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.

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.

CONSTANS, a key-player connecting day length to seed size.

Plants sense oscillation in the day length as a reliable seasonal cue to drive optimal vegetative and reproductive growth. A recent study by Yu et al. has revealed how day length regulates seed size through CONSTANS. The CONSTANS-APETALA2 module enables plants to optimize their reproductive growth based on their photoperiod response type.

Seed germination variability: why do genetically identical seeds not germinate at the same time?

For survival in the natural environment, plants have evolved a 'bet-hedging' strategy where individual variation is high and a range of phenotypes is produced. When faced with unpredictable environmental conditions, fluctuation in seed behaviour is a beneficial trait that allows plant species to survive, particularly if seedlings from early-germinated seeds die. However, this is not a desired trait from an agricultural perspective, where a set of uniformly growing seedlings is required. Whilst variability in seed behaviour is unavoidable, over the centuries humans have attempted to select seeds with minimum variability for agricultural use. In the model plant Arabidopsis, even non-stratified seeds in the same silique germinate variably, and it remains elusive how this variability is manifested from genes to a physiological outcome and what molecular mechanisms of bet-hedging facilitate this diversity. Will the re-introduction of valuable wild alleles into domesticated crops contribute to this variability between individual seeds by promoting bet-hedging? Recent advances have shed light on possible molecular pathways of germination that are affected at the level of single seeds and single cells. Here, we review the hormonal, molecular, and cellular mechanisms that might affect the germination outcome of individual genetically identical seeds.

Protein l-isoAspartyl Methyltransferase (PIMT) and antioxidants in plants.

All life forms, including plants, accumulate reactive oxygen species (ROS) as a byproduct of metabolism; however, environmental stresses, including abiotic stresses and pathogen attacks, cause enhanced accumulation of ROS in plants. The increased accumulation of ROS often causes oxidative damage to cells. Organisms are able to maintain levels of ROS below permissible limits by several mechanisms, including efficient antioxidant systems. In addition to antioxidant systems, recent studies suggest that protein l-isoaspartyl methyltransferase (PIMT), a highly conserved protein repair enzyme across evolutionary diverse organisms, plays a critical role in maintaining ROS homeostasis by repairing isoaspartyl-mediated damage to antioxidants in plants. Under stress conditions, antioxidant proteins undergo spontaneous isoaspartyl (isoAsp) modification which is often detrimental to protein structure and function. This reduces the catalytic action of antioxidants and disturbs the ROS homeostasis of cells. This chapter focuses on PIMT and its interaction with antioxidants in plants, where PIMT constitutes a secondary level of protection by shielding a primary level of antioxidants from dysfunction and permitting them to guard during unfavorable situations.

Phy meets ERFs to regulate seed germination.

The complex process of seed germination is impacted heavily by environmental cues, such as light, mediated via photosensory systems and phytochromes. This pathway was discovered a long time ago, but the underlying molecular mechanisms are not fully understood. Li et al. recently showed how ETHYLENE RESPONSE FACTORs (ERFs) modulate phytochrome-mediated regulation of germination.

Abiotic Stress Tolerance in Plants: Brassinosteroids Navigate Competently.

Brassinosteroid hormones (BRs) multitask to smoothly regulate a broad spectrum of vital physiological processes in plants, such as cell division, cell expansion, differentiation, seed germination, xylem differentiation, reproductive development and light responses (photomorphogenesis and skotomorphogenesis). Their importance is inferred when visible abnormalities arise in plant phenotypes due to suboptimal or supraoptimal hormone levels. This group of steroidal hormones are major growth regulators, having pleiotropic effects and conferring abiotic stress resistance to plants. Numerous abiotic stresses are the cause of significant loss in agricultural yield globally. However, plants are well equipped with efficient stress combat machinery. Scavenging reactive oxygen species (ROS) is a unique mechanism to combat the deleterious effects of abiotic stresses. In light of numerous reports in the past two decades, the complex BR signaling under different stress conditions (drought, salinity, extreme temperatures and heavy metals/metalloids) that drastically hinders the normal metabolism of plants is gradually being untangled and revealed. Thus, crop improvement has substantial potential by tailoring either the brassinosteroid signaling, biosynthesis pathway or perception. This review aims to explore and dissect the actual mission of BRs in signaling cascades and summarize their positive role with respect to abiotic stress tolerance.

Raffinose family oligosaccharides (RFOs): role in seed vigor and longevity.

