Planta

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. The 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 both by phylogenetic relatedness and lineage-specific adaptation. HighlightVariance of intermediate levels points to poising of the Calvin-Benson cycle varying between closely-related species in the tomato clade Solanum sect. lycopersicon subsect. Lycopersicum

Cadmium is a very toxic heavy metal. We studied Cd-treated barley plants with especial focus on rare atypical plants with signs of chlorosis. Cd treatment decreased the maximal photochemical activities of both photosystems while the activity of photosystem I decreased more than activity of photosystem II. In photosystem II, Cd treatment inhibited non-photochemical quenching that increased portion of unquenched "closed" complexes of photosystem II. The latter effect increased balance of limitations between the acceptor side of photosystem II (qC) and the donor side of photosystem I (Y(ND)) and raised the ratio qC/Y(ND). All these effects were enhanced in the atypical more damaged plants. Cd treatment reduced K content in the first leaves; in atypical plants, K content decreased even more. Cd treatment changed a pattern of stomatal conductance possibly by means of reducing K content in leaves. The untreated barley plants kept different stomatal conductance at adaxial and abaxial sides of leaves and fulfilled a complicated diurnal dynamics with large ups and downs of stomatal conductance. The typical Cd-treated plants were less flexible and demonstrated medium values. Stomatal conductance in the untreated plants were higher or lower than in the typical Cd-treated plants depending on a particular time; average daytime stomatal conductance was equal in both variants. At 10.00, stomatal conductance in the atypical Cd-treated plants was smaller than in the typical ones. Levels of 13 chloroplast mRNAs remained unchanged, while psbD decreased in both types of Cd-treated plants. HighlightsO_LISeveral Cd effects were enhanced in more damaged (atypical) chlorotic plants C_LIO_LICd treatment decreased activity of photosystem I and non-photochemical quenching C_LIO_LIRatio of limitations between photosystems II and I [qC/Y(ND)] was rather constant C_LIO_LICd treatment reduced K content in the first leaves C_LIO_LICd treatment changed pattern of stomatal conductance C_LI

CoverCress is a new winter annual oilseed crop developed from field pennycress within the past 20 years. Field pennycress is commonly considered to be self-pollinated but little basic research has been published and there is some misalignment of conclusions. Our experience working with pennycress plant growth in greenhouse and field conditions over the past 13 years suggests that outcrossing is uncommon. We conducted lab, greenhouse, and field experiments to strengthen the body of work. Pollen viability kinetics analysis showed that longevity of pollen viability is negatively impacted by increasing temperatures and by direct exposure to light. Samples treated at 4C declined to 50% viability in 12 hours while it took just 2.5 hrs at 37C, and 1.6 hrs in full sunlight on a cool early April day. Cross-pollination was absent among greenhouse-grown plants flowering inside an agitated plastic pollen-containment covering. Across greenhouse tests, high rates of cross-pollination occurred only in an emasculation treatment that rendered flowers male sterile and opened the pistil to cross-fertilization. Field trials designed to measure pollen flow distance using a trackable fae1 knockout reporter gene failed to show detectable movement of pollen under field conditions in two locations. This data strongly suggests that domesticated field pennycress may be considered a self-pollinated crop and managed as such.

The nucleus is the characteristic organelle for eukaryotic organisms. Unlike the classic textbook view of static two-dimensional nuclei, nuclear shape is dynamic inside the live cell. The alteration or deformed nuclear shape is the hallmark of cancer in animal cells and environmental stress in plants. The nuclear envelope proteins interact with chromatin to regulate gene expression. Unfortunately, we have limited knowledge about the impact of abiotic stress on nuclear shape, movement, and chromatin dynamics. To circumvent this issue, we are utilizing a dual fluorescently tagged marker lines - nuclear envelope protein and chromatin - to perform live cell imaging in the model plant Arabidopsis thaliana root. The live cell imaging was performed in control and salt-stressed conditions. We utilized these captured movies to analyze through open-source image processing software Fiji/ImageJ with the help of the TrackMate plugin. Using this method, we have demonstrated that chromatin velocity is decreased in salt-treated conditions. This method will be widely applied to quantitative live cell imaging of nuclear shape and chromatin dynamics during plant development and environmental stress. SummaryThis process aims to simultaneously record nucleus and chromatin dynamics in Arabidopsis thaliana roots and investigate changes in these dynamics in response to developmental and environmental cues.

