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Lipopolysaccharide-induced tumor necrosis factor-α (LITAF) is a membrane protein that is highly dependent on correct location to exert transcription factor activity and protein quality control. In humans, LITAF, PIG7 (p53-inducible gene 7), and SIMPLE (small integral membrane protein of the lysosome/late endosome) refer to the same gene, which acts as a tumor suppressor. Several studies have shown that the transcription factor activity and nuclear translocation of LITAF protein are critical for the induction of several immune cells via classical pathways. In plants, LITAF protein corresponds to the plasma membrane protein AtGILP ( Arabidopsis thaliana GSH-induced LITAF domain protein). The conservation of LITAF proteins across species and their putative role is still unclear. In this study, we investigate the LITAF-containing proteins, which we call GILP proteins, in Viridiplantae. We identified a total of 59 genes in 46 species, whose gene copies range from one to three. Phylogenetic analysis showed that multiple copies were originated via block duplication posteriorly to monocot and eudicot separation. Analysis of the LITAF domain of GILP proteins allowed the identification of a putative domain signature in Viridiplantae, containing a CXXCX41HXCPXC motif. The subcellular location for the majority of GILP proteins was predicted to be in the plasma membrane, based on a transmembrane domain positioned within the LITAF domain. In silico analysis showed that the GILP genes are neither tissue-specific nor ubiquitously expressed, being responsive to stress conditions. Finally, investigation of the GILP protein network resulted in the identification of genes whose families are known to be involved with biotic and/or abiotic stress responses. Together, the expression modulation of GILP genes associated with their plasma membrane location suggests that they could act in the signaling of biotic/abiotic stress response in plants. [ABSTRACT FROM AUTHOR]

As a central component of the hydrogen peroxide detoxifying system in plant cells, ascorbate peroxidases (APX) play an essential role in the control of intracellular reactive oxygen species (ROS) levels. To characterise the function of cytosolic APX isoforms ( OsAPX1 and OsAPX2) in the mechanisms of plant defence, OsAPX1/2 knockdown rice plants were previously obtained. OsAPX1/2 knockdown plants (APx1/2s) exhibited a normal phenotype and development, even though they showed a global reduction of APX activity and increased hydrogen peroxide accumulation. To understand how rice plants compensate for the deficiency of cytosolic APX, expression and proteomic analyses were performed to characterise the global expression pattern of the APx1/2s mutant line compared with non-transformed plants. Our results strongly suggest that deficiencies in cytosolic APX isoforms markedly alter expression of genes associated with several key metabolic pathways, especially of genes involved in photosynthesis and antioxidant defence. These metabolic changes are compensatory because central physiological processes such as photosynthesis and growth were similar to non-transformed rice plants. Our analyses showed modulation of groups of genes and proteins related to specific metabolic pathways. Among the differentially expressed genes, the largest number corresponded to those with catalytic activity. Genes related to oxidative stress, carbohydrate metabolism, photosynthesis and transcription factor-encoding genes were also modulated. These results represent an important step toward understanding of the role played by cytosolic APX isoforms and hydrogen peroxide in the regulation of metabolism by redox modulation in monocots. [ABSTRACT FROM AUTHOR]

Abstract Background Triacylglycerides (TAGs) are a class of neutral lipids that represent the most important storage form of energy for eukaryotic cells. DGAT (acyl-CoA: diacylglycerol acyltransferase; EC 2.3.1.20) is a transmembrane enzyme that acts in the final and committed step of TAG synthesis, and it has been proposed to be the rate-limiting enzyme in plant storage lipid accumulation. In fact, two different enzymes identified in several eukaryotic species, DGAT1 and DGAT2, are the main enzymes responsible for TAG synthesis. These enzymes do not share high DNA or protein sequence similarities, and it has been suggested that they play non-redundant roles in different tissues and in some species in TAG synthesis. Despite a number of previous studies on the DGAT1 and DGAT2 genes, which have emphasized their importance as potential obesity treatment targets to increase triacylglycerol accumulation, little is known about their evolutionary timeline in eukaryotes. The goal of this study was to examine the evolutionary relationship of the DGAT1 and DGAT2 genes across eukaryotic organisms in order to infer their origin. Results We have conducted a broad survey of fully sequenced genomes, including representatives of Amoebozoa, yeasts, fungi, algae, musses, plants, vertebrate and invertebrate species, for the presence of DGAT1 and DGAT2 gene homologs. We found that the DGAT1 and DGAT2 genes are nearly ubiquitous in eukaryotes and are readily identifiable in all the major eukaryotic groups and genomes examined. Phylogenetic analyses of the DGAT1 and DGAT2 amino acid sequences revealed evolutionary partitioning of the DGAT protein family into two major DGAT1 and DGAT2 clades. Protein secondary structure and hydrophobic-transmembrane analysis also showed differences between these enzymes. The analysis also revealed that the MGAT2 and AWAT genes may have arisen from DGAT2 duplication events. Conclusions In this study, we identified several DGAT1 and DGAT2 homologs in eukaryote taxa. Overall, the data show that DGAT1 and DGAT2 are present in most eukaryotic organisms and belong to two different gene families. The phylogenetic and evolutionary analyses revealed that DGAT1 and DGAT2 evolved separately, with functional convergence, despite their wide molecular and structural divergence.

We review the newly classified ascorbate peroxidase-related (APX-R) proteins, which do not use ascorbate as electron donor to scavenge H 2 O 2 . We summarize recent discoveries on the function and the characterization of the APX-R protein of the green unicellular alga Chlamydomonas reinhardtii and the land plant Arabidopsis thaliana . Additionally, we conduct in silico analyses on the conserved MxxM motif, present in most of the APX-R protein in different organisms, which is proposed to bind copper. Based on these analyses, we discuss the similarities between the APX-R and the class III peroxidases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Caccamo, Lazzarotto, Margis-Pinheiro, Messens and Remacle.)

