The Study On Formaldehyde Absorption, Metabolism And Formaldehyde Stress Response Genes In Ornamental Plants

Recently, formaldehyde (HCHO) has become one of the main indoor air pollutants because of a variety of insulation materials applied in buildings. Ornamental plants are thought to be the best choice to purify indoor formaldehyde, which is a simple, long-term effective ecological control method. Though, some studies have confirmed that plants have abilities to absorb and metabolism formaldehyde, plant genetic engineering on formaldehyde metabolism is still in an infancy stage, and lacking the understanding of the mechanism of formaldehyde metabolism in the plant. In order to obtain clues for plant's own metabolic strategies, expand the work of formaldehyde phytoremediation, we carried an investigation on formaldehyde absorption, metabolism and Formaldehyde Stress Response Genes in Ornamental Plants in the present study.The capacity of several ornamental plants to absorb gaseous and aqueous formaldehyde was determined and the factors influencing formaldehyde-absorption capacity were evaluated in these ornamental plants. The formaldehyde metabolism capacity and pathway was examed by labeling experiments with 14C formaldehyde and 13C formaldehyde. In addition, to investigate the physiological responses to formaldehyde stress in these plants, total protein, amino acid, soluble sugar and malondialdehyde contents were measured after the plant leaves were treated with formaldehyde. In order to understand the mechanism of plant response to formaldehyde stress at the molecular level, SSH method was adopted to construct a forward SSH-cDNA library for Rohdea japonica, to analysize the genes induced by formaldehyde stress. Smart technology was used to construct Smart-cDNA library for Cymbidium grandifiorium and the formaldehyde stress responsive genes was analysized by RT-PCR. The results were as following:1. The selected ornamental plants could absorb both gaseous and aqueous formaldehyde. The results showed that the abilities of selected five ornamental plants to absorb 7mM formaldehyde are as follows:R. japonica> Petunia hybrida> Pteris multifida Poir> Begonia semperflorens> Pilea peperomioides. The abilities of selected four ornamental plants to absorb 2mM formaldehyde were as follows:Rohdea japonica> Caulis Hederae Sinensis> C. grandifiorium> Philodendron pandurifor.2. The abilities of ornamental plants to absorb formaldehyde were affected by circumstance factors and microorganisms on the leaf surface. Low temperature (15℃) or alkaline pH (pH9.0) might inhibit the formaldehyde-absorption of the plants. The microorganisms on the leaf surface contributed on the absorption of formaldehyde by the plants and the contribution depends on the structure of leaf surface.3. The abilities of the selected four ornamental plants to metabolize 14C formaldehyde were as follows:C. grandifirium>R. japonica>P. pandurifor>C. Hederae Sinensis. After 24h treated with 14C formaldehyde,14C isotope activity of the soluble fraction in the four plants was higher than that in the insoluble fraction. Most 14C activity was presented in the soluble fraction of the plants.4.13CNMR spectrum of C. grandifiorium, R. japonica,P. pandurifor and C. Hederae Sinensis labeled with 13Cformaldehyde and 13Cmethanol showed that formaldehyde and methanol metabolism pathways are different in these plants. NMR spectrums of C. grandifiorium and R. japonica labeled with 13C formaldehyde suggested that their 13Cformaldehyde and 13Cmethanol metabolic pathway might be similar. Parts of formaldehyde was metabolized to 5,10-methylenetetrahydrofolate directly, and then metabolized to serine and subsequently entered photorespiratory pathway. Small parts of formaldehyde was metabolized to formic acid, which might be oxidased as CO2 or metabolized to 5,10-methylenetetrahydrofolate, and then to serine and finally also entered the photorespiratory pathway. Methanol was not present in the metabolites of formaldehyde metabolism.5. After treated with formaldehyde, the contents of amino acids in C. grandifiorium, R. japonica and P. pandurifor were all changed. In C. grandifiorium leaves, Glu content was firstly decreased then increased dramatically. Asp content was also increased largely and Ser content was also highered sighnificantly. In R. japonica leaves, Pro content increased dramatically in the initial 12 hours then decreased dramatically. Ser level only showed slightly increased. In P. pandurifor leaves, Glu content decreased dramatically to the lowest level in the initial 2 hours then increased while His level showed a large decrease in the initial 2 hours then increased smoothly. Ser level showed a markely increase trend.6. After treating with formaldehyde, physiological changes occurred in C. grandifirium,R. japonica and P. pandurifor to response to formaldehyde stress. Trend of protein content first decreased then increased, which showed that protein synthesis was reduced in the beginning of formaldehyde stree, some proteins may be broken down or consumed. In the initial stage of formaldehyde treatment, soluble sugar and malonaldehyde (MDA) were significantly increased, which suggested that membrane lipid oxidation was occurred because of formaldehyde stress, to improve the ability of formaldehyde detoxification, the synthesis of carbohydrates was increased.7. Leaves of R. japonica treated with 2mM formaldehyde were used as tester and untreated leaves were used as driverto conduct a forward SSH cDNA library which included 1248 positive clones. The insertion fragments of positive clones were ranged from 200bp to 800bp and its average length was about 500bp.8. The positive clones in the SSH cDNA library with insert larger than 500bp was selected to sequence. Sequence analysis generatae 296 ESTs among which 61% had significant homology with known function genes, including 12% related to metabolism and energy, 10% related to stress and defense,9% related to protein synthesis and processing,7% related to signal transduction,6% related to transcription and regulation,6% related to transporter,3% related to photosynthesis and cytoskeleton as well as 2% related to protein degradation and cell grown; In the remaining 39% ESTs,18% ESTs were unknown functions and 21% are new ESTs.9. Expression profiling analysis of the selected 18 genes from the SSH cDNA library was performed with RT-PCR. The results showed that the 18 genes were all induced by formaldehyde stress. The MDA content in R. japonica leaves was very higher after exposed to with 2mM formaldehyde, indicating that strong oxidative stress occurred after formaldehyde treatment. In corresponding to such physiological change, the expression of many stress-defense related genes, such as heat shock protein, thioredoxin, glutaredoxin, oxidoreductase and so on were up-regulated to protect leaf cells from damage by oxidative stress and enhance its detoxification ability to formaldehyde.10. Smart technology was used to construct Smart-cDNA library for C. grandifiorium, leaves which included 672 positive clones. The insertion fragments of the library were ranged from 200bp to 800bp and the average length was about 500bp. Sequence analysis for 136 positive clones with insert larger than 500bp generated 95 ESTs.11. Expression profiling anslysis for the 15 genes selected from the Smart-cDNA library was performed by RT-PCR. The results showed that transcription of five selected genes was up-regulated including senescence-associated protein, chloroplast glyceraldehydes-3-phosphate dehydrogenase B subunit, LHCII type I chlorophyll a/b binding protein, heat shock protein and L-isoaspartate-O-methyl transferase and transcription of six selected genes was down-regulated including light harvesting chlorophyll a/b binding protein, stress-responsive protein, ferredoxin-nadp reductase, photosystem I subunit IV, protochlorophyllide reductase B and pre-plastocyanin. The data suggested that the expression of photosynthesis related genes was repressed by formaldehyde stress.