Iron deficiency is a major abiotic constraint that limits soybean growth, nodulation, symbiotic nitrogen fixation, and yield, yet objective criteria for evaluating low-Fe tolerance and the regulatory mechanisms linking root–nodule responses with shoot adaptation remain insufficiently defined. Here, we established an entropy-weight-based evaluation system using 62 soybean accessions and identified Wanhuang506 (Wh506) as a highly tolerant cultivar and Flyer as a highly sensitive cultivar. Physiological validation showed that Wh506 maintained higher Fe accumulation, chlorophyll retention, antioxidant enzyme activities, and nodule development than Flyer under low-Fe stress. To explore the molecular basis of this contrast, integrated transcriptomic and metabolomic profiling was performed in leaves and root–nodule complexes (RNCs). Compared with Flyer, Wh506 exhibited stronger RNC-centered transcriptional and metabolic reprogramming involving Fe-related redox processes, secondary metabolism, and brassinosteroid (BR) biosynthesis. Multi-omics integration prioritized GmCYP90A1, a BR biosynthetic cytochrome P450 gene, as a candidate component associated with low-Fe tolerance, while the MYB transcription factor GmMYB093 was specifically induced in Wh506 RNCs. Yeast one-hybrid and dual-luciferase assays demonstrated that GmMYB093 directly binds to the GmCYP90A1 promoter and activates its transcription. Hairy-root overexpression of GmMYB093 or GmCYP90A1 increased endogenous BR levels, improved Fe accumulation, enhanced antioxidant capacity, reduced lipid peroxidation, and alleviated chlorosis and growth inhibition under low-Fe stress. Exogenous BR application further mitigated Fe-deficiency-induced chlorosis, particularly in sensitive accessions. These findings support a model in which the GmMYB093–GmCYP90A1–BR module contributes to soybean low-Fe adaptation by coordinating Fe homeostasis, redox protection, and root–nodule performance, providing candidate targets for breeding Fe-efficient soybean cultivars.
Plants establish intimate associations with rhizosphere microorganisms that profoundly influence their growth, development, and stress resilience. Among these, plant-growth-promoting rhizobacteria (PGPR) enhance nutrient acquisition, modulate phytohormone homeostasis and reshape root system architecture, thereby improving plant fitness. Despite extensive evidence supporting their beneficial effects, the molecular and cellular mechanisms underlying microbe-driven modulation of specific root traits remain incompletely understood. Through extensive phenotypic investigation, we established that Pseudomonas sp. M25, a previously described PGPR strain, produces a significant increase in leaf relative water content and evapotranspiration of Arabidopsis thaliana without impacting on rosette growth or photosynthetic parameters. Inoculation with M25 leads to enhanced drought tolerance, and this is associated not with changes in root architecture but with a marked increase in root hair (RH) abundance and length. The stimulation of RH development by this Pseudomonas strain is based on the genetic requirement for RH-related basic helix-loop-helix family transcription factors, including ROOT HAIR DEFECTIVE 6 (RHD6) and RHD6-LIKE 1 (RSL1), which regulate RH development via RHD6-LIKE 4 (RSL4) and RHD6-LIKE 2 (RSL2). Pseudomonas sp. M25 can partially circumvent the lack of RHD6 but requires RSL1 and the downstream transcription factors RSL2 and RSL4 to induce RH growth. These findings indicate that this bacterium can circumvent RHD6 to activate RSL4, which subsequently promotes RH growth. Our investigation identifies some essential signaling components regulated by Pseudomonas sp. M25 to optimize RH responses.
