Fusion of gametes possessing meiotically reduced (haploid) chromosome complements is the main pathway of propagation among eukaryotes. However, duckweeds, the smallest angiosperms, propagate mainly vegetatively, and meio...Fusion of gametes possessing meiotically reduced (haploid) chromosome complements is the main pathway of propagation among eukaryotes. However, duckweeds, the smallest angiosperms, propagate mainly vegetatively, and meiosis has not yet been documented in detail for this plant family. The more surprising was the recent evidence of rather frequent interspecific hybrids and triploid clonal accessions, which became obvious by genome size measurements, genomic in situ hybridization (GISH), and combined plastid and nuclear DNA markers. These observations indicated sexual propagation involving reduced as well as unreduced male and female gametes in Lemna minor and L. turionifera, leading to allodiploid and allotriploid hybrids (MT, MMT, MTT) and autotriploid L. minor (MMM) accessions. Here, we i) documented the meiotic stages of Lemna species for the first time photographically; ii) provided evidence of unreduced male gametes through fluorescent in situ hybridization (FISH) with single locus probes; iii) determined their abundance in different individuals and iv) discussed possible reasons for unreduced male gamete formation. These findings open new insights into the modes of sexual reproduction and evolution of duckweeds, which may be useful for future breeding efforts in this emerging crop.
Haghan M, Chau TN, Alajoleen R
… +13 more, Chen CY, Dip SA, Gurung V, Hsu CW, Kittivorawong C, Kundu S, Li X, Luo J, Salehin M, Simenc M, Yun J, Bargmann B, Li S
Single-cell genomics is rapidly reshaping plant biology, yet broader adoption is limited by plant-specific technical constraints, fragmented tools, and inconsistent analytical practices. Here we report outcomes from the...Single-cell genomics is rapidly reshaping plant biology, yet broader adoption is limited by plant-specific technical constraints, fragmented tools, and inconsistent analytical practices. Here we report outcomes from the 2025 Summer Workshop for Plant Single-Cell Analysis, which convened researchers to define community needs and design shared solutions. Participants identified five priority challenge areas: (1) improving data quality through imputation, simulation, and deep generative modeling; (2) developing automated, phylogenetically aware cell-type annotation frameworks; (3) reconstructing developmental trajectories and gene regulatory networks from single-cell and single-nucleus profiles; (4) creating visualization approaches that embed transcriptional states into anatomically grounded plant organ contexts; and (5) using Artificial Intelligence (AI) agents and foundation models to orchestrate end-to-end single-cell workflows. In response, we established PlantSCHub, a community-curated web portal that aggregates protocols, datasets, and tutorials to support reproducible plant single-cell analysis. We outline conceptual roadmaps for cross-species integration, multimodal trajectory and Gene Regulatory Networks (GRNs) inference, spatially anchored visualization, and AI scientist agents that dynamically coordinate analytical tools and literature. We discuss both scRNA-seq and scATAC-seq that are important for regulatory inference and cross-species analysis. Together, these efforts aim to transform isolated plant single-cell studies into an interoperable, evolving ecosystem that accelerates discovery and crop improvement.
As an important economic oil crop, the quality of Camellia drupifera (C. drupifera) is closely linked to its triterpenoid content, notably squalene, which exhibits significant potential in pharmaceutical applications. To...As an important economic oil crop, the quality of Camellia drupifera (C. drupifera) is closely linked to its triterpenoid content, notably squalene, which exhibits significant potential in pharmaceutical applications. To elucidate the regulatory mechanism of squalene biosynthesis in C. drupifera and to support its quality improvement and efficient production, this study constructed a MeJA-induced cDNA yeast library of C. drupifera. Using the promoter of squalene synthase (CdSQS) as bait, two zinc finger-homeodomain transcription factors, ZF-HD1 and ZF-HD9, were identified. Under MeJA treatment, the expression of ZF-HD1/9, MYC2, and SQS was upregulated, promoting squalene synthesis. Dual-luciferase reporter (DLR) and electrophoretic mobility shift assays (EMSA) confirmed that ZF-HD1/9 directly bind to and activate the CdSQS promoter. Using nanoparticle-mediated dsRNAi transient silencing, we demonstrated that knockdown of ZF-HD1/9 downregulates key structural genes(SQS, SQE, bAS, CYP716A17, and UGT73B2) and significantly reduces the levels of squalene, 2,3-oxidosqualene, and teasaponins. Furthermore, by establishing an Agrobacterium rhizogenes-mediated hairy root transformation system in C. drupifera, we conducted functional analyses of ZF-HD1/9, confirming their role as positive regulators of squalene synthesis. This study is the first to identify ZF-HD1/9 as novel transcription factors involved in the complex regulatory network of squalene biosynthesis in C. drupifera, expanding the functional scope of this transcription factor family in plants. The hairy root induction system developed herein also provides a foundational platform for gene functional studies and high-quality germplasm development in C. drupifera.
