Submitted to: Plant Journal
Publication Type: Peer reviewed journal
Publication Acceptance Date: 11/15/2009
Publication Date: 12/22/2009
Citation: Ligaba, A., Kochian, L.V., Pineros, M. 2009. Phosphorylation at S384 regulates the activity of the TaALMT1 malate transporter that underlies aluminum resistance in wheat. Plant Journal. 60:411-423. Interpretive Summary: Over 20% of the US land area and approximately 50% of the world’s arable lands are acidic (pH < 5). On these acid soils, aluminum (Al) toxicity is the primary factor limiting crop production as Al is toxic to plant roots, leading to a damaged and stunted root system. As a large proportion of the acid soils are in the tropics/subtropics where many developing countries are located, Al toxicity limits crop production in the very areas where food security is most tenuous. Because of the importance of this problem to agriculture worldwide, there is considerable interest and research effort by researchers at universities, government agencies, and international agriculture organizations in identifying genes that provide tolerance to Al toxicity in order to improve crop Al tolerance via molecular breeding and biotechnology. In this study we investigated the regulation of the wheat Al tolerance protein, TaALMT1, which confers tolerance by transporting malic acid out of the root in response to exposure to toxic Al ions. The malic acid comes out of the root and binds and detoxifies Al ions in the soil, allowing the root to grow in acid soils. We found that the activation of this transporter by Al ions in the soil requires a biochemical step whereby specialized enzymes called kinases in the root cell add a phosphate ion to the TaALMT1 transport protein. This process, known as phosphorylation, is important for the regulation of a number of proteins in all organisms. In this study we were able to identify the specific amino acids that are phosphorylated. Our goal is to better understand the function of specific portions of this TaALMT1 protein in order to engineer new versions of this protein that confer a higher degree of Al tolerance.
Technical Abstract: In this study we examined the role of protein phosphorylation & dephosphorylation in the transport properties of the wheat root malate efflux transporter underlying Al resistance, TaALMT1. Preincubation of Xenopus laevis oocytes expressing TaALMT1 with protein kinase inhibitors (K252a and staurosporine) strongly inhibited both basal and Al3+-enhanced TaALMT1-mediated inward currents (malate efflux). Preincubation with phosphatase inhibitors (okadaic acid and cyclosporine A) resulted in a significantly smaller inhibition of the TaALMT1-mediated currents. In contrast, exposure to the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), enhanced TaALMT1-mediated inward currents. Since these observations suggest that TaALMT1 transport activity is regulated by PKC-mediated phosphorylation, we proceeded to modify candidate amino acids in the TaALMT1 protein in an effort to identify structural motifs underlying the phosphorylation regulatory process. The transport properties of eight single point mutations (S56A, S183A, S324A, S337A, S351-352A, S384A, T323A and Y184F) were generated in amino acid residues predicted to be phosphorylation sites and examined electrophysiologically. The basic transport properties of mutants S56A, S183A, S324A, S337A, S351-352A, T323A and Y184F were not altered, relative to the wildtype TaALMT1. Likewise the sensitivity of these mutants to staurosporine resembled that observed for the wild-type transporter. However, the mutation S384A was noticeable as in oocytes expressing this mutant protein, TaALMT1-mediated basal and Al-enhanced currents were significantly inhibited, and the currents were insensitive to staurosporine or PMA. These findings indicate that S384 is an essential residue regulating Al-activation of transport activity in TaALMT1 via direct protein phosphorylation.