Physiological Response And Molecular Mechanisms Of Acid Invertases In Copper-mine And Non-copper Mine Populations Of Kummerowia Stipulacea Under Copper Stress

Metallophyte is a kind of special plant living in heavy metal contaminated areas and they evolve a series mechanism to tolerant heavy metals. This kind of plants is considered as a novel and green alternative solution to the problem of heavy metal pollution. The soils where metallophyte plants grew were generally poor, under such bad conditions metallophyte plants could normally grow and develope, so they might have special physiological and molecular mechanisms. Kummerowia stipulacea (Maxim.) Makino is a pseudometallophyte identified in recent years. The recent researches show that the matallophyte had some special patterns of resource allocation: under heavy metal stress conditions, they could allocate more resource to roots. This allocation pattern maybe a necessary resistant strategy adapting to the heavy metal contaminated sites.The plant materials of this study were metallophyte of Kummerowia stipulacea (Maxim.) Makino, one population was from a copper mine at Tonglushan hill (Cu-tolerant population), the other population from a non-cooper-contaminated site in Wuhan university, Hubei, China (non-tolerant population). Hydroponic cultures were conducted to comparatively investigate the difference of in biomass growth, and its mechanisms of photosynthesis and sucrose metabolism of Cu-mine and non-Cu-mine plants of K stipulacea under excessive copper conditions. Simultaneously, we clone acid invertase cDNA from the root of K stipulacea from Cu-mine and non-Cu-mine sites and analysis the transcript level by real-time PCR. Finally, we conducts compare enzyme characteristics study by Pichia pastoris expression system. From the results, we conclude the following conclusions:(1) The difference in physiological response of K. stipulacea from Cu-mine and non-copper-mine sites under the Cu treatmentThe root biomass, shoot biomass and root architecture of K. stipulacea from non-copper-mine sites were all significantly inhibited after Cu treatment, while those from copper sites were not change much and even some physiological indexes were elevated. Compared to the shoot growth, the root growth parameters were more sensitive to copper. The net photosynthetic rates of K. stipulacea from non-copper-mine sites were all significantly inhibited after Cu treatment, while that from copper sites was inhibited slightly. The water use efficiency of K. stipulacea from non-copper-mine sites was slight elevated while that from copper sites was significantly elevated. The net photosynthesis rate and water use efficiency in copper-mine population were higher than that in non-copper-mine population under Cu stress, which were useful for the plant growth in the elevated Cu and water deficiency soils due to the enough supply of nutrient components and energy in the contaminated population. It was the result that resistant plant had developed to adapt to the habitation.(2) Differences in sucrose metabolism of MP population and NMP of K. stipulacea under the copper treatmentThe sugar content, acid invertase activities in the root of K. stipulacea from copper-mine sites elevated significantly after Cu treatment, while the sugar content, acid invertase activities in the root of K. stipulacea from non-copper-mine sites not changed much. The sucrose content and acid invertase activities in the root of K. stipulacea from copper-mine site is significantly than that in non-copper-mine site. This is beneficial for copper-mine plants allocate more biomass to the root. The elevated acid invertase activities in the root of K. stipulacea from copper-mine site made for the sucrose transport from leaves into roots while the non-copper-mine plants did not have such mechanism.(3) Differences in acid invertase gene sequences between K. stipulacea from copper-mine and non-copper-mine sitesThe cDNA sequences coding for vacuolar invertase of K. stipulacea from non-copper-mine site has99.53%identity to that from copper-mine site and cDNA sequences coding for cell wall invertase from non-copper-mine site has98.76%identity to that from copper-mine site. In our study, we observed two and four divergent amino acids in vacuolar invertases and cell wall invertases, respectively, between the MP and NMP of K. stipulacea. The variations in amino acids between the two populations may lead to the variation in enzyme characteristics between the two populations.(4) Differences in expression level of acid invertases of K. stipulacea from copper-mine and non-copper-mine sites under the copper treatmentThe expression level of acid invertase in the root of K stipulacea from copper-mine sites elevated significantly after Cu treatment, while that in the root of K. stipulacea from non-copper-mine sites decreased. The transcript level of acid invertase in the root of K. stipulacea from copper-mine site is significantly higher than that in non-copper-mine site. There exists positive correlation between the enzyme activities and transcript levels of acid invertase in the root of K. stipulacea from copper-mine and non-copper-mine sites, which indicate there exist some relationship between enzyme activities and transcript levels of acid invertase in the root of K. stipulacea from copper-mine and non-copper-mine sites.(5) The expression in Pichia pastoris of acid invertases of K. stipulacea from copper-mine and non-copper-mine sitesThe mature peptides of the acid invertase coding region of K. stipulacea from the two sites linked to shuttle vector pPICZaeA and got the recombinants expression vector pPICZaeNCWinv, pPICZaeCWinv, pPICZaeeNCVinv and pPICZaeCVinv. By electroporation, these linearized vectors were transformed successfully into Pichia pastoris X33. The four acid invertases gene were expressed successfully in Pichia pastoris X33and got the bioactive of four acid invertases. The results show that optima pH and temperature of vacuolar and cell wall invertase from two sites almost keep the same. Km and Vmax of vacuolar and cell wall invertase from two sites have no significant differences, which indicate the enzyme characteristics of vacuolar and cell wall invertase from the two sites have no essence differences.