Regulating Inositol Biosynthesis in Plants: Myo-Inositol Phosphate Synthase and Myo-Inositol Monophosphatase

Simple item page
dc.contributor.authorStyer, Jean Christineen
dc.contributor.committeechairGillaspy, Glenda E.en
dc.contributor.committeememberHess, John L.en
dc.contributor.committeememberGrabau, Elizabeth A.en
dc.contributor.departmentBiochemistry and Anaerobic Microbiologyen
dc.date.accessioned2011-08-06T16:01:22Zen
dc.date.adate2000-03-17en
dc.date.available2011-08-06T16:01:22Zen
dc.date.issued2000-06-05en
dc.date.rdate2004-03-17en
dc.date.sdate2000-03-14en
dc.description.abstractInositol is important for normal growth and development in plants. The regulation of the inositol biosynthetic enzymes, <I>myo</I>-inositol phosphate synthase (MIPS) and <I>myo</I>-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, <I>Arabidopsis thaliana (At)</I> and potentially other plant species and (2) to determine the spatial expression patterns of <I>Lycopersicon esculentum</I> IMP-2 (<I>Le</I>IMP-2) at the cellular level. <I>Myo-inositol phosphate synthase (mips)</I> genes have been identified in plants, animals, fungi and bacteria. Alignment of the predicted amino acid sequences of <I>At</I>MIPS-1, -2 and <I>Glycine max</I> MIPS (<I>Gm</I>MIPS) indicated that <I>At</I>MIPS-1 and <I>Gm</I>MIPS are 87% identical, and <I>At</I>MIPS-2 and <I>Gm</I>MIPS are 89% identical. Based on these data, a <I>Gmmips</I> 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 <I>E. coli</I>. The fusion protein, HISMIPS, was extracted using denaturing conditions and purified using Ni²⁺ affinity chromatography. Anti-<I>Gm</I>MIPS antiserum from rabbit detected the recombinant HISMIPS protein (76 kD), and <I>Gm</I>MIPS (64 kD). Affinity purification by subtractive chromatography yielded anti-<I>Gm</I>MIPS antibody that detected <At</I>MIPS (66 kD) and a protein (34 kD) of unknown function. <I>At</I>MIPS 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 <I>inositol monophosphatase (Leimp)</I> genes are a developmentally regulated multigene family. From analysis of sequences, <I>Leimp</I>-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 <I>Leimp</I>-2::<I>uid</I>A plants were produced using the existing construct of the <I>Leimp</I>-2 promoter fused to the <I>uid</I>A gene (J. Keddie, University of California at Berkeley). Seedlings were preserved and sectioned. Using histological techniques, the analysis of the <I>Leimp</I>-2 promoter::<I>uid</I>A transgenic seedlings revealed that the <I>Leimp</I>-2 promoter causes expression at the base of the shoot apex and within leaflets of the first set of fully expanded leaves. Further, <I>Leimp</I>-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.en
dc.description.degreeMaster of Scienceen
dc.format.mediumETDen
dc.identifier.otheretd-03142000-15520040en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-03142000-15520040en
dc.identifier.urihttp://hdl.handle.net/10919/9870en
dc.publisherVirginia Techen
dc.relation.haspartETDMarch14.pdfen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectmyo-inositol-1L-phosphate synthaseen
dc.subjectMyo-inositolen
dc.subjectinositol monophosphataseen
dc.titleRegulating Inositol Biosynthesis in Plants: Myo-Inositol Phosphate Synthase and Myo-Inositol Monophosphataseen
dc.typeThesisen
thesis.degree.disciplineBiochemistry and Anaerobic Microbiologyen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.levelmastersen
thesis.degree.nameMaster of Scienceen

Files

Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
ETDMarch14.pdf
Size:
507.76 KB
Format:
Adobe Portable Document Format
Download

Collections

Masters Theses