CTR1 (Constitutive Triple Response 1) is a Raf-like serine/threonine protein kinase that functions downstream of ethylene receptors in plants. Despite this well-established role, the substrate spectrum of CTR1 kinases remains largely unexplored, with only a small number of direct substrates, such as EIN2 (Ethylene Insensitive 2) and MEK (Mitogen-Activated Protein Kinase), having been identified thus far. Using tomato as a research model, we identified SlSnRK2.4 (Sucrose Non-Fermenting 1-Related Protein Kinase 2.4) as a direct in vitro phosphorylation substrate of SlCTR1.3. Biochemical assays verified that SlSnRK2.4 physically interacts with SlCTR1.3 and undergoes direct phosphorylation by this kinase. These findings reveal a previously unrecognized CTR1-SnRK2 connection at the biochemical level in tomato, providing a foundation for future functional studies of this kinase-substrate pair.
Plant cuticles are lipophilic surface coatings that protect above-ground organs against environmental stress. An important cuticle component is wax, typically composed of very-long-chain hydrocarbons with terminal or in-chain functional groups. The current study aimed to provide a comprehensive analysis of leaf and stem cuticular waxes of Foeniculum vulgare, with special emphasis on characteristic wax components with in-chain hydroxyls or carbonyls. The stem and leaf waxes had similar compositions, including common compound classes such as fatty acids, aldehydes, alcohols and alkyl esters. The wax mixtures were dominated by ketones, with mainly C27 and C29 chains and keto groups on C-10 and C-12. Other compounds with mid-chain functionalities were identified by GC-MS analyses, including sets of (i) free as well as esterified ketoacids and primary/secondary ketols, and (ii) sec. alcohols, prim./sec. diols and sec./sec. ketols. The chain length and isomer patterns of these diverse wax compounds have implications for potential biosynthesis mechanisms.
Alternative splicing (AS) greatly expands transcriptome and proteome diversity in eukaryotes. Intron retention (IR) is the predominant AS type in plants, with critical roles in environmental adaptation and developmental control. Long viewed as splicing noise, IR is now established as a precisely regulated mechanism that modulates RNA stability, translation, subcellular localization, and protein function. Here, we review recent advances in plant IR research, covering technical progress in accurate IR identification using long-read and single-cell sequencing, cis- and trans-regulatory mechanisms, epigenetic coupling with transcription, and signal integration pathways. We highlight key functions of IR in flowering time regulation and stress responses, including the IR-nonsense-mediated mRNA decay (NMD) axis, functional protein isoforms, and nuclear transcript reservoirs for rapid stress memory. Finally, we discuss unresolved questions and future directions toward single-cell spatiotemporal dynamics, phase separation, and synthetic IR modules for crop improvement. This review provides an integrated framework for understanding IR as a central regulatory hub in plant post-transcriptional control.
Minimizing the accumulation of polycyclic aromatic hydrocarbons (PAHs) in crops is crucial for human health. While OsNRT2.3b overexpression reduced phenanthrene (Phe) in rice, the underlying mechanisms and its response to complex conditions remained unclear. This study first confirmed a negative correlation between OsNRT2.3b expression and Phe utilizing 16 varieties. Further, to investigate whether the pH-sensing site H167 of OsNRT2.3b is the key, the wild-type (WT), OsNRT2.3b-overexpression (Ox), and H167-mutated OsNRT2.3b overexpression (H167R) were employed to expose to Phe under different nitrogen (N) and pH. Individual N and pH experiments demonstrated that H167 played a decisive role in suppressing Phe, with Phe in H167R being significantly higher than Ox. Moreover, both -N compared to + N, and pH 4.0 compared to 7.0, increased Phe while simultaneously enhancing H167-mediated regulation. Phe in Ox was significantly lower than H167R by 24.6% (-N) and 22.3% (pH 4.0). Combined pH and N treatments revealed that H167 was most effective under acidic and N-deficient conditions. Under hydroponic pH 4.0 & -N and pot soil pH 5.4 & LN, Phe in the roots of Ox were 19.7% and 17.3% lower than H167R. Effect size analysis indicated that an acidic environment exerted a stronger effect on H167-mediated Phe reduction than N under the conditions tested. Notably, in grains, the pH-specific mediation by H167 decreased Phe by 16.6% under acidic and N-deficient soil. This study provides a basis for mitigating Phe accumulation and associated PAH-related risks in rice through molecular strategies and soil pH and N management.
