Planta

The production of reactive oxygen species (ROS) in response to cadmium (Cd) has been extensively studied, demonstrating that they play a key role in the plants response to this heavy metal. While the role of enzymes like RBOHs has been thoroughly studied, the function of other ROS-producing enzymes, such as peroxisomal glycolate oxidase (GOX), remains largely overlooked. Peroxisomal GOX is a core metabolic enzyme of the photorespiratory pathway occurring in chloroplasts, mitochondria and peroxisomes. Using Arabidopsis (Arabidopsis thaliana) mutants lacking the main peroxisomal GOX genes, GOX1 (gox1-1) and GOX2 (gox2-1) we explored their function in plant response to Cd. Although photosynthetic capacity appears to be affected to the same extent in both mutants under control and Cd stress conditions, GOX2 seems to play a greater role in ROS production in response to the metal. Transcriptomic analyses on WT and gox2-1 pointed to the mitochondrial electron transport chain (mETC) as a target of Cd stress. We further investigated the individual GOX1 and GOX2 functions in mETC regulation and redox state. Although oxidative ratio of mitochondria was higher in both mutants, it was more pronounced in the absence of GOX1. Furthermore, the mETC is affected in both mutants but the regulation of its components differs in each mutant. These results point out the different functions of the two photorespiratory GOX isoforms in Arabidopsis, leading to a better understanding of the photorespiratory pathway.

Under natural growth conditions, plants are not usually exposed to the high-energy ultraviolet C range (UV-C, 100-280 nm) of the solar spectrum, as this is absorbed by the ozone layer. However, low doses of UV-C radiation can trigger stress responses in plants. Nevertheless, it is not yet fully understood how UV-C light affects plant development at the hormonal level. Here we show that a single one-min UV-C light pulse (20 W/m2) alters gibberellin (GA) homeostasis in Arabidopsis in two phases: initially, the level of GA12 - a key precursor of the final part of gibberellin biosynthesis - is reduced. Consistent with this, the transcript levels of the CPS, KS and KAO2 genes, which encode enzymes involved in the initial parts of gibberellin biosynthesis, decrease. The level of the plant hormone GA4 also decreases initially, probably due to the reduced GA12 precursor levels. However, in a second phase, the endogenous GA4 levels rise in UV-C treated plants relative to control plants. This increase leads to an early onset of flowering, as well as increased growth and fertility, in UV-C-treated Arabidopsis plants. The GA signalling mutant gdella does not exibit wild-type phenotypic responses to UV-C treatment, indicating that GA signalling is essential for the UV-C response. To further narrow down the responsible steps in the GA-signalling pathway, we tested the kao1 and kao2 mutants, which are both impaired in early gibberellin biosynthesis. Neither mutant displays phenotypic responses to the UV-C treatment, indicating that both genes are required for mediating the UV-C response. In contrast, the quintuple 2-oxidase mutant C19--2oxqM exhibits responses to UV-C treatment similar to the wild-type, suggesting that the five catabolic 2-oxidases that act on C19-GAs play a negligible role in regulation GA-hormone levels for growth and development in this case. HighlightUV-C pulse triggers biphasic gibberellin dynamics, delaying early development but ultimately enhancing growth and fertility in Arabidopsis thaliana.

Hemp (Cannabis sativa) produces a wide array of medicinally significant compounds, including cannabidiol (CBD). These compounds are predominantly synthesized in female hemp inflorescences. The proposed research utilizes next-generation sequencing-based transcriptome analysis using a 3{square}-end-directed approach to identify differentially expressed genes between male and female hemp plants at the early vegetative stage. 886 differentially expressed genes (DEGs) were identified, a majority of which were upregulated in males compared to females. We hypothesized that alternative RNA processing contributes to sex-specific gene expression. To this end, 932 genes were identified that exhibited significant changes in poly(A) site usage when comparing males and females. These genes were much more likely to be differentially expressed, supportive of this hypothesis. Males tend to have longer 3 UTRs with canonical motifs found in the Near-Upstream Elements (NUE), compared to the shorter 3 UTRs in females, which have A-rich motifs near the cleavage site. This suggests that polyadenylation remodels hemp mRNAs with distal poly(A) sites being preferred in males. To further investigate when this sex-specific gene expression program is established, RNA was isolated from plants at various developmental stages, such as developing seeds, four-day-old seedlings, and different developmental stages up to four weeks after sowing. Diagnostic male-specific genes were analyzed using RT/PCR. The results indicate that sex-specific gene expression is not evident in seeds but rather is set during or after germination. SignificanceO_LIHemp males tend to have longer 3 UTRs with canonical motifs found in the Near-Upstream Elements (NUE), compared to the shorter 3 UTRs in females, which have A-rich motifs near the cleavage site. C_LIO_LIThe sex-specific gene expression program is not yet established in mature seed but is set in the time between germination and 4 days of growth. C_LI

