| Vacuolar processing enzyme(VPE) is one kind of cysteine proteases which exists in vacuole of higher plants, a member of the C13 family and exhibits caspase-1-like characteristics; it plays an important regulation role when the plant suffered to drought, salt,heavy metals and other abiotic stress. Malus hupehensis(Pamp) Rehd. var pinyiensis Jiang is originated in Mengshan region of China and it has a high disease resistance. In this study,MhVPEγ expression levels, enzyme activity and the changes of related physiological indicatorsin Malus hupehensis seedlings and MhVPEγ transgenic Arabidopsis plants under high temperature, drought, high salt, cadmium sulfate and exogenous hydrogen peroxide stress treatment were under detected and analyzed for studying the function of VPE, the relationship betweenm MhVPEγ gene expression levels and cell death and revealing the mechanism of VPE in cell death. In addition, the results provide basis for improved the resistance of apple rootstocks.(1) Under normal growth conditions, the changes of root activity, ATP content,caspase-3-like activity and cell death quantity in absorption root were more activity and all significantly higher than those in extensive roots. While changes of extensive roots were relatively stable, especially there was no significant change of the rear extension roots.(2) Under 0.1-3.0 mmol·L-1H2O2 treatment the expression level of MhVPEγ, the activities of VPE, caspase-1-like and caspase-3-like, as well as the cell death quantity all significantly increased with the increasing of H2O2 concentration; however, the ATP content and root activity was decreased. On the other side, the activities of caspase-1-like and caspase-3-like were reduced, the oxidative damage was significantly alleviated, and the cell death rate, root activity and expression level of MhVPEγ was decreased in the root of M.hupehensis Rehd. under H2O2 stress while the VPE specificity inhibitor Ac-ESEN-CHO was added. VPE and caspase-1-like could share the same substrate and VPE inhibitors significantly inhibited caspase-1-like activity in the root extract of M. hupehensis Rehd..These results indicated that the relationship between VPE, caspase-1-like, caspase-3-like and cell death was closely connected; VPE was similar to caspase-1-like in the activity and function, they could be involved in responses to oxidative stress by promoting cell death in the root of M. hupehensis Rehd..(3) Real-time PCR showed that MhVPEγ expression levels in the leaves of M.hupehensis was higher than that in roots and stems. In addition, cell death rate in the leaves of M. hupehensis was also higher than that in roots and stems, indicating that there is closely relationship between MhVPEγ expression level and cell death in plants.(4) We got three MhVPEγ-overexpressing Arabidopsis lines(T3-1, T3-2, and T3-3) by screening of kanamycin medium. VPE activity and MhVPEγ expression levels in transgenic lines were significantly higher than that of the wild-type Arabidopsis thaliana.(5) Under high temperature treatment, VPE activity and MhVPEγ expression level significantly increased with caspase-3-like activity, H2O2 content, lipid peroxidating and the increase of cell death quantity and apoptosis rate in all types of plants; whereas the changes of these indicators in MhVPEγ-overexpressing transgenic plants were much higher than that of the wild type, while ATP content and cell activity was significantly lower than that of wild-type, indicating that MhVPEγ reduce the resistance of transgenic plants to abiotic stress.(6) The leaves of transgenic plants lines turned smaller, yellow and seed number was significantly reduced and its growth cycle was also significantly shorter than that of the wild type. Drought stress can significantly accelerate the development process of Arabidopsis to earlier flowering and seed-setting. |
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