Dietary protein is essential for health, yet its association with mortality risk across various causes remains unclear. This study examines the associations between total, animal-, and plant-based dietary protein intake and macronutrient substitution, as well as all-cause, cardiovascular (CVD), and cancer-related mortality. We used data from the Golestan Cohort Study (GCS), a population-based prospective study initiated in 2004 in Golestan Province, Iran, comprising 43,050 adults aged 40-75 years. Dietary intakes were assessed using validated Food Frequency Questionnaires (FFQ). Mortality outcomes were tracked annually over a mean follow-up period of 15.05 years. Cox proportional hazard regression models adjusted for demographic, lifestyle, and clinical factors were employed. Substitution analysis and restricted cubic spline functions explored the association between different ranges of protein intake and substitution with carbohydrates or fats and mortality risks. During follow-up, 9,309 deaths were documented, including 3,707 from CVD and 1,917 from cancer. Higher total protein intake was associated with increased all-cause (HR for 10 g intake 1.02, 95% CI 1.01-1.03) and CVD mortality (HR for 10 g intake 1.02, 95% CI 1.00-1.03), while animal protein showed similar associations (HR for 10 g intake 1.01, 95% CI 1.01-1.02 for all-cause mortality; HR for 10 g intake 1.02, 95% CI 1.00-1.03 for CVD mortality), plant protein intake was associated with a reduced risk of all-cause mortality in the total population (HR for 10 g intake 0.97, 95% CI 0.95-1.00). The associations were slightly more significant in female and rural participants. Substituting total protein or animal protein with carbohydrates or fat was associated with a lower risk of total and CVD mortality. Protein intake showed a non-linear association with mortality. Balanced protein intake, comprising an appropriate mix of animal and plant sources, appears to support longevity and reduce mortality risk, particularly from cardiovascular causes.
Plant defensins (PDFs) are small, cysteine-rich antimicrobial peptides that play central roles in plant innate immunity. Despite their importance, a systematic characterization of the PDF gene family in soybean (Glycine max) has been lacking. Here, we report a genome-wide identification and comprehensive characterization of 27 GlyPDF genes encoding 31 protein isoforms in the soybean Williams82 v4 genome, identified through combined BLASTP and Hidden Markov Model (HMM) profiling using the plant defensin domain (PF00304). Phylogenetic analysis classified the GlyPDF family into four subfamilies (GlyPDF1-4), with GlyPDF2 being the largest (11 genes, accounting for > 40% of members). The 27 genes are unevenly distributed across 13 of the 20 soybean chromosomes, with chromosome 8 harboring the highest number of loci (6 genes). Three tandem duplication clusters and 19 intra-genomic collinear pairs were identified, indicating that both tandem duplication and ancestral whole-genome duplication (WGD) have driven family expansion. Ka/Ks analysis confirmed purifying selection across all duplicated gene pairs (Ka/Ks < 1.0). Physicochemical characterization revealed that the majority of GlyPDF proteins are basic (pI > 7.0), consistent with their roles as cationic antimicrobial effectors. Promoter analysis identified enrichment of jasmonate-responsive elements (22/27 genes), abscisic acid-responsive elements (19/27), and salicylate-responsive elements (16/27), as well as WRKY-binding W-box motifs (14/27), suggesting multifaceted hormonal and stress regulation. Tissue-specific qRT-PCR revealed broad but variable expression across roots, stems, leaves, flowers, and pods. Upon inoculation with Fusarium oxysporum, 18 of 27 GlyPDF genes were differentially expressed, with GlyPDF2.7, GlyPDF2.8, GlyPDF2.9, and GlyPDF3.3 displaying the strongest induction kinetics. These findings provide a useful genetic resource for the functional characterization of soybean defensins and their potential application in breeding disease-resistant varieties.
Date palm (Phoenix dactylifera L.) is an essential fruit crop in arid and semi-arid regions, where tissue culture is widely used for large-scale clonal propagation. However, this technique often generates somaclonal variants that deviate from original cultivar traits, largely due to epigenetic modifications such as DNA methylation. Here, whole genome bisulfite sequencing (WGBS) was applied to two widely cultivated Qatari cultivars, Khalas and Kheneizi, to compare cytosine methylation profiles between tissue culture-derived plants and offshoot-derived controls. Thousands of differentially methylated regions (DMRs) were identified. Khalas exhibited a predominance of hypomethylated regions, whereas Kheneizi showed slightly more hypermethylated regions. Across both cultivars, CG sites tended to lose methylation, while CHG sites more frequently gained methylation in tissue culture-derived plants. Genes overlapping with DMRs (DMGs) displayed cultivar-specific enrichment patterns. In Khalas, hypermethylated DMGs were mainly linked to nuclear organization, chromatin remodeling, and stress signaling, while hypomethylated genes were associated with defense responses and intercellular communication. In Kheneizi, hypermethylated DMGs were enriched in RNA metabolism, translation, and hormone signaling, whereas hypomethylated DMGs were associated with metabolism, phosphorylation-mediated regulation, and stress responses. KEGG enrichment further revealed shared involvement of RNA degradation, mRNA surveillance, aminoacyl-tRNA biosynthesis, and MAPK signaling pathways. These results demonstrate that tissue culture induces DNA methylation changes in date palm, potentially affecting development and contributing to off-type phenotypes. Understanding these epigenetic alterations provides valuable insights for improving clonal fidelity and optimizing tissue culture propagation in perennial crops.
