Transition metal homeostasis in Arabidopsis thaliana : the role of AtHMA4 and AtHMA1

Baccarini, Paul Joseph (2004) Transition metal homeostasis in Arabidopsis thaliana : the role of AtHMA4 and AtHMA1. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

This study has focused on a number of HMA transporters present in Arabidopsis thaliana, with particular emphasis being placed on AtHMA4 and AtHMA1. This work was part of a study that was the first to report on a HMA of the Zn/Cd/Pb/Co sub-class in any higher plant species. This work reports on the cloning of AtHMA4 and its functional characterisation. The cDNA has an open-reading frame of 3516 bp encoding a polypeptide of 1172 amino acids. Analysis of the 5’ region of AtHMA4 using RACE-PCR highlighted the presence of two alternative transcripts within the plant resulting from differential splicing of an intron in the 5’ untranslated region. AtHMA4 contains the conserved motifs found in all P-type ATPases and also motifs characteristic of heavy-metal ATPases (also known as P_IB-type ATPases or CPx-ATPases). AtHMA4 clusters with the Zn/Cd/Pb/Co subclass of HMAs in phylogenetic analysis and is closely related to AtHMA2 and AtHMA3 but more distantly related to AtHMA1.

AtHMA4 was expressed in all organs of Arabidopsis with highest levels in the roots. AtHMA1 was also expressed widely with highest levels in leaves, followed by stems and roots. AtHMA4 expression was up-regulated in the roots when plants were exposed to Zn, Cu and Mn, but down-regulated when exposed to Cd and Co. AtHMA1 expression in roots was reduced by Zn, Cu and Cd, whilst Mn, and to a lesser extent Co, tended to up-regulate expression. The expression profile in leaves was difficult to determine, but Zn was observed to cause up-regulation of both genes. A concentration range for Zn revealed that AtHMA4 transcript abundance increased markedly in roots and leaves with increasingly levels of Zn, whilst AtHMA1 in roots displayed consistent and gradual reductions. AtHMA2 showed no response to Zn while AtHMA3 showed strong down-regulation and AtHMA5 was up-regulated.