Understanding the evolution and the effect of plasticity in plant responses to environmental changes is crucial to combat global climate change. It is particularly interesting in species that survive in distinct environments, such as Eugenia uniflora, which thrives in contrasting ecosystems within the Atlantic Forest (AF). In this study, we combined transcriptome analyses of plants growing in nature (Restinga and Riparian Forest) with greenhouse experiments to unveil the DEGs within and among adaptively divergent populations of E. uniflora . We compared global gene expression among plants from two distinct ecological niches. We found many differentially expressed genes between the two populations in natural and greenhouse-cultivated environments. The changes in how genes are expressed may be related to the species' ability to adapt to specific environmental conditions. The main difference in gene expression was observed when plants from Restinga were compared with their offspring cultivated in greenhouses, suggesting that there are distinct selection pressures underlying the local environmental and ecological factors of each Restinga and Riparian Forest ecosystem. Many of these genes engage in the stress response, such as water and nutrient transport, temperature, light intensity, and gene regulation. The stress-responsive genes we found are potential genes for selection in these populations. These findings revealed the adaptive potential of E. uniflora and contributed to our understanding of the role of gene expression reprogramming in plant evolution and niche adaptation.

Anther development is a complex process essential for plant reproduction and crop yields. In recent years, significant progress has been made in the identification and characterization of the bHLH transcription factor family involved in anther regulation in rice and Arabidopsis, two extensively studied model plants. Research on bHLH transcription factors has unveiled their crucial function in controlling tapetum development, pollen wall formation, and other anther-specific processes. By exploring deeper into regulatory mechanisms governing anther development and bHLH transcription factors, we can gain important insights into plant reproduction, thereby accelerating crop yield improvement and the development of new plant breeding strategies. This review provides an overview of the current knowledge on anther development in rice and Arabidopsis, emphasizing the critical roles played by bHLH transcription factors in this process. Recent advances in gene expression analysis and functional studies are highlighted, as they have significantly enhanced our understanding of the regulatory networks involved in anther development.

We have previously shown that rice plants silenced for peroxisomal ascorbate peroxidase (OsAPX4-RNAi) display higher resilience to photosynthesis under oxidative stress and photorespiratory conditions. However, the redox mechanisms underlying that intriguing response remain unknown. Here, we tested the hypothesis that favorable effects triggered by peroxisomal APX deficiency on photosynthesis resilience under CAT inhibition are dependent on the intensity of photorespiration associated with the abundance of photosynthetic and redox proteins. Non-transformed (NT) and OsAPX4-RNAi silenced rice plants were grown under ambient (AC) or high CO 2 (HC) conditions and subjected to 3-amino-1,2,4-triazole (3-AT)-mediated CAT activity inhibition. Photosynthetic measurements evidenced that OsAPX4-RNAi plants simultaneously exposed to CAT inhibition and HC lost the previously acquired advantage in photosynthesis resilience displayed under AC. Silenced plants exposed to environment photorespiration and CAT inhibition presented lower photorespiration as indicated by smaller Gly/Ser and Jo/Jc ratios and glycolate oxidase activity. Interestingly, when these silenced plants were exposed to HC and CAT-inhibition, they exhibited an inverse response compared to AC in terms of photorespiration indicators, associated with higher accumulation of proteins. Multivariate and correlation network analyses suggest that the proteomics changes induced by HC combined with CAT inhibition are substantially different between NT and OsAPX4-RNAi plants. Our results suggest that the intensity of photorespiration and peroxisomal APX-mediated redox signaling are tightly regulated under CAT inhibition induced oxidative stress, which can modulate the photosynthetic efficiency, possibly via a coordinated regulation of protein abundance and rearrangement, ultimately triggered by crosstalk involving H 2 O 2 levels related to CAT and APX activities in peroxisomes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Masson SAS.)

Intrinsically disordered proteins (IDPs) have numerous dynamic conformations. Given the difficulties in tracking temporarily folded states of this kind of protein, methods such as molecular modeling and molecular dynamics (MD) simulations make the process less costly, less laborious, and more detailed. Few plant IDPs have been characterized so far, such as proteins from the Abscisic acid, Stress and Ripening (ASR) family. The present work applied, for the first time, the two above-mentioned tools to test the feasibility of determining a three-dimensional transition model of OsASR5 and to investigate the relationship between OsASR5 and zinc. We found that one of OsASR5's conformers contains α-helices, turns, and loops and that the metal binding resulted in a predominance of α-helix. This stability is possibly imperative for the transcription factor activity. The promoter region of a sugar transporter was chosen to test this hypothesis and free energy calculations showed how the ion is mandatory for this complex formation. The results produced here aim to clarify which conformation the protein in the bound state assumes and which residues are involved in the process, besides developing the understanding of how the flexibility of these proteins can contribute to the response to environmental stresses., (© 2023 Wiley Periodicals LLC.)

Chloroplast ascorbate peroxidases exert an important role in the maintenance of hydrogen peroxide levels in chloroplasts by using ascorbate as the specific electron donor. In this work, we performed a functional study of the stromal APX in rice (OsAPX7) and demonstrated that silencing of OsAPX7 did not impact plant growth, redox state, or photosynthesis parameters. Nevertheless, when subjected to drought stress, silenced plants (APX7i) show a higher capacity to maintain stomata aperture and photosynthesis performance, resulting in a higher tolerance when compared to non-transformed plants. RNA-seq analyses indicate that the silencing of OsAPX7 did not lead to changes in the global expression of genes related to reactive oxygen species metabolism. In addition, the drought-mediated induction of several genes related to the proteasome pathway and the down-regulation of genes related to nitrogen and carotenoid metabolism was impaired in APX7i plants. During drought stress, APX7i showed an up-regulation of genes encoding flavonoid and tyrosine metabolism enzymes and a down-regulation of genes related to phytohormones signal transduction and nicotinate and nicotinamide metabolism. Our results demonstrate that OsAPX7 might be involved in signaling transduction pathways related to drought stress response, contributing to the understanding of the physiological role of chloroplast APX isoforms in rice.

Ascorbate peroxidase (APX), Monodehydroascorbate Reductase (MDAR), Dehydroascorbate Reductase (DHAR) and Glutathione Reductase (GR) enzymes participate in the ascorbate-glutathione cycle, which exerts a central role in the antioxidant metabolism in plants. Despite the importance of this antioxidant system in different signal transduction networks related to development and response to environmental stresses, the pathway has not yet been comprehensively characterized in many crop plants. Among different eudicotyledons, the Euphorbiaceae family is particularly diverse with some species highly tolerant to drought. Here the APX, MDAR, DHAR, and GR genes in Ricinus communis , Jatropha curcas , Manihot esculenta , and Hevea brasiliensis were identified and characterized. The comprehensive phylogenetic and genomic analyses allowed the classification of the genes into different classes, equivalent to cytosolic, peroxisomal, chloroplastic, and mitochondrial enzymes, and revealed the duplication events that contribute to the expansion of these families within plant genomes. Due to the high drought stress tolerance of Ricinus communis, the expression patterns of ascorbate-glutathione cycle genes in response to drought were also analyzed in leaves and roots, indicating a differential expression during the stress. Altogether, these data contributed to the characterization of the expression pattern and evolutionary analysis of these genes, filling the gap in the proposed functions of core components of the antioxidant mechanism during stress response in an economically relevant group of plants.