The continuous growth of the global population is intensifying the challenge of sustaining future food production. Among the major constraints to agricultural productivity, heavy metal (HM) contamination of soils has emerged as a serious environmental problem that adversely affects crop growth and yield. Environmentally friendly strategies are therefore needed to mitigate HM stress in plants. Endophytic fungi have gained attention for their potential to enhance plant tolerance to abiotic stresses. In this study, the endophytic fungus Paecilomyces lilacinus was evaluated for its ability to produce phytohormones and alleviate lead (Pb) and cobalt (Co) stress in maize (Zea mays L.). Firstly, culture filtrate of P. lilacinus was analyzed for phytohormone production under Pb and Co stress conditions. The fungus produced significant amounts of gibberellic acid (43.01 µg mL⁻¹), salicylic acid (2192.1 µg mL⁻¹), and abscisic acid (35.4 µg mL⁻¹), along with measurable protein content (170.06 µg mL⁻¹) in cobalt contaminated filtrate. Secondly, pot experiments were conducted to evaluate the effect of P. lilacinus inoculation on maize plants grown under different concentrations of Pb and Co. Inoculated plants showed increased endogenous levels of GA₃, SA, and ABA, along with significantly higher chlorophyll content compared to non-inoculated controls. The fungal association also enhanced antioxidant capacity, as indicated by increased 2,2-diphenyl-1-picrylhydrazyl (DPPH) inhibition activity, which reached 90.9% under Pb (90 mg) and 89.1% under Co (90 mg). Similarly, the highest ABTS inhibition activity (96%) was recorded under Pb (90 mg). Moreover, P. lilacinus inoculation significantly increased the activities of different antioxidant enzymes, including catalase, ascorbate acid oxidase, and peroxidase. Enhanced uptake of Pb and Co from the soil was also observed in inoculated maize plants compared with control plants. The findings demonstrate that Paecilomyces lilacinus mitigates heavy metal toxicity in maize by enhancing phytohormone production and strengthening antioxidant defense mechanisms in a symbiotic association with the host plant. This endophytic interaction improves plant tolerance to Pb and Co stress and promotes metal uptake, highlighting the potential of P. lilacinus as a sustainable biological approach for reducing heavy metal toxicity and improving crop productivity in contaminated soils.
Environmental factors such as low temperature and water stress often hinder the normal growth of sugarcane (Saccharum spp.), which threatens the supply of raw materials for sugar and biomass provided by sugarcane, resulting in severe economic development losses. Improving the stress resistance of sugarcane has always been one of the important directions in sugarcane breeding. Erianthus fulvus, a wild relative of sugarcane, exhibits remarkable stress tolerance. Therefore, fully exploring the excellent stress-tolerance genes in E. fulvus can provide important candidate genes for improving the stress resistance of sugarcane in future molecular breeding programs. The evolutionary characteristics of EfMYB124, a gene induced by low temperature and drought, were analysed. Moreover, the gene was successfully transferred into Arabidopsis thaliana, and the degree of damage, physiological responses, and stress-induced gene expression of the transgenic plants under stress were investigated. EfMYB124 is most closely related to At1R-MYB in group A1, and the members of this group have the potential to function in response to adverse stress. Numerous syntenic genes of EfMYB124 were identified in the sugarcane genome. Under drought and low-temperature treatments, the EfMYB124-transgenic plants exhibited relatively mild external damage. Moreover, the activities or contents of three antioxidant enzymes (CAT, POD, and SOD) and Pro in these plants increased rapidly, while the production of MDA decreased. Further analysis revealed that EfMYB124 could specifically regulate the expression of cold stress-related genes (CBF1, CBF3, COR15a, and COR4) and drought stress-related genes (RD29A, P5CS1, SOD1, and CAT2). Additionally, EfMYB124 represses the basal expression levels of key ABA signaling-related genes, including NCED3, ABI5, ABF2, and RD29B. In summary, this study has initially confirmed that EfMYB124 plays a positive role in regulating plants' resistance to drought and low temperatures. These key findings lay a solid foundation for us to further analyze the function of EfMYB and study its upstream and downstream interaction relationships. Meanwhile, they provide the necessary scientific reference for the future application of this gene in the genetic improvement of stress tolerance trait breeding in sugarcane.
