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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

A Second Protein L-Isoaspartyl Methyltransferase Gene in Arabidopsis Produces Two Transcripts Whose Products Are Sequestered in the Nucleus^1,[w]

Department of Horticulture (Q.X., A.B.D.), University of Kentucky Agriculture Biotechnology Undergraduate Program (M.P.B.), Department of Agronomy (R.D.D.), Plant Physiology/Biochemistry/Molecular Biology Program (Q.X.), Seed Biology Program (Q.X., A.B.D.), and University of Kentucky Agriculture Experiment Station, S129, Agriculture Science Center North (Q.X., R.D., A.B.D.), University of Kentucky, Lexington Kentucky, 40546–0312; and Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095–1569 (S.T.V., S.G.C.)

The spontaneous and deleterious conversion of L-asparaginyl and L-aspartyl protein residues to L-iso-Asp or D-Asp occurs as proteins age and is accelerated under stressful conditions. Arabidopsis (Arabidopsis L. Heynh.) contains two genes (At3g48330 and At5g50240) encoding protein-L-isoaspartate methyltransferase (EC 2.1.1.77; PIMT), an enzyme capable of correcting this damage. The gene located on chromosome 5 (PIMT2) produces two proteins differing by three amino acids through alternative 3' splice site selection in the first intron. Recombinant protein from both splicing variants has PIMT activity. Subcellular localization using cell fractionation followed by immunoblot detection, as well as confocal visualization of PIMT:GFP fusions, demonstrated that PIMT1 is cytosolic while a canonical nuclear localization signal, present in PIMT2 and the shorter PIMT2, is functional. Multiplex reverse transcription-PCR was used to establish PIMT1 and PIMT2 transcript presence and abundance, relative to -TUBULIN, in various tissues and under a variety of stresses imposed on seeds and seedlings. PIMT1 transcript is constitutively present but can increase, along with PIMT2, in developing seeds presumably in response to increasing endogenous abscisic acid (ABA). Transcript from PIMT2 also increases in establishing seedlings due to exogenous ABA and applied stress presumably through an ABA-dependent pathway. Furthermore, cleaved amplified polymorphic sequences from PIMT2 amplicons determined that ABA preferentially enhances the production of PIMT2 transcript in leaves and possibly in tissues other than germinating seeds.

² Present address: University of Kentucky Medical School, 800 Rose Street, Lexington, KY, 40536.

^[w] The online version of this article contains Web-only data.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.046094.

^* Corresponding author; e-mail adownie{at}uky.edu; fax 859–257–7874.

Received May 10, 2004; returned for revision May 31, 2004; accepted June 7, 2004.

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