Ascorbate peroxidases (APXs) are heme peroxidases involved in the control of hydrogen peroxide levels and signal transduction pathways related to development and stress responses. Here, a total of 238 APX, 30 APX-related (APX-R), and 34 APX-like (APX-L) genes were identified from 24 species from the Poaceae family. Phylogenetic analysis of APX indicated five distinct clades, equivalent to cytosolic (cAPX), peroxisomal (pAPX), mitochondrial (mitAPX), stromal (sAPX), and thylakoidal (tAPX) isoforms. Duplication events contributed to the expansion of this family and the divergence times. Different from other APX isoforms, the emergence of Poaceae mitAPXs occurred independently after eudicot and monocot divergence. Our results showed that the constitutive silencing of mitAPX genes is not viable in rice plants, suggesting that these isoforms are essential for rice regeneration or development. We also obtained rice plants silenced individually to sAPX isoforms, demonstrating that, different to plants double silenced to both sAPX and tAPX or single silenced to tAPX previously obtained, these plants do not show changes in the total APX activity and hydrogen peroxide content in the shoot. Among rice plants silenced to different isoforms, plants silenced to cAPX showed a higher decrease in total APX activity and an increase in hydrogen peroxide levels. These results suggest that the cAPXs are the main isoforms responsible for regulating hydrogen peroxide levels in the cell, whereas in the chloroplast, this role is provided mainly by the tAPX isoform. In addition to broadening our understanding of the core components of the antioxidant defense in Poaceae species, the present study also provides a platform for their functional characterization.

The diversity of diacylglycerol acyltransferases (DGATs) indicates alternative roles for these enzymes in plant metabolism besides triacylglycerol (TAG) biosynthesis. In this work, we functionally characterized castor bean (Ricinus communis L.) DGATs assessing their subcellular localization, expression in seeds, capacity to restore triacylglycerol (TAG) biosynthesis in mutant yeast and evaluating whether they provide tolerance over free fatty acids (FFA) in sensitive yeast. RcDGAT3 displayed a distinct subcellular localization, located in vesicles outside the endoplasmic reticulum (ER) in most leaf epidermal cells. This enzyme was unable to restore TAG biosynthesis in mutant yeast; however, it was able to outperform other DGATs providing higher tolerance over FFA. RcDAcTA subcellular localization was associated with the ER membranes, resembling RcDGAT1 and RcDGAT2, but it failed to rescue the long-chain TAG biosynthesis in mutant yeast, even with fatty acid supplementation. Besides TAG biosynthesis, our results suggest that RcDGAT3 might have alternative functions and roles in lipid metabolism.

Ascorbate peroxidases (APXs) are heme peroxidases that remove hydrogen peroxide in different subcellular compartments with concomitant ascorbate cycling. Here, we analysed and discussed phylogenetic and molecular features of the APX family. Ancient APX originated as a soluble stromal enzyme, and early during plant evolution, acquired both chloroplast-targeting and mitochondrion-targeting sequences and an alternative splicing mechanism whereby it could be expressed as a soluble or thylakoid membrane-bound enzyme. Later, independent duplication and neofunctionalization events in some angiosperm groups resulted in individual genes encoding stromal, thylakoidal and mitochondrial isoforms. These data reaffirm the complexity of plant antioxidant defenses that allow diverse plant species to acquire new means to adapt to changing environmental conditions., (© 2022 Federation of European Biochemical Societies.)

Although mitochondria have a central role in energy transduction and reactive oxygen species (ROS) production, the regulatory mechanisms and their involvement in plant stress signaling are not fully established. The phytohormone salicylic acid (SA) is an important regulator of mitochondria-mediated ROS production and defense signaling. The role of SA and adenine nucleotides in the regulation of the mitochondrial succinate dehydrogenase (SDH) complex activity and ROS production was analyzed using WT, RNAi SDH1-1 and disrupted stress response 1 (dsr1) mutants, which show a point mutation in SDH1 subunit and are defective in SA signaling. Our results showed that SA and adenine nucleotides regulate SDH complex activity by distinct patterns, contributing to increased SDH-derived ROS production. As previously demonstrated, SA induces the succinate-quinone reductase activity of SDH complex, acting at or near the ubiquinone binding site. On the other hand, here we demonstrated that adenine nucleotides, such as AMP, ADP and ATP, induce the SDH activity provided by the SDH1 subunit. The regulation of SDH activity by adenine nucleotides is dependent on mitochondrial integrity and is prevented by atractyloside, an inhibitor of adenine nucleotide translocator (ANT), suggesting that the regulatory mechanism occurs on the mitochondrial matrix side of the inner mitochondrial membrane, and not in the intermembrane space, as previously suggested. On the other hand, in the intermembrane space, ADP and ATP limit mitochondrial oxygen consumption by a mechanism that appears to be related to cytochrome bc1 complex inhibition. Altogether, these results indicate that SA signaling and adenine nucleotides regulate the mitochondrial electron transport system and mitochondria-derived ROS production by direct effect in the electron transport system complexes, bringing new insights into mechanisms with direct implications in plant development and responses to different environmental responses, serving as a starting point for future physiological explorations., (Copyright © 2022 Elsevier B.V. All rights reserved.)

There is large diversity among glutathione peroxidase (GPx) enzymes regarding their function, structure, presence of the highly reactive selenocysteine (SeCys) residue, substrate usage, and reducing agent preference. Moreover, most vertebrate GPxs are very distinct from non-animal GPxs, and it is still unclear if they came from a common GPx ancestor. In this study, we aimed to unveil how GPx evolved throughout different phyla. Based on our phylogenetic trees and sequence analyses, we propose that all GPx encoding genes share a monomeric common ancestor and that the SeCys amino acid was incorporated early in the evolution of the metazoan kingdom. In addition, classical GPx and the cysteine-exclusive GPx07 have been present since non-bilaterian animals, but they seem to have been lost throughout evolution in different phyla. Therefore, the birth-and-death of GPx family members (like in other oxidoreductase families) seems to be an ongoing process, occurring independently across different kingdoms and phyla.