ASR genes are a class of genes that are expressed in plants in response to abscisic acid, abiotic stress, and fruit ripening. While they have been successfully cloned and functionally characterized in various plant species, research on this gene in Tamarix hispida remains limited. In this study, six ThASR genes were cloned from Tamarix hispida. Expression analysis revealed that ThASR genes respond to salt stress. Specifically, ThASR4 was found to be highly induced in both shoots and roots of Tamarix hispida under salt stress, thus prompting its selection for further functional characterization in salt stress responses. ThASR4 is targeted to the nucleus and possesses transcriptional activity. Under salt stress conditions, compared with the wild-type (WT) lines, ThASR4-overexpressing Arabidopsis thaliana exhibited enhanced germination rate, longer primary roots, and higher fresh weight, indicating a marked improvement in salt tolerance. Correspondingly, both ThASR4-overexpressing T. hispida and A. thaliana plants exhibited significantly diminished levels of reactive oxygen species (ROS), malondialdehyde (MDA), and electrolyte leakage, along with elevated activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), as well as increased proline (Pro) content, compared to Control plants. In contrast, ThASR4 RNA interference (RNAi) transgenic T. hispida displayed the opposite phenotypic and physiological outcomes. Furthermore, ThASR4 was found to upregulate the expression of ROS scavenger-associated genes (ThPOD1, ThSOD1 and ThCAT3) and salt Stress-Responsive genes (ThSOS3, ThPIP2;5, and ThDREB). ThASR4 enhances salt tolerance in T. hispida and A. thaliana by improving ROS scavenging capacity. The present study provides a theoretical foundation for further investigation into the regulatory mechanism of ThASR4 in the salt stress adaptation of T. hispida.
Drought and nitrogen (N) deposition significantly impact plant growth in northern regions. The balance between carbon (C) and N metabolism is crucial for plant adaptation to global environmental change. Salix gordejevii is widely distributed across the northern temperate zone and is characterized by rapid growth and high adaptability. However, the molecular mechanisms underlying its adaptation to drought and N deposition remain largely unclear. Sucrose non-fermenting 1-related protein kinases (SnRKs) represent a class of serine/threonine protein kinases that are extensively involved in plant C and N metabolism, as well as in responses to abiotic stress. We hypothesised that the SnRK gene family was essential for willow adaptation to drought and N deposition, with its evolutionary and structural features being closely linked to its stress-responsive expression patterns. A total of 43 SpSnRK genes were identified and classified into three subfamilies: SpSnRK1 (2 members), SpSnRK2 (11 members), and SpSnRK3 (30 members). These genes exhibited an uneven distribution across the genome. Among them, 37 genes contained both coding and non-coding regions, while 6 consisted solely of coding regions. Most SpSnRK proteins were predicted to localize in the cytoplasm. The promoters of SpSnRK genes were found to be enriched with numerous stress- and hormone-responsive cis-acting elements. Furthermore, the SnRK genes expression profiles in leaves and roots were analyzed under four treatments (control, drought, N deposition, and a combination of drought and N deposition). The expression profile analysis revealed that, under drought stress or N deposition, a greater number of responsive SgSnRK genes were detected in the roots. However, under the combined conditions of drought and N deposition, the number of responsive SgSnRK genes increased significantly in the leaves. The SnRK gene family in willow consisted of three subfamilies that exhibit structural differences and were unevenly distributed across the chromosomes. These genes played critical roles in multiple stress signaling pathways. Furthermore, their expression in response to drought and N deposition showed tissue specificity and varies according to the mode of stress combination.These findings provide a basis for further exploration of the molecular mechanisms underlying SnRK-associated abiotic stress responses.