Fusarium head blight (FHB) threatens wheat production worldwide, and major-effect resistance sources remain limited. Elymus repens (2n = 6x = 42, StStStStHH) is a perennial Triticeae species with potential for wheat impr...Fusarium head blight (FHB) threatens wheat production worldwide, and major-effect resistance sources remain limited. Elymus repens (2n = 6x = 42, StStStStHH) is a perennial Triticeae species with potential for wheat improvement. Here, we characterized the genomic constitution of the wheat-E. repens partial amphidiploid P1142-1-2, which shows strong FHB resistance. P1142-1-2 carries the complete wheat genome plus seven pairs of E. repens-derived chromosomes or chromosome fragments containing St chromatin from 1St-7St. From crosses between P1142-1-2 and common wheat cv. Chuannong16, we identified two resistant derivative lines carrying either a 1StL isochromosome or a 1StL telosome. Genomic sequencing enabled the development of 17 1StL-specific PCR markers for precise detection of alien chromatin. Genetic analysis demonstrated that 1StL confers strong resistance to FHB in wheat. Diagnostic assays did not detect previously reported alien FHB resistance genes, indicating that 1StL carries a novel resistance locus, designated Fhb.Er-1StL. These materials and markers broaden the genetic basis of FHB resistance in wheat and provide useful resources for wheat improvement.
Ethylene has been recognized as a plant hormone that impacts growth, development, and stress responses for over a century. Hallmarks of hormone receptors are that they have saturable and specific ligand-binding activity...Ethylene has been recognized as a plant hormone that impacts growth, development, and stress responses for over a century. Hallmarks of hormone receptors are that they have saturable and specific ligand-binding activity and the binding of the ligand regulates cellular functions. Using these characteristics, several reports over the past decade have discovered and characterized plant-like ethylene receptors in bacteria. These studies show ethylene signals via these receptors to have a profound impact on the metabolism and physiology of the bacteria. Several studies also raise the possibility that some fungi contain functional plant-like ethylene receptors. These receptors in non-plant species have ethylene-binding domains similar to plants, but diverge in other domains and downstream signaling pathways. This review summarizes recent advances characterizing plant-like ethylene receptors and signal transduction in microbes and discusses the possibility that there are also noncanonical ethylene receptors that are evolutionarily distinct from those found in plants.
The functionality of the UV-B photoreceptor UVR8 (UV RESISTANCE LOCUS8) has widely been demonstrated in angiosperms under controlled conditions, but studies in nature or using bryophytes are scarce. To fill this gap, we...The functionality of the UV-B photoreceptor UVR8 (UV RESISTANCE LOCUS8) has widely been demonstrated in angiosperms under controlled conditions, but studies in nature or using bryophytes are scarce. To fill this gap, we compared the UV responses of two genotypes of the model liverwort Marchantia polymorpha, the wild type (WT) and a mutant lacking a functional UVR8, to provide for the first time an evolutionary perspective of UVR8 performance in both controlled and field conditions. In the laboratory, UVR8 was essential to induce gene expression (CHS1, FNSI, HY5, SIG5, and ELIP2) and metabolite accumulation (apigenin and luteolin derivatives, particularly apigenin 7-O-glucuronide) in WT plants, contributing to conserve photosynthetic performance. In the mutant, gene and metabolite responses were much weaker, occurred only under high UV-B in a UVR8-independent response, and did not confer photoprotection to the photosynthetic apparatus. In a field seasonal cycle, differences between UV treatments and genotypes were attenuated and the UVR8 functionality was less clear, probably due to the low UV-B levels required to trigger plant responses, and the interaction of other abiotic factors. Given the similarity of UVR8 functionality in M. polymorpha and Arabidopsis thaliana under both controlled and field conditions, UVR8 has been conserved across plant evolution.