The bHLH family is considered as the second largest class of transcription factors in plants, playing important roles in various biological processes, particularly in color regulation. Previous research indicated that bHLH family genes were significantly involved in screening for genes associated with color variation in chili fruits. Further analysis of the predicted bHLH family genes revealed a high correlation between CabHLH118 and delphinidin, and subsequent assays showed that CabHLH118 was localized in the nucleus, possessed transcriptional activation activity, but lacked self-activation activity in yeast. Silencing or transient overexpression of CabHLH118 in pepper CS03 fruits resulted in significant phenotypic changes, accompanied by a marked decrease or increase in anthocyanin accumulation, respectively. Consistently, the expression of CabHLH118 was significantly down- or up-regulated, and the structural genes in anthocyanin biosynthesis pathway showed corresponding changes. Yeast one-hybrid (Y1H) and dual-luciferase assay demonstrated that CabHLH118 can bind to the upstream promoter of CaF3'H and promote anthocyanin formation. Furthermore, CabHLH118 may respond to blue light to enhance anthocyanin biosynthesis, although the underlying mechanism requires further verification. Collectively, these findings contribute to the understanding of anthocyanin regulation in Solanaceae plants.
The ascorbate-glutathione (AsA-GSH) antioxidant system is essential for plant growth, fruit ripening, and abiotic stress adaptation, while the functional mechanisms of genes participating in the AsA recycling pathway in blackcurrant remain largely unclear. A candidate differentially expressed gene, RnAPX6, which potentially regulates AsA biosynthesis and recycling, was screened via transcriptome profiling of blackcurrant cultivars with high- and low-total AsA contents at four distinct fruit developmental stages. Transient overexpression and VIGS silencing in blackcurrant fruit, as well as stable heterologous transformation in tomato, were performed to explore the biological roles of RnAPX6. RnAPX6 reshaped AsA redox balance by coordinating AsA recycling and biosynthetic pathways. This gene acted as a negative regulator of fruit ripening; its overexpression delayed peel pigment accumulation, maintained fruit firmness, suppressed ethylene production and MDA generation, and reduced total soluble solids. In addition, heterologous RnAPX6 overexpression in tomato alleviated cold- and salt-induced injury by boosting antioxidant enzyme activity, eliminating excess ROS, and stabilizing photosystems. This work uncovers the dual roles of RnAPX6 in fruit quality modulation and stress defense, offering a valuable gene resource for breeding superior horticultural crops.
Flower development involves dynamic cell wall changes driven by coordinated synthesis, breakdown, and remodeling processes that support cell division, differentiation, and expansion. Endo-1,4-β-glucanases (EGases or cellulases), belonging to the Glycosyl Hydrolase family 9 (GH9), are key enzymes involved in cell wall disassembly and metabolism, as well as cell expansion and differentiation, fruit ripening, and organ abscission. However, their roles during female reproductive development remain poorly understood. Here, we studied the cellulase SlGH9-16 during gynoecium and fruit development in tomato. Expression profiling of SlGH9-16 and its orthologs AtGH9B2 and AtGH9B13 showed conserved and dynamic spatio-temporal expression patterns in female reproductive organs for these three GH9 members. We also generated CRISPR-Cas9 mutants and overexpression (OE) lines for SlGH9-16 and showed that this enzyme is necessary for normal cellular patterning and morphogenesis of the tomato gynoecium. Furthermore, SlGH9-16 serves as a dose-dependent modifier influencing both fruit architecture and texture, with its loss-of-function significantly extending post-harvest shelf life. FT-IR and HPTLC analyses suggest that these phenotypic changes are correlated with distinct chemical changes in the polysaccharide matrix of the pericarp cell wall. In summary, these findings highlight a role for GH9 cellulases in reproductive development and the importance of endo-1,4-β-glucanases in the morphogenesis of tomato gynoecium and fruit.
Auxin, a key plant hormone, regulates growth and development through two distinct receptor systems. The well-established transport inhibitor response 1 (TIR1)/auxin-signaling F-box protein (AFBs) pathway mediates transcriptional responses in the nucleus, whereas a distinct non-transcriptional pathway involving plasma membrane-associated co-receptors auxin binding protein 1 (ABP1)/ABP1-like protein 1 and 2 (ABL1/2) and transmembrane kinases (TMKs) mediates rapid cellular responses. Recent breakthroughs have resolved long-standing debates regarding the apoplastic auxin signaling, highlighting the TMK kinase family and its auxin-binding co-receptors ABP1/ABL1/2 as the core of this rapid auxin signaling system. This review explores how this signaling complex has evolved across different plant species and how it enables the perception of extracellular auxin to trigger fast cellular responses, including global phosphorylation, apoplastic acidification, and cytoskeletal changes, within seconds. By examining these interactions and their evolutionary origins, we present a unified framework for understanding how the integration of intracellular and extracellular auxin signals drives the remarkable versatility of plant development.