Several laboratories have shown that even UV-naive model plants can acclimate to relatively low doses of supplemental broad-band UV radiation. On the other hand, responses to supplemental narrow-band 311 nm UV-B radiation (outside of the main absorption of UVR8) was applied to study antioxidants responses that were unlike our previous findings in plants exposed to broad-band UV-B or sunlight. The results suggest that signalling pathways activated by 311 nm radiation are distinct from those stimulated by other wavelengths. In this study we are examining the photoreceptor dependence of photosynthetic, antioxidants and epidermal pigment responses. To investigate this, we used photoreceptor deficient Arabidopsis thaliana on Columbia-0 and Wassilewskija backgrounds (uvr8-6 and uvr8-7). Both the broad- and narrow-band UV-B (∼7 kJ m-2 d-1 biologically effective dose) was applied to supplement 110 μmol m-2 s-1 photosynthetically active radiation for 4 h in the middle of the light period for four days. Our results showed that the photosynthetic yields were modified by broad-band UV, but they were unaffected to 311 nm radiation. However, both uvr8 mutants exhibited altered yields, although to varying degrees. Antioxidant enzyme results show that base values (in the absence of UV) are different not just between wild types, but between the mutants as well. After both UV treatments, enzyme activities displayed significant differences between the wild type and mutant groups. The outcome of our experiments highlights the necessity of studying action spectra of separate UV-B wavelengths in order to understand the complexity of responses to UV radiation.
Optimizing nutrient use efficiency while maintaining fruit quality is a central challenge for sustainable tomato breeding. This study leverages 53 chromosome segment substitution lines (CSSLs) derived from a cross between cultivated tomato and the wild relative Solanum habrochaites (LA1777) to explore the genetic and metabolic basis of adaptation to varying nutrient availability. The CSSLs were evaluated under high-input (HI) and low-input (LI) regimes and characterized for their agro-qualitative performances and primary metabolome profiles. Gas chromatography–mass spectrometry identified 63 compounds revealing a significant metabolic variability driven by input conditions. The plastic response of metabolites highlighted a shift in the accumulation of fruit's carbon- and nitrogen-related compounds: HI conditions enhanced amino acids levels, whereas the LI regime prioritized carbon-rich compounds, resulting in increased sugar content. Using a 51K SNP array, the population was mapped at high resolution across 2614 BINs covering 68% of the genome. Metabolic QTL (mQTL) analysis using stepwise regression and 1000 permutations identified 81 high-confidence loci, including pleiotropic regions on chromosomes 1, 3, and 4 that were associated with amino and organic acid metabolism, corresponding to conserved regions across wild tomato species. Analysis of candidate genes revealed that specific genomic regions responsive to LI govern abiotic stress resistance, while stable hotspots across nutrient regimes highlight the presence of highly canalized genetic hubs. The integration of mapping data with previously identified mQTL and transcriptomic studies validates the biological relevance of these identified loci in coordinating nutrient stress responses and fruit metabolic adaptation. Several lines demonstrated superior metabolite accumulation under stress and varying degrees of plasticity, making them ideal candidates for breeding. These findings provide a high-resolution framework for marker-assisted selection to develop climate-resilient, high-quality tomato cultivars optimized for sustainable agriculture.