Understanding the subcellular localization of RNA and proteins is critical to dissecting gene regulation in eukaryotic organisms. However, this task is elusive as existing fractionation methods often rely on protoplast i...Understanding the subcellular localization of RNA and proteins is critical to dissecting gene regulation in eukaryotic organisms. However, this task is elusive as existing fractionation methods often rely on protoplast isolation or commercial kits, that are labor-intensive, costly, and can introduce stress-induced transcriptomic and proteomic changes. Here, we present a simple, rapid, and cost-effective protocol for the fractionation of nuclear and cytoplasmic components directly from diverse plant tissues, that does not require protoplastization. This Subcellular-fractionation protocol in 3 steps (a.k.a. "bueno, bonito y barato" -spanish for "good, nice and cheap"-), referred to as "SuB3", yields nuclear- and cytoplasmic- enriched subcellular fractions suitable for downstream applications such as RT-PCR, RNA/cDNA sequencing, and Western blotting. The procedure is based on sequential detergent-assisted extraction and centrifugation and enables the simultaneous isolation of RNA and protein from the same biological material. Due to its simplicity, speed, and broad compatibility, this protocol is a valuable tool for plant molecular biology laboratories investigating subcellular dynamics of gene expression.
Even though the canonical role of the transcription factor LEAFY (LFY) is in establishing the identity of angiosperm floral meristems, homologs of this gene are found across all land plants, predating the evolution of fl...Even though the canonical role of the transcription factor LEAFY (LFY) is in establishing the identity of angiosperm floral meristems, homologs of this gene are found across all land plants, predating the evolution of flowers. It has been hypothesized that the ancestral role of LFY was more broadly meristematic, regulating cell division, and that it acquired its reproductive function more recently. Here, we review mounting evidence from LFY orthologs in non-flowering plants that support the hypothesis that the reproductive role of LFY arose earlier in the land plant lineage than previously thought, in the gametophyte of haploid-dominant plants, and that it was co-opted to the sporophyte as land plants evolved towards diploid-dominant life cycles. Additionally, we examine how recent insights into LFY's mechanism of action inform the reconstruction of its functional evolution, including its recognition as a member of the elite class of plant pioneer transcription factors.
Published studies have reported species-variance between profiles of Calvin-Benson cycle (CBC) intermediates, not only between C4 species and C3 species, but also within C3 species (Arrivault et al., 2019, Borghi et al....Published studies have reported species-variance between profiles of Calvin-Benson cycle (CBC) intermediates, not only between C4 species and C3 species, but also within C3 species (Arrivault et al., 2019, Borghi et al. 2019). It was proposed that this variance reflects lineage-dependent changes in the balance between different reactions, or poising, of the CBC. These earlier studies investigated phylogenetically-unrelated C3 species. In the current study, CBC intermediates were profiled in five closely-related species from Solanum sect. lycopersicon subsect. Lycopersicum. Levels of individual CBC intermediates showed many significant differences. In a principal component analysis, whilst three species (Solanum lycopersicum, Solanum cheesmaniae, Solanum neorickii) overlapped, Solanum pimpinellifolium and especially Solanum pennellii grouped separately, and were at opposing ends of the distribution. When combined with published data, whilst the separation between Solanum species was retained, they formed a group that was separated from five other C3 species, as well as two C4 species. It is discussed that the observed variation in CBC metabolites profiles within Solanum, together with their separation from other C₃ species, supports the idea that CBC evolution is shaped by phylogenetic relatedness and, by implication, lineage-specific adaptation.
Plants constantly experience partial hypoxia due to varying external oxygen availability and the existence of hypoxic niches, such as the shoot apical meristem, phloem or root nodules. Waterlogging experiments indicate t...Plants constantly experience partial hypoxia due to varying external oxygen availability and the existence of hypoxic niches, such as the shoot apical meristem, phloem or root nodules. Waterlogging experiments indicate that hypoxic stress at the root does not only lead to a local metabolic response, such as the accumulation of hypoxia-related metabolites, but also causes metabolic alterations in the normoxic shoot. Moreover, hypoxia-related metabolites are exported from the hypoxic root towards the normoxic shoot, where they can be recycled. Maintaining import of glycolytic substrates from the normoxic shoot into the hypoxic root is suggested to play a crucial role in managing hypoxic stress in waterlogged roots. These findings indicate that locally confined hypoxic stress induces systemic responses. The apparent metabolic interplay between hypoxic and normoxic tissue can facilitate the plant to endure differing oxygen availabilities between tissues and organs without active oxygen circulation. Here, we define this mechanism as 'metabolic snorkeling'. Beyond waterlogging, metabolic snorkeling might also occur between hypoxic niches and the adjacent normoxic tissue. In this review, the role of metabolic snorkeling in waterlogging-endurance and its applicability to hypoxic niches is described and discussed.