Plants simultaneously experience temperature and light, yet whether these cues interact at the chromatin level to generate distinct regulatory states remains unresolved. This review establishes a hierarchical evidence framework, ranging from single-factor responsiveness to causal functional validation, to critically evaluate claims of photothermal epigenetic integration. We synthesize evidence across major regulatory modules (phyB–PIF, COP1–SPA, ELF3) and chromatin mechanisms (H2A.Z dynamics, histone modifications, DNA methylation). Our analysis reveals substantial convergence of temperature and light at signaling and transcriptional nodes, and robust evidence that each cue independently influences chromatin. However, direct evidence for a non-additive temperature × light chromatin state with demonstrated causal function is scarce, primarily due to the rarity of factorial experiments that can distinguish interaction effects from additive responses. The rice ACT1 study exemplifies rigorous causal epigenetic validation but investigates cold stress alone, highlighting the need for photothermal factorial designs. We extend this framework to crop improvement and vegetatively propagated species such as enset, where clonal reproduction offers opportunities to investigate epigenetic persistence. Crucially, we emphasize that persistence alone cannot establish adaptive inheritance—stability, phenotypic effects, transmission, and fitness consequences require independent evaluation. Resolving when combined temperature and light produce functionally consequential chromatin states will determine whether photothermal epigenetic regulation represents a bona fide mechanism of environmental plasticity or primarily a correlate of broader physiological responses. Although plants clearly exhibit heat-stress memory and integrate temperature and light through interconnected signaling and chromatin-regulatory networks, there is still limited direct evidence that combined heat and light exposure generates a distinct, integrated epigenetic memory that cannot be explained by the individual or interacting effects of each cue. More controlled factorial experiments are needed to determine whether combined temperature–light histories produce unique and persistent molecular states. This review provides a rigorous interpretive lens for evaluating evidence and identifies priority experimental approaches needed to establish or refute photothermal epigenetic integration.
Water-deficit severely limits cotton (Gossypium hirsutum) yield, yet how plants coordinate cuticle barrier formation with membrane lipid remodeling under water-deficit remains unknown. Through WGCNA of water-deficit transcriptomes, we identified GhLTP1, a lipid transfer protein, as the hub gene of the water-deficit-responsive module. GhLTP1 encodes a secreted protein localized to the epidermal cell wall and apoplast. Silencing GhLTP1 impaired water-deficit tolerance and caused intracellular accumulation of cutin precursors (O-acyl-ω-hydroxy fatty acids) coupled with reduced epicuticular waxes, particularly C29 and C31 n-alkanes, indicating that GhLTP1 is required for cuticular lipid accumulation. Notably, GhLTP1 silencing also triggered adaptive remodeling of cellular and thylakoid membrane lipids, by increasing unsaturation, very-long-chain fatty acid incorporation, and adjusting phospholipid-to-galactolipid ratios, consistent with cuticle defects exacerbating water loss while redirecting carbon flux from wax synthesis toward membrane lipids. We further show that GhMYB94 directly binds the GhLTP1 promoter to activate its expression, and GhMYB94-silenced plants phenocopy GhLTP1-RNAi lines. Our findings establish the GhMYB94–GhLTP1 module as a regulatory node that coordinates cuticle formation with membrane lipid remodeling to confer water-deficit tolerance in cotton.
