Physiological Responses And Screening Of Differentially Expressed Genes Of Typha Orientalis Presl Under Pb²⁺ Stress

Lead （Pb）, a ubiquitous but highly toxic heavy metal, is harmful to human health through various pathways. Typha orientalis Presl offers a new way to restore lead pollution in the environment as phytoremediation, which was regarded as a new Pb-tolerant and Pb-accumulating plant. However, it is not very clear of the mechanisms of heavy metal tolerance in plants at present. Therefore, hydroponic experiments were conducted to research the effect of growth, physiological changes of Typha orientalis Presl under Pb²⁺ stress in different concentrations. Meanwhile, we explored the root cell gene in response to Pb²⁺stress. The main research results as follow:（1） Effects of different concentrations of Pb²⁺on physiological responses and subcellular structure changes of Typha orientalis Presl were studied in this experiment. With the increase of exogenous Pb²⁺concentrations, the growth of cattail seedlings were more significantly inhibited, and poisoning symptoms were more obvious. Pb²⁺treatment enhanced the generation rate of O2- in leaves, but lowered in roots. However contents of hydrogen peroxide （H2O2） in leaves and roots were significantly higher than that of the control. Photosynthetic pigments in leaves increased first and then decreased. There were no significant changes of MDA contents in leaves compared with the control, whereas MDA contents in roots significantly declined. In leaves, superoxide dismutase （SOD） activity reached the peak and then decreased compared to the control, which was always higher than that of control. However, peroxydase （POD） and catalase （CAT） activities in Pb²⁺treatment groups were all lower than the control group. In roots, superoxide dismutase （SOD） activity decreased significantly compared to the control except 1.00 mmol·L-1 Pb²⁺ treatment, both catalase （CAT） and peroxydase （POD） activities reached their peak and then decreased with the increase of exogenous Pb²⁺concentrations. By means of transmission electron microscope technology, it was observed that Pb²⁺induced damages of cell ultrastructure:chloroplast envelope was damaged, thylakoid swelled and broke in leaves cells; In leaves and roots cells, mitochondria were vacuolated and the envelope was broken; nuclear membrane was damaged, nucleolus lost and chromatin condensed. It was concluded that Pb²⁺stress induced imbalance of physiological metabolism and irreversible damage of subcellular structure.（2） Effects of different concentrations of Pb²⁺on lead particles subcellular localization and mineral elements were studied. With the increase of exogenous Pb²⁺concentrations, Typha orientalis Presl was able to accumulate Pb²⁺, and Pb²⁺accumulation remarkablely increased. The bioconcentration factor was 122-813, but all translocation factors were less than 1. Subcellular fraction analysis revealed that the contents of lead increased first and then slightly decreased in cell wall, which reached the peak value 5.55 mg·g-1 FW in 1.00mmol·L-1 Pb²⁺treatment. However, the contents of lead increased gradually in organelles and soluble fraction, but the distribution ratio was obviously unequal, and the Pb²⁺levels occurred in different parts of root cell with the following sequence cell wall> organelles> soluble fraction. Subcellular localization observation showed that lead mainly appeared in cell wall, and was also found in cell membrane structure and cell matrix. The effects of Pb²⁺on mineral elements in root cells were different, of which the absorption of macroelements Mg and microelements Fe, Cu, Zn were promoted, on the contrary, the absorption of macroelements K, Ca were inhibited. It was concluded that root cell of cattail resisted Pb²⁺stress by regulating the balance of cell ions, the regional distribution of lead particles and so on.（3） In order to investigate genes which were expressed differently in Typha orientalis Presl under Pb²⁺stress. We got lots of differentially expressed unigenes with different treatments, and then to classify and annotate different unigenes by GO, KEGG in order to study on unigenes corresponding function and metabolic pathways. Among of Go function enrichment, metabolic process, single-organism process, cell part, organelle, catalytic activity and binding were the most enriched entries. Among of KEGG metabolic pathways, phenylalanine metabolism, phenylpropanoid biosynthesis and starch and sucrose metabolism were the most enriched pathways. The analytical results indicated that the mainly differentially expressed genes related to reinforce material synthesis of cell wall structure and some key enzymes of antioxidant metabolism genes.