Abstract
P1B-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.
Highlights
Hyperaccumulator plants actively take up metals or metalloids and store them in above ground parts in the vacuoles of large epidermal cells
The metal activation of the NcHMA4 ATPase activity with various concentrations of Cd and Zn (Leitenmaier et al, 2011) was measured in the current work to verify the activity of protein for the further steps of characterization
Metal binding was analyzed via the change of a characteristic ligand to metal charge transfer (LMCT) band at 375 nm (Figure 1)
Summary
Hyperaccumulator plants actively take up metals or metalloids and store them in above ground parts in the vacuoles of large epidermal cells. Enhanced xylem loading of the metal ion in the root, increased root-to-shoot transport and highly efficient metal detoxification are the common attributes of metal hyperaccumulation (Lasat et al, 1996; Küpper et al, 1999; Krämer et al, 2000) It involves active pumping of the metals into specific storage sites, such as vacuoles of the epidermal cells, where the concentrations of heavy metals can reach up to several hundred mmol.l−1 (Küpper et al, 1999, 2001). The second structure further clarified the importance of two conserved cysteine residues in the high affinity metal binding site, and via comparison of two crystallized states of the enzymatic cycle it provided better insight into the pumping mechanism (Wang et al, 2014) Such crystal structures cannot reveal, all the biochemical characteristics and features that are required for the enzymatic function of these proteins, such as in which metal concentration range they are really active, what are the activation energies, etcetera. Since the two hyperaccumulators have contrasting metal storage mechanisms, the comparative study would reveal new aspects of the molecular biological mechanisms leading to metal hyperaccumulation
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