Phosphate (Pi) homeostasis is critical for rice growth and development. Previous studies have shown that OsHMGB1, a chromatin-associated protein, positively regulates Pi accumulation by modulating the chromatin accessibility of phosphate starvation-responsive (PSR) genes. OsHMGB1 is induced under Pi-deficient conditions, and its protein level declines more rapidly under Pi-sufficient conditions, suggesting the existence of post-translational regulation. However, the mechanism underlying OsHMGB1 protein degradation remains unclear. Here, we demonstrate that the degradation of OsHMGB1 under Pi-sufficient conditions depend on the ubiquitin-26S proteasome pathway. OsHMGB1 interacts with OsPHO2, an E2 ubiquitin-conjugating enzyme and a key negative regulator of Pi signaling in rice. The protein abundance of OsHMGB1 is increased in the pho2 mutant due to reduced protein degradation. Furthermore, in vivo ubiquitination assays indicate that OsPHO2 facilitates the ubiquitination of OsHMGB1. Genetic analysis shows that the hmgb1pho2 double mutant partially suppresses the excessive Pi accumulation phenotype of pho2 in older leaves, indicating a genetic interaction between OsHMGB1 and OsPHO2 in Pi homeostasis. In addition, transcriptome analysis reveals that OsPHO2 and OsHMGB1 oppositely regulate the expression of a series of PSR genes, as validated by RT-qPCR for OsPT1, OsPT4, and OsRNS3. Collectively, these findings suggest that OsPHO2 maintains Pi homeostasis in rice partially by mediating the ubiquitin-dependent degradation of OsHMGB1.
Safflower has recently garnered increasing attention as a promising oil crop. The oil content of safflower achenes and the efficiency of subsequent oil extraction are significantly influenced by the hull/pericarp percentage (the ratio of the pericarp weight to the total achene weight). However, the genetic regulation mechanism controlling the hull development remains largely unexplored. In this study, we conducted phenotypic, metabolomic, and transcriptomic analyses on two safflower varieties with distinct hull percentage: normal hull (NH) and thin hull (TH). Our results revealed that the higher hull percentage in the NH was attributed to larger cell size and a thicker secondary cell wall (SCW) in the hypodermis. Metabolomic analysis showed that metabolites involved in lignin biosynthesis accumulated at higher levels during the early achene developmental stage in the NH. Transcriptomic data further indicated that differentially expressed genes (DEGs) were enriched in the phenylpropanoid biosynthesis pathway, with genes related to lignin biosynthesis exhibiting significantly higher expression levels in the NH variety than those in TH. Weighted gene co-expression network analysis (WGCNA) identified the turquoise module as the most correlated gene group, and transcription factors CtNAC043, CtNAC073a, and CtMYB46 as key candidate regulators of lignin biosynthesis during safflower hull development. Overexpression of CtNAC043, CtNAC073a, and CtMYB46 in transgenic safflower hairy roots significantly increased lignin content. Moreover, the expression of lignin biosynthesis genes was induced by CtNAC043, CtNAC073a, and CtMYB46. Notably, CtNAC073a and CtMYB46 were upregulated in CtNAC043-overexpressing hairy roots. These findings provide insights into the molecular mechanisms underlying lignin biosynthesis during safflower hull development and offer a foundation for breeding safflower varieties with desired hull percentage.
Arabidopsis thaliana seeds release large capsules of mucilaginous polysaccharides, whose adherence depends not only cellulose and hemicellulose biosynthesis but also their interactions. However, the key proteins regulating the connection within this polysaccharide network in the seed coat mucilage remain unclear and require further investigation. Through reverse genetics, we demonstrate that UBIQUITIN-PROTEIN LIGASE 3 (UPL3), a nucleus-localized, HECT E3 ligase protein, is required for the proper organization of cellulosic rays in seed coat mucilage. Disruption of UPL3 results in an enlarged seed mucilage halo due to elongated, diffusely stained cellulose structures, while not affecting the biosynthesis, structure, and chemical composition of pectic polymers. Furthermore, the distribution, rather than the biosynthesis, of xylans is significantly affected in upl3 seeds. Genetic analysis revealed that upl3 partially suppresses the mucilage defects of cellulose synthase 5 (cesa5) and completely rescues those of irregular xylem 7 (irx7), but not mucilage modified 5 (mum5) and irx14. Our findings suggest that UPL3 plays a regulatory role in xylan organization (and possibly in xylan synthesis in the irx7 background), and is essential for maintaining the proper co-organization of cellulose and xylans, which is likely important for their functional interplay in mucilage architecture.
