The team, led by Sanjeev Kumar of Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu, with collaborators from the Bhabha Atomic Research Centre in Mumbai and ICRISAT in Hyderabad, began with a high-yielding but cold-susceptible variety called GNG1958, released for India’s northwestern plain zone. Seeds were irradiated at the Bhabha Atomic Research Centre with gamma rays at doses of 250, 300, 350, 400, and 450 gray. Pivotal analysis identified two key doses—254.90 gray, slightly below the lethal dose for 50 percent of seeds, and 335.74 gray, the lethal dose itself—and 500 seeds were treated at each level.

From those irradiated seeds, the researchers developed 257 mutant lines in the second generation, or M2, comprising 100 lines from the lower dose and 157 from the higher one. These were sown in November 2023 at the university’s experimental farm, located at 32.68 degrees north latitude and 281 meters above sea level, where winter conditions are ideal for cold screening. Alongside the mutants, the team planted three reference lines: the untreated control GNG1958, the nationally susceptible check ICC12968, and the nationally cold-tolerant check ICC92944. Field data were recorded from 25 plants per mutant, while laboratory analyses of electrolyte leakage, chlorophyll, and carotenoids used samples from 10 plants each.

The screening protocol was deliberately harsh. Plants were only evaluated for cold tolerance after the susceptible check ICC12968 had died in the field, ensuring that the surviving mutants had faced genuinely lethal temperatures. Electrolyte leakage index, a measure of membrane damage, was calculated by measuring the electrical conductivity of leaf tissue soaked in deionized water, then again after autoclaving at 120 degrees Celsius to destroy all cells. The ratio between the two readings reveals how much of the cell’s internal electrolytes had already leaked out through damaged membranes—a lower percentage means the membrane stayed intact under cold stress.

Chlorophyll and carotenoid pigments were extracted using dimethyl sulphoxide and quantified by spectrophotometry at 665, 645, and 454 nanometers, following established equations for chlorophyll a, chlorophyll b, and carotenoids. In the field, each mutant was scored on a cold tolerance rating scale from 1 to 9, where 1 means no damage and 9 means most seedlings dead or dying. A frost resistance ratio was also calculated by dividing the number of plants surviving at harvest by the number present after emergence but before the first frost, with values approaching 1 indicating resistance.

Across two growing seasons—the Rabi seasons of 2023–24 and 2024–25—the pooled data revealed 50 promising cold-tolerant mutants. Of these, 26 showed electrolyte leakage below 10 percent combined with carotenoid content above 0.50 milligrams per gram of fresh weight, a combination the authors interpret as resistance to cold stress at the vegetative stage during periods of high cold. Three mutants stood out as highly promising: 335.74 Gy-Ch-M-45 with an electrolyte leakage index of just 6.79 percent, total chlorophyll of 2.48 milligrams per gram, and a seed yield of 48.78 grams per plant; 335.74 Gy-Ch-M-155 with leakage of 7.24 percent and the highest yield among the three at 59.22 grams per plant; and 335.74 Gy-Ch-M-66 with leakage of 7.30 percent and chlorophyll of 2.49 milligrams per gram.

For comparison, the susceptible check ICC12968 recorded an electrolyte leakage of 42.77 percent and a cold tolerance rating of 6, meaning it succumbed to cold injury in the field. The control GNG1958 leaked 31.23 percent and scored 4 on the rating scale, while the national tolerant check ICC92944 leaked 25.94 percent and scored 2.50. Against these benchmarks, the elite mutants performed dramatically better, with several showing cold tolerance ratings of 1.0 to 1.5—scores indicating essentially no visible damage—while simultaneously maintaining higher chlorophyll and carotenoid levels than the parent variety.

Correlation analysis revealed a tight relationship between membrane integrity and visible cold damage: the electrolyte leakage index and the cold tolerance rating were positively correlated with a coefficient of 0.87, significant at the 1 percent level. Seed yield correlated positively with total chlorophyll, carotenoid content, and frost resistance ratio, but negatively with electrolyte leakage and cold tolerance rating. In practical terms, mutants that kept their membranes sealed and their pigments intact also produced more seed—a coherent physiological signature of cold tolerance rather than a coincidence of unrelated traits.

Biochemical profiling of the top 15 mutants in the third generation added another layer. Proline, an osmoprotectant that stabilizes proteins and membranes under stress, ranged from 0.90 to 3.44 micromoles per gram of fresh weight, with mutant 335.74 Gy-Ch-M-153 accumulating the most. Sugar content, measured by the anthrone method, ranged from 6.64 to 46.16 milligrams per gram, with 335.74 Gy-Ch-M-45 and 335.74 Gy-Ch-M-65 exceeding even the cold-tolerant check. Protein percentage, estimated non-destructively by near-infrared spectroscopy, varied narrowly between 21.26 and 23.08 percent. Two mutants in particular—254.90 Gy-Ch-M-01 and 254.90 Gy-Ch-M-32—showed higher sugar, proline, and protein content than the control along with superior values on eight and nine cold tolerance parameters respectively, plus higher seed yield.

Genetic analysis of the mutant population revealed that heritability was high for all traits but genetic advance was low, except for seed yield, where high heritability paired with high genetic advance indicated additive gene action amenable to selection. For the cold tolerance traits themselves, the pattern of high heritability with low genetic advance pointed to non-additive gene action—epistasis, dominance, and gene-by-environment interaction—meaning direct selection on those traits alone would yield disappointing gains. The authors suggest that the wide variation in seed yield among mutants may reflect stable gene mutations that will persist in subsequent generations, making yield itself the more reliable selection target.