Celery (Apium graveolens L.), an important green leafy vegetable in the Apiaceae family, is rich in various nutrients including dietary fiber. Cellulose is a major component of the celery cell walls and serves as the primary substrate for saccharification. Here, we characterized a cellulose synthase, AgCESA5, that positively regulates cellulose accumulation in celery. The AgCESA5 protein was localized in the plasma membrane. Homologous overexpression of AgCESA5 in 'Jinnan Shiqin' celery revealed increased cellulose content in the petioles of transgenic plants and a looser structural texture of parenchyma cell walls. Furthermore, we identified two R2R3-MYB transcription factors, designated AgMYB59 and AgMYB6, that physically bind to the AgCESA5 promoter, as validated by yeast one-hybrid and dual-luciferase reporter assays. Both AgMYB6 and AgMYB59 exhibited transcriptional activation activity and were localized to the nucleus. In the petioles of five celery cultivars, the relative expression levels of AgMYB59 and AgMYB6 showed expression patterns highly consistent with that of AgCESA5 at both 70 and 100 days after germination. Collectively, these results indicate that AgCESA5 is involved in celery cellulose synthesis. AgMYB59 and AgMYB6 may play important roles in cellulose biosynthesis as regulators of AgCESA5.
Upward migration of plant species is a widespread phenomenon in mountain ecosystems under current global warming. Mount Teide (Canary Islands) offers a unique opportunity to study it because of its harsh environment and longstanding botanical studies. Since 1800, current distribution of two endemic species of its summit-ecosystem has shifted very distinctly, more strongly in the case of the generalist Pterocephalus lasiospermus than in the case of the summit specialist Viola cheiranthifolia. In this study we compared the physiological and biochemical responses to environmental stresses (particularly high UV and visible radiation) at two elevations where they currently coexist (2300 and 3500 m). Both species showed different composition of UV-absorbing foliar compounds, but a similar high photosynthetic efficiency at elevated radiation, with no symptoms of photoinhibition even at high irradiance. At high elevation, V. cheiranthifolia showed higher chlorophyll content and remarkable carbon assimilation, with elevation-related changes in physiology but not in leaf mass area (LMA). By contrast, P. lasiospermus showed stress symptoms at higher elevation: higher photoprotection demand in terms of visible and UV radiation and a higher predawn-to-midday responsivity. These results reveal contrasting strategies for coping with extreme mountain environments: high specialization to summit conditions in the endemic violet and greater, but less effective, physiological plasticity in the recent colonizer.
Waterlogging constrains terrestrial plants by limiting gas diffusion and altering the hydraulic and chemical environment of roots. Yet it remains unclear which whole-plant responses arise from oxygen limitation alone and which require the broader physical context of excess water. Using high-resolution gravimetric lysimetry in tomato, we compared N₂-induced hypoxia under near-field-capacity conditions with root-zone waterlogging. N₂ injection reduced root-zone O₂ from approximately 18%-19% to below 1% and altered pH, redox potential, and mineral relations, but whole-plant transpiration declined only after sustained exposure. Waterlogging caused a faster, genotype-dependent transpiration decline in M82, IL11-4, and IL8-1. Adventitious-root emergence at the soil-air interface coincided with transient partial recovery of transpiration, whereas stronger adventitious-root development occurred in plants with larger transpiration losses. The renewed decline in transpiration after drainage was consistent with a partial contribution of surface-associated adventitious roots, although restoration of drainage also altered root-zone aeration, water availability, and primary-root conditions and therefore did not isolate their specific contribution. Together, the distinct response kinetics and root phenotypes show that rapid N₂-induced hypoxia did not reproduce the full waterlogging response. Adventitious roots were induced most strongly under severe stress and were temporally associated with partial, but not complete, recovery of whole-plant transpiration.
