PROTEIN L-ISOASPARTYL METHYLTRANSFERASE protects enolase dysfunction by repairing isoaspartyl-induced damage and is positively implicated in agronomically important seed traits.

The protein-repairing enzyme (PRE) PROTEIN L-ISOASPARTYL METHYLTRANSFERASE (PIMT) influences seed vigor by repairing isoaspartyl-mediated protein damage in seeds. However, PIMTs function in other seed traits, and the mechanisms by which PIMT affects such seed traits are still poorly understood. Herein, through molecular, biochemical, and genetic studies using overexpression and RNAi lines in Oryza sativa and Arabidopsis thaliana, we demonstrate that PIMT not only affects seed vigor but also affects seed size and weight by modulating enolase (ENO) activity. We have identified ENO2, a glycolytic enzyme, as a PIMT interacting protein through Y2H cDNA library screening, and this interaction was further validated by BiFC and co-immunoprecipitation assay. We show that mutation or suppression of ENO2 expression results in reduced seed vigor, seed size, and weight. We also proved that ENO2 undergoes isoAsp modification that affects its activity in both in vivo and in vitro conditions. Further, using MS/MS analyses, amino acid residues that undergo isoAsp modification in ENO2 were identified. We also demonstrate that PIMT repairs such isoAsp modification in ENO2 protein, protecting its vital cellular functions during seed maturation and storage, and plays a vital role in regulating seed size, weight, and seed vigor. Taken together, our study identified ENO2 as a novel substrate of PIMT, and both ENO2 and PIMT in turn implicate in agronomically important seed traits.

Initiation of B-type starch granules in wheat endosperm requires the plastidial α-glucan phosphorylase PHS1.

The plastidial α-glucan phosphorylase (PHS1) can elongate and degrade maltooligosaccharides (MOSs), but its exact physiological role in plants is poorly understood. Here, we discover a specialized role of PHS1 in establishing the unique bimodal characteristic of starch granules in wheat (Triticum spp.) endosperm. Wheat endosperm contains large A-type granules that initiate at early grain development and small B-type granules that initiate in later grain development. We demonstrate that PHS1 interacts with B-GRANULE CONTENT1 (BGC1), a carbohydrate-binding protein essential for normal B-type granule initiation. Mutants of tetraploid durum wheat (Triticum turgidum) deficient in all homoeologs of PHS1 had normal A-type granules but fewer and larger B-type granules. Grain size and starch content were not affected by the mutations. Further, by assessing granule numbers during grain development in the phs1 mutant and using a double mutant defective in both PHS1 and BGC1, we demonstrate that PHS1 is exclusively involved in B-type granule initiation. The total starch content and number of starch granules per chloroplast in leaves were not affected by loss of PHS1, suggesting that its role in granule initiation in wheat is limited to the endosperm. We therefore propose that the initiation of A- and B-type granules occurs via distinct biochemical mechanisms, where PHS1 plays an exclusive role in B-type granule initiation.

Gene expression profile of the developing endosperm in durum wheat provides insight into starch biosynthesis.

BACKGROUND: Durum wheat (Triticum turgidum subsp. durum) is widely grown for pasta production, and more recently, is gaining additional interest due to its resilience to warm, dry climates and its use as an experimental model for wheat research. Like in bread wheat, the starch and protein accumulated in the endosperm during grain development are the primary contributors to the calorific value of durum grains. RESULTS: To enable further research into endosperm development and storage reserve synthesis, we generated a high-quality transcriptomics dataset from developing endosperms of variety Kronos, to complement the extensive mutant resources available for this variety. Endosperms were dissected from grains harvested at eight timepoints during grain development (6 to 30 days post anthesis (dpa)), then RNA sequencing was used to profile the transcriptome at each stage. The largest changes in gene expression profile were observed between the earlier timepoints, prior to 15 dpa. We detected a total of 29,925 genes that were significantly differentially expressed between at least two timepoints, and clustering analysis revealed nine distinct expression patterns. We demonstrate the potential of our dataset to provide new insights into key processes that occur during endosperm development, using starch metabolism as an example. CONCLUSION: We provide a valuable resource for studying endosperm development in this increasingly important crop species.

The Arabidopsis F-box protein SKP1-INTERACTING PARTNER 31 modulates seed maturation and seed vigor by targeting JASMONATE ZIM DOMAIN proteins independently of jasmonic acid-isoleucine.

