Comparison and Characterization of a Cell Wall Invertase Promoter from Cu-Tolerant and Non-Tolerant Populations of Elsholtzia haichowensis

Cell wall invertase (CWIN) activity and the expression of the corresponding gene were previously observed to be significantly elevated in a Cu-tolerant population of Elsholtzia haichowensis relative to a non-tolerant population under copper stress. To understand the differences in CWIN gene regulation between the two populations, their CWIN promoter β-glucuronidase (GUS) reporter vectors were constructed. GUS activity was measured in transgenic Arabidopsis in response to copper, sugar, and phytohormone treatments. Under the copper treatment, only the activity of the CWIN promoter from the Cu-tolerant population was slightly increased. Glucose and fructose significantly induced the activity of CWIN promoters from both populations. Among the phytohormone treatments, only salicylic acid induced significantly higher (p < 0.05) activity of the Cu-tolerant CWIN promoter relative to the non-tolerant promoters. Analysis of 5′-deletion constructs revealed that a 270-bp promoter fragment was required for SA induction of the promoter from the Cu-tolerant population. Comparison of this region in the two CWIN promoters revealed that it had 10 mutation sites and contained CAAT-box and W-box cis-elements in the Cu-tolerant promoter only. This work provides insights into the regulatory role of SA in CWIN gene expression and offers an explanation for differences in CWIN expression between E. haichowensis populations.

cDNA cloning, heterologous expression and characterization of a cell wall invertase from copper tolerant population of Elsholtzia haichowensis

The main objective of the present study was to clone, heterologously express and characterize a novel cell wall invertase (FCWI) from a Cu tolerant population of Elsholtzia haichowensis. The full-length FCWI cDNA contained an open reading frame (ORF) of 1671 bp which encoded a 556-amino-acid protein. The theoretical molecular mass and pI of the deduced protein were 62.5 kDa and 9.29, respectively. Phylogenetic analysis showed that FCWI had a closer evolutionary relationship to cell wall invertase of dicot. FCWI was expressed in methylotrophic yeast Pichia pastoris and purified to near homogeneity. Recombinant FCWI enzyme had pH optima of 4.0 and temperature optima of 50◦C. Activity analyses in the presence of various metal cations indicated that FCWI was completely inhibited by Hg

Copper and Inhibitors Induced Changes on Subcellular Antioxidant Enzyme Activities in the Leaves of Elsholtzia haichowensis

Under hydroponics, shoots of the copper (Cu) accumulator Elsholtzia haichowensis after 100 μM CuSO4 treatment 6 day were additionally treated with different inhibitors, then the effects of Cu and inhibitors on antioxidant enzyme activities in leave cells of E. haichowensis were investigated. Our results showed that Cu treatment significantly increased the activities of plasma membrane–bound NADPH oxidase, apoplastic peroxidase, apoplastic superoxide dismutase (SOD), symplastic SOD and symplastic ascorbate peroxidase in leaves of E. haichowensis. When additional treatment with N-N-diethyldithiocarbamate as an inhibitor of SOD, NaN3 as an inhibitor of peroxidase, diphenyleneiodonium as an inhibitor of NADPH oxidase and1,2-dihydroxybenzene-3,5-disulphonic acid as an O2•– scavenger, the activities of Cu-induced antioxidant enzymes in leave cells of E. haichowensis were significantly inhibited. The results showed that Cu-induced increase in the activities of various antioxidant enzymes in different cell compartments may represent a defense mechanism against oxidative stress.