Climate change-induced heatwaves threaten the sustainable cultivation of Camellia reticulata, a valuable oil-producing species adapted to cool climates. Despite its economic importance, the mechanisms underlying its response to elevated temperatures are poorly understood. We hypothesized that C. reticulata activates distinct physiological and lipidomic adjustments to cope with different intensities of heat stress. To test this hypothesis, we subjected plants to three temperature regimes: 25 °C (control), 35 °C, and 40 °C, and conducted an integrated analysis of physiological traits and lipid metabolism. Our results revealed distinct response strategies in C. reticulata under moderate versus extreme heat stress. Both 35 °C and 40 °C treatments led to significant decreases in chlorophyll content compared with the control, with a much more drastic reduction under 40 °C. Exposure to 35 °C enhanced antioxidant systems and shifted membrane lipids toward a more thermostable composition, effectively preventing visible injury despite mild PSII photoinactivation. In contrast, plants exposed to 40 °C displayed severe PSII photodamage and progressive leaf browning during recovery, even with elevated flavonoid accumulation and enhanced antioxidant capacity. Buds, however, remained viable. This physiological disruption was closely linked to deleterious remodeling of membrane lipids that compromises membrane stability. Concomitantly, significant accumulation of unsaturated neutral glycerolipids was observed, suggesting a cellular protective mechanism that sequesters excess unsaturated fatty acids into storage pools. The preferential survival of buds over leaves indicates that plants prioritize meristem protection. This study identifies the adaptive mechanisms under moderate heat and characterizes the physiological breakdown under extreme heat, highlighting lipid metabolism as a key target for improving thermotolerance in C. reticulata.
Maintaining developmental stability despite sex-linked gene dosage imbalance represents a fundamental challenge for dioecious species. In the dioecious plant Silene latifolia, floral sexual dimorphism is associated with the sex-linked WUSCHEL (SlWUS1) and a CLAVATA3-like gene (GSFY) resulting in sex-specific differences in WUS-CLV3 copy number. Because the WUS-CLV3 feedback loop is a central regulator of stem cell homeostasis in the shoot apical meristem (SAM), such dosage imbalance would be expected to destabilise meristem maintenance. We performed a quantitative analysis of SAM size in male and female Silene latifolia plants across vegetative developmental stages. Seedlings were grown in liquid half-strength Murashige and Skoog (½ MS) medium, and SAM morphology was examined using differential interference contrast microscopy. SAM diameter and area were subsequently measured using ImageJ. SAM size did not differ between male and female plants throughout vegetative development, despite differences in WUS copy number. This robustness persisted following exogenous application of the GSFY peptide (GSFYp) and cytokinin (CK), both of which significantly modulated SAM size and WUS expression. Expression analyses showed that the X-linked SlWUS1 is weakly expressed in the SAM, whereas the autosomal SlWUS2 exhibits high and sex-independent expression and responds similarly to peptide and CK signals in both sexes. These findings indicate that stem cell homeostasis in S. latifolia is primarily maintained by the autosomal SlWUS2, which buffers sex-linked dosage variation without relying on classical gene dosage compensation. Our results suggest the presence of a regulatory architecture that protects essential developmental processes from destabilisation during the evolution of dioecy.
Beetles (Coleoptera) display exceptional dietary diversity and occupy a wide range of ecological niches, often involving close associations with plants and microbes. Ambrosia beetles (Curculionidae; Scolytinae and Platypodinae) exemplify ecological specialization by cultivating mutualistic fungi within galleries excavated in their host trees' xylem, with the fungi serving as their main food source. The striped ambrosia beetle Trypodendron lineatum is a pest of conifers, relying on its nutritional mutualist Phialophoropsis ferruginea for survival. This fungiculture-based lifestyle provides a system for exploring how specialized mutualism is reflected at the genomic level. Hence, we performed a comparative genomics analysis between T. lineatum and nine other beetle species with different ecological specializations. We hypothesized that fungiculture is associated with specific genomic adaptations, including changes in gene family composition related to nutrition, detoxification, and immunity. The small genome of T. lineatum (74.4-83.6 Mb) exhibits comparatively low levels of repetitive DNA (19.9%), including a reduced proportion of transposable elements. Annotation generated 14,830 high-quality gene predictions, most of which were supported by transcript evidence or functional domains. Comparative orthology analysis across ten beetle species identified 13,896 orthogroups, with T.lineatum having 78 species-specific orthogroups comprising 238 genes. Gene family evolution analyses revealed 33 families with significant size changes in T. lineatum, including 16 expansions and 17 contractions. Notably, gene families associated with digestion, detoxification, and immunity were contracted. These included glycoside hydrolase 28, cytochrome P450, serpin, and trypsin families, which may reflect the fungus-based, rather than plant-based, diet of T. lineatum, and reduced reliance on broad-spectrum immune defenses. In contrast, expansions in the THAP and CD80-like immunoglobulin domain families indicate diversification of genes involved in genomic regulation and immune recognition. Our results suggest that the genome of T. lineatum is characterized by low repeat content and compact gene architecture. The observed contractions in key gene families involved in plant digestion, detoxification, and immunity may represent genomic signatures of its obligate mutualistic specialization and narrow ecological niche. Our findings provide the first insights into the genomic adaptations of fungus-farming ambrosia beetles, suggesting that co-evolved insect-microbe mutualisms may lead to reductions in a variety of gene families.