Virus induced gene silencing (VIGS) is a method that exploits plant antiviral defence mechanisms to downregulate endogenous genes. The technique is versatile, rapid, and widely used for functional genomics studies. Here we report a method for VIGS in the medicinal plant, Calendula officinalis (pot marigold). This species produces anti-inflammatory triterpenoids and has also been bred and cultivated as an ornamental plant. We describe a method for the injection of Agrobacterium tumefaciens cultures into leaf midribs and compare visual marker genes for tracking VIGS utilising constructs that simultaneously target visual marker and target genes. We use these tools to demonstrate that silencing a gene encoding cycloartenol synthase results in changes to leaf phytosterols. This method could be used to further investigate the genetic basis of specialised metabolism in this species and could be adapted to other members of the Asteraceae family, many of which are of economical and chemical value.

Quinoa (Chenopodium quinoa Willd.) is a genetically diverse Andean crop valued for its nutrition and adaptability to varied agroclimatic conditions with potential for cultivation in European and Mediterranean, particularly on marginal lands. Low temperatures during early sowing can impair germination, while delayed sowing increases the risk of poor maturation due to unfavorable autumn weather. To assess the adaptation of quinoa to cold stress, we evaluated germination and phenotypic variation in 60 accessions from highland and coastal ecotypes across three sowing dates in South-Western Germany: late winter (S1), early spring (S2), and spring (S3). Cold stress in S1 delayed seedling-emergence and reduced emergence percentages, yet these plants produced the highest average seed yield per plot (64 g) compared to S2 (46 g) and S3 (35 g). Highland accessions showed earlier seedling-emergence and with higher emergence percentages, while coastal types matured earlier and gave higher yields across sowing dates. A complementary laboratory experiment assessed germination under cold (4.4 {degrees}C) and control (18.3 {degrees}C) conditions, using both manual scoring and image analysis via a Mask R Convolutional Neural Network, to track seedling growth. This confirmed the beneficial germination performance of highland accessions under cold stress, with strong agreement between manual and automated scoring. Our findings suggest that quinoa demonstrates resilience to cold stress with highland quinoa exhibiting superior germination traits, and early sowing, despite reduced emergence, can lead to higher yields. We conclude that combining favorable traits such as faster maturity and higher yield of coastal ecotypes with superior germination traits of highland accessions is a promising avenue for breeding improved quinoa varieties for cold climatic regions.

BackgroundNon-optimal temperatures have become a major constraint on plant development under rapidly changing climatic conditions. Both sub- and supra-optimal temperatures reduce physiological activity, alter plant morphology, lead to plant mortality, and ultimately decrease crop productivity. Temperature-tolerant plants employ physiological and morphological mechanisms to mitigate such stress. In this study, we aimed to identify the source of temperature tolerance in warm-climate adapted melon (Cucumis melo L.). ResultsSuboptimal temperature-tolerant accession (Ananas Yoqneam; AY) and susceptible accession (PI414723) were reciprocally grafted and grown under controlled temperature regimes of 16 {degrees}C, 25 {degrees}C, and 35 {degrees}C. Physiological and morphological traits were measured to characterize tolerance mechanisms and whole-plant responses. Temperature emerged as the dominant factor governing plant performance. Whereas non-grafted parental lines maintained consistent differences across all temperature regimes, reciprocal graft combinations diverged mainly under suboptimal (16 {degrees}C) conditions. Under these temperatures, scion identity strongly determined whole-plant performance through biochemical limitations. ConclusionThese results highlight the importance of scion-derived traits in abiotic stress tolerance and their downstream influence on root function.