Ascorbate peroxidases (APX) are class I members of the Peroxidase-Catalase superfamily, a large group of evolutionarily related but rather divergent enzymes. Through mining in public databases, unusual subsets of APX homologs were identified, disclosing the existence of two yet uncharacterized families of peroxidases named ascorbate peroxidase-related (APX-R) and ascorbate peroxidase-like (APX-L). As APX, APX-R harbor all catalytic residues required for peroxidatic activity. Nevertheless, proteins of this family do not contain residues known to be critical for ascorbate binding and therefore cannot use it as an electron donor. On the other hand, APX-L proteins not only lack ascorbate-binding residues, but also every other residue known to be essential for peroxidase activity. Through a molecular phylogenetic analysis performed with sequences derived from basal Archaeplastida, the present study discloses the existence of hybrid proteins, which combine features of these three families. The results here presented show that the prevalence of hybrid proteins varies among distinct groups of organisms, accounting for up to 33% of total APX homologs in species of green algae. The analysis of this heterogeneous group of proteins sheds light on the origin of APX-R and APX-L and suggests the occurrence of a process characterized by the progressive deterioration of ascorbate-binding and catalytic sites towards neofunctionalization.

Iron (Fe) toxicity is one of the most common mineral disorders affecting rice (Oryza sativa) production in flooded lowland fields. Oryza meridionalis is indigenous to northern Australia and grows in regions with Fe-rich soils, making it a candidate for use in adaptive breeding. With the aim of understanding tolerance mechanisms in rice, we screened a population of interspecific introgression lines from a cross between O. sativa and O. meridionalis for the identification of quantitative trait loci (QTLs) contributing to Fe-toxicity tolerance. Six putative QTLs were identified. A line carrying one introgression from O. meridionalis on chromosome 9 associated with one QTL was highly tolerant despite very high shoot Fe concentrations. Physiological, biochemical, ionomic, and transcriptomic analyses showed that the tolerance of the introgression lines could partly be explained by higher relative Fe retention in the leaf sheath and culm. We constructed the interspecific hybrid genome in silico for transcriptomic analysis and identified differentially regulated introgressed genes from O. meridionalis that could be involved in shoot-based Fe tolerance, such as metallothioneins, glutathione S-transferases, and transporters from the ABC and MFS families. This work demonstrates that introgressions of O. meridionalis into the O. sativa genome can confer increased tolerance to excess Fe., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Different environmental conditions can lead plants to a condition termed oxidative stress, which is characterized by a disruption in the equilibrium between the production of reactive oxygen species (ROS) and antioxidant defenses. Glutathione peroxidase (GPX), an enzyme that acts as a peroxide scavenger in different organisms, has been identified as an important component in the signaling pathway during the developmental process and in stress responses in plants and yeast. Here, we demonstrate that the mitochondrial isoform of rice (Oryza sativa L. ssp. Japonica cv. Nipponbare) OsGPX3 is induced after treatment with the phytohormone abscisic acid (ABA) and is involved in its responses and in epigenetic modifications. Plants that have been silenced for OsGPX3 (gpx3i) present substantial changes in the accumulation of proteins related to these processes. These plants also have several altered ABA responses, such as germination, ROS accumulation, stomatal closure, and dark-induced senescence. This study is the first to demonstrate that OsGPX3 plays a role in ABA signaling and corroborate that redox homeostasis enzymes can act in different and complex pathways in plant cells. SIGNIFICANCE: This work proposes the mitochondrial glutathione peroxidase (OsGPX3) as a novel ABA regulatory pathway component. Our results suggest that this antioxidant enzyme is involved in ABA-responses, highlighting the complex pathways that these proteins can participate beyond the regulation of cellular redox status., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Peroxidases are enzymes that catalyze the reduction of hydrogen peroxide, thus minimizing cell injury and modulating signaling pathways as response to this reactive oxygen species. Using a phylogenetic approach, we previously identified a new peroxidase family composed of a small subset of ascorbate peroxidase (APx) homologs with distinguished features, which we named ascorbate peroxidase-related (APx-R). In this study, we showed that APx-R is an ascorbate-independent heme peroxidase. Despite being annotated as a cytosolic protein in public databases, transient expression of AtAPx-R-YFP in Arabidopsis thaliana protoplasts and stable overexpression in plants showed that the protein is targeted to plastids. To characterize APx-R participation in the antioxidant metabolism, we analyzed loss-of-function mutants and AtAPx-R overexpressing lines. Molecular analysis showed that glutathione peroxidase 7 (GPx07) is specifically induced to compensate the absence of APx-R. APx-R overexpressing lines display faster germination rates, further confirming the involvement of APx-R in seed germination. The constitutive overexpression of AtAPx-R-YFP unraveled the existence of a post-translational mechanism that eliminates APx-R from most tissues, in a process coordinated with photomorphogenesis. Our results show a direct role of APx-R during germinative and post-germinative development associated with etioplasts differentiation.

Anther development is a complex process regulated by a myriad of transcription factors belonging to distinct protein families. In this study, we focus on the functional characterization of OsbHLH35, a basic Helix-Loop-Helix (bHLH) TF that regulates anther development in rice. Plants overexpressing OsbHLH35 presented small and curved anthers, leading to a reduction of 72 % on seed production. Rice transgenic plants expressing GUS reporter gene under the control of OsbHLH35 promoter (pOsbHLH35::GUS) showed that this TF specifically accumulates in anthers at the meiosis stage and in other spikelet tissues. Yeast one-hybrid screening identified three members of the Growth-Regulating Factor (GRF) family, OsGRF3, OsGRF4, and OsGRF11, as transcriptional regulators of OsbHLH35. Transactivation assay showed that OsGRF11 negatively regulates OsbHLH35 expression in Arabidopsis protoplasts. This regulation was also observed in planta through the analysis of transgenic plants overexpressing OsGRF11 (OsGRF11OE), confirming that OsGRF11 is a negative regulator of OsbHLH35 in rice. Our data suggest that OsbHLH35 plays an essential role in anther development in rice and the fine control of its expression is crucial to ensure proper seed production., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Programmed cell death (PCD) is a genetically controlled process that leads to cell suicide in both eukaryotic and prokaryotic organisms. In plants PCD occurs during development, defence response and when exposed to adverse conditions. PCD acts controlling the number of cells by eliminating damaged, old, or unnecessary cells to maintain cellular homeostasis. Unlike in animals, the knowledge about PCD in plants is limited. The molecular network that controls plant PCD is poorly understood. Here we present a review of the current mechanisms involved with the genetic control of PCD in plants. We also present an updated version of the AtLSD1 deathosome, which was previously proposed as a network controlling HR-mediated cell death in Arabidopsis thaliana. Finally, we discuss the unclear points and open questions related to the AtLSD1 deathosome., (Copyright © 2020. Published by Elsevier B.V.)