Rice is a short-day plant whose flowering is critically regulated by photoperiod. Through long-term domestication and selective breeding, rice cultivation has adapted to photoperiod variations across different latitudes, resulting in varieties with broad photoperiod sensitivity (PS). PS is essential for rice to adapt to different geographical regions and a wide range of cultivation environments. This study compared the panicle differentiation phenotypes of four rice materials with different photoperiod sensitivities under long-day (LD) and short-day (SD) conditions. Results showed that highly photoperiod-sensitive varieties exhibit a stronger response to photoperiod shifts, leading to accelerated young panicle differentiation and earlier heading under SD conditions. For example, the highly photoperiod-sensitive material H26 headed 56 days earlier under SD than under LD conditions. Correspondingly, under SD conditions, the expression of key flowering genes Hd3a and RFT1 was significantly upregulated in H20, H17, and H26. Transcriptome analysis of leaves treated on day 10 revealed that SD conditions significantly upregulated the expression of key flowering genes such as RFT1, Hd3a, and Se5, while downregulating flowering repressor genes such as Ghd7 and Ghd8, thereby promoting photoperiod-induced early heading. Functional enrichment analysis indicated that these DEGs were primarily associated with starch and sucrose metabolism, plant hormone signal transduction, and circadian rhythm-plant pathway. Furthermore, we identified 125 upregulated and 56 downregulated genes that were uniquely co-expressed across all three photoperiod-sensitive materials. Additionally, five uniquely expressed DEGs related to flowering were identified in the highly photoperiod-sensitive material H26. Our study identified DEGs specifically associated with photoperiod-sensitive materials. These genes represent promising candidates for further investigation into the molecular mechanisms underlying photoperiod responsiveness in rice. Our research helps clarify how rice responds to changes in photoperiod, elucidates the expression pattern of photoperiod-induced genes, and provides valuable insights for breeding rice varieties that are better adapted to changing climatic conditions.
Drought stress severely limits soybean productivity, including during early vegetative growth, highlighting the importance of identifying cultivars with favorable early-stage drought responses. Because drought evaluation based on single traits may not adequately capture coordinated plant responses, integrative multivariate approaches are needed to characterize drought-response patterns among soybean cultivars. Six soybean cultivars were evaluated under greenhouse conditions following a five-day water-withholding treatment at the V3 stage. Morphological, physiological, and biochemical traits were assessed and integrated using a standardized Drought Resistance Index (DRI) and composite D-value framework. Multivariate analyses, including hierarchical clustering, correlation analysis, principal component analysis (PCA), and partial least squares regression (PLS-R), were used to evaluate coordinated drought-response patterns among cultivars. Drought stress reduced most physiological and morphological traits, particularly stomatal conductance, photosynthetic performance, plant growth, and leaf water status. In contrast, proline accumulation increased under drought stress across cultivars, whereas increases in several root-related traits and transpiration responses were observed only in certain cultivars despite substantial reductions in stomatal conductance. Clear differences in drought-response profiles were observed among cultivars. Dering1 exhibited the highest D-value and, together with Dega1, was grouped within the drought-resistant response category, whereas Biosoy1 showed the lowest D-value and was classified as drought-sensitive. Correlation analysis indicated coordinated associations among plant water status, photosynthetic traits, and root-related characteristics under drought conditions. Principal component analysis revealed distinct multivariate response profiles among tolerant cultivars, with Dering1 more closely aligned with root-related and water-status-associated traits, whereas Dega1 was more strongly associated with photosynthetic and biomass-related indices. Partial least squares regression identified several traits, including net photosynthetic rate, stomatal density, instantaneous carboxylation efficiency, and root volume, as showing relatively stronger associations with D-value variation within the analytical framework. The integrative DRI-D-value framework, combined with multivariate analyses, provided an exploratory approach to evaluating coordinated drought-response patterns during early vegetative growth under short-term greenhouse drought conditions. The findings further suggest that early vegetative drought responses in soybean involve coordinated variation among water-status, photosynthetic, and root-related traits. Dering1 and Dega1 may therefore serve as promising candidates for further evaluation under broader developmental stages and environmental conditions.