F-box proteins have diverse functions in eukaryotic organisms, including plants, mainly targeting proteins for 26S proteasomal degradation. Here, we demonstrate the role of the F-box protein SKP1-INTERACTING PARTNER 31 (SKIP31) from Arabidopsis (Arabidopsis thaliana) in regulating late seed maturation events, seed vigor, and viability through biochemical and genetic studies using skip31 mutants and different transgenic lines. We show that SKIP31 is predominantly expressed in seeds and that SKIP31 interacts with JASMONATE ZIM DOMAIN (JAZ) proteins, key repressors in jasmonate (JA) signaling, directing their ubiquitination for proteasomal degradation independently of coronatine/jasmonic acid-isoleucine (JA-Ile), in contrast to CORONATINE INSENSITIVE 1, which sends JAZs for degradation in a coronatine/JA-Ile dependent manner. Moreover, JAZ proteins interact with the transcription factor ABSCISIC ACID-INSENSITIVE 5 (ABI5) and repress its transcriptional activity, which in turn directly or indirectly represses the expression of downstream genes involved in the accumulation of LATE EMBRYOGENESIS ABUNDANT proteins, protective metabolites, storage compounds, and abscisic acid biosynthesis. However, SKIP31 targets JAZ proteins, deregulates ABI5 activity, and positively regulates seed maturation and consequently seed vigor. Furthermore, ABI5 positively influences SKIP31 expression, while JAZ proteins repress ABI5-mediated transactivation of SKIP31 and exert feedback regulation. Taken together, our findings reveal the role of the SKIP31-JAZ-ABI5 module in seed maturation and consequently, establishment of seed vigor.

Methionine sulfoxide reductase B5 plays a key role in preserving seed vigor and longevity in rice (Oryza sativa).

Oxidation of methionine leads to the formation of methionine S-sulfoxide and methionine R-sulfoxide, which can be reverted by two types of methionine sulfoxide reductase (MSR): MSRA and MSRB. Though the role of MSR enzymes has been elucidated in various physiological processes, the regulation and role of MSR in seeds remains poorly understood. In this study, through molecular, biochemical, and genetic studies using seed-specific overexpression and RNAi lines of OsMSRB5 in Oryza sativa, we demonstrate the role of OsMSRB5 in maintaining seed vigor and longevity. We show that an age-induced reduction in the vigor and viability of seeds is correlated with reduced MSR activity and increased methionine sulfoxide (MetSO) formation. OsMSRB5 expression increases during seed maturation and is predominantly localized to the embryo. Further analyses on transgenic lines reveal the role of OsMSRB5 in modulating reactive oxygen species (ROS) homeostasis to preserve seed vigor and longevity. We show that ascorbate peroxidase and PROTEIN l-ISOASPARTYL METHYLTRANSFERASE undergo MetSO modification in seeds that affects their functional competence. OsMSRB5 physically interacts with these proteins and reverts this modification to facilitate their functions and preserve seed vigor and longevity. Our results thus illustrate the role of OsMSRB5 in preserving seed vigor and longevity by modulating ROS homeostasis in seeds.

Arabidopsis ABSCISIC ACID INSENSITIVE4 targets PROTEIN L-ISOASPARTYL METHYLTRANSFERASE1 in seed.

MAIN CONCLUSION: Arabidopsis ABSCISIC ACID INSENSITIVE4 (ABI4) positively regulates the protein repairing enzyme (PRE) PROTEIN L-ISOASPARTYL METHYLTRANSFERASE1 (PIMT1) in seed for its implication in seed vigor and longevity. PROTEIN L-ISOASPARTYL METHYLTRANSFERASE (PIMT) is a protein repairing enzyme (PRE) and is implicated in seed vigor and longevity. PIMT has been shown to be induced by ABA, however, its detailed regulation by ABA signaling components is unknown. Herein, we report that ABSCISIC ACID INSENSITIVE4 (ABI4) directly binds to the PIMT1 promoter and regulates its expression in Arabidopsis seeds. AtPIMT1 promoter analysis demonstrated the presence of putative ABI4 binding sites. Our Y1H analysis revealed that AtABI4 transcription factor binds to the AtPIMT1 promoter. Dual luciferase assay also demonstrated the binding of the AtABI4 transcription factor to the AtPIMT1 promoter. Subsequently, we have generated AtPIMT1 promoter GUS lines and revealed that ABA induced expression of GUS in Arabidopsis thaliana. Expression analyses exhibited reduced accumulation of PIMT1 protein and transcript with significant reduction in total PIMT activity in abi4-1 mutants as compared to that of the wild type. The AtPIMT1 promoter GUS expression in abi4-1 mutants was also found to be severely affected in both the control and ABA treatment. Hence, through molecular and genetic evidences we show that the AtABI4 plays a central role in regulating the expression of AtPIMT1 to impart seed vigor and longevity to orthodox seeds.