- Growth Regulating Factors (GRFs) comprise a transcription factor family with important functions in plant growth and development. They are characterized by the presence of QLQ and WRC domains, responsible for interaction with proteins and DNA, respectively. The QLQ domain is named due to the similarity to a protein interaction domain found in the SWI2/SNF2 chromatin remodeling complex. Despite the occurrence of the QLQ domain in both families, the divergence between them had not been further explored. Here, we show evidence for GRF origin and determined its diversification in angiosperm species. Phylogenetic analysis revealed 11 well-supported groups of GRFs in flowering plants. These groups were supported by gene structure, synteny, and protein domain composition. Synteny and phylogenetic analyses allowed us to propose different sets of probable orthologs in the groups. Besides, our results, together with functional data previously published, allowed us to suggest candidate genes for engineering agronomic traits. In addition, we propose that the QLQ domain of GRF genes evolved from the eukaryotic SNF2 QLQ domain, most likely by a duplication event in the common ancestor of the Charophytes and land plants. Altogether, our results are important for advancing the origin and evolution of the GRF family in Streptophyta.

Key Message: MdoDHN11 acts in the nucellus layer to protect the embryo and the endosperm from limited water availability during apple seed development. Dehydrins (DHNs) are protective proteins related to several plant developmental responses that involve dehydration such as seed desiccation and abiotic stresses. In apple (Malus × domestica Borkh.), the seed-specific MdoDHN11 was suggested to play important roles against dehydration during seed development. However, this hypothesis has not yet been evaluated. Within this context, several experiments were performed to functionally characterize MdoDHN11. In situ hybridization analysis during apple seed development showed that MdoDHN11 expression is confined to a maternal tissue called nucellus, a central mass of parenchyma between the endosperm and the testa. The MdoDHN11 protein was localized in the cytosol and nucleus. Finally, transgenic Arabidopsis plants expressing MdoDHN11 were generated and exposed to a severe water-deficit stress, aiming to mimic a situation that can occurs during seed development. All transgenic lines showed increased tolerance to water deficit in relation to wild-type plants. Taken together, our results provide evidences that MdoDHN11 plays important roles during apple seed development by protecting the embryo and the endosperm from limited water availability, and the mechanism of action probably involves the interaction of MdoDHN11 with proteins and other components in the cell.

Retrograde signalling pathways that are triggered by changes in cellular redox homeostasis remain poorly understood. Transformed rice plants that are deficient in peroxisomal ascorbate peroxidase APX4 (OsAPX4-RNAi) are known to exhibit more effective protection of photosynthesis against oxidative stress than controls when catalase (CAT) is inhibited, but the mechanisms involved have not been characterized. An in-depth physiological and proteomics analysis was therefore performed on OsAPX4-RNAi CAT-inhibited rice plants. Loss of APX4 function led to an increased abundance of several proteins that are involved in essential metabolic pathways, possibly as a result of increased tissue H2O2 levels. Higher photosynthetic activities observed in the OsAPX4-RNAi plants under CAT inhibition were accompanied by higher levels of Rubisco, higher maximum rates of Rubisco carboxylation, and increased photochemical efficiencies, together with large increases in photosynthesis-related proteins. Large increases were also observed in the levels of proteins involved in the ascorbate/glutathione cycle and in other antioxidant-related pathways, and these changes may be important in the protection of photosynthesis in the OsAPX4-RNAi plants. Large increases in the abundance of proteins localized in the nuclei and mitochondria were also observed, together with increased levels of proteins involved in important cellular pathways, particularly protein translation. Taken together, the results show that OsAPX4-RNAi plants exhibit significant metabolic reprogramming, which incorporates a more effective antioxidant response to protect photosynthesis under conditions of impaired CAT activity.

Triacylglycerols (TAG) are the major form of energy storage in plants. TAG are primarily stored in seeds and fruits, but vegetative tissues also possess a high capacity for their synthesis and storage. These storage lipids are essential to plant development, being used in seedling growth during germination, pollen development, and sexual reproduction, for example. TAG are also an important source of edible oils for animal and human consumption, and are used for fuel and industrial feedstocks. The canonical pathway leading to TAG synthesis is the glycerol-3-phosphate, or Kennedy, pathway, which is an evolutionarily conserved process in most living organisms. The enzymatic machinery for synthesizing TAG is well known in several plant species, and the genes encoding these enzymes have been the focus of many studies. Here, we review recent progress on the understanding of evolutionary, functional and biotechnological aspects of the glycerol-3-phosphate pathway enzymes that produce TAG. We discuss current knowledge about their functional aspects, and summarize valuable insights into genetically engineered plants for enhancing TAG accumulation. Also, we highlight the evolutionary history of these genes and present a meta-analysis linking positive selection to gene family and plant diversification, and also to the domestication processes in oilseed crops., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

PLAC8 is a cysteine-rich protein described as a central mediator of tumor evolution in mammals; as such, it represents a promising candidate for diagnostic and therapeutic targeting. The human PLAC8 gene is also involved in contact hypersensitivity response and presents a role in psoriatic skin. In plants, PLAC8 motif-containing proteins are involved in the determination of organ size and growth, response to infection, Ca 2+ influx, Cd resistance, and zinc detoxification. In general, PLAC8 motif-containing proteins present the conserved CCXXXXCPC or CLXXXXCPC region. However, there is no devised nomenclature for the PLAC8 motif-containing proteins. Here, through the analysis of 445 sequences, we show that PLAC8 motif-containing proteins constitute a unique gene family, and we propose a unified nomenclature. This is the first report indicating the existence of different groups of PLAC8 proteins, which we have called types I, II, and III. Type I genes are found in mammals, fungi, plants, and algae, and types II and III are exclusive to plants. Our study describes for the first time PLAC8 type III proteins. Whether these sequences maintain their known functional role or possess distinct functions of types I and II genes remains unclear.

