Protein repair L-isoaspartyl methyltransferase 1 is involved in both seed longevity and germination vigor in Arabidopsis

Abstract

The formation of abnormal amino acid residues is a major source of spontaneous age-related protein damage in cells. The protein l-isoaspartyl methyltransferase (PIMT) combats protein misfolding resulting from l-isoaspartyl formation by catalyzing the conversion of abnormal l-isoaspartyl residues to their normal l-aspartyl forms. In this way, the PIMT repair enzyme system contributes to longevity and survival in bacterial and animal kingdoms. Despite the discovery of PIMT activity in plants two decades ago, the role of this enzyme during plant stress adaptation and in seed longevity remains undefined. In this work, we have isolated Arabidopsis thaliana lines exhibiting altered expression of PIMT1, one of the two genes encoding the PIMT enzyme in Arabidopsis. PIMT1 overaccumulation reduced the accumulation of l-isoaspartyl residues in seed proteins and increased both seed longevity and germination vigor. Conversely, reduced PIMT1 accumulation was associated with an increase in the accumulation of l-isoaspartyl residues in the proteome of freshly harvested dry mature seeds, thus leading to heightened sensitivity to aging treatments and loss of seed vigor under stressful germination conditions. These data implicate PIMT1 as a major endogenous factor that limits abnormal l-isoaspartyl accumulation in seed proteins, thereby improving seed traits such as longevity and vigor. The PIMT repair pathway likely works in concert with other anti-aging pathways to actively eliminate deleterious protein products, thus enabling successful seedling establishment and strengthening plant proliferation in natural environments.

Figure 1.

Mechanism of Spontaneous l-Isoaspartyl Formation and Enzymatic Repair by PIMT. Deamidation of Asn (top right) and isomerization of Asp (top left) lead to the formation of an unstable succinimidyl ring that is spontaneously hydrolyzed to generate a mixture of Asp (15 to 30%) and of abnormal isoAsp (70 to 85%) residues at physiological pH. The peptide backbone is shown in gray to emphasize the kink in the polypeptide chain that occurs in the isoAsp bond and the linear nature of the chain in the normal Asp bond. PIMT catalyzes the first step of the conversion of isoAsp into Asp. The formed l-isoAsp-OMe spontaneously gives rise to Asp (15 to 30%) and isoAsp (70 to 85%). Thus, several enzymatic cycles are needed to fully repair proteins. AdoHcy, S-adenosyl homocystein; l-Asp, l-aspartyl; l-Asn, l-asparaginyl; l-isoAsp, l-isoaspartyl; l-isoAsp-OMe, l-isoaspartyl-O-methylester.

Figure 2.

Characterization of the pimt1-1 Mutant Allele. (A) Schematic representation of the PIMT1 locus. The arrow indicates the translational start site, black rectangles represent exons, and introns are indicated between exons. The black triangle indicates the position of the T-DNA insertion 187 bp upstream of the ATG initiation codon. (B) PIMT1 sequence flanking the T-DNA insertion. The black triangle marks the T-DNA insertion site. LB and RB indicate the left and right borders of the T-DNA insert, respectively. The sequence deletion in PIMT1 caused by the insertion of the T-DNA is underlined. The three putative ABRE motifs previously described by Mudgett and Clarke (1996) are highlighted in gray, and the first motif that encompasses the 13-bp deleted sequence is called MT1a (Mudgett and Clarke, 1996). (C) RT-PCR comparison of PIMT1 transcript accumulation in the wild type (black) and pimt1-1 mutant (gray) freshly harvested dry mature seeds and in 5-d-old seedlings cultivated on water (−ABA) or on water for 4 d and transferred the fourth day to water containing 100 μM ABA for an additional day (+ABA). Means ± sd are shown (n = 3). (D) PIMT1 and PIMT2 protein accumulation levels were compared in wild-type and pimt1-1 dry mature seeds and corresponding 1 d imbibed seeds. Proteins (30 μg) were separated by SDS-PAGE and immunodetected with polyclonal antibodies specifically raised against PIMT1 or PIMT2. Recombinant rPIMT1-His or rPIMT2-His proteins (30 ng) were used as controls. Parts of the Coomassie blue–stained gels are presented below the protein gel blot as loading controls. Total protein extracts of wild-type and pimt1-1 dry seeds display a PIMT enzyme activity of 0.36 ± 0.05 pmol min⁻¹ mg⁻¹ protein and 1.2 ± 0.09 pmol min⁻¹ mg⁻¹ protein, respectively. (E) Quantitation of l-isoaspartyl-containing methyl-accepting substrates in wild-type and pimt1-1 dry mature seeds and corresponding 1 d imbibed seeds. Values are from three repetitions each using 75 mg of seeds (mean ± sd).

Figure 3.

Sensitivity of pimt1-1 Mutant Seeds to Storage Treatments Conducted at 40°C and 15 to 20% or 8% Water Content. Wild-type (black) and pimt1-1 (gray) dry mature seeds were submitted to a storage treatment performed for 12 d either at 15 to 20% (40°C, 75% RH) or 8% (40°C, 35% RH) water content (WC). Germination percentages from these seed samples were measured 4 d after sowing. Values are from four repetitions of 100 seeds (4× 100) (mean ± sd). (A) Germination performance of seed samples submitted to the high-RH storage treatment. (B) Germination performance of seed samples submitted to the low-RH storage treatment.