The broader context makes the finding significant. Chickpea is the second most important cool-season food legume in the world after dry beans, and India is its largest producer, consumer, and importer. Yet productivity hovers around one ton per hectare, constrained by biotic and abiotic stresses. Cold is a particularly costly problem: winter-sown chickpea in northern South Asia and parts of Australia faces temperatures between 0 and 15 degrees Celsius during the reproductive stage, causing flower and pod loss and reducing yield potential by 30 to 40 percent. Economic losses from cold in chickpea have been estimated at 186 million US dollars, compared to 1.3 billion from drought and heat and 354 million from salinity. Climate change may intensify cold spells in temperate regions, making cold-resilient cultivars more urgent, not less.

Mutation breeding offers a way forward precisely because cultivated chickpea has a narrow genetic base. Unlike wide crosses with wild relatives, mutagenesis can be applied directly to elite cultivars without dragging along unwanted linked traits. India has a long history with the technique—the first micro-mutant chickpea varieties in the world were developed at the Indian Agricultural Research Institute in New Delhi and released in 1985 following gamma irradiation. The new mutants from Jammu join that lineage, and the authors propose them as donor parents for introgressing cold tolerance into well-adapted varieties, as parental genotypes for mapping populations, and as subjects for differential gene expression studies aimed at identifying the genes underlying cold tolerance.

What makes the work notable is the multi-year, multi-generation discipline behind it. Rather than declaring victory after one season, the team carried 217 of the original 257 mutants into a second winter, re-screened them in the M3 generation, and only then shortlisted 50 putative mutants and, from those, 15 elite lines based on pooled mean performance across both years and both field and laboratory traits. The 15 top performers—including 254.90 Gy-Ch-M-01, 254.90 Gy-Ch-M-09 with a yield of 74.20 grams per plant, and 335.74 Gy-Ch-M-140 at 72.69 grams per plant—were stable across generations, which is exactly what a breeding program needs from a donor parent.

The study was funded by the Board of Research in Nuclear Sciences of the Government of India, grant number 3758, reflecting the institutional partnership between the agricultural university and the Bhabha Atomic Research Centre that made the irradiation work possible. As climate variability tightens its grip on subtropical agriculture, the Jammu mutants offer a concrete, field-validated resource: lines that have already survived the coldest nights the northwestern Himalayas can deliver, and that carry the physiological and biochemical machinery to keep photosynthesis running, membranes sealed, and seeds filling when the temperature drops. For breeders worldwide trying to push chickpea yields upward against a backdrop of increasingly erratic weather, these gamma-induced lines may prove to be exactly the genetic raw material they have been looking for.

Subject of Research: Gamma-induced cold-tolerant chickpea mutants screened across physiological, biochemical, and agronomic traits in northwestern India

Article Title: Characterization of physiological, biochemical, and agronomical traits of elite induced chickpea mutants from the northwestern Himalayan region of the UT of J & K, India

Article References: Kumar, S., Saha, A. J., Padha, R., Chand, G., Kashyap, S. C., Srivastava, R. K., Singh, A. K., & Mahajan, A. (2026). Characterization of physiological, biochemical, and agronomical traits of elite induced chickpea mutants from the northwestern Himalayan region of the UT of J & K, India. Frontiers in Plant Physiology, 4, Article 1766618. https://doi.org/10.3389/fphgy.2026.1766618

Image Credits: AI Generated

DOI: 10.3389/fphgy.2026.1766618

Keywords: chickpea, cold stress, mutation breeding, gamma irradiation, electrolyte leakage, chlorophyll, carotenoids, proline, frost resistance, seed yield, Cicer arietinum, plant physiology

The DVL family belongs to a broader class of small peptide-encoding genes that have attracted growing attention in plant biology. Unlike classic protein-coding genes that produce enzymes or structural components, small peptide genes typically produce short molecules that act as signals, relaying information between cells and fine-tuning developmental programs. In Arabidopsis, the founding member of the family was named ROTUNDIFOLIA3, later grouped with DEVIL, because mutations in these genes visibly reshape leaves and organs. Since then, DVL-like genes have been identified in many plant species, where they influence everything from organ shape to responses to environmental challenges. What remained unclear was how many of these genes rice carries, where they sit in the genome, and whether their natural variation has any bearing on the traits farmers care about.

To answer those questions, the team carried out a systematic bioinformatic survey of the rice reference genome. The search identified five DVL genes, which the authors designate as members of the OsDVL family. Five may sound like a small number, but small peptide families are often compact, and each member can carry substantial functional weight. The researchers characterized the physical properties of the encoded peptides and examined the structure of each gene, establishing a baseline catalog that other rice geneticists can now build on. Identifying the complete membership of a gene family is a deceptively simple but essential step: without it, expression data and genetic associations can be misassigned or missed entirely.

With the family cataloged, the team turned to the regulatory regions upstream of each gene. Promoters, the DNA sequences that sit before a gene and control when and where it is switched on, are rich in short motifs called cis-regulatory elements that serve as binding sites for transcription factors. Scanning the OsDVL promoters revealed a wealth of stress-responsive elements, including motifs typically associated with responses to salinity, cold, drought and hormonal signals. This regulatory architecture suggests that rice DVL genes are not passive developmental housekeepers but are wired to respond when the plant encounters adverse conditions. In an era when saline soils and erratic temperatures increasingly threaten rice paddies across Asia, that wiring is of more than academic interest.

Expression profiling reinforced that suspicion. By examining transcript levels across different tissues, the researchers found that OsDVL genes show tissue-specific patterns, meaning individual family members are active in particular organs or developmental stages rather than uniformly throughout the plant. More strikingly, when the team looked at how selected OsDVL genes behaved under salt and cold stress, transcript levels changed significantly. Genes whose expression rises or falls under stress are prime candidates for mediators of tolerance, because a plant’s ability to survive salinity or chilling often depends on how quickly and appropriately it reprograms its gene activity. The data position OsDVL genes as plausible contributors to abiotic stress responses, though the authors are careful to frame this as a role suggested by the evidence rather than one proven by direct functional experiments.

