The prerequisite for breeding a plant to be used in phytoremediation is its high tolerance to grow normally in soil contaminated by certain heavy metals. As mechanisms of plant uptake and transport of nickel (Ni) are not fully understood, it is of significance to utilize exogenous genes for improving plant Ni tolerance. In this study, rcnA from Escherichia coli encoding an exporter of Ni and cobalt was overexpressed constitutively in Arabidopsis thaliana, and the performance of transgenic plants was assayed under Ni stress. The subcellular localization of rcnA in plant cells was found to be the plasma membrane. Constitutive overexpression of rcnA in Arabidopsis rendered better growth of either seedlings on agar medium containing 85, 100, and 120 μM NiCl₂ or adult plants in a nutrient solution with 5 mM NiCl₂ added. Compared to the wildtype, rcnA-OE transgenic plants under Ni stress accumulated lower levels of reactive oxygen species (i.e., superoxide and hydrogen peroxide) in leaves and exhibited less oxidative damage in shoots, as demonstrated by less electrolyte leakage and the lower malondialdehyde content. Notably, rcnA-OE transgenic plants retained a higher content of Ni in roots and had a lower content of Ni in shoots. Therefore, our findings indicated that the bacterial rcnA gene may be utilized to improve plant Ni tolerance through genetic transformation.

Anthocyanin biosynthesis determines the leaf color of Acer palmatum as a widely-planted landscape tree. Previously, ApMYB1 has been characterized as a positive regulator of anthocyanin biosynthesis. To further elucidate the mechanism of leaf coloration, the present study identified a basic helix-loop-helix (bHLH) transcription factor (ApbHLH1) through the phylogenetic analysis of 156 putative bHLH proteins in Acer palmatum and eight reference bHLHs which were known to be involved in the anthocyanin biosynthesis of selected plants. Protein structure comparison showed that ApbHLH1 has a conserved bHLH domain, and its N-terminal contains an MYB-interacting region. The expression of ApbHLH1 in leaves was found to not be correlated with anthocyanin contents either in green, semi-red leaves or during leaf autumnal senescence when anthocyanin content increased. ApbHLH1 expression in detached leaves was induced by exogenous senescence-promoting chemicals, including H₂O₂, SA, MeJA, ACC and ABA, with certain durations. In particular, either high light or low temperature induced ApbHLH1 expression significantly, and combination of high light and low temperatures seemed more effective in inducing ApbHLH1 expression. Luciferase complementation imaging assays confirmed the physical interaction between ApbHLH1 and ApMYB1, which could be abolished by either the truncating MYB-interacting region of ApbHLH1 or the deleting bHLH interacting domain of ApMYB1. The transient expression of ApbHLH1 could not induce anthocyanin production, while the co-expression of ApbHLH1 and ApMYB1 resulted in a higher accumulation of anthocyanins compared to the expression of ApMYB1 alone in tobacco leaves. Collectively, our results revealed that ApbHLH1 participated in leaf coloration through binding with ApMYB1 and enhancing the ApMYB1 function of promoting anthocyanin biosynthesis during leaf autumnal reddening in Acer palmatum. ApbHLH1 could have the potential for breeding color-leafed plants through co-transformation with ApMYB1.