During plant development and in response to stress conditions, autophagy contributes to the intracellular degradation of cellular components and subsequent nutrient recycling. As this process is highly connected to the n...During plant development and in response to stress conditions, autophagy contributes to the intracellular degradation of cellular components and subsequent nutrient recycling. As this process is highly connected to the nutrient status of the plant, autophagy also contributes to the mobilisation of sulfur from source to sink tissues as well as the maintenance of primary sulfate assimilation. In turn, sulfur signals regulate autophagy, with sulfide (an intermediate of primary sulfate assimilation) exerting a repressive effect and sulfur deficiency having a stimulatory effect. In addition to a sulfur deficiency response in the plant resulting from low external sulfate availability, stresses such as metal exposure also perturb sulfur metabolism and can induce a 'functional sulfur deficiency' response through a surge in the production of thiol-rich metal chelators. As autophagy is increasingly linked to metal stress responses, this review proposes potential pathways through which metal-induced autophagy is linked to perturbations in sulfur metabolism, focusing on redox alterations and sucrose non-fermenting 1 (SNF1)-related kinase (SnRK)/target of rapamycin (TOR)-mediated nutrient signalling. Lastly, the connection between autophagy and sulfur status to plant stress tolerance is also discussed in terms of potential valorisation strategies to maximise plant growth on metal-contaminated soils.
Hard red spring wheat (HRSW) is an important market class of wheat in North America. We genotyped 1,013 HRSW lines representing a century of breeding using SNP array and targeted KASP genotyping to assess changes in gene...Hard red spring wheat (HRSW) is an important market class of wheat in North America. We genotyped 1,013 HRSW lines representing a century of breeding using SNP array and targeted KASP genotyping to assess changes in genetic diversity and Rht genes. Our results suggest that early breeding efforts broadened an initially narrow gene pool derived from few founders and subsequently maintained genetic diversity. Analysis of Rht genes revealed that Rht-D1b was the predominant semi-dwarf allele following the introduction of gibberellic acid (GA)-insensitive alleles (Rht-B1b and Rht-D1b) during the Green Revolution of the 1960s. However, wheat scab epidemics of 1990s coincided with most breeding programs shifting to Rht-B1b. Adoption of Rht-B1b and Rht-D1b remains low in some regions of the Great Plains, where breeding programs have utilized alternative dwarfing GA-sensitive alleles at Rht24 and Rht25 loci. We analyzed the effect of Rht genes on plant height in the HRSW growing region and found that the effect of Rht-B1b and Rht-D1b diminishes in drier environments. Combining dwarfing alleles at Rht24 and Rht25 was associated with a significant reduction in plant height even in the absence of Green Revolution genes. This study offers insights to guide future breeding strategies for HRSW in North America.
Wheat is a staple crop vital to global food security, yet its productivity is increasingly threatened by drought stress. The extent to which contrasting wheat genotypes exhibit stress memory, and how this influences morp...Wheat is a staple crop vital to global food security, yet its productivity is increasingly threatened by drought stress. The extent to which contrasting wheat genotypes exhibit stress memory, and how this influences morpho-physiological adjustments during repeated drought exposures, remains unclear. Through integrated analyses of morpho-physiological responses, gene expression patterns, and key regulatory pathways, we examined intergenerational and transgenerational drought memory in drought-tolerant "Intro" and drought-sensitive "Sonalika". Under repeated stress, seedlings of both genotypes with drought history showed enhanced resilience. Transcriptome profiling identified drought memory genes exhibiting consistent differential expression between initial and repeated stress exposures in seeds and leaves. Gene ontology analysis revealed enrichment of "Intro" seed memory genes in transcripts linked to H3K36me3, while its leaf memory genes were enriched in pathways related to ABA signaling, osmoprotectant accumulation, protein synthesis, and antioxidant activity. By contrast, "Sonalika" showed enrichment of seed transcripts associated with H3K9ac and H3K14ac, and leaf memory genes linked to transcriptional activity, proteostasis, and immune-related pathways. The functional drought memory exhibited by "Sonalika", despite the absence of transcripts associated with canonical methylation-based chromatin marks, suggests alternative regulatory strategies. Together, these results indicate that wheat seedlings exhibit genotype-specific drought stress memory involving distinct transcriptional programs and putative epigenetic regulators. Although histone modifications were not directly assessed, the enrichment of histone-related genes suggests their potential involvement in mediating these responses. Direct chromatin-level analyses will be required to validate these mechanisms and support future efforts to enhance drought resilience through targeted breeding.