The most ambitious part of the study, and the part with the clearest implications for breeding, is the haplotype analysis. A haplotype is a specific combination of genetic variants at a locus, inherited together, and haplotype diversity within a species reflects its evolutionary history and the selective pressures breeders have applied. The researchers mined sequence data from 3,010 rice accessions, a panel that spans the remarkable genetic breadth of cultivated rice, including the indica and japonica subspecies and landraces collected across decades of germplasm exploration. At the OsDVL loci, they uncovered substantial allelic diversity, with multiple distinct haplotypes segregating across the panel.

That diversity tells a story. The distribution of haplotypes across the accession panel appears to have contributed to population differentiation, meaning different rice groups carry different favored versions of these genes. Even more intriguingly, the patterns suggest that genetic improvement during rice breeding has left its mark on OsDVL loci. When breeders select plants for higher yield, better grain quality or stress tolerance, they inadvertently select the haplotypes carried by those plants. Detecting the signature of such selection at DVL loci implies that these small peptide genes have been riding along with, or possibly contributing to, the agronomic progress of the past century of rice improvement.

The connection to agronomy became explicit when the team linked haplotypes to measurable traits. At most OsDVL loci, the predominant haplotypes were significantly associated with key yield-related traits, the suite of characteristics such as grain number, panicle architecture and plant height that together determine how much harvestable rice a plant produces. In practical terms, this means a breeder could, in principle, look at which haplotype a rice line carries at an OsDVL locus and gain information relevant to its yield potential. That is the essence of marker-assisted breeding, a technique in which DNA markers are used to guide the selection of plants carrying favorable alleles long before those traits can be seen in the field. The authors highlight DVL-associated haplotypes as potential targets for exactly this kind of marker-assisted selection.

It is worth pausing on why small peptides keep surfacing in studies of yield and stress. Development and environmental response are not separate programs in a plant; they are deeply intertwined. A rice plant facing salinity must decide whether to keep growing, slow down, or redirect resources, and those decisions are mediated by signaling networks in which small peptides act as messengers. A peptide that influences cell proliferation or organ growth could easily have downstream consequences for how many grains a panicle bears, and the same signaling pathway could be co-opted under stress to adjust growth to available resources. The OsDVL findings fit this logic: a compact gene family, stress-responsive promoters, tissue-specific expression, and haplotypes correlated with yield traits all point toward genes that sit at the junction of development and adaptation.

The study also illustrates a methodological trend reshaping plant genetics. Rather than focusing on a single gene in a single variety, the work integrates genome-wide identification, promoter analysis, expression profiling and population-scale haplotype mining into one pipeline. This systems-level approach allows researchers to move from gene discovery to breeding relevance within a single study, and it depends on the availability of massive public datasets such as the 3,010-accession rice panel, which has become a workhorse for resequencing-based association analyses. As similar panels accumulate for other crops, expect more gene families to receive the same treatment, revealing hidden layers of diversity that single-reference-genome studies cannot see.

There are, of course, open questions. The evidence linking OsDVL genes to stress tolerance and yield is associative and expression-based; direct functional validation, through gene knockout, overexpression or CRISPR editing followed by phenotyping under stress, will be needed to establish causation and to determine which of the five genes matters most for which trait. The molecular receptors and downstream partners of the OsDVL peptides in rice also remain to be identified. Still, the study delivers a valuable and actionable starting point. It catalogs the family, maps its regulatory logic, documents its natural diversity across three thousand rice varieties, and flags haplotypes that breeders can begin testing. For a gene family that has lived in the shadow of rice’s better-known developmental regulators, the DEVIL genes have now stepped into the light, and the path from small peptide to bigger harvests looks considerably shorter than it did before.

Subject of Research: Identification, expression and haplotype diversity of DEVIL (DVL) small peptide-encoding genes in rice

Article Title: Identification and haplotype diversity analysis of DVL family genes in rice

Article References: Liu, X., Zhou, Y., Zhu, M., Lv, W., Wang, C., Zhang, M., Tang, C., Yang, S., & Shi, Y. (2026). Identification and haplotype diversity analysis of DVL family genes in rice. BMC Plant Biology. https://doi.org/10.1186/s12870-026-10051-5

Image Credits: AI Generated

DOI: 10.1186/s12870-026-10051-5

Keywords: rice, DVL genes, DEVIL family, small signaling peptides, haplotype diversity, abiotic stress, salt stress, cold stress, yield traits, marker-assisted breeding, genetic diversity, plant genetics

The name SPX is an acronym drawn from three distant relatives that share the domain: the Suppressor of Yeast gpa1, the yeast phosphate regulator PHO81, and the human retrovirus receptor XPR1. That spread alone hints at how ancient and versatile the module is. The SPX domain is a hydrophilic stretch of roughly 165 amino acids at the N-terminus of these proteins, and it has been conserved across eukaryotic lineages precisely because it solves a universal problem: how does a cell know how much inorganic phosphate it contains? In plants, the answer turned out to be elegant. SPX domains do not primarily bind phosphate itself. Instead, they recognize inositol pyrophosphates, a family of high-energy signaling molecules derived from inositol hexakisphosphate, the familiar phytic acid that seeds stockpile as a phosphorus reserve.