O_LIRationale: Plants in urban environments often experience heat stress and responses to heat stress often include vegetative and reproductive traits like rosette width and fruit morphology. However, our understanding of natural variation in vegetative and reproductive traits in urban environments is severely limited. C_LIO_LIMethods: We grew an urban weed, Capsella bursa-pastoris, in common garden environments that simulate an urban heat gradient to determine how heat affected growth, survival and reproduction. Additionally, we used geometric morphometric techniques alongside deterministic techniques to quantify variation in C. bursa-pastoris fruit shape and investigated the predictive relationship between fruit shape and seed production. C_LIO_LIKey results: We found that temperatures above 30C act as an environmental constraint on both C. bursa-pastoris fruit shape and reproduction, resulting in malformed fruits and no seed production. However, leaf number and plant survival were unaffected by high urban heat. C_LIO_LIMain conclusions: While plants may grow and survive in the high urban heat, heat could still limit population persistence. C_LI

RNA interference (RNAi) shows great potential to protect crops against fungal diseases, yet reported protection efficiencies vary greatly, and our understanding of the factors responsible for this variance remains limited. In this meta-analysis, we evaluated 89 studies that compare the efficiency of host-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS) in controlling fungal diseases, focusing on biotrophic, hemibiotrophic, and necrotrophic fungi, the use of formulations, and the dsRNA design as explanatory factors for differences between reported efficiency values. Our results indicate that SIGS is slightly more effective, particularly in biotrophs. Surprisingly, SIGS studies using formulations did not outperform those applying naked dsRNA. We also assessed parameters of RNA design. Differences in dsRNA length and the number of constructs, and number of targets showed no consistent significant effect on resistance in either HIGS or SIGS. Interestingly, however, HIGS studies reported significantly higher efficiency when targeting genes closer to the 3 end and SIGS when targeting genes closer to the 5 end. We discuss potential reasons for the reported patterns, such as variability in dsRNA uptake mechanisms, intercellular trafficking and Dicer processing, and conclude that more research is needed to understand the biological mechanisms determining RNAi efficiency for fungal control.

G-protein coupled receptors (GPCRs) are responsible for translating environmental signals of various types into cellular signals. Over 40 thousand GPCR orthologs have been discovered in the supergroup Unikonta, and around 800 genes encode for GPCRs in the human genome. In contrast to this astonishing variety, only a handful of GPCR-related genes have been reported in vascular plants, a major group within land plants. In an attempt to advance our understanding of plant GPCRs as well as their role in plant cellular signaling, here we present comprehensive bioinformatic analysis that includes phylogenetic hypotheses, in silico structural analysis, and tissue distribution of transcripts. Altogether, our work strongly suggests that GCR1 is the sole genuine GPCR expressed in Embriophyta. Finally, we briefly discuss the potential role of GCR1 in root hairs, the tubular outgrowths in root epidermal cells that are involved in nutrient absorption, environmental interaction, and root development.

Spinach (Spinacia oleraceae) is a principal vegetable crop commercially grown in Controlled Environment Agriculture (CEA). Recent research suggests that root morphological and architectural differences among crop species influence yield, resource use efficiency, and environmental stress tolerance. These root traits may be exploited to increase yield, promote efficient nutrient use, and mitigate environmental stressors. This study measured differences between various spinach cultivars in CEA systems to reveal morphological and anatomical variation. We grew three spinach cultivars with different reported growing rates ( Income, Darkside, and El-Majestic) under NFT hydroponic and substrate-based systems in a controlled greenhouse environment over 45 days with destructive harvests at days 15, 30, and 45. Supplemental light (250 {micro}mol/m2/s) with 12-hour photoperiod and periodic fertigation was used. Harvests included the collection of leaf and root biomass, and scanning of root systems in WinRhizo software, measuring ten variables. On day 45, root cross-sections from orders 1-5 were embedded in JB-4 resin, sectioned, stained, and analyzed for diameter, vasculature, and rhizodermis characteristics. Results indicate that in spinach, differences in root system morphology are linked to cultivation systems over cultivar identity. Vascular and root anatomical alterations are minor compared to morphological differences in response to the cultivation system. Hydroponic-style growth systems are associated with the proliferation of fine-root ideotypes compared with substrate-based conditions. Such findings affirm previous studies, which suggest plastic root morphology in response to growth systems, and may be used to help create more resilient, resource-efficient cultivars. HighlightsO_LIIn spinach, root system morphology differences are linked to cultivation systems. C_LIO_LIRoot vascular and anatomical alterations are minor in response to cultivation system. C_LIO_LIHydroponic growth systems are linked to fine-root ideotype proliferation in spinach. C_LIO_LIFine-root ideotype proliferation may be a breeding target for CEA spinach. C_LI

