PROTEIN l-ISOASPARTYL METHYLTRANSFERASE2 Is Differentially Expressed in Chickpea and Enhances Seed Vigor and Longevity by Reducing Abnormal Isoaspartyl Accumulation Predominantly in Seed Nuclear Proteins  

Abstract

PROTEIN L-ISOASPARTYL METHYLTRANSFERASE (PIMT) is a widely distributed protein-repairing enzyme that catalyzes the conversion of abnormal L-isoaspartyl residues in spontaneously damaged proteins to normal aspartyl residues. This enzyme is encoded by two divergent genes (PIMT1 and PIMT2) in plants, unlike many other organisms. While the biological role of PIMT1 has been elucidated, the role and significance of the PIMT2 gene in plants is not well defined. Here, we isolated the PIMT2 gene (CaPIMT2) from chickpea (Cicer arietinum), which exhibits a significant increase in isoaspartyl residues in seed proteins coupled with reduced germination vigor under artificial aging conditions. The CaPIMT2 gene is found to be highly divergent and encodes two possible isoforms (CaPIMT2 and CaPIMT29) differing by two amino acids in the region I catalytic domain through alternative splicing. Unlike CaPIMT1, both isoforms possess a unique 56-amino acid amino terminus and exhibit similar yet distinct enzymatic properties. Expression analysis revealed that CaPIMT2 is differentially regulated by stresses and abscisic acid. Confocal visualization of stably expressed green fluorescent protein-fused PIMT proteins and cell fractionation-immunoblot analysis revealed that apart from the plasma membrane, both CaPIMT2 isoforms localize predominantly in the nucleus, while CaPIMT1 localizes in the cytosol. Remarkably, CaPIMT2 enhances seed vigor and longevity by repairing abnormal isoaspartyl residues predominantly in nuclear proteins upon seed-specific expression in Arabidopsis (Arabidopsis thaliana), while CaPIMT1 enhances seed vigor and longevity by repairing such abnormal proteins mainly in the cytosolic fraction. Together, our data suggest that CaPIMT2 has most likely evolved through gene duplication, followed by subfunctionalization to specialize in repairing the nuclear proteome.

ESCRT-III-Associated Protein ALIX Mediates High-Affinity Phosphate Transporter Trafficking to Maintain Phosphate Homeostasis in Arabidopsis

“…Stained embryos were observed under a Leica LMD-6000 microscope (Verma et al, 2013). To genotype ungerminated seeds, genomic DNA was isolated from pools of 15 embryos released from their integuments using needles.…”

An Arabidopsis ATP-Dependent, DEAD-Box RNA Helicase Loses Activity upon IsoAsp Formation but Is Restored by PROTEIN ISOASPARTYL METHYLTRANSFERASE

“…An additional degree of complexity would be introduced in dehydrated, quiescent, or dormant seeds when, upon imbibition, a substantial fraction of all DEAD-box helicase proteins in a cell simultaneously suffers isoAsp formation, detrimentally affecting their activity. This may be one reason why the longevity of PIMT-deficient seeds is compromised (Ogé et al, 2008;Verma et al, 2013).…”

“…In this partially dehydrated state, proteins can undergo spontaneous degradation to form the succinimide derivative of Asn and Asp residues (Desfougères et al, 2011) that is then largely converted to the isomerized isoAsp derivative upon imbibition. IsoAsp formation is often detrimental to proper protein function and, if unrepaired, can be lethal (Ogé et al, 2008;Verma et al, 2013).…”

Genomics: A Hallmark to Monitor Molecular and Biochemical Processes Leading Toward a Better Perceptive of Seed Aging and ex-situ Conservation

“…Recently, PIMT2 was reported to have a similar function as PIMT1 in both Arabidopsis and chickpea. The major difference is their localization, with PIMT1 functioning in the cytosol and PIMT2 in the nucleus (Verma et al, 2013). In dehydrated orthodox seeds, proteins spontaneously degrade into succinimide derivatives of Asp and Asn residues, which are isomerized upon imbibition into derivatives of isoAsp (Nayak et al, 2013), which is deleterious to appropriate protein function and, if unrepaired, will compromise seed vigor.…”