The key messenger is InsP8, produced when ITPK1 converts InsP6 to InsP7 and the kinases VIH1 and VIH2 add a pyrophosphate group. Structural studies have shown that the SPX domain folds into a three-helix bundle topped by an N-terminal alpha-hairpin, creating a strongly basic, positively charged surface pocket that is electrostatically complementary to these polyanionic molecules. The pocket can bind free phosphate only weakly, with affinities in the low millimolar range, but inositol pyrophosphates bind with nanomolar to micromolar affinity. In effect, the SPX domain ignores the noise of bulk phosphate and listens to a dedicated chemical signal whose concentration tracks cellular phosphorus status. When InsP8 levels rise under phosphate-replete conditions, it docks into the SPX pocket and triggers a conformational change that enables SPX proteins to grab their downstream targets.

Those targets are the PHR transcription factors, the master switches of the phosphate starvation response. Under sufficient phosphate, InsP8-loaded SPX proteins bind the coiled-coil domain of PHR1 or its equivalents, sequestering them in the cytoplasm or blocking their binding to the P1BS DNA motif in the promoters of starvation-induced genes. When phosphate runs short, ATP-dependent kinase activity falls, InsP8 production drops, the SPX-PHR complex falls apart, and PHR dimers stream into the nucleus to switch on an entire adaptive program: high-affinity phosphate transporters, the transporter facilitator PHF1, the microRNA miR399, the non-coding RNA IPS1, and genes that remodel root architecture and secrete phosphatases into the soil. Recent crystal structures of the rice SPX1-PHR2 complex showed the mechanism in atomic detail: InsP6 binding stabilizes a helix that sterically interferes with PHR2 dimerization, while SPX1 simultaneously contacts the MYB DNA-binding domain, competing directly with the promoter. Mutations that disrupt the InsP6-binding site abolish the interaction and cause plants to over-accumulate phosphate.

The review also traces how this core module diversified over roughly a billion years of plant evolution. Phylogenetic analysis across chlorophyte algae, streptophyte algae, bryophytes, ferns, gymnosperms, and angiosperms resolves four subfamilies defined by their C-terminal domains. The SPX-only and SPX-RING lineages trace back to the green alga Volvox carteri, while SPX-EXS proteins first appear in streptophyte algae and SPX-MFS proteins emerge only with the bryophytes, the first land plants. Each subfamily took on a distinct job. SPX-only proteins such as Arabidopsis SPX1 and SPX2 are the sensors. SPX-EXS proteins, exemplified by PHO1, load phosphate into the xylem for the long journey from root to shoot; a recent cryo-electron microscopy structure of Arabidopsis PHO1;H1 bound to InsP6 revealed that inositol phosphate binding promotes dimerization and activates transport. SPX-MFS proteins such as the vacuolar transporters PHT5/VPT stash phosphate in the vacuole, using a clever intramolecular switch in which InsP binding releases the SPX domain’s autoinhibition of the transport domain. SPX-RING proteins such as NLA act as E3 ubiquitin ligases that tag plasma membrane phosphate transporters for degradation when supplies are ample.

Even within the sensing subfamily, evolution has produced surprising variety. In rice, SPX4 holds PHR2 in the cytoplasm until phosphate starvation triggers its rapid destruction by RING-type E3 ligases, releasing the transcription factor. In tomato, the picture changes again: the interaction between SlSPX5 and the transcription factor SlPHL1 is constitutive and independent of phosphate, but under starvation a cis-natural antisense RNA transcribed from the SPX5 locus causes RNA polymerase II to pause, recruiting the cleavage and polyadenylation machinery and truncating the SPX5 transcript. Less SPX5 protein means less sequestration of PHL1, and the starvation response proceeds. It is a post-transcriptional twist on the canonical theme, and it illustrates how the same regulatory logic can be rewired at entirely different levels of gene expression.

Perhaps the most consequential part of the review is its account of SPX proteins as integration hubs rather than single-purpose sensors. In rice, the nitrate transporter NRT1.1B recruits an E3 ligase to degrade SPX4, simultaneously releasing both PHR2 for phosphate signaling and NLP3 for nitrate signaling, thereby coupling nitrogen and phosphorus nutrition in one cascade. A 2025 study pushed this further: NRT1.1B turns out to bind not only nitrate but also the drought hormone abscisic acid, and under low-nitrate conditions this binding promotes formation of an NRT1.1B-SPX4 complex that frees the transcription factor NLP4 to activate ABA-responsive genes. Nitrate availability, phosphate status, and drought signals converge on a single SPX-dependent node, and the mechanism appears conserved across Arabidopsis, maize, and wheat.

The integrative reach extends to symbiosis and immunity. Most flowering plants partner with arbuscular mycorrhizal fungi, which trade soil phosphate for plant carbon. When phosphate is plentiful, SPX proteins repress PHR activity and with it the mycorrhiza-specific phosphate transporters, suppressing fungal colonization; knockout mutants are colonized more heavily, overexpressors barely at all. In Medicago, SPX1 and SPX3 go further, promoting strigolactone synthesis under low phosphate and later acting as timers that limit the lifespan of the arbuscules, the fungal feeding structures inside root cells. In rice, SPX1 and SPX2 bind the brassinosteroid signaling factor BZR1, and their availability depends on whether PHR2 has sequestered them. Under ample phosphate, BZR1 stays active, promoting growth and the antifungal phytoalexin sakuranetin; under starvation, freed SPX proteins restrain BZR1, trading growth and basal immunity for phosphate scavenging. Cold responses are implicated too: OsSPX1 overexpression improves chilling tolerance, cotton GhSPX9 is required for cold adaptation, and in maize the E3 ligase NLA coordinates jasmonate-mediated cold signaling with phosphate transporter turnover under low temperature.