ABI transcription factors and PROTEIN L-ISOASPARTYL METHYLTRANSFERASE module mediate seed desiccation tolerance and longevity in Oryza sativa.

In contrast to desiccation-tolerant orthodox seeds, recalcitrant seeds are desiccation sensitive and are unable to survive for a prolonged time. Here, our analyses of Oryza species with contrasting seed desiccation tolerance reveals that PROTEIN L-ISOASPARTYL METHYLTRANSFERASE (PIMT), an enzyme that repairs abnormal isoaspartyl (isoAsp) residues in proteins, acts as a key player that governs seed desiccation tolerance to orthodox seeds but is ineffective in recalcitrant seeds. We observe that, unlike the orthodox seed of Oryza sativa, desiccation intolerance of the recalcitrant seeds of Oryza coarctata are linked to reduced PIMT activity and increased isoAsp accumulation due to the lack of coordinated action of ABA and ABI transcription factors to upregulate PIMT during maturation. We show that suppression of PIMT reduces, and its overexpression increases, seed desiccation tolerance and seed longevity in O. sativa. Our analyses further reveal that the ABI transcription factors undergo isoAsp formation that affect their functional competence; however, PIMT interacts with and repairs isoAsp residues and facilitates their functions. Our results thus illustrate a new insight into the mechanisms of acquisition of seed desiccation tolerance and longevity by ABI transcription factors and the PIMT module.

A conserved NAG motif is critical to the catalytic activity of galactinol synthase, a key regulatory enzyme of RFO biosynthesis.

Galactinol synthase (GolS) catalyzes the key regulatory step in the biosynthesis of Raffinose Family Oligosaccharides (RFOs). Even though the physiological role and regulation of this enzyme has been well studied, little is known about active site amino acids and the structure-function relationship with substrates of this enzyme. In the present study, we investigate the active site amino acid and structure-function relationship for this enzyme. Using a combination of three-dimensional homology modeling, molecular docking along with a series of deletion, site-directed mutagenesis followed by in vitro biochemical and in vivo functional analysis; we have studied active site amino acids and their interaction with the substrate of chickpea and Arabidopsis GolS enzyme. Our study reveals that the GolS protein possesses GT8 family-specific several conserved motifs in which NAG motif plays a crucial role in substrate binding and catalytic activity of this enzyme. Deletion of entire NAG motif or deletion or the substitution (with alanine) of any residues of this motif results in complete loss of catalytic activity in in vitro condition. Furthermore, disruption of NAG motif of CaGolS1 enzyme disrupts it's in vivo cellular function in yeast as well as in planta. Together, our study offers a new insight into the active site amino acids and their substrate interaction for the catalytic activity of GolS enzyme. We demonstrate that NAG motif plays a vital role in substrate binding for the catalytic activity of galactinol synthase that affects overall RFO synthesis.

PROTEIN l-ISOASPARTYL METHYLTRANSFERASE (PIMT) in plants: regulations and functions.

Proteins are essential molecules that carry out key functions in a cell. However, as a result of aging or stressful environments, the protein undergoes a range of spontaneous covalent modifications, including the formation of abnormal l-isoaspartyl residues from aspartyl or asparaginyl residues, which can disrupt the protein's inherent structure and function. PROTEIN l-ISOASPARTYL METHYLTRANSFERASE (PIMT: EC 2.1.1.77), an evolutionarily conserved ancient protein repairing enzyme (PRE), converts such abnormal l-isoaspartyl residues to normal l-aspartyl residues and re-establishes the protein's native structure and function. Although originally discovered in animals as a PRE, PIMT emerged as a key PRE in plants, particularly in seeds, in which PIMT plays a predominant role in preserving seed vigor and viability for prolonged periods of time. Interestingly, higher plants encode a second PIMT (PIMT2) protein which possesses a unique N-terminal extension, and exhibits several distinct features and far more complexity than non-plant PIMTs. Recent studies indicate that the role of PIMT is not restricted to preserving seed vigor and longevity but is also implicated in enhancing the growth and survivability of plants under stressful environments. Furthermore, expression studies indicate the tantalizing possibility that PIMT is involved in various physiological processes apart from its role in seed vigor, longevity and plant's survivability under abiotic stress. This review article particularly describes new insights and emerging interest in all facets of this enzyme in plants along with a concise comparative overview on isoAsp formation, and the role and regulation of PIMTs across evolutionary diverse species. Additionally, recent methods and their challenges in identifying isoaspartyl containing proteins (PIMT substrates) are highlighted.