Seedlessness in grapes is a desirable trait, especially for in natura consumption. Previously, we showed that VviAGL11 is the main responsible gene for seed morphogenesis in grapevine. Here we tested the function of this gene in grapevine with the use of plant plasmids. VviAGL11 was cloned into silencing and overexpression versions of p28iIR plasmid. Reproductive grapevine bunches from different seeded and seedless cultivars were separately treated with VviAGL11-harboring plasmids, along with controls. Plasmids were detected in leaves after a month of treatment, and berries, leaves, stems and seeds were analyzed for ectopic gene expression by RT-qPCR after 90 days of plasmid injection. Fruits from the seedless 'Linda' treated with the VviAGL11-overexpression plasmid showed high expression levels of VviAGL11 and exhibited small seeds that were not found in the untreated control samples. Mature grapes from seeded 'Italia' and 'Ruby' bunches treated with the VviAGL11-silencing plasmid showed decreased VviAGL11 expression, reduced number of seeds and increased number of seed traces. The present study confirms that VviAGL11 is a key master regulator of seed morphogenesis in grapevine and corroborates with the applicability of plant plasmids as promising biotechnological tools to functionally test genes in perennial plants in a rapid and confident way., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Galactinol synthase (GolS) is a key enzyme in the biosynthetic pathway of raffinose family oligosaccharides (RFOs), which play roles in carbon storage, signal transduction, and osmoprotection. The present work assessed the evolutionary history of GolS genes across the Rosaceae using several bioinformatic tools. Apple (Malus × domestica) GolS genes were transcriptionally characterized during bud dormancy, in parallel with galactinol and raffinose measurements. Additionally, MdGolS2, a candidate to regulate seasonal galactinol and RFO content during apple bud dormancy, was functionally characterized in Arabidopsis. Evolutionary analyses revealed that whole genome duplications have driven GolS gene evolution and diversification in Rosaceae speciation. The strong purifying selection identified in duplicated GolS genes suggests that differential gene expression might define gene function better than protein structure. Interestingly, MdGolS2 was differentially expressed during bud dormancy, concomitantly with the highest galactinol and raffinose levels. One of the intrinsic adaptive features of bud dormancy is limited availability of free water; therefore, we generated transgenic Arabidopsis plants expressing MdGolS2. They showed higher galactinol and raffinose contents and increased tolerance to water deficit. Our results suggest that MdGolS2 is the major GolS responsible for RFO accumulation during apple dormancy, and these carbohydrates help to protect dormant buds against limited water supply.

sn-Glycerol-3-phosphate 1-O-acyltransferase (GPAT) is an important enzyme that catalyzes the transfer of an acyl group from acyl-CoA or acyl-ACP to the sn-1 or sn-2 position of sn-glycerol-3-phosphate (G3P) to generate lysophosphatidic acids (LPAs). The functional studies of GPAT in plants demonstrated its importance in controlling storage and membrane lipid. Identifying genes encoding GPAT in a variety of plant species is crucial to understand their involvement in different metabolic pathways and physiological functions. Here, we performed genome-wide and evolutionary analyses of GPATs in plants. GPAT genes were identified in all algae and plants studied. The phylogenetic analysis showed that these genes group into three main clades. While clades I (GPAT9) and II (soluble GPAT) include GPATs from algae and plants, clade III (GPAT1-8) includes GPATs specific from plants that are involved in the biosynthesis of cutin or suberin. Gene organization and the expression pattern of GPATs in plants corroborate with clade formation in the phylogeny, suggesting that the evolutionary patterns is reflected in their functionality. Overall, our results provide important insights into the evolution of the plant GPATs and allowed us to explore the evolutionary mechanism underlying the functional diversification among these genes.

H 2 O 2 , which is continually produced by aerobic metabolism, is a cytotoxic molecule when in high levels. However, low levels can act as a signaling molecule able to regulate the expression of stress responses, senescence, programmed cell death, plant growth, and development. Ascorbate peroxidase (APX) enzyme plays an essential role in the control of intracellular H 2 O 2 levels. Here, the function of a gene encoding a peroxisomal APX (OsAPX4) from rice (Oryza sativa L.) was studied. OsAPX4 gene expression can be detected in roots and panicles, but the highest expression level occurs in leaves. Silencing of OsAPX4 and OsAPX3 expression in RNAiOsAPX4 did not affect the growth of plants under growth chamber conditions, but aging transgenic plants interestingly displayed an early senescence phenotype. Leaf fragments from silenced plants were also more sensitive to induced senescence conditions. RNAiOsAPX4 plants did not present detectable changes in intracellular H 2 O 2 levels, but biochemical analyses showed that transgenic plants displayed some decreased APX activity in the chloroplastic fraction. Also, the peroxisomal enzyme glycolate oxidase exhibited lower activity, whereas catalase activity was similar to non-transformed rice. The results imply that OsAPX4 gene has an important role in leaf senescence pathway mediated by ROS signaling., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Fumarate and succinate are known to be present in prebiotic systems essential for the origin of life. The fumarate and succinate interconversion reactions have been conserved throughout evolution and are found in all living organisms. The fumarate and succinate interconversion is catalyzed by the enzymes succinate dehydrogenase (SDH) and fumarate reductase (FRD). In this work we show that SDH and FRD are part of a group of enzymes that we propose to designate "fumarate reductase superfamily". Our results demonstrate that these enzymes emerged from a common ancestor and were essential in the development of metabolic pathways involved in energy transduction., (Copyright © 2017 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Despite the wide appreciation of seedless grapes, little is known about the molecular mechanisms that drive the stenospermocarpic seedless-type phenotype in grapevine. In order to address the molecular mechanisms that control seedlessness in grapevine, our study aimed to characterize VviAGL11, a class D MADS-box transcription factor gene that has been proposed as the major candidate gene involved in Vitis vinifera seed morphogenesis. VviAGL11 allelic variations in seeded and seedless grapevine cultivars were determined, and its correlations with allele-specific steady-state mRNA levels were investigated. VviAGL11 relative expression was significantly higher in seeds at 2, 4, and 6 weeks after fruit set, whereas in the seedless grape its transcript levels were extremely low in all stages analyzed. In situ hybridization revealed transcript accumulation specifically in the dual endotesta layer of the seeds, which is responsible for elongation and an increase of cell number, a necessary step to determine the lignification and the final seed size. No hybridization signals were visible in the seedless grapevine tissues, and a morphoanatomical analysis showed an apparent loss of identity of the endotesta layer of the seed traces. Ectopic expression of VviAGL11 in the Arabidopsis SEEDSTICK mutant background restored the wild-type phenotype and confirmed the direct role of VviAGL11 in seed morphogenesis, suggesting that depletion of its expression is responsible for the erroneous development of a highly essential seed layer, therefore culminating in the typical apirenic phenotype., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Heavy metals are natural non-biodegradable constituents of the Earth's crust that accumulate and persist indefinitely in the ecosystem as a result of human activities. Since the industrial revolution, the concentration of cadmium, arsenic, lead, mercury and zinc, amongst others, have increasingly contaminated soil and water resources, leading to significant yield losses in plants. These issues have become an important concern of scientific interest. Understanding the molecular and physiological responses of plants to heavy metal stress is critical in order to maximize their productivity. Recent research has extended our view of how plant hormones can regulate and integrate growth responses to various environmental cues in order to sustain life. In the present review we discuss current knowledge about the role of the plant growth hormones abscisic acid, auxin, brassinosteroid and ethylene in signaling pathways, defense mechanisms and alleviation of heavy metal toxicity.