The paper presents the results of a comparative analysis of gene networks activated by water stress and low temperatures in extensive (Saratovskaya 29, S29) and intensive (Yanetskis Probat, YP) wheat varieties during the seedling development stage. It is concluded that the creation of the S29 variety, which occurred through complex stepwise hybridization and selection for morphological traits, productivity, and grain quality traits, resulted in the emergence and inheritance of regulatory gene networks involving proteins with the CC domain, as well as the BTB/POZ and TAZ domains, which have an increased affinity for transcription factors involved in the transcriptomic response to changing external conditions. It was established that, at the transcriptomic level, the S29 variety is characterized by a transition to an energy saving mode to maintain the activity of the Calvin-Benson cycle under the water deficit conditions and the inhibition of proteolytic processes at low temperatures. The transcriptional response of the high-yielding YP variety to 24-hour low-temperature treatment was more active and involved a larger number of genes compared to the S29 variety. Identifying varietal variability in molecular genetic mechanisms of resistance to abiotic stressors facilitates the development of marker-assisted and genomic selection technologies for common wheat. Key messageThe extensive S29 variety was characterized by its transition to energy-saving mode to maintain the Calvin-Benson cycle under water deficit and a reduction in proteolytic processes under low temperature.

BackgroundOn-site food production will be required to achieve NASAs goal of a sustainable Lunar habitat. Toward this end, the use of fine, soil-like material on the Lunar surface, known as regolith, has been proposed as a plant growth substrate. However, how this substrate may affect plant growth is not well understood. Lunar regolith is devoid of the organic materials that make soils on earth fertile for plant growth, and has been weathered by solar winds, cosmic rays, and micrometeorite impacts. Additionally, regolith at certain lunar sites may contain heavy metals. These metal ions may leech, thus posing challenges with accumulation in plant material. To address and verify the efficacy of regolith-based crop production, we used lunar regolith simulants (LRS). We investigated the effects of LRS on potato (Solanum tuberosum cv Modoc) plant and tuber development, gene expression, and nutrition profiles. ResultsGrowth in LRS negatively impacted the potato plant size and tuber yield. While the degree of impact differed between simulants, all plants grown in LRS were statistically significantly shorter in height than plants grown in control soil. Further experiments with the lunar mare simulant 1E (LMS-1E) show that these effects can be ameliorated through the addition of vermicompost, an organic component, with a 70:30 v/v ratio of LMS-1E to compost being virtually indistinguishable from controls. Changes in gene expression profiles also differed between simulants, with genes related to photosynthesis, biotic and abiotic stress responses, signaling, and terpenes and flavonoids metabolism being commonly altered. Despite these observed differences in transcription, broad changes in metabolite profiles were not observed. ConclusionsLRS are clearly stressful on plants. However, amendment of the substrate with composted materials appears to be a viable strategy to alleviate stress. Given these observations, regolith-based agriculture may not be viable for very early food production when organic matter content is low. However, this would improve over time with continual incorporation of organic matter to regolith. As such, we believe regolith-based agriculture is a viable long-term strategy.

Tacca chantrieri, black bat flower, has showy flowers often appearing almost black. Here, we present the genome sequence and corresponding annotation to identify the genetic basis of the pigmentation. Candidate genes associated with the anthocyanin biosynthesis were identified based on this genome sequence and investigated with respect to their properties. The best dihydroflavonol 4-reductase (DFR) candidate, which harbours all amino acid residues believed to be required for DFR activity, shows a threonine in the substrate preference determining position where most characterized DFRs display asparagine or aspartate. This amino acid residue appears to be frequent in the Dioscoreaceae family as a comprehensive investigation revealed.