Pollen is vital for reproduction of flowering plants. Several nutritional and biological benefits for humans and pollinators are described and related to its richness and diversity of metabolites. However, the chemical composition of pollen from several horticulturally significant plant species, such as those in the genus Petunia, has not been thoroughly characterised. Here, we present the first comprehensive description of the chemical profile of two distinct P. hybrida lines: V26 (violet flowers) and W115 (white flowers) using untargeted metabolomics. Our workflow started from pollen sampling and isolation, followed by a 3-in-1 liquid phase extraction for wide-range metabolite recovery, and data acquisition by ultra-high-performance liquid chromatography coupled to high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) using three chromatographic columns (C8, C18, and HILIC) with positive and negative ionisation. For data analysis, we implemented a user-friendly and reproducible data processing pipeline based on open-source computational tools. The P. hybrida pollen is rich in glycosylated flavonoids, phenolamides, and lipids, which were detected mostly in non-polar phase extracts analysed by reversed-phase chromatography. Simple phenylpropanoids, fatty acids, amino acids, and terpenoids were annotated to a lesser extent. Statistical analyses integrated with molecular networking demonstrated a distinct metabolic dichotomy: phenolamide derivatives are predominantly present in the pollen of the V26 line, while flavonoids are accumulated in the pollen of W115. This finding suggests different regulation of the phenylpropanoid metabolism in pollen of P. hybrida lines with differing flower colours and sheds light on hypotheses of ecological roles of pollen secondary metabolites, for example, in plant-pollinator interactions. Our findings suggest a metabolic trade-off in the phenylpropanoid pathway in the two studied P. hybrida lines. These cultivar-specific chemical signatures may have significant implications for pollen viability and interactions with pollinators. Furthermore, our analytical and computational workflow serves as a robust template for the deep metabolic profiling of other under-characterised and complex natural matrices.
Salt stress is widely recognized as a major abiotic constraint that limits plant growth and productivity. Alfalfa (Medicago sativa L.), a globally important forage legume, exhibits considerable variability in salt toll,erance across its diverse cultivars. The present study aims to provide a comprehensive evaluation of eight alfalfa varieties by examining their morphological, physiological, and biochemical responses at four NaCl levels (0, 60, 120, and 180 mM NaCl). Salt tolerance is quantified using a combination of the membership function method, correlation analysis, principal component analysis, and different indices of salt tolerance. High salt stress significantly reduced plant height (PH), shoot dry weight (SDW), root dry weight (RDW), and number of leaves (NOL) of all alfalfa varieties by 47.21%, 61.36%, 64.19%, and 59.91%, respectively, compared with the control. Similarly, physiological traits including relative water content (RWC) and stomatal conductance (SC) declined by 43.93% and 60.87%, respectively, whereas electrolyte leakage (EL) and malondialdehyde (MDA) increased by 267.35% and 178.48%, respectively. Salt stress also markedly altered antioxidant responses among varieties, with superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities increasing by 111.27%, 170.87%, and 125.87%, while V5 (A'erjienong) and V2 (Weinasi) consistently showed tolerance, whereas V4 (Fudina) and V8 (Qianjing) exhibited susceptibility across varying stress levels. Principal component analysis (PCA) classified the traits into three main groups, which explained 93.15% of the total variation, and identified SOD, POD, CAT, SS, and Car as crucial factors affecting salt tolerance. Comprehensive membership function analysis indicated that variety V5 (A'erjienong) was the most tolerant, while variety V4 (Fudina) remained the most sensitive. This study highlights the importance of morpho-physiological and biochemical traits, along with stress tolerance indices, as reliable indicators for evaluating salt tolerance in alfalfa. Future integration of these traits with advanced genetic and breeding approaches may facilitate the development of high-yielding, salt-tolerant alfalfa cultivars.
Chromium (Cr) contamination severely hinders plant growth and biomass production. Salicylic acid (SA) and gamma-aminobutyric acid (GABA) play a vital role in improving plant stress resilience. So for, their synergistic effects in mitigating Cr toxicity remain unexplored. Thus, this research aimed to examine the effects of SA and GABA in mitigating the toxic effects of Cr in barley. The study included Cr stress (0 and 40 mg kg- 1), two barley cultivars (Sultan-17 and Haider-93), and foliar sprays of SA (1 mM) and GABA (1 mM). Chromium toxicity significantly decreased growth and yield in both cultivars by increasing hydrogen peroxide (H2O2: 84%), Cr accumulation, and decreasing leaf water content (37-42%), chlorophyll synthesis (36-40%), and nutrient uptake. The foliar spray of SA + GABA markedly retrieved the toxic impacts of Cr by minimizing the Cr accumulation in roots (54-58%), leaves (46-50%), H2O2 (55-62%), increasing the chlorophyll synthesis, leaf water contents (52-59%), osmolytes synthesis, and antioxidants activities (9-91%), thereby improving the growth and yield. Additionally, SA + GABA improved the secondary metabolites production and maintained the ionic balance by increasing the nutrient uptake, thereby improving the barley growth and yield. Overall, cultivar Sultan-17 performed appreciably well compared to Haider-93 owing to its better potential to counteract Cr. Thus, co-applied SA and GABA mitigate Cr toxicity by improving antioxidant defense, photosynthetic pigments, secondary metabolites production, and restricting the Cr uptake. These findings provides foundation to develop eco-friendly and economical measures to enhance crop productivity and contaminated soils.