Drought limits wheat production in the Brazilian Cerrado biome. In order to search for candidate genes associated to the response to water deficit, we analyzed the gene expression profiles, under severe drought stress, in roots and leaves of the cultivar MGS1 Aliança, a well-adapted cultivar to the Cerrado. A set of 4,422 candidate genes was found in roots and leaves. The number of down-regulated transcripts in roots was higher than the up-regulated transcripts, while the opposite occurred in leaves. The number of common transcripts between the two tissues was 1,249, while 2,124 were specific to roots and 1,049 specific to leaves. Quantitative RT-PCR analysis revealed a 0.78 correlation with the expression data. The candidate genes were distributed across all chromosomes and component genomes, but a greater number was mapped on the B genome, particularly on chromosomes 3B, 5B and 2B. When considering both tissues, 116 different pathways were induced. One common pathway, among the top three activated pathways in both tissues, was starch and sucrose metabolism. These results pave the way for future marker development and selection of important genes and are useful for understanding the metabolic pathways involved in wheat drought response.

Since the first diacylglycerol acyltransferase (DGAT) gene was characterized in plants, a number of studies have focused on understanding the role of DGAT activity in plant triacylglycerol (TAG) biosynthesis. DGAT enzyme is essential in controlling TAGs synthesis and is encoded by different genes. DGAT1 and DGAT2 are the two major types of DGATs and have been well characterized in many plants. On the other hand, the DGAT3 and WS/DGAT have received less attention. In this study, we present the first general view of the presence of putative DGAT3 and WS/DGAT in several plant species and report on the diversity and evolution of these genes and its relationships with the two main DGAT genes (DGAT1 and DGAT2). According to our analyses DGAT1, DGAT2, DGAT3 and WS/DGAT are very divergent genes and may have distinct origin in plants. They also present divergent expression patterns in different organs and tissues. The maintenance of several types of genes encoding DGAT enzymes in plants demonstrates the importance of DGAT activity for TAG biosynthesis. Evolutionary history studies of DGATs coupled with their expression patterns help us to decipher their functional role in plants, helping to drive future biotechnological studies.

Phytocystatins are well-known inhibitors of C1A cysteine proteinases. However, previous research has revealed legumain (C13) protease inhibition via a carboxy-extended phytocystatin. Among the 12 phytocystatins genes in rice, OcXII is the only gene possessing this carboxy-terminal extension. The specific legumain inhibition activity was confirmed, in our work, using a recombinant OcXII harboring only the carboxy-terminal domain and this part did not exhibit any effect on papain-like activities. Meanwhile, rice plants silenced at the whole OcXII gene presented higher legumain and papain-like proteolytic activities, resulting in a faster initial seedling growth. However, when germinated under stressful alkaline conditions, OcXII-silenced plants exhibited impaired root formation and delayed shoot growth. Interestingly, the activity of OcXII promoter gene was detected in the rice seed scutellum region, and decreases with seedling growth. Seeds from these plants also exhibited slower growth at germination under ABA or alkaline conditions, while maintaining very high levels of OcXII transcriptional activation. This likely reinforces the proteolytic control necessary for seed germination and growth. In addition, increased legumain activity was detected in OcXII RNAi plants subjected to a fungal elicitor. Overall, the results of this study highlight the association of OcXII with not only plant development processes, but also with stress response pathways. The results of this study reinforce the bifunctional ability of carboxy-extended phytocystatins in regulating legumain proteases via its carboxy-extended domain and papain-like proteases by its amino-terminal domain.

The maintenance of H2O2 homeostasis and signaling mechanisms in plant subcellular compartments is greatly dependent on cytosolic ascorbate peroxidases (APX1 and APX2) and peroxisomal catalase (CAT) activities. APX1/2 knockdown plants were utilized in this study to clarify the role of increased cytosolic H2O2 levels as a signal to trigger the antioxidant defense system against oxidative stress generated in peroxisomes after 3-aminotriazole-inhibited catalase (CAT). Before supplying 3-AT, silenced APX1/2 plants showed marked changes in their oxidative and antioxidant profiles in comparison to NT plants. After supplying 3-AT, APX1/2 plants triggered up-expression of genes belonging to APX (OsAPX7 and OsAPX8) and GPX families (OsGPX1, OsGPX2, OsGPX3 and OsGPX5), but to a lower extent than in NT plants. In addition, APX1/2 exhibited lower glycolate oxidase (GO) activity, higher CO2 assimilation, higher cellular integrity and higher oxidation of GSH, whereas the H2O2 and lipid peroxidation levels remained unchanged. This evidence indicates that redox pre-acclimation displayed by silenced rice contributed to coping with oxidative stress generated by 3-AT. We suggest that APX1/2 plants were able to trigger alternative oxidative and antioxidant mechanisms involving signaling by H2O2, allowing these plants to display effective physiological responses for protection against oxidative damage generated by 3-AT, compared to non-transformed plants., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

