the differentially changed metabolites in (GlcN)7 treatment were mostly involved in carbon fixation in photosynthetic organisms, TCA cycle, pyruvate metabolism, and alanine, aspartate and glutamate metabolism in wheat seedlings. (GlcN)8 mainly induced the carbon fixation in photosynthetic organisms, starch and sucrose metabolism and galactose metabolism. However, (GlcN)6 mainly activated the fructose and manose metabolism. It seemed that (GlcN)7 was more effective in activating the metabolic response of wheat seedlings relative to primary C and N metabolism.
3. In order to further validate the results of metabolite profiles and give a more comprehensive understanding of growth-promotion effect of COS, we therefore selected (GlcN)7 as a representative to investigate its impacts on primary C and N metabolism at metabolites, enzyme activities and transcript levels. Our results showed that (GlcN)7 could promote the light reaction in photosynthesis of wheat seedlings. A higher accumulation of sucrose content was also observed after (GlcN)7 treatment, accompanied by an increase in sucrose phosphate synthase (SPS) and fructose 1, 6-2 phosphatase (FBPase) activities as well as their up-regulation of relative expression level. Several metabolites associated with TCA cycle, including oxaloacetate and malate, were also improved along with an elevation of phosphoenolpyruvate carboxylase (PEPC) and pyruvate dehydrogenase (PDH) activities and their transcription expression levels. On the other hand, (GlcN)7 could also enhance the N reduction and N assimilation. Glutamate, aspartate and some other amino acids were higher in (GlcN)7-treated plants, accompanied by the activation of key enzymes of N reduction and N assimilation, including nitrate reductase (NR), glutamate synthase (GOGAT), glutamine synthetase (GS), glutamate dehydrogenase (GDH) and glutamic oxalacetic transaminase (AAT). Together, these results suggested COS could induce a pleiotropic modulation of carbon and nitrogen metabolism in wheat seedlings.
4. In order to further reveal the metabolic response mechanism induced by COS on plant and how COS regulate the metabolic pathwsays, we simultaneously investigated mRNAs and microRNAs (miRNAs) expression profiles of wheat seedlings in response to (GlcN)7. We identified 400 chitosan-responsive differentially expressed genes, including 268 up-regulated and 132 down-regulated mRNAs. According to GO analysis and KEGG analysis, we found (GlcN)7 could regulate the genes expression involved in photosynthesis, primary carbon and nitrogen metabolism, defense responses and transcription factors. Moreover, miRNAs also participate in (GlcN)7-mediated metabolic regulation on plant growth. Compared with the control, the length distribution of small RNAs (sRNAs) was significantly changed in (GlcN)7 library. We identified 87 known and 21 novel miRNAs, among which 56 miRNAs were induced or repressed by chitosan heptamer. Specialy, miRNA156, miRNA159a, miRNA164, miRNA171a were significantly down-reguated, while miR167c, miRNA319 and miRNA1127 were up-regulated obviously.