Brassica napus L. is one of the world's most important edible oil crops, and its productivity is severely affected by abiotic stresses, including heavy metal contamination. Vanadium (V) toxicity disrupts plant growth, impairs photosynthesis, and induces excessive reactive oxygen species (ROS) accumulation. Melatonin (MT) is an emerging plant regulatory molecule known to enhance stress tolerance; however, its physiological and transcriptomic roles in mitigating V-induced toxicity in B. napus remain poorly understood. In this study, B. napus seedlings were subjected to four treatments: control (CK), MT (100 µM), V (100 mg L⁻¹), and MT + V (MTV). V stress significantly inhibited seedling growth, biomass accumulation, chlorophyll content, chlorophyll fluorescence (Fv/Fm), and water status, while increasing oxidative damage and ROS accumulation. MT supplementation markedly alleviated V-induced phytotoxicity by improving growth performance, chlorophyll accumulation, antioxidant enzyme activities (POD, CAT, and APX), and ROS scavenging capacity. MT treatment also reduced overall V accumulation in seedlings under V stress conditions. Transcriptomic analysis identified numerous differentially expressed genes (DEGs) associated with stress responses, antioxidant regulation, photosynthesis, and phenylpropanoid biosynthesis. Weighted Gene Co-expression Network Analysis (WGCNA) identified four key co-expression modules (turquoise, blue, brown, and yellow) strongly associated with MT-mediated stress responses. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed significant enrichment of genes involved in phenylpropanoid biosynthesis, glutathione metabolism, antioxidant defense, and transporter-related pathways. Our findings demonstrate that MT alleviates V-induced toxicity in B. napus through coordinated physiological and transcriptomic regulation involving antioxidant defense, photosynthetic protection, and stress-responsive metabolic pathways. This study provides new insight into the molecular mechanisms underlying MT-mediated heavy metal stress tolerance and highlights the potential application of MT for improving crop resilience under vanadium-contaminated conditions.
Ubiquitination modification is a core regulatory mechanism of plant growth, development and stress response. The CHY zinc finger domain-containing RING-type E3 ubiquitin ligase (RZFP) subfamily plays a unique role in plant signal integration, but its systematic research in sugarcane remains unreported. Tillering is a key agronomic trait determining sugarcane yield, while its molecular regulatory mechanism is yet to be fully elucidated. This study aimed to systematically characterize the RZFP gene family in sugarcane and explore its association with tillering regulation. A total of 47 ShRZFP genes were identified from the sugarcane XTT22 genome. Phylogenetic analysis divided ShRZFP proteins into 3 groups with independent expansion characteristics; segmental duplication was the main driver of family expansion, dominated by purifying selection during evolution. ShRZFP proteins harbored 2-10 conserved motifs, with consistent gene structures within the same evolutionary branch. Light-responsive elements were the most abundant cis-acting elements in ShRZFP promoters. Weighted Gene Co-expression Network Analysis (WGCNA) screened 6 candidate tillering-related ShRZFP genes enriched in the yellow module, whose co-expressed genes were significantly enriched in the photosynthesis pathway. Upstream transcriptional regulatory network prediction revealed that candidate ShRZFP genes may be regulated by transcription factors (TFs) including WRKY, AP2/ERF, TCP and DBB. qRT-PCR validation showed these 6 candidate ShRZFP genes were significantly responsive to key tillering-related phytohormones, suggesting their putative involvement in the negative regulation of sugarcane tillering. This study is the first systematic analysis of the ShRZFP gene family in sugarcane, which fills the research gap of this gene family in sugarcane, and provides valuable candidate genes and a preliminary theoretical basis for molecular improvement of sugarcane tillering traits.
