Title: FUNCTIONAL CHARACTERIZATION AND EXPRESSION ANALYSIS OF THE NRAMP GENE FAMILY IN RICE Authors
|Narayanan, Narayanan - BAYLOR COLLEGE MED|
|Vasconcelos, Marta - BAYLOR COLLEGE MED|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: March 1, 2006
Publication Date: March 1, 2006
Citation: Naryanan, N.N., Vasconcelos, M.W., Grusak, M.A. 2006. Functional characterization and expression analysis of the NRAMP gene family in rice. In: Proceedings of the 31st Rice Technical Working Group Meeting, February 26 – March 1, 2006, The Woodlands, Texas. p. 10. Technical Abstract: In plants, several groups of gene families are responsible for the uptake, transport and storage of different transition metals, such as iron, zinc, copper and manganese. In this work, we used different molecular resources such as NCBI, TIGR and RGRC to identify what appears to be the entire Nramp gene family in rice. The Nramp genes are widely distributed throughout living organisms and are involved in the transport of a broad range of divalent metal cations. We characterized the OsNramp genes in terms of sequence similarity using ClustalW and Tree View, and we analyzed their respective protein composition, in terms of amino acid number, number of transmembrane domains, and putative localization in the cell using bioinformatic tools such as PSORT and Target P. Bioinformatic analysis revealed that in rice (Oryza sativa L.), there are 8 distinct, putative Nramp genes (OsNramps). These range from 216 to 515 amino acids, and have from 3 to 11 putative transmembrane domains. Target P analysis indicated that OsNRAMP3 may be a chloroplast thylakoid membrane protein, whereas OsNRAMP8 seems to be localized to the mitochondrial membrane. PSORT analysis indicated that all OsNRAMPs are most likely plasma membrane proteins, not ruling out the possibility of some being present in the mitochondria, the chloroplast, the golgi body or the endoplasmic reticulum (ER). Sequence alignment with Clustal W and SPRINT revealed that the characteristic motif 5 of the natural resistance-associated macrophage proteins (NRAMPs) is present in OsNRAMP1-7. We also conducted semi-quantitative RT PCR in different rice tissues in order to understand the specific expression profile of this gene family in distinct plant tissues. Our analysis indicated that most OsNramp genes have a wide distribution of expression in different plant tissues, with the exception of OsNramp 4, whose expression seems to be specific to the panicle branches. OsNramps 3, 5 and 7 were the only OsNramps showing expression in rice embryos. Finally, the role of the OsNramps in metal transport in rice was studied using heterologous expression and functional analysis in yeast. In yeast, expression of OsNRAMP3 complements the phenotype of a yeast strain defective in zinc uptake. Expression of OsNRAMP2, 3, 4 and 8 restored the growth of the smf yeast mutant, which has impaired manganese transport activity. The fet3fet4 mutant, a yeast mutant defective in both low and high affinity iron uptake system, was also used to study the possible role of the OsNramps in Fe transport, and results will be presented. It has previously been suggested that NRAMPs are able to transport Cd. However, when the OsNramps were expressed in yeast, there was no apparent increase in cadmium sensitivity. These data indicate that the rice OsNramp genes encode metal transporters with multiple specificities.