The translational implications are considerable. Because SPX proteins govern how efficiently crops acquire, move, store, and recycle phosphorus, they are obvious targets for engineering phosphorus use efficiency. The review outlines candidate strategies: tuning vacuolar storage via SPX-MFS proteins, silencing negative regulators such as NLA to prolong transporter activity, editing the NRT1.1B-SPX4 module to balance nitrogen and phosphorus fertilizer use, and relaxing SPX-mediated repression to boost mycorrhizal colonization on poor soils. The authors are candid about the obstacles. Constitutive activation of the starvation response carries metabolic costs and potential pleiotropy, demanding inducible or tissue-specific expression; SPX gene families are redundant, so multiplexed genome editing will be needed; and most functional work has been done in Arabidopsis and rice rather than the staple crops that matter most for food security. Post-translational modifications of SPX proteins remain almost unexplored, and the structural basis for functional differences among subfamilies is only beginning to emerge from crystallography and cryo-EM.

What emerges from the synthesis is a unifying model the authors call the SPX-centered regulatory network: a layered architecture in which InsP8 acts as the currency of phosphate information, SPX-only proteins read it and gate the PHR transcriptional program, and the effector subfamilies execute transport, storage, and recycling from the organelle to the whole plant, all while feeding signals back into nitrogen, stress, symbiotic, and immune pathways. For a problem as old as land plants themselves, the solution has turned out to be remarkably modular, and remarkably exploitable. If structure-guided engineering of SPX modules delivers even part of its promise, the humble domain named for a yeast suppressor, a yeast kinase inhibitor, and a mammalian virus receptor could become one of the most important tools in the effort to grow more food with less phosphorus.

Subject of Research: SPX domain proteins as phosphate sensors and multifunctional signaling hubs in plants

Article Title: The SPX protein family in plants: from phosphate sensors to multifunctional signaling hubs

Article References: Ge, S., Yuan, K., & Lei, M. (2026). The SPX protein family in plants: from phosphate sensors to multifunctional signaling hubs. Stress Biology, 6(1), Article 36. https://doi.org/10.1007/s44154-026-00307-3

Image Credits: AI Generated

DOI: 10.1007/s44154-026-00307-3

Keywords: SPX proteins, phosphate signaling, inositol pyrophosphates, InsP8, PHR transcription factors, phosphorus use efficiency, arbuscular mycorrhizal symbiosis, nitrogen-phosphorus crosstalk, plant immunity, cold stress, vacuolar phosphate storage, plant evolution

The review, led by Ikram Ullah and Naveed Ahmad, who contributed equally, together with Aashaq Hussain Bhat, Adil Hussain, Fan Wei, and Wu Hongzhi, draws together evidence spanning cold stress perception, signal transduction, transcriptional regulation, metabolic reprogramming, and phytohormone interactions in pepper. Its central argument is that pepper’s intrinsic cold tolerance has been constrained not by the absence of defensive machinery, but by its incomplete deployment. The crop possesses endogenous cold-responsive regulators such as CaNAC035 and CabHLH035, along with antioxidant defense systems, yet these remain insufficiently activated when temperatures drop, leaving plants exposed to damage they are nominally equipped to resist.

At the heart of the problem lies a cluster of biological complications that conventional breeding has struggled to disentangle. Key cold-response regulators show low transcriptional activation under stress, meaning the genetic instructions for defense are present but poorly executed. Functional redundancy among cold-responsive genes further complicates matters: multiple genes can substitute for one another, so knocking out or enhancing a single factor often produces negligible phenotypic change. Cold tolerance itself is polygenic, governed by many genes of small effect scattered across the genome. Layered on top of these complexities are low genetic diversity within elite pepper germplasm and linkage drag, the troublesome tendency of undesirable traits to hitchhike along with desirable ones during crossing. Together, these obstacles have stymied decades of conventional cold-resistance breeding.

To overcome them, the authors champion what they call Precision Breeding 2.0, an integrated innovation that couples multi-omics-based target identification with next-generation genome-editing techniques. Rather than editing one gene at a time, this approach allows precise and multiplex engineering of complex, interconnected regulatory networks. The distinction matters because cold tolerance is not a switch but a symphony: calcium signatures, phosphorylation cascades, hormone signals, and transcriptional circuits must all be tuned in concert. By mapping these networks through transcriptomics, proteomics, and metabolomics before editing, breeders can identify the highest-leverage nodes and modify several of them simultaneously.

Among the most promising strategies highlighted in the review is the engineering of the DREB/CBF pathway, the canonical cold-response cassette first characterized in Arabidopsis and now recognized across crops. In pepper, cold-inducible CBF transcription factors interact with homeodomain leucine zipper proteins, and genome-wide analyses of the C-repeat binding factor family in related Capsicum species have revealed functional diversity in low-temperature responses. The review also outlines allele-specific editing, which lets researchers swap inferior versions of genes for superior ones without altering anything else in the genome, and targeted disruption of negative regulators, transcriptional brakes that suppress cold responses and whose removal can unlock latent tolerance.

The molecular detail underpinning these strategies is impressive. Recent work has shown that CaSnRK2.4-mediated phosphorylation of CaNAC035 regulates abscisic acid synthesis in pepper under cold stress, linking the ABA hormone pathway directly to cold acclimation. The bHLH transcription factor CabHLH035 promotes cold resistance and reactive oxygen species homeostasis, while the NAC factor CaNAC064 has been identified as a cold tolerance regulator, and CaMYB80 enhances cold tolerance by directly targeting CaPOA1. Newer discoveries continue to expand the toolkit: CaPDX1, a novel protein that positively regulates cold tolerance through interaction with CaSnRK2.4, and CaDoF10, a DNA-binding-with-one-finger transcription factor recently shown to act as a positive regulator of cold stress tolerance. CBL-interacting protein kinase CaCIPK13 reinforces defense mechanisms against cold, and the dehydrin CaDHN3 contributes to membrane stabilization, while CaPIF8 links light and cold signaling through phytochrome-interacting factors.