Low temperature stress causes significant damage to the strawberry plant. During cold stress, plants undergo morphological and physiological changes often regulated at the genetic and/or epigenetic levels. Some strawberry cultivars are more cold-hardy than others. Using the diploid woodland strawberry as a model, we analyzed the effects of cold acclimation on methylome and transcriptome dynamics in the crowns and leaves of three ecotypes with contrasting cold tolerance. Alta, which was the most cold-tolerant ecotype, exhibited the highest genetic and epigenetic plasticity in response to cold. CHH-context methylation dominated the differentially methylated regions (DMRs) with more hypomethylation in crowns and hypermethylation in leaves. CG methylation was enriched in gene bodies, while non-CG methylation was prevalent in upstream and downstream regions. Our study revealed that less than a quarter of differentially methylated genes (DMGs) showed changes in transcript accumulation levels. This finding indicates that universal cold response in Fragaria vesca, as reflected by gene expression, cannot be mechanistically attributed to DNA methylation. The majority of differentially expressed differentially methylated genes (DEDMGs) were ecotype- and tissue-specific. Enrichment analysis revealed that these genes were involved in pathways related to stress tolerance, such as carbohydrate metabolism, lipid metabolism, ATP hydrolysis, and cellular detoxification. Each ecotype responded to cold through mobilization of its own set of differentially expressed genes (DEGs), DMGs, and DEDMGs, and variation in expression and methylation patterns exhibited by Alta, FDP817, and NCGR1363 suggest that cold signaling processes and survival depend on the tissue, ecotype, and geographical origin of the plants exposed to cold stress. Therefore, this study highlights the potential of both genetic markers and epialleles as molecular markers for the development of cold-tolerant octoploid strawberry cultivars that are better suited for propagation in Nordic climates.

Improving regeneration of ribulose-1,5-bisphosphate (RUBP) is a promising approach to improve photosynthesis and plant growth. In addition to transgenic overexpression of target genes, it could be possible to directly overexpress endogenous target genes, through transcriptional enhancements. As shown by the recent discovery of a short sequence motif, that resembles the known octopine synthase (ocs) enhancer, transcriptional enhancement is achievable by relatively short endogenous sequences. In this study, we query the genome of several model and crop plant genomes for the presence of short enhancer motifs. We find hits across all genomes including some in promoter regions of genes. By using derivatives of these motifs in a transient fluorescence assay, we show that several of these are capable of inducing target gene expression in different promoter contexts. A motif scan of the created constructs, for the presence of known transcription factor binding sites, shows that the insertion of these motifs has created binding sites for different TGA-, NAC- and bZIP-transcription factors. Taken together our study shows the feasibility of finding enhancer sequences in the genomes of different plants. With advancement in gene-editing technologies, like prime editing, using such endogenous enhancer sequences, could allow for precise cisgenic promoter engineering of target genes.

Apples (Malus x domestica) are popular fruits grown in temperate regions of the world. The various genotypes must meet a specific threshold amount of cold exposure before they are competent to break dormancy, a quantity approximated as "chill hours". Several varieties have been identified that exhibit an ultra-low-chill requirement, or more precisely shallow dormancy, breaking vegetative and floral buds early in spring in response to minimal cold exposure. These ultra-low-chill genotypes originated from the Bahamas ( Dorsett Golden,1960s), Israel ( Anna, 1950s) and Alabama, USA ( Shell of Alabama, 1880s). The separation in time and space implies that each would feature distinct genetic lesions that govern dormancy control, providing discrete mechanisms to incorporate a low-chill trait in variety improvement. However, analysis of microsatellites and ultimately genome sequence indicates that Dorsett Golden and Anna share strong concordance with the Shell of Alabama genotype, as well as other ultra-low-chill varieties. Kinship analysis confirms that all are closely related, despite differences in year and place of origin. All three low-chill genotypes share common mutations in the DORMANCY ASSOCIATED MADS-BOX1(DAM1) gene, a known repressor of vegetative growth during dormancy. Genomic sequence diversity is observed among Shell of Alabama individuals, including differences in DAM1 that match differences in flowering time. The results of this study call into question the pedigrees of the ultra-low-chill apple germplasm and indicate variation in an otherwise narrow genetic base for use in future breeding efforts.

In vitro regeneration of Citrus plants is a widely used method, however, induction of adventitious roots from regenerated shoots remains a major bottleneck, limiting the recovery of healthy plants for commercial production and genomic research for crop improvement. We established an in vitro regeneration system producing profuse, healthy roots for sweet orange (Citrus sinensis cv. Benyenda) by optimising combinations and concentrations of auxins. Prior to optimising the rooting media (RTMs), we obtained a shoot regeneration rate of 90.6% from sweet orange epicotyl explants using a cytokinin, 6-benzylaminopurine (BAP). Across twelve auxin-supplemented RTMs containing different concentrations of indole-3-butyric acid (IBA) and/or 1-naphthaleneacetic acid (NAA), rooting percentages ranged from 8 - 87.5%. The combination of IBA 1.0 mg L-1 and NAA 0.1 mg L-1 promoted the best overall performance, 75 {+/-} 7.2% rooting percentage with healthy, callus-free roots ([≥]5 cm in length), whereas other RTMs with other auxin combinations induced callus and limited root elongation. The best-performing SRM and RTM were subsequently used for selection and recovery of transgenic sweet orange lines carrying an empty CRISPR/Cas9 construct, resulting in an 4.8% transformation efficiency. