Protein Biochemistry and Expression Regulation of Cadmium/Zinc Pumping ATPases in the Hyperaccumulator Plants Arabidopsis halleri and Noccaea caerulescens

Seema Mishra; Archana Mishra; Hendrik Küpper

doi:10.3389/fpls.2017.00835

Protein Biochemistry and Expression Regulation of Cadmium/Zinc Pumping ATPases in the Hyperaccumulator Plants Arabidopsis halleri and Noccaea caerulescens

Front Plant Sci. 2017 May 22:8:835. doi: 10.3389/fpls.2017.00835. eCollection 2017.

Authors

Seema Mishra^{1

2

3}, Archana Mishra², Hendrik Küpper^{1

2

4}

Affiliations

¹ Fachbereich Biologie, Mathematisch-Naturwissenschaftliche, Universität KonstanzKonstanz, Germany.
² Department of Biophysics and Biochemistry of Plants, Institute of Plant Molecular Biology, Biology Centre of the ASCRČeské Budějovice, Czechia.
³ CSIR-National Botanical Research Institute, Plant Ecology and Environmental Science DivisionLucknow, India.
⁴ Department of Experimental Plant Biology, Faculty of Science, University of South BohemiaČeské Budějovice, Czechia.

Abstract

P_1B-ATPases are decisive for metal accumulation phenotypes, but mechanisms of their regulation are only partially understood. Here, we studied the Cd/Zn transporting ATPases NcHMA3 and NcHMA4 from Noccaea caerulescens as well as AhHMA3 and AhHMA4 from Arabidopsis halleri. Protein biochemistry was analyzed on HMA4 purified from roots of N. caerulescens in active state. Metal titration of NcHMA4 protein with an electrochromic dye as charge indicator suggested that HMA4 reaches maximal ATPase activity when all internal high-affinity Cd²⁺ binding sites are occupied. Although HMA4 was reported to be mainly responsible for xylem loading of heavy metals for root to shoot transport, the current study revealed high expression of NcHMA4 in shoots as well. Further, there were additional 20 and 40 kD fragments at replete Zn²⁺ and toxic Cd²⁺, but not at deficient Zn²⁺ concentrations. Altogether, the protein level expression analysis suggested a more multifunctional role of NcHMA4 than previously assumed. Organ-level transcription analysis through quantitative PCR of mRNA in N. caerulescens and A. halleri confirmed the strong shoot expression of both NcHMA4 and AhHMA4. Further, in shoots NcHMA4 was more abundant in 10 μM Zn²⁺ and AhHMA4 in Zn²⁺ deficiency. In roots, NcHMA4 was up-regulated in response to deficient Zn²⁺ when compared to replete Zn²⁺ and toxic Cd²⁺ treatment. In both species, HMA3 was much more expressed in shoots than in roots, and HMA3 transcript levels remained rather constant regardless of Zn²⁺ supply, but were up-regulated by 10 μM Cd²⁺. Analysis of cellular expression by quantitative mRNA in situ hybridisation showed that in A. halleri, both HMA3 and HMA4 mRNA levels were highest in the mesophyll, while in N. caerulescens they were highest in the bundle sheath of the vein. This is likely related to the different final storage sites for hyperaccumulated metals in both species: epidermis in N. caerulescens, mesophyll in A. halleri.

Keywords: QISH; RT-qPCR; cadmium; heavy metal ATPase; metal hyperaccumulator plants; zinc.