Beyond transcription factors, the review emphasizes metabolic reprogramming as a critical dimension of cold adaptation. Soluble sugars, glycine betaine, proline, and antioxidant flavonoids accumulate under chilling conditions to protect membranes and scavenge reactive oxygen species. Comparative transcriptomic and metabolomic analyses of cold-tolerant and cold-sensitive pepper species have pinpointed essential metabolic pathways that distinguish resilient genotypes from vulnerable ones. Melatonin application has been shown to bolster photosynthetic performance and redox homeostasis in cold-stressed pepper seedlings, and exogenous glycine betaine improves tolerance of combined low-temperature and low-light stress by enhancing antioxidant capacity. These findings suggest that editing genes governing compatible solute biosynthesis and flavonoid accumulation could complement transcription-factor engineering.

Hormonal crosstalk emerges as another central theme. Abscisic acid and low temperatures act synergistically to induce CBF/DREB1 expression in other species, and ABA core signaling components are active in pepper seed responses. The review situates cold responses within a broader web of phytohormone interactions involving jasmonic acid, cytokinins, and brassinosteroids, each modulating stress outcomes in tissue- and timing-specific ways. Circadian regulation adds yet another layer, since cold responses are gated by the clock, meaning that the same genetic intervention may yield different results depending on the time of day it is deployed, a consideration the authors argue should inform both experimental design and field deployment.

Accelerating the pipeline from discovery to delivery, the review proposes integrating transcriptomics, proteomics, metabolomics, high-throughput phenomics, and speed breeding to compress the identification, validation, and deployment of superior alleles into a fraction of the traditional timeline. High-throughput phenomics allows researchers to quantify chilling injury, membrane leakage, chlorophyll fluorescence, and recovery kinetics across thousands of seedlings, while speed breeding shortens generation cycles so that edited lines can be advanced and field-tested rapidly. Epigenetic memory and systemic priming, through which plants retain a trained state after mild stress exposure, offer an additional lever: chromatin-level modifications could potentially be exploited or engineered to keep cold-response genes in a poised, readily activated configuration.

The practical stakes are considerable. Cold snaps damage seedlings in open fields and greenhouses alike, and postharvest chilling injury degrades fruit quality during cold storage, imposing losses across the entire supply chain. As climate volatility increases, the window of safe cultivation for a crop with such narrow thermal tolerance is shrinking in key production regions. By connecting functional genomics with precision genome engineering, the authors contend, pepper breeders can move beyond the incremental gains of conventional crossing and build cultivars with fundamentally rewired stress responses. The review’s roadmap, spanning DREB/CBF pathway engineering, multiplex editing of regulatory networks, negative-regulator disruption, and omics-guided allele deployment, positions Capsicum annuum as a test case for how Precision Breeding 2.0 could reshape the resilience of horticultural crops more broadly, ensuring that one of humanity’s favorite spices keeps thriving in a colder, less predictable world.

Subject of Research: Functional genomics and precision genome-editing strategies for engineering cold stress resilience in Capsicum annuum

Article Title: Engineering cold stress resilience in capsicum annuum through functional genomics and precision breeding

Article References: Ullah, I., Ahmad, N., Bhat, A. H., Hussain, A., Wei, F., & Hongzhi, W. (2026). Engineering cold stress resilience in capsicum annuum through functional genomics and precision breeding. Plant Cell Reports, 45(10), Article 288. https://doi.org/10.1007/s00299-026-03965-9

Image Credits: AI Generated

DOI: 10.1007/s00299-026-03965-9

Keywords: Capsicum annuum, cold stress, chilling tolerance, CRISPR/Cas, Precision Breeding 2.0, DREB/CBF pathway, multi-omics, transcriptional regulation, abscisic acid signaling, climate resilience, plant biotechnology, genome editing

The study, published in Plant Cell Reports, set out to answer two intertwined questions. First, where did a family of plant stress genes known as ERF-VII come from in the deep history of plant evolution? Second, what do these genes actually do in the tea plant when the temperature drops? To resolve both, the researchers combined broad comparative genomics across 14 plant lineages with detailed analyses of 20 tea plant cultivars, creating one of the most comprehensive portraits of this gene family in any woody beverage crop.

The ERF-VII family belongs to the larger AP2/ERF superfamily of transcription factors, master regulators that bind specific DNA sequences and switch suites of target genes on or off. In the model plant Arabidopsis thaliana, ERF-VII proteins are famous for their role in oxygen sensing: they carry a conserved N-terminal motif, the MCGGA/I motif, that tags them for destruction by the oxygen-dependent branch of the N-degron protein degradation pathway. When oxygen is scarce, as during flooding or submergence, these proteins escape degradation, accumulate in the nucleus and activate survival programs. The new work shows that this regulatory architecture is ancient and evolutionarily telling.

By reconstructing phylogenetic trees of ERF-VII proteins across lineages ranging from algae to flowering plants, the team found that the family originated after the divergence of vascular plants, and that its emergence coincided precisely with the appearance of the MCGGA/I motif linked to oxygen-dependent degradation. This suggests that the oxygen-sensing capacity of ERF-VII factors was built into the family at its inception. Gymnosperms, the conifers and their relatives, retained only a few conserved members, while angiosperms underwent dramatic, lineage-specific expansion. Monocots, the grasses and their kin, expanded the family extensively, apparently driven by whole-genome duplication events, whereas eudicots showed more moderate expansion accompanied by functional diversification, with duplicated genes taking on specialized roles in stress and development.