Seed vigor and longevity are important agronomic attributes, as they are essentially associated with crop yield and thus the global economy. Seed longevity is a measure of seed viability and the most essential property in gene bank management since it affects regeneration of seed recycling. Reduced seed life or storability is a serious issue in seed storage since germplasm conservation and agricultural enhancement initiatives rely on it. The irreversible and ongoing process of seed deterioration comprises a complex gene regulatory network and altered metabolism that results in membrane damage, DNA integrity loss, mitochondrial dysregulation, protein damage, and disrupted antioxidative machinery. Carbohydrates and/or sugars, primarily raffinose family oligosaccharides (RFOs), have emerged as feasible components for boosting or increasing seed vigor and longevity in recent years. RFOs are known to perform diverse functions in plants, including abiotic and biotic stress tolerance, besides being involved in regulating seed germination, desiccation tolerance, vigor, and longevity. We emphasized and analyzed the potential impact of RFOs on seed vigor and longevity in this review. Here, we comprehensively reviewed the molecular mechanisms involved in seed longevity, RFO metabolism, and how RFO content is critical and linked with seed vigor and longevity. Further molecular basis, biotechnological approaches, and CRISPR/Cas applications have been discussed briefly for the improvement of seed attributes and ultimately crop production. Likewise, we suggest advancements, challenges, and future possibilities in this area.

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.

RNA Interference for Improving Disease Resistance in Plants and Its Relevance in This Clustered Regularly Interspaced Short Palindromic Repeats-Dominated Era in Terms of dsRNA-Based Biopesticides.

RNA interference (RNAi) has been exploited by scientists worldwide to make a significant contribution in the arena of sustainable agriculture and integrated pest management. These strategies are of an imperative need to guarantee food security for the teeming millions globally. The already established deleterious effects of chemical pesticides on human and livestock health have led researchers to exploit RNAi as a potential agri-biotechnology tool to solve the burning issue of agricultural wastage caused by pests and pathogens. On the other hand, CRISPR/Cas9, the latest genome-editing tool, also has a notable potential in this domain of biotic stress resistance, and a constant endeavor by various laboratories is in progress for making pathogen-resistant plants using this technique. Considerable outcry regarding the ill effects of genetically modified (GM) crops on the environment paved the way for the research of RNAi-induced double-stranded RNAs (dsRNA) and their application to biotic stresses. Here, we mainly focus on the application of RNAi technology to improve disease resistance in plants and its relevance in today's CRISPR-dominated world in terms of exogenous application of dsRNAs. We also focused on the ongoing research, public awareness, and subsequent commercialization of dsRNA-based biocontrol products.

Chromatin-Based Transcriptional Reprogramming in Plants under Abiotic Stresses.

Plants' stress response machinery is characterized by an intricate network of signaling cascades that receive and transmit environmental cues and ultimately trigger transcriptional reprogramming. The family of epigenetic regulators that are the key players in the stress-induced signaling cascade comprise of chromatin remodelers, histone modifiers, DNA modifiers and regulatory non-coding RNAs. Changes in the histone modification and DNA methylation lead to major alterations in the expression level and pattern of stress-responsive genes to adjust with abiotic stress conditions namely heat, cold, drought and salinity. The spotlight of this review falls primarily on the chromatin restructuring under severe abiotic stresses, crosstalk between epigenetic regulators along with a brief discussion on stress priming in plants.

Emerging roles of the ubiquitin-proteasome pathway in enhancing crop yield by optimizing seed agronomic traits.

KEY MESSAGE: Ubiquitin-proteasome pathway has the potential to modulate crop productivity by influencing agronomic traits. Being sessile, the plant often uses the ubiquitin-proteasome pathway to maintain the stability of different regulatory proteins to survive in an ever-changing environment. The ubiquitin system influences plant reproduction, growth, development, responses to the environment, and processes that control critical agronomic traits. E3 ligases are the major players in this pathway, and they are responsible for recognizing and tagging the targets/substrates. Plants have a variety of E3 ubiquitin ligases, whose functions have been studied extensively, ranging from plant growth to defense strategies. Here we summarize three agronomic traits influenced by ubiquitination: seed size and weight, seed germination, and accessory plant agronomic traits particularly panicle architecture, tillering in rice, and tassels branch number in maize. This review article highlights some recent progress on how the ubiquitin system influences the stability/modification of proteins that determine seed agronomic properties like size, weight, germination and filling, and ultimately agricultural productivity and quality. Further research into the molecular basis of the aforementioned processes might lead to the identification of genes that could be modified or selected for crop development. Likewise, we also propose advances and future perspectives in this regard.

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.

JA Shakes Hands with ABA to Delay Seed Germination.

Seed germination is a multifaceted process, controlled by many cues, wherein phytohormones play a central role. Despite extensive studies, it remains obscure how hormonal balance and crosstalk between hormones regulate seed germination. Here we highlight new findings showing that crosstalk between jasmonates (JA) and abscisic acid (ABA) delays seed germination.

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.