Exploring the roles of membrane proteins in Manganese homeostasis of higher plants

Manganese (Mn) is an essential heavy metal micronutrient in plant growth and development, but becomes toxic when present in excess, with potential agricultural yield losses under both nutritional extremes. Membrane transporters play a key role in enabling plant growth under Mn deficiency and toxicity. This study directly compares the roles of members from different ubiquitous transporter families in Mn homeostasis: Group 8/9 members of the Metal Tolerance Proteins (MTPs), At MTP8–MTP11; At ECA3, a P2A-type ATPase; and Natural Resistance Associated Macrophage Proteins, At NRAMP1 and At NRAMP2. MTP8 plays a significant role in Mn detoxification at the tonoplast, conferring Mn hypertolerance when overexpressed; it is also the only Group 8/9-MTP involved in alleviating Mn/iron antagonism. A role in Mn detoxification is also assigned to MTP10, identified through comparison of novel double and triple mtp knockout mutants. Expression in yeast provides evidence that these proteins can transport Mn, with greater Mn tolerance conferred by MTP8 and MTP10 than MTP11. ECA3 is also shown to play a minor role in conferring tolerance to Mn toxicity in planta, but this is only apparent when MTP11 is non-functional; its major role appears to lie in alleviating Mn deficiency. This thesis also clarifies the disputed subcellular localisations of ECA3 and MTP11, demonstrating targeting to the trans- and cis-Golgi, respectively. Additionally, NRAMP2 is shown to alleviate Mn deficiency at the cis-Golgi. Characterisation of double and triple mutants has begun to clarify the relative contribution of NRAMP1, NRAMP2 and ECA3 in Mn deficiency. The use of mutants in this study has also highlighted the antagonistic effects of calcium in Mn homeostasis. Overall, this study provides a more comprehensive understanding of how key transporters function together in Mn homeostasis, identifying targets that may be useful for crop improvement.