Soil salinity severely limits plant growth and productivity. Carex rigescens, a low-maintenance turfgrass species native to China, exhibits remarkable tolerance to abiotic stresses. Previous studies have highlighted the importance of the phenylalanine metabolic pathway in salt stress defense in C. rigescens; however, the specific mechanisms remain poorly understood. To elucidate the downstream metabolic and molecular components of this pathway, we analyzed flavonoid metabolism in two contrasting C. rigescens varieties-salt-sensitive 'Lvping No. 1' and salt-tolerant 'Lvping No. 2'-and functionally characterized the 4-hydroxyphenylpyruvate dioxygenase (CrHPPD) gene. Salt stress altered the abundance of several flavonoid metabolites, including 2'-hydroxygenistein, genistin, kaempferol, taxifolin, myricetin, and eriodictyol, identifying them as candidate salt-responsive metabolites. Naringenin and apigenin showed genotype- and tissue-dependent abundance patterns between the two varieties. We cloned CrHPPD, characterized its encoded protein, and found that CrHPPD-GFP displayed a cell periphery-associated fluorescence pattern in transient expression assays, although precise localization requires marker-based validation. CrHPPD expression was induced by NaCl and ABA, whereas PEG treatment elicited a weaker and more transient response. Furthermore, overexpression of CrHPPD in Arabidopsis thaliana enhanced germination rate, root length, catalase activity, and chlorophyll retention under salt stress, with the chlorophyll effect being most evident in OE6. Collectively, these findings reveal genotype- and tissue-specific flavonoid remodeling in C. rigescens under salt stress and demonstrate that CrHPPD positively contributes to salt tolerance when overexpressed in Arabidopsis. These results suggest that flavonoid metabolism and the HPPD-associated homogentisate/tocopherol antioxidant branch may represent two stress-responsive components of phenylalanine/tyrosine-derived metabolism, although their direct mechanistic connection requires further validation.
Nitrogen nutrition remains a major challenge for sustainable crop production, particularly under the current need to reduce fertilizer inputs. This study evaluated whether inoculation with arbuscular mycorrhizal fungi (AMF) improves the physiological, nutritional, and metabolic performance of escarole (Cichorium endivia L.) plants under nitrogen-limited conditions. Escarole plants were grown under two nitrogen regimes, full nitrogen supply and 25% of the optimal nitrogen concentration, either inoculated or non-inoculated with Rhizophagus irregularis. Plant growth, mineral composition, soluble sugars, proteins, pigments, and non-targeted LC-MS metabolomic profiles were analyzed. The results showed that mycorrhizal symbiosis mitigated the negative effects of nitrogen deficiency, maintaining shoot development and overall productivity, while reducing root growth, likely due to improved nutrient acquisition via fungal symbionts. Although total nitrogen content decreased under limiting conditions, AMF-inoculated plants exhibited higher levels of glucose, soluble proteins, and chlorophylls, alongside sustained or improved uptake of other essential minerals. Metabolomic profiling revealed AMF-dependent changes in the chemical profile and in multiple metabolic pathways, with 7165 metabolic features altered under nitrogen limitation and 58% of them increased in mycorrhizal plants. Principal component analysis further separated mycorrhizal and non-mycorrhizal plants, with PC1 explaining 49% of the chemical variability under nitrogen limitation. This metabolic response led to the accumulation of bioactive compounds such as riboflavin, L-citrulline, sarcosine, cyclic pyranopterin monophosphate, and 5-hydroxy-L-tryptophan. Overall, these findings demonstrate that AMF enhance plant performance under nitrogen limitation not by increasing nitrogen content per se, but by optimizing nitrogen utilization and reshaping metabolic networks associated with C/N balance.