With the evolutionary framework established, the researchers turned to the tea plant itself. A pan-genome analysis, comparing genome sequences from 20 different tea cultivars, revealed that the ERF-VII gene complement is not identical across varieties. Some cultivars carry genes that others lack, and the family members fall into categories that differ in number and distribution between accessions. This varietal variation is significant because it provides raw material for breeders: genes present in hardy landraces but absent or divergent in elite cultivars could underpin differences in freezing tolerance that breeders can exploit through marker-assisted selection.

To identify which ERF-VII members respond to cold, the team mined transcriptome data from the Tea Plant Information Archive, a public repository of gene expression profiles. One member, named CsRAP2.2, stood out. Its expression was strongly induced by low temperature and, crucially, remained sustained throughout cold treatment rather than flickering briefly and fading. The name is telling: CsRAP2.2 is an ortholog of Arabidopsis RAP2.2, a factor previously implicated in hypoxia survival and low-oxygen, oxidative and osmotic stress responses. The tea version appears to have been recruited into the cold-response network, hinting at an evolutionary link between oxygen sensing and chilling tolerance in this species.

Correlation alone does not prove function, so the researchers ran a series of direct tests. When they silenced CsRAP2.2 in tea leaves, the plants became measurably more vulnerable to cold. Conversely, when they overexpressed the gene in tea leaves and introduced it heterologously into Arabidopsis, both systems showed enhanced cold tolerance. The physiological basis of this protection was traced to three pillars of cellular defense. Transgenic and overexpressing material maintained photosystem II efficiency, the quantum engine of photosynthesis that cold typically damages; they suffered less membrane lipid peroxidation, the oxidative rancidity of cellular membranes that accompanies freezing injury; and they showed improved antioxidant capacity, the enzymatic and chemical scavenging of reactive oxygen species that otherwise accumulate as toxic byproducts of cold stress.

To place CsRAP2.2 within the wider regulatory landscape, the team applied weighted gene co-expression network analysis, a computational method that clusters thousands of genes into modules based on coordinated expression across samples and conditions. This analysis positioned CsRAP2.2 as a hub, a highly connected node integrating cold signaling, hormone pathways and oxygen-sensing machinery. Gene Ontology enrichment of its co-expressed network highlighted processes ranging from protein transport to the detection of oxygen and hypoxia, consistent with the idea that cold, hormone and low-oxygen signals converge on ERF-VII factors in tea. In practical terms, CsRAP2.2 may act less like a lone switch and more like a control tower, coordinating multiple stress-response programs simultaneously.

The broader implications extend well beyond tea biology. Global tea production is concentrated in subtropical and highland regions where frost events are becoming increasingly erratic as the climate shifts. A validated cold-tolerance gene with demonstrated function in both tea and a heterologous model gives breeders a concrete molecular target. Marker-assisted breeding or genome editing approaches could introduce or amplify favorable CsRAP2.2 alleles in susceptible cultivars, potentially protecting harvests without the long timelines of conventional crossing. The pan-genome perspective adds another layer: because ERF-VII gene content varies among cultivars, screening germplasm collections for the most protective variants becomes a tractable strategy.

The study also enriches our understanding of transcription factor family evolution. By anchoring the origin of ERF-VII genes to the rise of vascular plants and tying it to a specific protein motif, the work illustrates how a single molecular innovation, a degron recognized by the N-end rule pathway, can seed an entire regulatory system that plants later repurposed for drought, flooding, hypoxia and now cold tolerance. For the humble tea bush, it seems, survival in the cold may hinge on an ancient oxygen-sensing trick repurposed by millions of years of evolution. For the scientists and growers betting on climate-resilient agriculture, CsRAP2.2 offers a promising lead, and possibly the genetic key to keeping the world’s favorite infusion flowing through frosts to come.

Subject of Research: Evolution and cold-stress function of the ERF-VII gene family in tea plant (Camellia sinensis)

Article Title: Genome-wide identification and evolutionary analysis of the ERF-VII gene family in the tea plant (Camellia sinensis) and functional characterization of CsRAP2.2 in response to cold stress

Article References: Huang, Q., Pan, R., Wu, L., Chen, S., Hu, J., Long, Z., Zhao, J., Hao, X., & Tang, H. (2026). Genome-wide identification and evolutionary analysis of the ERF-VII gene family in the tea plant (Camellia sinensis) and functional characterization of CsRAP2.2 in response to cold stress. Plant Cell Reports, 45(10), Article 290. https://doi.org/10.1007/s00299-026-03973-9

Image Credits: AI Generated

DOI: 10.1007/s00299-026-03973-9

Keywords: tea plant, Camellia sinensis, ERF-VII, CsRAP2.2, cold stress, transcription factors, pan-genome, oxygen sensing, N-degron pathway, plant evolution, molecular breeding, abiotic stress

The research, led by Jingbing Zhao and colleagues at the College of Agriculture of Ningxia University in Yinchuan, China, takes an approach that has become one of the most powerful ideas in modern plant genomics: the pan-genome. Rather than mapping genes against a single reference genome, the team surveyed 26 genetically diverse maize inbred lines, capturing the full spectrum of genetic variation that a single reference would miss. Out of this panoramic view, they identified 13 Zm4CL genes, of which nine were classified as core genes, present in essentially all lines, and four were near-core genes, absent from a small subset of genotypes. That distinction matters more than it might sound, because genes that vary in their presence across the species are often the very ones that help plants adapt to particular environments.

Phylogenetic analysis sorted the 13 Zm4CL genes into three evolutionary clades, hinting at an ancient history of gene duplication and divergence within the grass lineage. The team then probed the evolutionary forces acting on these genes by calculating the ratio of nonsynonymous to synonymous substitution rates, the widely used Ka/Ks metric. For most members of the family, the results pointed to purifying selection, the genomic equivalent of a strict quality-control regime in which harmful mutations are weeded out because the protein’s function is too important to compromise. But a handful of genes showed signs of relaxed evolutionary constraint, suggesting they may have been free to explore new or specialized functions during maize’s diversification, a pattern consistent with the way duplicated genes often partition ancestral roles or acquire novel ones.