Reynoutria japonica is both an important industrial source of resveratrol and a medicinal plant notable for its strong environmental adaptability. However, its responses to hydrogen sulfide (H2S), a key environmental signaling molecule, remain largely unexplored. Lysine β-hydroxybutyrylation (Kbhb) is a newly identified short-chain fatty acylation, but its role in regulating plant stress responses is unclear. Here, we performed the first proteome-wide profiling of Kbhb in R. japonica, identifying 17,102 high-confidence sites across 6185 proteins. Motif analysis revealed six conserved motifs. Functional enrichment analysis revealed that Kbhb-modified proteins are predominantly involved in core carbon and energy metabolism, with extensive modifications on enzymes related to acetyl-CoA flux, β-hydroxybutyrate metabolism, and the biosynthesis of resveratrol and anthraquinones. Under NaHS-imposed H2S stress, short-term exposure (3 h) triggered ROS accumulation and increased global Kbhb levels, particularly on histones H3 and H2A, suggesting an early signaling or chromatin-associated response; prolonged exposure reduced chlorophyll content and decreased Kbhb abundance. Pharmacological inhibition and in planta transient assays identified RjapHDA5L and RjapHDA9L as potential candidate regulators that regulate Kbhb modification's homeostasis. Furthermore, functional validation in a yeast heterologous expression system demonstrated that increasing Kbhb levels via β-hydroxybutyrate (BHB) or HDAC inhibitor treatment significantly enhanced the enzymatic activity of isocitrate dehydrogenase (IDH), a key TCA cycle enzyme. These findings indicate that Kbhb serves as a functional activator of core metabolic enzymes. Our study provides the first global landscape of Kbhb in R. japonica and establishes a molecular foundation for understanding how Kbhb dynamics regulate plant metabolism and epigenetic adaptation under environmental stress.
Fruit softening is key for strawberry quality and shelf-life. Cell wall structure is closely associated with texture, and its assembly/disassembly takes place during fruit development and ripening. Softening requires the coordinated spatiotemporal action of sets of cell wall modifying enzymes and therefore a transcriptional regulation is expected. Members of DOF (DNA-binding with One Finger) transcription factor (TF) family have been extensively studied in plant development and stress responses; however, their direct involvement in fruit softening remains poorly understood, particularly in non-climacteric fruits. In this study, the functional characterization of FchDOF1 from Fragaria chiloensis was accomplished and its role during fruit ripening was investigated. FchDOF1 localizes in the nucleus and displays a fruit-specific expression pattern, with a marked induction during ripening. Transient modulation of FchDOF1 expression in fruits reveals that overexpression significantly increases transcript levels of key cell wall-related genes, including FchPL, FchPG, FchXTH1 and FchEXP5, whereas RNAi-mediated silencing leads to reduction of FchPL and FchPG transcripts. Dual-luciferase assay further demonstrates that FchDOF1 specifically transactivates the FchPL promoter and EMSA assays confirmed its specific binding to DOF-recognition motifs within the FchPL promoter, supporting a direct regulatory interaction. In silico promoter analysis identified differential distributions of DOF-responsive elements among cell wall genes, suggesting promoter architecture-dependent regulatory specificity. Together, these results provide functional evidence of FchDOF1 acting as positive regulator of a subset of genes involved in cell wall disassembly during fruit ripening. This study expands current knowledge on functional diversification of DOFs and highlights their contribution to fruit softening.