Recent advances in plant breeding increasingly move beyond binary manipulation of gene expression toward the precise modulation of biological function. In this Perspective, we highlight how subtle genetic variation, particularly naturally occurring single nucleotide polymorphisms, can be leveraged to fine-tune enzyme kinetics, substrate specificity, and metabolic fluxes. Using the recently published example of spermidine hydroxycinnamoyl transferases (OsSHT1/2) in rice, we illustrate how natural haplotypes can modulate phenylpropanoid metabolism and pathogen resistance without compromising growth. While this example primarily operates through regulatory variation rather than direct modification of enzyme catalytic properties, it demonstrates the broader potential of allele-aware manipulation of metabolic pathways for crop improvement. We place this concept in a broader context by discussing how allele-aware breeding, which exploits existing natural variation, can be complemented by targeted genome editing approaches to recreate or refine beneficial variants. We further argue that integrating these strategies with data-driven breeding frameworks, combining genomics, phenomics, envirotyping, and machine learning, will enable predictive selection of optimal allele combinations across diverse environments. Importantly, we emphasize that the loss of natural variants during domestication reflects historical trade-offs rather than functional redundancy, and that such "lost" alleles can serve as valuable resources for modern crop improvement. Together, we propose that shifting the focus from enzyme quantity to enzyme quality provides a powerful conceptual and practical framework for plant breeding. Adoption of this approach will facilitate more precise, efficient, and sustainable crop improvement, bridging natural variation, molecular design, and predictive breeding in the era of precision agriculture.
Saline-alkaline stress imposes combined ionic and high-pH constraints, yet its integrated effects across developmental stages in rice remain poorly understood. This study evaluated a gamma-induced mutant line (SAT-9) derived from the salt-sensitive cultivar Pathum Thani 1 (PTT1) in comparison with Pokkali under controlled hydroponic conditions with defined salinity and saline-alkaline treatments. Responses were assessed at both seedling and booting stages, including ion homeostasis, physiological performance, oxidative status, gene expression, and yield-related traits. SAT-9 maintained low Na+ accumulation and stable Na+/K+ ratios across tissues, indicating effective ion homeostasis under stress. This was associated with coordinated upregulation of key transporters (OsHKT1;5, OsSOS1) and proton pump activity (OsAHA7), supporting maintenance of electrochemical gradients under elevated pH. In parallel, enhanced antioxidant capacity in SAT-9 limited H2O2 and malondialdehyde accumulation, preserving membrane integrity and photosynthetic efficiency. These responses were sustained at the booting stage, where SAT-9 maintained stable growth, reduced spikelet sterility, and minimal changes in grain yield and quality under saline-alkaline conditions. In contrast, PTT1 showed excessive Na+ accumulation, disrupted ionic balance, and severe oxidative damage, while Pokkali exhibited partial tolerance that declined under high pH. This study suggests that saline-alkaline tolerance is governed by the integration of ion transport, proton-coupled regulation, and redox homeostasis across developmental stages. This study provides an integrative, stage-resolved framework linking ion homeostasis, proton transport, and redox regulation to yield stability under saline-alkaline stress. The results highlight the potential of mutation-derived germplasm for developing rice adapted to saline-alkaline environments.
Glucose-6-phosphate dehydrogenase (G6PDH), a key regulatory enzyme of the oxidative pentose phosphate pathway, plays a central role during seed germination through the generation of NADPH and metabolic intermediates required for biosynthetic processes, including DNA synthesis. Therefore, cell proliferation must be linked to OPPP activity to ensure the supply of metabolites and reducing equivalents necessary for cell cycle initiation. Cell cycle progression is regulated by heterodimeric complexes of cyclins (Cyc) and cyclin-dependent kinases (CDKs), able to phosphorylate target proteins, thus we hypothesized that G6PDH could be an integral part of this regulatory network. To test this, we investigated the interaction of the cytosolic G6PDH from maize (ZmCy-G6PDH) with ZmCycs or ZmCDKs, its phosphorylation by Cyc/CDK complexes, and if this phosphorylation affects its activity. Pull-down assays showed that ZmCy-G6PDH interacted with CycD2; 2, CycD3; 1 or CycB2; 1 and CDKA; 1, but not with CycB1; 2 or CDKB1; 1. ZmCy-G6PDH was phosphorylated in vitro and semi-in vivo by different Cyc/CDK complexes exclusively in its homodimeric and homotetrameric conformations, leading to significant increase in ZmCy-G6PDH activity. Unexpectedly, in vitro treatment with the CDK-specific and ATP-competitive inhibitor RO-3306, also enhanced ZmCy-G6PDH activity. G6PDH activity increased as germination advanced in maize embryo axes, but a parallel treatment with RO-3306 decreased this activity, it being more evident at 24 and 36 h. Incidentally, the main reactive oxygen species (ROS) contention barriers, played by glutathione reductase and catalase, also showed reduced activity in embryo axes treated with the inhibitor. These results suggest a correlation between cell cycle machinery and the oxidative pentose phosphate pathway via ZmCy-G6PDH phosphorylation during germination, and this might become relevant to the contention of the oxidative stress.