Perhaps the most provocative finding came from the team’s examination of structural variations, large-scale differences in DNA sequence that include deletions, insertions, and rearrangements. Long overshadowed by single-letter changes in the genetic code, structural variants have emerged in recent years as major drivers of trait variation in crops. In this study, structural variations were significantly associated with the expression levels of two key family members, Zm4CL2 and Zm4CL3. In other words, the same gene can behave very differently in different maize lines simply because the surrounding or intervening DNA architecture differs. Sequence comparisons went a step further, revealing that in some genotypes structural variations were linked to alterations in conserved protein domains, the functional workhorses of the enzyme itself. A change of that kind could plausibly alter not just how much enzyme a plant produces, but what that enzyme can actually do.

Why does any of this matter for the plant? The 4CL enzymes occupy a pivotal junction in the phenylpropanoid pathway, the metabolic assembly line that converts phenylalanine into an astonishing array of compounds, including lignin, flavonoids, and assorted defensive molecules. Lignin, the tough polymer that stiffens cell walls, is indispensable for structural integrity and water transport, but it also influences how digestible maize stover is for livestock and how easily cellulose can be extracted for biofuels. Previous work on maize brown midrib mutants, in which 4CL1 function is disrupted, showed that tweaking this pathway can increase cell wall digestibility. Beyond development, the phenylpropanoid pathway is a first responder to stress, churning out protective compounds when the plant is attacked by pathogens or battered by harsh weather. Understanding the genetic controls over 4CL enzymes therefore opens a window onto both agronomic quality and stress resilience.

To see those controls in action, the researchers turned to transcriptome data, examining where and when the Zm4CL genes are switched on across different tissues and in response to various challenges. The picture that emerged was one of remarkable specialization. Individual genes showed distinct tissue-specific expression patterns, implying that the family members have divided the labor of phenylpropanoid production across roots, stems, leaves, and reproductive structures. Under stress, the responses diverged further: the genes displayed varied transcriptional reactions to both abiotic and biotic pressures, with cold and drought standing out as particularly potent triggers of dynamic expression changes. This kind of regulatory diversification is a hallmark of gene family evolution, allowing a plant to fine-tune its metabolism with far more nuance than a single gene could achieve.

The team did not stop at gene expression. Enzyme activity assays measured the actual biochemical output of 4CL under five different stress treatments, and the results were strikingly stress-specific. Cold stress significantly increased 4CL enzymatic activity at the twelve-hour mark, a rapid mobilization consistent with an urgent need for protective phenylpropanoid compounds when temperatures plunge. Heat, salt, and alkali stresses told a different story: activity initially dipped and then rebounded, suggesting the plant absorbs an immediate metabolic shock before restoring its enzymatic machinery. Drought, notably, had no significant effect on enzyme activity, even though it clearly altered the expression of representative Zm4CL genes in time-course RT-qPCR experiments. That dissociation between transcript levels and enzyme output is a well-recognized phenomenon in plant biology, a reminder that messenger RNA abundance and protein function do not always march in lockstep, and that post-transcriptional and post-translational regulation can shape the final phenotype.

For breeders, the study delivers a comprehensive framework that could accelerate the development of stress-tolerant maize. Knowing which Zm4CL genes are core and which are near-core tells researchers where genetic diversity is likely to harbor useful alleles. Knowing that structural variations modulate the expression of specific family members suggests that these variants could serve as molecular markers in marker-assisted selection, or even as targets for genome editing approaches that aim to rewire stress responses without introducing foreign DNA. The links between 4CL function, lignin content, and cell wall digestibility add a further incentive, since varieties optimized for both resilience and feedstock quality would carry substantial economic value.

The broader significance of the work lies in its demonstration that pan-genome analysis can transform our understanding of even well-studied gene families. Earlier cataloging efforts, constrained by single reference genomes, inevitably undercounted genes that are absent from the reference line. By embracing the full diversity of 26 inbred lines, the Ningxia University team captured the near-core genes that would otherwise have slipped through the net, and connected their variation to real differences in gene expression, protein architecture, and stress physiology. As structural variation continues to be recognized as a dominant force shaping crop genomes, studies of this kind are likely to multiply, extending the pan-genome lens to other metabolic pathways and other staple crops.

The study was supported by the General Project of the Ningxia Natural Science Foundation. All data generated during the research are included in the published article and its supplementary files, giving the wider plant science community immediate access to a resource that promises to inform functional studies and genetic improvement efforts for years to come. For a crop that must feed a growing population on a warming, increasingly unpredictable planet, every hidden layer of genetic flexibility matters, and the maize 4CL family has just revealed a good deal more of its own.

Subject of Research: Pan-genome analysis of the 4-coumarate:CoA ligase gene family in maize and its responses to abiotic stress

Article Title: Pan-genome characterization of the maize 4CL gene family and its dynamic responses to abiotic stress

Article References: Zhao, J., Ren, L., Li, L., & Shao, D. (2026). Pan-genome characterization of the maize 4CL gene family and its dynamic responses to abiotic stress. Plant Molecular Biology, 116(5), Article 96. https://doi.org/10.1007/s11103-026-01760-4

Image Credits: AI Generated

DOI: 10.1007/s11103-026-01760-4

Keywords: maize, pan-genome, 4CL gene family, phenylpropanoid pathway, structural variation, abiotic stress, lignin biosynthesis, gene expression, purifying selection, drought stress, cold stress, crop improvement