Potato tuberization is controlled by photoperiod, temperature, and kinase-mediated signaling, but the mitogen-activated protein kinase kinase (MAPKK) components involved in the stolon-to-tuber transition remain poorly defined. In this study, a transcriptomic analysis of the potato cultivar 'Favorita' across eight stages of tuber formation identified 6, 2982, 723, 2579, 4673, 5028, and 6015 differentially expressed genes (DEGs) when comparing each of the seven developmental stages (Sto1, Sto2, Tu1, Tu2, Tu3, Tu4, and Tu5) to the early stolon stage (Sto), respectively. Among them, StMKK9 emerged as a candidate regulator of the transition from stolon to tuber. Subcellular localization showed that StMKK9 is predominantly localized in the nucleus. In the mid-late maturing potato cultivar 'Desiree', StMKK9 overexpression accelerated tuber initiation, increased tuber number and weight per plant, and altered the expression of early-maturity-related genes. StMKK9-overexpressing (StMKK9-OE) lines also exhibited alleviated cold-induced oxidative damage, as indicated by reduced ROS accumulation, MDA content, and electrolyte leakage. Yeast two-hybrid (Y2H) and luciferase complementation imaging (LCI) assays further showed that StMKK9 interacts with the cold-responsive transcription factor StCBF3. Together, these results identify StMKK9 as a positive regulator associated with potato tuber development and low-temperature adaptation. They support a presumptive regulatory module in which MAPK-associated signaling may connect StCBF3-mediated cold responses with photoperiod-related tuberization programs, providing a potential target for breeding early-maturing and climate-resilient potato cultivars.
The efficacy of indigenous microbial isolates (IMIs), namely Trichoderma atroviride AMUTATROV-31, T. asperelloides AMUTASPD-51, T. harzianum AMUTHZ-72, T. viride AMUTVR-61, Purpureocillium lilacinum AMUPL-31, Pochonia chlamydosporia AMUPC-31, Bacillus subtilis AMUBS-80, and Pseudomonas fluorescens AMUPF-80 isolated from the rhizosphere of green gram soils in Aligarh, India, was evaluated for their potential to mitigate root-knot disease caused by Meloidogyne incognita. The isolates were purified using single-spore or single-colony techniques and identified through morphological and molecular techniques. The effectiveness of seed treatments (4 mL kg-1 seeds; 2.0 to 3.2 × 106 CFU/mL), the spore suspension (SS), and culture filtrate (CF) of IMIs was evaluated under pot conditions. Inoculation with M. incognita (2000 J2 per pot) significantly reduced the plant growth (20-24%), yield (16-36%), functional nodules (18%), leghaemoglobin (21%), leaf pigments (28-36%), seed protein (21%) and leaf photosynthesis (28%) of green gram cv. SML-668, while increased the non-functional nodulation (19%), phenolic compounds (9-27%), defense enzymes (17-20%), stress-related metabolites (36-38%), stomatal conductance and transpiration (21-25%) over control. The treatments with the SS of P. lilacinum AMUPL-31 and P. chlamydosporia AMUPC-31 significantly reduced the galling (54-61%), egg mass production (36-58%), fecundity (34-44%), reproduction factor (46-64%), multiplication factor (58-77%), and soil population (46-66%) of M. incognita over control. The microbial treatments promoted the above variables of plant growth, grain yield (18-32%), nodulation (20-42%), and photosynthesis rate (14-20%) in nematode infected green gram compared to the infected control. The SS of T. harzianum AMUTHZ-72 and T. viride AMUTVR-61 demonstrated potential for commercial application, as these treatments significantly suppressed the root galling and nematode reproduction (14-70%), and improved the yield of green gram (12-28%).
