| In the one-carbon (C1) metabolic network of higher plants, the folate-independent metabolism of three Cl compounds including methanol (CH3OH), formaldehyde (HCHO) and formate (HCOOH) is the most obscure sector. Application of exogenous methanol on a series of plants has been reported to be able to enhance their growth and yields. However, the effect of CH3OH stimulation on plant growth was affected by various environmental conditions. As the metabolites downstream CH3OH metabolism in higher plants, both HCHO and HCOOH are toxic to plants but the toxicity of HCOOH is weaker than that of HCHO. In this study, foliar application of these three C1compounds was firstly performed on pot-grown Arabidopsis plants to provide a preliminary exploration on whether foliar application of C1compounds at very low concentration exerted an influence on plant growth, physiological characteristics as well as expressions of genes related to the defense-responsive systems, C1metabolic pathways and photosynthesis. As a ubiquitous environmental pollutant, comprehensive studies have been carried out to investigate the molecular mechanism of HCHO toxicity in medical field, while limited researches were performed to explore the toxicity mechanism of HCHO in plants. Arabidopsis and tobacco are the most widely used model plants in the fields of plant science. Our previous studies indicated that Arabidopsis and tobacco displayed different tolerance to exogenous HCHO due to their radically different HCHO-metabolic mechanisms. In this study, the reverse suppression subtractive hybridization (SSH) cDNA library was constructed in Arabidopsis and tobacco under2mM HCHO stress, respectively, combining with cDNA microarray analysis in Arabidopsis, to identify the potential HCHO-repressive genes in Arabidopsis and tobacco. The main purpose of this section is to reveal the HCHO toxic mechanisms in the two model plants and to interpret the similarities and differences in mechanisms of HCHO inhibition on the growth of Arabidopsis and tobacco at the transcriptional level as well as to provide new candidate genes for genetic manipulation to improve plant HCHO-resistant ability. Although previous studies have already provided some clues on the possible metabolic flux of CH3OH in plants, the detailed metabolic pathway of CH3OH in plants is still unclear. Both Arabidopsis and tobacco were treated with13CH3OH to obtain their13C-NMR metabolic profiles for further clarification of their CH3OH-metabolic mechanisms. In addition, using a photorespiration mutant of Arabidopsis and different compounds related to CH3OH metabolism, further analysis were performed to clarity the correlation of CH3OH metabolism and the stimulatory effects of CH3OH on plant growth. In the end, the effects of CH3OH on the NaH13CO3metabolic profiles and the expression of photosynthesis-related genes were comprehensively analyzed to clarify the mechanism of CH3OH stimulation on the growth of the two model plants at the metabolic and transcriptional levels. The major results and conclusions are as follows.Effects of foliar spraying different C1compounds on Arabidopsis grown under greenhouse conditions were analyzed. The results indicated that methanol stimulated the growth of Arabidopsis, accompanied by activation on expression of photosynthesis-related genes. On the contrary, formaldehyde and formic acid inhibited its growth and the inhibition of formaldehyde was much remarkable. Formaldehyde treatment elevated the levels of soluble sugars, H2O2and carbonyl-proteins in leaves, suggesting occurrence of oxidative stress. Meanwhile, formaldehyde activated the stress-responsive system in Arabidopsis. Interestingly, three C1compounds had no remarkable impacts on expressions of most selected genes involved in C1metabolism but all repressed the expression of5,10-methylene-THF reductase.Comprehensive analyses at physiological and transcriptional levels were performed to analyze toxic effects of HCHO exposure on Arabidopsis and tobacco. Leaf chlorosis and bleach were identified as the most representative symptoms when plants were exposed to HCHO. HCHO treatment exerted impacts on photosynthesis by repressing expressions of photosynthesis and chloroplast structure-related genes. Exposure to2mM HCHO led to a remarkable increase in levels of protein carbonyl and DNA-protein crosslinks in Arabidopsis, indicating the accumulation of oxidative damage to proteins and DNA. Up-regulation in many genes encoding heat shock proteins was suggested to be an important protective mechanism for Arabidopsis plants in response to the oxidative damage of proteins produced under HCHO stress. Previous investigations on HCHO toxicity in animals validated that inhalation of exogenous HCHO induced accumulation of oxidative damages on membrane lipids and proteins, which was suggested to be the typical toxic effects of HCHO in tobacco. This may be due to the relative weaker ability to metabolize exogenous HCHO by tobacco as compared with Arabidopsis. Meanwhile, down-regulations of HSPs (such as HSP70and DNAJ) might be a potential cause of the oxidative damage to proteins. Moreover, repressed expression of a large number of transcripts related to protein synthesis might be one of the molecular bases of the weak tolerance of tobacco to HCHO stress. Differing from cases in Arabidopsis, transcription of many key enzymes (such as SAMS, SAMDC, SAMMT) related to the activated methylation cycle in C1metabolic network were repressed in tobacco under HCHO stress. This might have impacts on synthesis of hormones and the components of membrane structure, which would lead to the oxidative damage to membrane lipids and the severe inhibition on the growth of tobacco. Repressive effects of HCHO on expressions of transcripts involved in detoxification metabolism in higher plants, such as CYP, GT and GST, were proposed to be the common toxic mechanism of HCHO in plants.The most important index indicating the stimulatory effect of CH3OH on plant growth is reflected as an increase in biomass, which is mainly attributed to photosynthesis. Using13C-NMR analysis, the relation between metabolism of13CH3OH and the effects of CH3OH stimulation on plant growth was investigated The results indicated that, when grown on MS agar medium, addition of2mM exogenous CH3OH had best effects on the growth of Arabidopsis and tobacco. However, the H13COOH produced from the metabolism of2mM13CH3OH in Arabidopsis preferred to flow into the C1metabolic pathways and produce large amount of [3-13C]Ser rather than oxidized as CO2which was then incorporated into the Calvin cycle and converted into carbohydrates. These results suggest that the stimulation effects of CH3OH on Arabidopsis growth are independent from its metabolism. Thus, it is hypothesized that the free CH3OH absorbed by Arabidopsis might act as a signal molecule to modulate the plant growth. Further analysis validated the presence of low concentration of CH3OH significantly induced the expression of most photosynthesis-related transcripts, which is suggested to be the main molecular basis for stimulation of CH3OH on plant growth. In tobacco,[3-13C]Ser was also identified to be a major product of13CH3OH metabolism. A comparison for metabolic profiles of13CH3OH with different concentration indicated no significant difference in the mechanism between lower and higher concentration of CH3OH metabolism in tobacco. Further analysis on the expression profiles of chloroplast proteins and photosynthesis-related key enzymes obtained a different result from those with Arabidopsis. CH3OH had no significant inducible effects on expressions of chloroplast structural proteins but pronouncedly stimulated expressions of key enzymes involved in CO2assimilation. Moreover, different concentrations of CH3OH showed almost same inducible effects on expression of these genes in tobacco. |
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