Combined salinity and waterlogging severely constrain barley productivity, yet the genetic basis of inherited resilience to this stress combination remains poorly understood. Here, we investigated intergenerational and transgenerational stress memory in a barley diversity panel of 138 genotypes evaluated across three successive generations under four treatment-history groups (C→C, C→S, S→C, and S→S). Morphophysiological, ionic, biochemical, and recovery-related traits were measured, and stress-memory indices were derived from first-stage accession best linear unbiased estimates (BLUEs) to avoid unnecessary shrinkage before GWAS. Primary stress exposure caused strong reductions in growth, water status, chlorophyll retention, and photosynthetic efficiency, together with increased Na+ accumulation, membrane injury, and oxidative damage. In contrast, progeny derived from stressed lineages and re-exposed to stress showed improved biomass, relative water content, SPAD, Fv/Fm, and K+/Na+ ratio, accompanied by reduced Na+, electrolyte leakage, malondialdehyde and H2O2. Stress-memory indices confirmed intergenerational and cumulative repeated-stress gains, with low mean carryover penalties under non-stress conditions. Correlation analysis resolved a tolerance module associated with growth, photosynthetic stability, and ion homeostasis, and a damage module associated with Na+ accumulation, oxidative stress, and membrane leakage. Genome-wide association analysis using 19 K genotyping-by-sequencing SNPs identified 128 candidate marker-trait associations at the exploratory threshold of -log10(P) ≥ 4. The strongest candidate signals were detected for transgenerational memory gain and intergenerational memory, particularly for Fv/Fm, proline, K+, and K+/Na+. Haplotype and linkage disequilibrium visualization supported the major candidate loci and their local haplotype structure. Candidate genes implicated calcium signalling, redox regulation, transcriptional control and ion transport as key components of stress-memory regulation. These findings indicate that combined salinity-waterlogging stress memory in barley is an inherited and genetically complex response; however, formal broad- or narrow-sense heritability was not estimated in the present study.
The mediator complex is a central hub in transcriptional regulation in eukaryotes, yet its biological function in woody plants remains largely unexplored. Here, we systematically identified and characterized the Mediator (MED) gene family in the forest model tree Populus alba × P. glandulosa and focused on PagMED20, a conserved subunit of the head module. Genome-wide analysis revealed significant diversity in the evolutionary history, protein structures, and cis-regulatory elements of the PagMED family. Among these subunits, PagMED20 was selected for further study based on its preferential expression in leaves and nuclear localization, suggesting a potential role in leaf-associated processes. Overexpression of PagMED20 in poplar resulted in enhanced vegetative growth, including increases in plant height, stem diameter, and biomass accumulation. Physiological analyses showed that these growth changes were accompanied by increased chlorophyll content, net photosynthetic rate, and stomatal conductance. Cytological observations further revealed increased stomatal density and enhanced secondary xylem development in the overexpression lines. At the molecular level, PagMED20 overexpression was associated with coordinated changes in the expression of genes related to photosystem components, carbon assimilation, and stomatal development. In addition, chlorophyll fluorescence analysis indicated increased electron transport rates and reduced non-photochemical quenching under the tested conditions, suggesting altered photosystem II energy utilization. Together, these results indicate that PagMED20 overexpression is associated with coordinated changes in growth, photosynthetic performance, and stomatal traits in poplar. This study provides a foundation for understanding the potential roles of Mediator subunits in woody plant growth and physiology and identifies PagMED20 as a candidate gene for improving biomass production in forest trees.