Fatty acyl-acyl carrier protein thioesterases B (FATB) plays a critical role in regulating fatty acid chain length and saturation in plants. However, the function of FATB genes in tobacco remains poorly understood. In this study, we identified and characterized NtFATB, a FATB homolog in tobacco. Phylogenetic analysis revealed that NtFATB is closely related to FATB proteins from Nicotiana tomentosiformis and several Solanaceae species. Sequence alignment and domain prediction identified conserved catalytic residues and two characteristic acyl-ACP thioesterase domains. Expression analysis showed that NtFATB is predominantly expressed in trichomes and flowers, peaks at the seedling stage, and is strongly induced by cold stress. Subcellular localization demonstrated that NtFATB-GFP localizes to the endoplasmic reticulum. To investigate its biological function, we generated NtFATB RNAi lines and performed untargeted lipidomic profiling. Comparative analysis revealed that silencing NtFATB reduced the levels of 7 glycerophospholipids and 3 free fatty acids, and that triacylglycerols and ceramides were the lipid classes exhibiting the most robust changes. Moreover, the unsaturation level of several major membrane lipids, including DGDG, PA, PI, PG, PC, and LPA, was significantly decreased. Silencing of NtFATB caused lipid-class-specific and statistically significant changes in acyl chain length (ACL), with small increases observed in PA and PE, and small decreases in DGDG and MGDG. Functionally, NtFATB RNAi lines displayed heightened sensitivity to cold stress, as evidenced by earlier wilting, elevated reactive oxygen species (O2-) and malondialdehyde accumulation, and reduced activities of antioxidant enzymes (SOD, POD, and CAT). Consistently, the expression levels of core cold-responsive genes (NtICE1, NtCBF1-4, NtLEA14, NtCOR413, and NtGOLS1) were markedly suppressed in RNAi lines under cold stress. Collectively, our findings establish that NtFATB is essential for maintaining lipid homeostasis, and it positively regulates cold stress tolerance in association with enhanced antioxidant capacity and elevated expression of ICE-CBF-COR pathway genes in tobacco.
Osmotic stress severely limits plant growth and agricultural productivity, primarily by disrupting cellular water balance. Although the CBL-CIPK signaling network is recognized as a central mediator of abiotic stress responses, the functions of CiCIPKs from pecan (Carya illinoinensis) in osmotic stress tolerance remain largely unclear.In this study, two osmotic stress-responsive genes, CiCIPK8 and CiCIPK11, were cloned and functionally characterized via heterologous expression in Arabidopsis. Under mannitol-induced osmotic stress, transgenic lines exhibited significantly enhanced seed germination and root elongation compared with the wild-type (WT) plants. Physiological analyses revealed that overexpression of CiCIPK8 and CiCIPK11 improved osmotic stress tolerance by increasing antioxidant enzyme activities, reducing oxidative damage, and promoting the accumulation of compatible osmolytes, including proline, soluble proteins, and soluble sugars. Transcriptomic profiling, combined with quantitative reverse transcription-polymerase chain reaction (qRT-PCR) validation, demonstrated that CiCIPK8 and CiCIPK11 coordinately regulate multiple stress-responsive pathways, including plant hormone signal transduction, mitogen-activated protein kinase (MAPK) cascades, cell wall dynamics, and metabolic regulation. In addition, yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays demonstrated that CiCIPK8 interacted with CiCBL1 and CiCBL8, whereas CiCIPK11 specifically interacted with CiCBL1, suggesting the involvement of a Ca2+-dependent CBL-CIPK signaling module.In conclusion, these findings reveal that CiCIPK8 and CiCIPK11 function as positive regulators of osmotic stress tolerance by integrating ROS scavenging, osmotic adjustment, and transcriptional reprogramming. This study provides valuable insights into stress signaling in pecan and offers candidate targets for the molecular breeding of osmotic stress-tolerant woody crops.
Oil palm (Elaeis guineensis Jacq.) is the most productive oil-bearing crop globally, yet the molecular basis of mesocarp development and lipid accumulation remains poorly understood. Ultra-deep data-independent acquisition mass spectrometry (DIA-MS) was applied to characterize proteome dynamics in two contrasting genotypes, seedless (KS) and thin-shelled (TS), across five developmental stages (P1-P5) spanning fruit development to mature oil accumulation. Phenotypic analysis revealed higher mesocarp proportion and oil content in KS during late maturation. A total of 137,615 peptides corresponding to 12,163 protein groups were identified, providing a temporal proteomic landscape of mesocarp development. Multivariate analysis indicated that developmental progression was the primary contributor to proteomic variation, whereas genotype-associated differences increased during lipid accumulation. Differentially abundant proteins were mainly associated with carbohydrate metabolism, photosynthesis, proteolysis, antioxidant responses, and lipid biosynthesis. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and KOG analyses suggested extensive remodeling of metabolic networks, including developmental changes in photosynthesis-associated proteins and increased representation of lipid-associated pathways during maturation. Weighted protein co-expression network analysis identified 17 modules associated with developmental progression and lipid accumulation, highlighting candidate proteins involved in carbon metabolism, energy production, and cellular protection. Genes encoding selected hub protein candidates were further examined by RT-qPCR. Biochemical analyses supported these proteomic patterns, showing increased acetyl-CoA availability, enhanced antioxidant enzyme activities (SOD, CAT, APX, and GR), improved GSH/GSSG balance, and reduced oxidative damage in KS. Together, these findings provide a temporal proteomic and biochemical framework for understanding genotype-associated differences in oil accumulation and identify candidate metabolic networks for functional studies.