In this study, the level of Rubisco protein was reduced in wheat using RNAi, to test the hypothesis that photosynthesis, growth, and grain yield could be maintained whilst improving nitrogen use efficiency. The RNAi Rubisco wheat plants, with a Rubisco activity of less than 70% of wild type (WT) plant levels, had reduced photosynthesis, reductions in leaf and stem biomass and decreased seed yield. Interestingly, in the wheat RNAi Rubisco lines that had a small (<30%) reduction in Rubisco activity, the seed number, total seed weight and harvest index were comparable to that of WT type plants. However, no improvement in photosynthetic nitrogen use efficiency (PNUE) was evident in any of the RNAi Rubisco lines. Notably, PNUE was lower than for WT wheat plants in the RNAi lines with more than a 30% reduction in Rubisco activity. This result was unexpected and caused by an accumulation of N in both the leaves and seeds. At present we do not have an explanation for this but one possible hypothesis is that it could be due to slower growth caused by a reduction in source strength in the RNAi plants, which in turn resulted in changes to carbon and nitrogen allocation. HighlightWheat RNAi plants with small reductions in the amount and activity of Rubisco had a similar biomass and total seed weight to that of untransformed controls but no improvement in nitrogen use efficiency was evident.

Soil salinity is a rapidly intensifying abiotic stress that significantly limits wheat productivity, particularly in coastal and irrigated agroecosystems. Although sodium (Na+) ion exclusion has been recognized as a key tolerance mechanism, the integration of physiological performance with Nax1-mediated molecular regulation among regionally adapted wheat genotypes remains insufficiently characterized. The present study aimed to dissect salinity tolerance by combining hydroponic phenotyping, multivariate trait analysis, molecular marker profiling, and quantitative expression analysis of the Na+ ion transporter gene Nax1. Seventeen spring wheat genotypes were evaluated under four salinity levels (0.0, 10, 12, and 14 dS m-{superscript 1}). Germination and survival rate, shoot and root growth, and biomass accumulation were measured. Principal component analysis (PCA) and hierarchical clustering were performed to classify genotypes, while SSR (simple sequence repeat) and Nax-linked markers assessed genetic diversity. Relative Nax1 expression was quantified using qRT-PCR (quantitative real-time polymerase chain reaction). Salinity significantly reduced germination, survival, elongation, and biomass, with strong genotype-dependent variation. Multivariate analyses clearly separated tolerant and sensitive genotypes, with biomass retention and survival contributing most to total variation. Marker analysis revealed moderate genetic polymorphism. Notably, tolerant genotypes exhibited 3-6-fold induction of Nax1 under severe salinity, positively correlating with biomass maintenance. These findings demonstrate that salinity tolerance in wheat is associated with coordinated physiological resilience and enhanced Nax1-mediated Na ion exclusion, thereby advancing mechanistic understanding and supporting molecular-assisted breeding for salt-affected environments.

Photosynthesis accounts for most of the final grain yield in rice, making improvements in radiation use efficiency (RUE) a key strategy for enhancing productivity. Agronomically, RUE is defined as the biomass produced per unit of total solar radiation or photosynthetically active radiation intercepted by the canopy. However, the interaction between carbon and nitrogen metabolism plays a critical role in determining plant growth and grain yield. Assimilated nitrogen is required for the synthesis of photosynthetic pigments and enzymes, while the reduction of nitrate (NOLL) and nitrite (NOLL), as well as the assimilation of ammonium (NHLL), depend on the reducing power and carbon skeletons generated by photosynthesis. In this study, two high-yielding rice (Oryza sativa) cultivars--an indica-type (El Paso 144) and a japonica-type (INIA Parao) were subjected to two nitrogen treatments (3 mM and 9 mM NOLL/NHLL) and two light intensities (850 and 1500 mol mL{superscript 2} sL{superscript 1}). A strong interaction between light intensity and nitrogen metabolism was observed, with contrasting responses between subspecies. These differences reflect a coordinated regulation of carbon assimilation and primary nitrogen metabolism. The results provide new insights into the metabolic strategies underlying nitrogen compound accumulation under variable irradiance. Such knowledge is essential for improving nitrogen fertilizer use efficiency and yield performance in elite rice genotypes cultivated under commercial field conditions.

