Inositol is important for normal growth and development in plants. The regulation of the inositol biosynthetic enzymes, myo-inositol phosphate synthase (MIPS) and myo-inositol monophosphatase (IMP) was investigated. The specific aims of this research were (1) to develop a tool to study MIPS protein accumulation in a model plant system, Arabidopsis thaliana (At) and potentially other plant species and (2) to determine the spatial expression patterns of Lycopersicon esculentum IMP-2 (LeIMP-2) at the cellular level.
Myo-inositol phosphate synthase (mips) genes have been identified in plants, animals, fungi and bacteria. Alignment of the predicted amino acid sequences of AtMIPS-1, -2 and Glycine max MIPS (GmMIPS) indicated that AtMIPS-1 and GmMIPS are 87% identical, and AtMIPS-2 and GmMIPS are 89% identical. Based on these data, a Gmmips cDNA was fused at the N-terminus to a 6X histidine tag (5' GAC GAC GAC GAC GAC GAC 3'), cloned into an overexpression vector and overexpressed in E. coli. The fusion protein, HISMIPS, was extracted using denaturing conditions and purified using Ni2+ affinity chromatography. Anti-GmMIPS antiserum from rabbit detected the recombinant HISMIPS protein (76 kD), and GmMIPS (64 kD). Affinity purification by subtractive chromatography yielded anti-GmMIPS antibody that detected MIPS (66 kD) and a protein (34 kD) of unknown function. AtMIPS accumulated to high levels in unopened flowers, opened flowers, and immature siliques (6 mm in length or less), but was not detectable in bolts, cauline or rosette leaves.
The tomato inositol monophosphatase (Leimp) genes are a developmentally regulated multigene family. From analysis of sequences, Leimp-2 is intron-less and has the putative start site of translation located at +108 bp downstream from the putative start site of transcription. Investigation of the 5’ UTR revealed the 3' end of a partial open reading frame (338 bp) highly homologous to the gene for calmodulin. Three light responsive elements and a cold responsive element were also identified in the 5' UTR.
Transgenic Leimp-2::uidA plants were produced using the existing construct of the Leimp-2 promoter fused to the uidA gene (J. Keddie, University of California at Berkeley). Seedlings were perserved and sectioned. Using histological techniques, the analysis of the Leimp-2 promoter::uidA transgenic seedlings revealed that the Leimp-2 promoter causes expression at the base of the shoot apex and within leaflets of the first set of fully expanded leaves. Further, Leimp-2 promoter expression was localized to epidermal and cortex cells on the abaxial side of the 1st and 2nd fully expanded compound leaves.
These studies of MIPS and IMP expression lay a foundation for a better understanding of the regulation of inositol biosynthesis in Arabidopsis, tomato, and other plant species.