Erwinia amylovora, the causative agent of fire blight, poses a significant threat to global pome fruit production. This study presents a comprehensive genomic analysis of 317 E. amylovora strains and 227 Erwinia phages to elucidate virulence evolution, phage-host dynamics, and the genomic signatures of the co-evolutionary arms race. Our analysis suggests that a substantial portion of E. amylovora's virulence factors (VFs) share evolutionary origins with diverse plant, human, and animal pathogens, underscoring widespread horizontal gene transfer. We identified bacterial phage hydrolases‑like proteins that share phylogenetic and domain-level similarities with phage endolysins. These observations are consistent with the possibility that some bacterial hydrolases originated from phage-derived ancestors, although functional repurposing remains to be experimentally validated. Crucially, our analysis identifies systematic, non-random associations between bacterial defense systems (e.g., RM, CRISPR-Cas, TA) and mobile anti-defense genes. Statistical correlations show strong patterns of co-occurrence and mutual exclusivity, which are consistent with an ongoing phage-bacteria arms race. These patterns provide a genomic basis for generating hypotheses about co-evolutionary dynamics. These findings may advance our understanding of E. amylovora pathogenicity and phage interactions, offering foundational insights for developing targeted phage-based biocontrol strategies against this devastating plant pathogen. Experimental validation of the predicted virulence factors and defense correlations is warranted to confirm their biological roles.
Silicon (Si) deficiency limits plant growth, physiological efficiency, and yield in high-value crops such as Coriandrum sativum L. A field experiment was conducted on Si-deficient soil at TNAU Coconut Farm, Coimbatore, India, using coriander variety CO (CR) 4. Seven treatments in a randomized block design with three replications evaluated calcium silicate (CaSiO3), and rice husk ash (RHA) at 225, and 275 kg Si ha- 1, alone, and combined with Bacillus altitudinis SSB4, across growth, physiological, biochemical, antioxidant, and yield parameters. Si + SSB4 integration significantly improved plant height (up to 60.9% over control), leaf area index, SPAD index, and shoot and root dry matter production. Leaf Si content increased by 84.1-88.4% over absolute control, and 21.7-24.5% over RDF alone. Antioxidant enzymes (CAT, POD, and SOD), and biochemical attributes (total soluble sugars, total soluble protein, total phenols, and ascorbic acid) were markedly enhanced, with total soluble protein recording the highest increase (194.8% over absolute control). RHA-based treatments marginally but non-significantly outperformed CaSiO3-based treatments. The highest leaf yield (5.46 t ha- 1) was recorded with RDF + RHA at 275 kg Si ha- 1 + SSB4 (53.8% over RDF), followed by RDF + CaSiO3 at the same level (52.1%). Strong positive correlations (r = 0.863-0.990) among Si content, antioxidant enzymes, biochemical parameters, and yield confirmed a coordinated Si-mediated improvement. Integrated application of CaSiO3 or RHA with B. altitudinis SSB4 synergistically enhanced growth, antioxidant defence, biochemical quality, and leaf yield of coriander in Si-deficient soils, supporting the adoption of combined chemical-biological Si management strategies.
Platycladus orientalis foliage, referring to the dried twigs and leaves of the Cupressaceae plant P. orientalis, is a traditional Chinese medicine known for its anti-inflammatory and sedative properties. The composition and concentration of metabolites in P. orientalis foliage vary depending on extraction solvents and the age of the tree, which makes the selection of efficient solvents and appropriate tree age crucial. This study evaluated solvent extraction efficiency for bioactive compounds from P. orientalis foliage and analyzed metabolic variations across trees aged 5-3000 years. Acetic acid (AcOH) achieved the highest extraction yield (14.17%), while Ethanol (EtOH) yielded the lowest (9.60%). EtOH extracts exhibited the highest total phenolic and flavonoid contents, with flavonoids increasing with tree age. Water and EtOH showed stronger DPPH scavenging than AcOH; water excelled in ·OH scavenging, whereas AcOH led in ABTS scavenging and total antioxidant activity. Older trees consistently outperformed younger ones in antioxidant assays. Both EtOH and AcOH inhibited E. coli and S. aureus; AcOH's effect against E. coli strengthened with tree age. Water and essential oil showed no antibacterial effects, though all extracts, especially essential oil, inhibited Aspergillus niger. GC-MS identified 88 metabolites and 14 bioactive compounds. β-pinene and geraniol characterized trees under 1000 years, while caryophyllene oxide and α-terpineol accumulated in super-aged trees. These compounds exhibited multi-target pharmacological synergy via anti-tumor, analgesic, anti-inflammatory, and neuroprotective pathways. UHPLC-QE-MS suggested upregulated phenylpropanoid and flavonoid biosynthesis in 3000-year-old trees, leading to accumulation of key antioxidants like isoeugenol, coniferin, myricetin, and quercetin. These findings confirm that even at 3000 years of age, P. orientalis maintains a robust capacity to synthesize active metabolites, underscoring its considerable potential as a valuable resource for pharmaceutical research and development. This study highlights the synergistic effects of solvent and tree age on the bioactive compounds in P. orientalis foliage, providing a theoretical basis for optimizing extraction processes and developing high-value natural products. It also underscores the unique value of ancient tree resources in pharmaceutical applications, offering new strategies for plant-derived drug development.