Arabidopsis protein l-ISOASPARTYL METHYLTRANSFERASE repairs isoaspartyl damage to antioxidant enzymes and increases heat and oxidative stress tolerance.

Stressful environments accelerate the formation of isoaspartyl (isoAsp) residues in proteins, which detrimentally affect protein structure and function. The enzyme PROTEIN l-ISOASPARTYL METHYLTRANSFERASE (PIMT) repairs other proteins by reverting deleterious isoAsp residues to functional aspartyl residues. PIMT function previously has been elucidated in seeds, but its role in plant survival under stress conditions remains undefined. Herein, we used molecular, biochemical, and genetic approaches, including protein overexpression and knockdown experiments, in Arabidopsis to investigate the role of PIMTs in plant growth and survival during heat and oxidative stresses. We demonstrate that these stresses increase isoAsp accumulation in plant proteins, that PIMT activity is essential for restricting isoAsp accumulation, and that both PIMT1 and PIMT2 play an important role in this restriction and Arabidopsis growth and survival. Moreover, we show that PIMT improves stress tolerance by facilitating efficient reactive oxygen species (ROS) scavenging by protecting the functionality of antioxidant enzymes from isoAsp-mediated damage during stress. Specifically, biochemical and MS/MS analyses revealed that antioxidant enzymes acquire deleterious isoAsp residues during stress, which adversely affect their catalytic activities, and that PIMT repairs the isoAsp residues and thereby restores antioxidant enzyme function. Collectively, our results suggest that the PIMT-mediated protein repair system is an integral part of the stress-tolerance mechanism in plants, in which PIMTs protect antioxidant enzymes that maintain proper ROS homeostasis against isoAsp-mediated damage in stressful environments.

Deciphering the structural basis of the broad substrate specificity of myo-inositol monophosphatase (IMP) from Cicer arietinum.

Myo-inositol monophosphatase (IMP) is a crucial enzyme in the inositol biosynthetic pathway that dephosphorylates myo-inositol 1-phosphate and other inositol phosphate derivative compounds to maintain the homeostasis of cellular inositol pool. In our previous research, we have biochemically and functionally characterized IMP enzyme from chickpea (CaIMP), which was able to catalyze diverse substrates. We cloned, overexpressed recombinant CaIMP protein and purified it and further characterized the CaIMP with its three main substrates viz. galactose 1-P, inositol 6-P and fructose 1,6-bisP. Homology model of CaIMP was generated to elucidate the factors contributing to the broad substrate specificity of the protein. The active site of the CaIMP protein was analysed with respect to its interactions with the proposed substrates. Structural features such as, high B-factor and flexible loop regions in the active site, inspired further investigation into the static and dynamic behaviour of the active site of CaIMP protein. The electrostatic biding of each of the key substrates was assessed through molecular docking. Furthermore, molecular dynamics simulations showed that these interactions indeed were stable for extended periods of time under physiological conditions. These experiments conclusively allowed us to establish the primary factors contributing to the promiscuity in substrate binding by CaIMP protein.

Arabidopsis SKP1-like protein13 (ASK13) positively regulates seed germination and seedling growth under abiotic stress.

SKP1 (S-phase kinase-associated protein1) proteins are key members of the SCF (SKP-cullin-F-box protein) E3 ligase complexes that ubiquitinate target proteins and play diverse roles in plant biology. However, in comparison with other members of the SCF complex, knowledge of SKP1-like proteins is very limited in plants. In the present work, we report that Arabidopsis SKP1-like protein13 (ASK13) is differentially regulated in different organs during seed development and germination and is up-regulated in response to abiotic stress. Yeast two-hybrid library screening and subsequent assessment of in vivo interactions through bimolecular fluorescence complementation analysis revealed that ASK13 not only interacts with F-box proteins but also with other proteins that are not components of SCF complexes. Biochemical analysis demonstrated that ASK13 not only exists as a monomer but also as a homo-oligomer or heteromer with other ASK proteins. Functional analysis using ASK13 overexpression and knockdown lines showed that ASK13 positively influences seed germination and seedling growth, particularly under abiotic stress. Taken together, our data strongly suggest that apart from participation to form SCF complexes, ASK13 interacts with several other proteins and is implicated in different cellular processes distinct from protein degradation.

Stress-Inducible Galactinol Synthase of Chickpea (CaGolS) is Implicated in Heat and Oxidative Stress Tolerance Through Reducing Stress-Induced Excessive Reactive Oxygen Species Accumulation.