The physiological role of plant mitochondrial glutathione peroxidases is scarcely known. This study attempted to elucidate the role of a rice mitochondrial isoform (GPX1) in photosynthesis under normal growth and salinity conditions. GPX1 knockdown rice lines (GPX1s) were tested in absence and presence of 100 mM NaCl for 6 d. Growth reduction of GPX1s line under non-stressful conditions, compared with non-transformed (NT) plants occurred in parallel to increased H2 O2 and decreased GSH contents. These changes occurred concurrently with photosynthesis impairment, particularly in Calvin cycle's reactions, since photochemical efficiency did not change. Thus, GPX1 silencing and downstream molecular/metabolic changes modulated photosynthesis differentially. In contrast, salinity induced reduction in both phases of photosynthesis, which were more impaired in silenced plants. These changes were associated with root morphology alterations but not shoot growth. Both studied lines displayed increased GPX activity but H2 O2 content did not change in response to salinity. Transformed plants exhibited lower photorespiration, water use efficiency and root growth, indicating that GPX1 could be important to salt tolerance. Growth reduction of GPX1s line might be related to photosynthesis impairment, which in turn could have involved a cross talk mechanism between mitochondria and chloroplast originated from redox changes due to GPX1 deficiency., (© 2016 Institute of Botany, Chinese Academy of Sciences.)

The E2 promoter binding factor (E2F) proteins are present in almost all eukaryotic organisms and are essential to control several processes, such as the cell cycle progression, cell division, DNA replication, and apoptosis. The E2F family comprises two different types of proteins: the typical E2Fs and atypical E2Fs, which differ structurally and have specific functions. The E2F gene family was described for the first time in plants in 1999, and since then several studies have focused on the functional aspects, but the evolutionary history of this gene family is still unknown. Here, we investigated the evolutionary history of the E2F gene family in plants. Our findings suggest that E2F proteins arose early after the emergence of the eukaryotic species, while DEL proteins appear to have arisen before the metazoan and plants origin probably through a partial duplication of an ancient E2F protein. Our data also suggest that E2Fs activators and repressors appeared twice during evolution, once in the metazoan lineage and again in the embryophyte lineage., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Rice is the most tolerant staple crop to aluminium (Al) toxicity, which is a limiting stress for grain production worldwide. This Al tolerance is the result of combined mechanisms that are triggered in part by the transcription factor ASR5. ASRs are dual target proteins that participate as chaperones in the cytoplasm and as transcription factors in the nucleus. Moreover, these proteins respond to biotic and abiotic stresses, including salt, drought and Al. Rice plants with silenced ASR genes are highly sensitive to Al. ASR5, a well-characterized protein, binds to specific cis elements in Al responsive genes and regulates their expression. Because the Al sensitive phenotype found in silenced rice plants could be due to the mutual silencing of ASR1 and ASR5, we investigated the effect of the specific silencing of ASR5. Plants with artificial microRNA silencing of ASR5 present a non-transformed phenotype in response to Al because of the induction of ASR1. ASR1 has the same subcellular localization as ASR5, binds to ASR5 cis-regulatory elements, regulates ASR5 regulated genes in a non-preferential manner and might replace ASR5 under certain conditions. Our results indicate that ASR1 and ASR5 act in concert and complementarily to regulate gene expression in response to Al., (© 2015 John Wiley & Sons Ltd.)

Lysophosphatidic acid acyltransferases (LPAATs) perform an essential cellular function by controlling the production of phosphatidic acid (PA), a key intermediate in the synthesis of membrane, signaling and storage lipids. Although LPAATs have been extensively explored by functional and biotechnological studies, little is known about their molecular evolution and diversification. We performed a genome-wide analysis using data from several plants and animals, as well as other eukaryotic and prokaryotic species, to identify LPAAT genes and analyze their evolutionary history. We used phylogenetic and molecular evolution analysis to test the hypothesis of distinct origins for these genes. The reconstructed phylogeny supported the ancient origin of some isoforms (plant LPAAT1 and LPAATB; animal AGPAAT1/2), while others emerged more recently (plant LPAAT2/3/4/5; AGPAAT3/4/5/8). Additionally, the hypothesis of endosymbiotic origin of the plastidic isoform LPAAT1 was confirmed. LPAAT genes from plants and animals mainly experienced strong purifying selection pressures with limited functional divergence after the species-specific duplications. Gene expression analyses of LPAAT isoforms in model plants demonstrated distinct LPAAT expression patterns in these organisms. The results showed that distinct origins followed by diversification of the LPAAT genes shaped the evolution of TAG biosynthesis. The expression pattern of individual genes may be responsible for adaptation into multiple ecological niches., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The Lesion Simulating Disease (LSD) genes encode a family of zinc finger proteins that play a role in programmed cell death (PCD) and other biological processes, such as plant growth and photosynthesis. In the present study, we report the reconstruction of the evolutionary history of the LSD gene family in Viridiplantae. Phylogenetic analysis revealed that the monocot and eudicot genes were distributed along the phylogeny, indicating that the expansion of the family occurred prior to the diversification between these clades. Sequences encoding proteins that present one, two, or three LSD domains formed separate groups. The secondary structure of these different LSD proteins presented a similar composition, with the β-sheets being their main component. The evolution by gene duplication was identified only to the genes that contain three LSD domains, which generated proteins with equal structure. Moreover, genes encoding proteins with one or two LSD domains evolved as single-copy genes and did not result from loss or gain in LSD domains. These results were corroborated by synteny analysis among regions containing paralogous/orthologous genes in Glycine max and Populus trichocarpa. The Ka/Ks ratio between paralogous/orthologous genes revealed that a subfunctionalization process possibly could be occurring with the LSD genes, explaining the involvement of LSD members in different biological processes, in addition to the negative regulation of PCD. This study presents important novelty in the evolutionary history of the LSD family and provides a basis for future research on individual LSD genes and their involvement in important pathway networks in plants.