Figure 4.

PIMT1 and IsoAsp Contents throughout Storage at 8% Water Content. Wild-type and pimt1-1 dry mature seeds were submitted for 0, 6, and 12 weeks to a storage treatment conducted at 40°C and 35% RH yielding a seed water content of 8%, similar to the water content of the dry mature seeds. PIMT1 accumulation levels were estimated by protein gel blotting following SDS-PAGE of protein extracts (30 μg). Quantitation of l-isoaspartyl–containing methyl-accepting substrates was also performed. (A) PIMT1 accumulation during storage treatment at 8% water content. Parts of the Coomassie blue–stained gels are presented below the protein gel blots as loading controls. (B) Quantitation of l-isoaspartyl–containing methyl-accepting substrates. Values are from three repetitions each using 100 mg of seeds (mean ± sd).

Figure 5.

Characterization of PIMT1 Overaccumulating Lines. PIMT1 and PIMT2 accumulation levels were compared in dry mature seeds or green siliques, respectively, for wild-type and three independent PIMT1 overaccumulating (O1, O2, and O3) lines. Proteins (30 μg) were separated by SDS-PAGE and immunodetected with polyclonal antibodies raised specifically against PIMT1 or PIMT2. Parts of the Coomassie blue–stained gels are presented below the protein gel blots as loading controls. PIMT2 accumulation was analyzed in green siliques because Xu et al. (2004) described the transient accumulation of PIMT2 transcript during Arabidopsis seed formation. Our detection of the PIMT2 protein is in agreement with these data and documents a constant accumulation of this protein independently of that of PIMT1. (A) Quantitation of PIMT1 accumulation levels. (B) Quantitation of PIMT2 accumulation levels. (C) Quantitation of l-isoaspartyl–containing methyl-accepting substrates in dry mature seeds. Values are from three repetitions each using 75 mg of seeds (mean ± sd). (D) Germination performance of the seed samples. Dry mature seeds from wild-type (black circles) and three independent PIMT1 overaccumulating lines (O1 [gray squares], O2 [gray circles], and O3 [gray triangles]) were submitted to a storage treatment conducted at 15 to 20% water content (WC; 40°C, 82% RH) for 7 d. Then, the germination percentages of these seeds were monitored 4 d after sowing. Values are from four repetitions of 100 seeds (4× 100) (mean ± sd). (E) Viability of the seed samples. Wild-type, O1, O2, and O3 dry mature seeds were submitted to a storage treatment conducted for 5 d at 15 to 20% water content (40°C, 82% RH). Seed viability was estimated using tetrazolium staining according to the protocol described by Wharton (1955). Dark-red staining indicates that seeds are viable; light-pink staining indicates reduced seed viability. Bar = 1 mm.

Figure 6.

Characterization of PIMT1 Underaccumulating Lines. PIMT1 and PIMT2 accumulation levels were compared in dry mature seeds or green siliques, respectively, for wild-type and three independent PIMT1 underaccumulating lines (U1, U2, and U3). Proteins (30 μg) were separated by SDS-PAGE and immunodetected with polyclonal antibodies raised specifically against PIMT1 or PIMT2. Parts of the Coomassie blue–stained gels are presented below the protein gel blots as loading controls. (A) Quantitation of PIMT1 accumulation levels. (B) Quantitation of PIMT2 accumulation levels. (C) Quantitation of l-isoaspartyl–containing methyl-accepting substrates in dry mature seeds. Values are from three repetitions each using 75 mg of seeds (mean ± sd). (D) Germination performance of the seed samples. Dry mature seeds from wild-type (black circles) and three independent PIMT1 underaccumulating lines (U1 [gray circles], U2 [gray triangles], and U3 [gray squares]) were submitted to a storage treatment conducted at 15 to 20% water content (WC, 40°C, 82% RH) for 6 d. The germination percentages of these seeds were monitored 4 d after sowing. Values are from four repetitions of 100 seeds (4× 100) (mean ± sd). (E) Viability of the seed samples. Dry mature seeds of wild-type, U1, U2, and U3 lines were submitted to a storage treatment conducted at 15 to 20% water content (40°C, 82% RH) for 4 d. Seed viability was then estimated using tetrazolium staining (Wharton, 1955). Dark-red staining indicates that seeds are viable; light-pink staining indicates reduced seed viability. Bar = 1 mm.

Figure 7.

Sensitivity of Germination to NaCl or Mannitol. The influence of 100 mM NaCl or 300 and 400 mM mannitol on germination percentages was examined using freshly harvested dry mature seed samples exhibiting varying accumulation of PIMT1. Germination percentages were monitored 4 d after sowing. Values are from four repetitions of 100 seeds (4× 100) (mean ± sd). Man, mannitol. (A) Seeds from the pimt1-1 mutant and respective wild-type controls. (B) Seeds from the three independent PIMT1 overaccumulating lines O1, O2, and O3 and respective wild-type controls. (C) Seeds from the three independent PIMT1 underaccumulating lines U1, U2, and U3 and respective wild-type controls.