Mineral reserves in perennial tree organs buffer crop nutrition across seasons, but their magnitude and location vary among species. We hypothesized that deciduous almond, constrained to a narrow growing season, would commit to building large mineral reserves in perennial organs. In contrast, evergreen olive, able to take up minerals year-round, would rely on continuous uptake. We grew deciduous almond and evergreen olive trees under a range of nitrogen (N, 10-150 mg L-1), phosphorus (P, 1-15 mg L-1), and potassium (K, 10-150 mg L-1) mineral concentrations in the irrigation water and monitored seasonal mineral composition in roots, wood, and leaves. Trees were later destructively harvested to determine organ biomass and mineral mass. Almond and olive differed in their mineral allocation (Fig. 8 - graphical abstract). Almond leaf concentrations stabilized after peak vegetative growth, whereas olive leaves accumulated minerals continuously. Almond fruit concentrations were elastic across the mineral application range. Almond leaf concentrations were largely insensitive to mineral application. Olive root and leaf concentrations increased across the tested range. Almond biomass peaked, then declined at high N. Olive biomass scaled continuously with mineral application. Whole-tree mass balance revealed mineral reserves equivalent to up to 1.5 years of annual removal in almond, where most of the reserve turns over via senescence. In contrast, in olive, minerals accumulate in the roots and leaves up to 10 seasons, contradicting our initial hypothesis. Leaf mineral concentration reliably classified a tree's mineral status in olive, but only for K in almond. Precise almond fertilization may therefore require whole-tree mass-balance approaches rather than leaf diagnostics.
Carotenoid cleavage dioxygenases (CCDs) play critical roles in plant growth, development, and abiotic stress responses, yet their genome-wide identification and drought response mechanisms remain unexplored in wheat. In this study, 34 TaCCD genes were identified in wheat, distributed across 15 chromosomes and phylogenetically classified into five subfamilies. Gene structure analysis indicated that members within each subfamily shared conserved motifs and similar intron-exon arrangements. Cis-regulatory element analysis suggested the potential roles of these genes in stress adaptation, developmental processes, and hormone signaling. Moreover, prediction of tertiary structures and protein-protein interactions revealed unique structural features and potential interacting partners of the TaCCD proteins. In addition, TaNCED9a, a member of the TaCCD family, showed the highest transcript level in wheat roots among all detected TaCCD genes and was significantly induced by drought stress. Subcellular localization assay indicated that TaNCED9a was located in chloroplasts. Downregulation of TaNCED9a expression led to reduced drought resistance in wheat, accompanied by an accumulation of reactive oxygen species and a decrease in endogenous abscisic acid levels. Using yeast one-hybrid, dual-luciferase, and tobacco transient co-expression assays, the upstream regulatory factor TaDREB-7A was identified, which can regulate the expression of TaNCED9a. Additionally, a KASP molecular marker was developed to identify the superior haplotype TaNCED9a-HapI, which exhibited a significantly higher germination rate compared to TaNCED9a-HapII under drought conditions, and was predominant in wheat. These results offer valuable insights into the TaCCD gene family's response mechanisms to drought stress in wheat, simultaneously identifying promising genetic resources for enhancing drought tolerance through molecular breeding.