Tagliabue AG, Friel J, Chiozzotto R
… +11 more, Baccichet I, Da Silva Linge C, Calastri E, Biffi G, Zaracho N, Gattolin S, Micali S, Eduardo I, Bassi D, Rossini L, Cirilli M
Fruit maturity date is a key developmental trait in fruit trees, as it determines the timing at which fruits enter the ripening phase, thereby shaping harvest calendar and fruit quality. In peach, ripening time varies ov...Fruit maturity date is a key developmental trait in fruit trees, as it determines the timing at which fruits enter the ripening phase, thereby shaping harvest calendar and fruit quality. In peach, ripening time varies over several months, yet the genetic and regulatory basis underlying this extensive phenological diversity has remained largely unresolved. Here, we dissect the long-standing chromosome 4 maturity date locus (qMD4.1) and show that a multi-allelic series of structural variants in the upstream regulatory region of the transcription factor NAC1 explains most of the observed variation in ripening time in cultivated peach. Across multiple independent populations and diverse genetic backgrounds, these variants (collectively termed Md) stratify maturity date from ultra-early to late cultivars with largely additive and dosage-dependent effects. Using recombinant-based genetic dissection, long-read sequencing, transcriptomic analyses and epigenomic data integration, we show that Md alleles are consistently associated with allele-dependent shifts in the temporal dynamics of NAC1 transcript accumulation, and that the affected region overlaps with chromatin features characteristic of regulatory activity. By resolving the genetic architecture of a major phenological locus, our results support a central role for cis-regulatory structural variation in modulating developmental timing in peach and provide biologically interpretable markers for predicting maturity date. More broadly, this work illustrates how non-coding structural variation can contribute to variation in fruit developmental trajectories, with implications for breeding strategies aimed at modulating harvest windows in peach and other stone fruit species.
Most angiosperm roots develop two apoplastic barriers: the endodermis and the exodermis. Although the exodermis is widespread among angiosperms, its functional role remains comparatively poorly understood. Here, we studi...Most angiosperm roots develop two apoplastic barriers: the endodermis and the exodermis. Although the exodermis is widespread among angiosperms, its functional role remains comparatively poorly understood. Here, we studied root apoplastic barrier formation in potato (Solanum tuberosum). Using histochemical staining, we mapped the spatiotemporal deposition of lignin/poly(phenolic) material and suberin in the endodermis and the exodermis. The exodermis develops an outer polar lignin/poly(phenolic) cap and rapidly establishes a continuous suberized layer close to the root tip, whereas endodermal cells form Casparian strips first and suberize later and discontinuously. The early and continuous suberization of the exodermis suggests a potential role in regulating the radial movement of mineral nutrients. To explore this possibility, we analyzed plants downregulating the suberin biosynthetic gene CYP86A33, which showed a 61% reduction in root suberin content. Suberin-deficient plants displayed altered element concentration and reduced growth. In addition, micro-PIXE analyses provided spatial information of elemental distribution within the roots and highlighted anatomical compartments defined by apoplastic barriers. Together, these results support the contribution of exodermal suberin to ion homeostasis and plant growth, and provide new insights into the role of root barriers in mineral nutrient regulation in a major crop species.
Cold acclimation of leaf respiration often increases respiration rates at a set temperature after temperature declines. Whether a similar response occurs in stems and contributes to frost tolerance remains unknown. We ev...Cold acclimation of leaf respiration often increases respiration rates at a set temperature after temperature declines. Whether a similar response occurs in stems and contributes to frost tolerance remains unknown. We evaluated stem CO2 efflux (Es) and cold acclimation in dormant Quercus robur trees in a climate chamber with day/night air temperatures weekly changing from 20/10oC to 5/-10oC and back to 20/10oC over eleven weeks. Cold hardening was evidenced by lower cell damage at subfreezing temperatures compared to trees kept in a greenhouse at 21.5/18.5oC. A hysteresis between Es and stem temperature (Ts) evidenced a time lag between Es and metabolic activity during chilly and subfreezing weeks. Stem CO2 efflux was strongly related to Ts down to -10oC. At a standardized Ts of 5oC, Es_5 decreased the day after weekly temperatures declined, suggesting a rapid down-regulation of respiratory metabolism to enhance cryoprotection. Accordingly, soluble sugar concentrations increased with decreasing Ts. However, upon subsequent warming from subfreezing temperatures, stems continued dehydrating and did not recover previous respiration rates, suggesting freeze/thaw embolism and cell damage reduced stem apparent "respiratory capacity". Moderate frost tolerance in cold-hardened plants appears to depend on stem dehydration and SS accumulation, not increased stem metabolic activity.