The objective of this study is to determine the quality and define the different classes of honeys produced in the Ivorian forest region according to their pollen content. This involves the analysis of five honey samples from the sub-prefecture of Cechi. Four of the honey samples were wild-harvested, and one was from experimental beekeeping in the Cechi reserve. A total of 54 pollen taxa were identified. The most represented botanical families are: Fabaceae (9 species, or 16.67%), Apocynaceae, and Combretaceae, each with 5 species, or 9.27%. The pollen taxon richness of the honeys varies from 18 to 34 taxa. Most are polyfloral honeys, with the exception of the honey from the reserve, which contains 66.13% Bridelia micrantha pollen (Euphorbiaceae), a monofloral honey. These samples contain highly variable pollen content and fall into three categories of honey: honeys rich in pollen, honeys very rich in pollen, and honeys extremely rich in pollen, attesting to their high quality and natural origin.

Betalamic acid is the chromophore of betalains, which are pigments of chemotaxonomic and physiological importance. This study involves RACE-PCR-based gene cloning, heterologous expression, protein purification, and steady-state kinetics of B. alba L. var. Rubra L-DOPA/dopamine-4,5-dioxygenase 1 (BrDOD1). BrDOD1 is a unique high betalamic acid-forming LigB homolog in plants having comparable affinity for both L-DOPA and dopamine (KM < 50 M). Ascorbic acid (10 mM) shifted the steady-state kinetics from inhibitory to activator at a particular substrate concentration of both L-DOPA and dopamine. This increased both KM and Vmax by more than 6.5-fold, indicating that ascorbic acid acted as a molecular crowding agent in the enzyme assay. BrDOD1s physiological substrate is L-DOPA as the reaction rate for L-DOPA was 6.6-fold higher than dopamine, L-DOPA was present in higher concentration than that of dopamine in the same plant, and molecular dynamic simulations showed better stability of BrDOD1-L-DOPA complex than that of dopamine. Further, two more LigB homologs from the same plant have also been cloned. Based on the betalamic acid-forming activity, molecular phylogeny, conserved structural regions, and theoretical pI, betalainic plant LigB homologs have been classified into three groups to better understand the evolutionary trajectory of the LigB homologs in plants.

BackgroundExtensin peroxidases (EPs) are class III plant peroxidases and are responsible for intermolecular covalent crosslinking of extensin (EXT) monomers to create scaffolds within plant cell walls. The formation of these scaffolds impacts plant development, mechanical wounding, and response to pathogen attacks. Therefore, elucidating the molecular mechanism controlling covalent crosslinking of EXT monomers is crucial for understanding cell wall deposition and potentially improving plant growth and adaptation. The focus of this work is to use in silico analysis to determine the structural characteristics of an EP from tomato (TomEP) to elucidate its specificity for crosslinking of EXT monomers. ResultsIn this study the two-dimensional (2D) and three-dimensional (3D) structures of TomEP were determined using several advanced bioinformatics tools and compared to two other peroxidases: GvEP1 (a known EP) and HRP-C (having a low affinity for EXT substrates). The results revealed that TomEP is a stable and hydrophilic protein with high thermal stability. The heme binding pockets of TomEP and GvEP1 have more hydrophobic residues and larger volume and pocket area compared to HRP-C. Molecular docking at the active site, which includes a heme heteroatom, showed that the ligands consisting of the hydrophobic Tyrosine-X-Tyrosine [-Y-X-Y-] motifs (i.e., [-Y-K-Y-], [-Y-V-Y-], and [-Y-Y-Y-] found in EXTs, and their derivatives, Isodityrosine (IDT), Pulcherosine (Pul), Di-Isodytirosine (diIDT), bind perfectly to the active site of TomEP with dominant interactions of Val54, Ser94, Ala96 and Phe196 residues. Pulcherosine had the highest binding affinity, while [-Y-K-Y-] showed the lowest binding affinity. Molecular dynamics simulations showed that [-Y-X-Y-] motifs (and the derivative substrate ligands) remain bound to the active site of TomEP throughout the 100 ns long simulation. Furthermore, the binding of these substrates stabilized the protein structure. ConclusionThese results may explain why TomEP is particularly well-suited for EXT crosslinking and will have significant implications on biochemistry, biotechnology, and the potential use of these EPs in crops improvement.