Raffinose family oligosaccharides (RFOs) participate in various aspects of plant physiology, and galactinol synthase (GolS; EC 2.4.1.123) catalyzes the key step of RFO biosynthesis. Stress-induced accumulation of RFOs, in particular galactinol and raffinose, has been reported in a few plants; however, their precise role and mechanistic insight in stress adaptation remain elusive. In the present study, we have shown that the GolS activity as well as galactinol and raffinose content are significantly increased in response to various abiotic stresses in chickpea. Transcriptional analysis indicated that the CaGolS1 and CaGolS2 genes are induced in response to different abiotic stresses. Interestingly, heat and oxidative stress preferentially induce CaGolS1 over CaGolS2. In silco analysis revealed several common yet distinct cis-acting regulatory elements in their 5'-upstream regulatory sequences. Further, in vitro biochemical analysis revealed that the CaGolS1 enzyme functions better in stressful conditions than the CaGolS2 enzyme. Finally, Arabidopsis transgenic plants constitutively overexpressing CaGolS1 or CaGolS2 exhibit not only significantly increased galactinol but also raffinose content, and display better growth responses than wild-type or vector control plants when exposed to heat and oxidative stress. Further, improved tolerance of transgenic lines is associated with reduced accumulation of reactive oxygen species (ROS) and consequent lipid peroxidation as compared with control plants. Collectively, our data imply that GolS enzyme activity and consequent galactinol and raffinose content are significantly increased in response to stresses to mitigate stress-induced growth inhibition by restricting excessive ROS accumulation and consequent lipid peroxidation in plants.

Differentially expressed galactinol synthase(s) in chickpea are implicated in seed vigor and longevity by limiting the age induced ROS accumulation.

Galactinol synthase (GolS) catalyzes the first and rate limiting step of Raffinose Family Oligosaccharide (RFO) biosynthetic pathway, which is a highly specialized metabolic event in plants. Increased accumulation of galactinol and RFOs in seeds have been reported in few plant species, however their precise role in seed vigor and longevity remain elusive. In present study, we have shown that galactinol synthase activity as well as galactinol and raffinose content progressively increase as seed development proceeds and become highly abundant in pod and mature dry seeds, which gradually decline as seed germination progresses in chickpea (Cicer arietinum). Furthermore, artificial aging also stimulates galactinol synthase activity and consequent galactinol and raffinose accumulation in seed. Molecular analysis revealed that GolS in chickpea are encoded by two divergent genes (CaGolS1 and CaGolS2) which potentially encode five CaGolS isoforms through alternative splicing. Biochemical analysis showed that only two isoforms (CaGolS1 and CaGolS2) are biochemically active with similar yet distinct biochemical properties. CaGolS1 and CaGolS2 are differentially regulated in different organs, during seed development and germination however exhibit similar subcellular localization. Furthermore, seed-specific overexpression of CaGolS1 and CaGolS2 in Arabidopsis results improved seed vigor and longevity through limiting the age induced excess ROS and consequent lipid peroxidation.

Rice PROTEIN l-ISOASPARTYL METHYLTRANSFERASE isoforms differentially accumulate during seed maturation to restrict deleterious isoAsp and reactive oxygen species accumulation and are implicated in seed vigor and longevity.

PROTEIN l-ISOASPARTYL O-METHYLTRANSFERASE (PIMT) is a protein-repairing enzyme involved in seed vigor and longevity. However, the regulation of PIMT isoforms during seed development and the mechanism of PIMT-mediated improvement of seed vigor and longevity are largely unknown. In this study in rice (Oryza sativa), we demonstrate the dynamics and correlation of isoaspartyl (isoAsp)-repairing demands and PIMT activity, and their implications, during seed development, germination and aging, through biochemical, molecular and genetic studies. Molecular and biochemical analyses revealed that rice possesses various biochemically active and inactive PIMT isoforms. Transcript and western blot analyses clearly showed the seed development stage and tissue-specific accumulation of active isoforms. Immunolocalization studies revealed distinct isoform expression in embryo and aleurone layers. Further analyses of transgenic lines for each OsPIMT isoform revealed a clear role in the restriction of deleterious isoAsp and age-induced reactive oxygen species (ROS) accumulation to improve seed vigor and longevity. Collectively, our data suggest that a PIMT-mediated, protein repair mechanism is initiated during seed development in rice, with each isoform playing a distinct, yet coordinated, role. Our results also raise the intriguing possibility that PIMT repairs antioxidative enzymes and proteins which restrict ROS accumulation, lipid peroxidation, etc. in seed, particularly during aging, thus contributing to seed vigor and longevity.