Drought tolerance is a critical component contributing to the overall drought resistance of grasses. Still, our understanding of tolerance mechanisms in this group remains limited. We sought to investigate the mechanisms...Drought tolerance is a critical component contributing to the overall drought resistance of grasses. Still, our understanding of tolerance mechanisms in this group remains limited. We sought to investigate the mechanisms contributing to drought tolerance in zoysiagrasses, an economically important group of turfgrass cultivated worldwide. Experiments were performed using four cultivars with contrasting tolerances. Declines in the integrity of leaf cells and photochemistry preceded the initiation of leaf embolism in drought-susceptible, but not in drought-tolerant cultivars. Lobo, the most drought-tolerant cultivar, experienced cellular and photochemistry damage only after extensive embolism. Empire, the most drought-susceptible cultivar, experienced considerable damage to cells and the photosynthetic apparatus before, or soon after, the initiation of embolism. The occurrence of cell damage independently of xylem embolism in some of these grasses contrasts with the typical pattern observed in woody species. Leaves of all zoysiagrasses were equally highly resistant to embolism, with large estimated stomatal safety margins. Although embolism resistance did not explain differences in tolerance among these cultivars, we cannot rule out the importance of such highly resistant xylem to their overall drought tolerance. These results describe critical tolerance mechanisms in grasses and have important implications for breeding programs improving drought resistance of cultivated grasses.
Legumes sanction root nodules containing rhizobial strains with low nitrogen fixation rates (less effectively fixing). Pea (Pisum sativum) nodules contain both undifferentiated bacteria and terminally differentiated nitr...Legumes sanction root nodules containing rhizobial strains with low nitrogen fixation rates (less effectively fixing). Pea (Pisum sativum) nodules contain both undifferentiated bacteria and terminally differentiated nitrogen-fixing bacteroids. It is critical to understand how sanctions act on both bacteria and bacteroids, and how they differ. In addition, less effective strains could potentially evade sanctioning by entering the same nodule as an effective strain i.e., piggybacking. P. sativum was co-inoculated with pairwise combinations of three strains of rhizobia with different effectiveness, to test whether ineffective strains can evade sanctions in this way. We assessed the effect of sanctions on nodule populations of bacteria and bacteroids using flow cytometry and the effects on nodule internal structure using confocal microscopy. We show that sanctioning lowered bacteroid populations and caused a reduction in the size of bacteria. Sanctions also precipitated an early change in nodule cell morphology. In nodules containing two strains that differed in their nitrogen-fixation ability, both were treated equally. Thus, peas sanction whole nodules based on their nitrogen output, but do not sanction at the cellular level. Our results demonstrate peas conditionally sanction at the whole nodule level, providing stability to the symbiosis by reducing the fitness of ineffective strains, but cannot target individual strains in a mixed nodule.
Glutaredoxins (GRXs) constitute a distinct family within the large thioredoxin superfamily. Based on the historic order of discoveries and structural similarities, GRXs were originally annotated as glutathione-dependent...Glutaredoxins (GRXs) constitute a distinct family within the large thioredoxin superfamily. Based on the historic order of discoveries and structural similarities, GRXs were originally annotated as glutathione-dependent oxidoreductases with a CxxC active site motif. These prototypical GRXs are now clustered as class I. Later, it was discovered that some GRXs, particularly those with the highly conserved active site motif CGFS, coordinate iron-sulfur clusters and exhibit little or no oxidoreductase activity. Throughout the course of evolution across kingdoms, the CGFS GRXs form the most highly conserved class II. Compared to non-plant species, the GRX family in spermatophytes has undergone significant enlargement and diversification, resulting in typically more than 30 isoforms that are separated into four classes. This expansion is largely due to the evolution of new plant-specific class III GRXs or ROXYs, characterized by a central CCxx active site motif. The increasing number of GRX copies in the genome, coupled with the modification of domains and addition of domains with different functions, has resulted in the functional diversification of plant GRXs. In this review, we aim to provide an overview of the established and emerging roles of GRXs in plant metabolism.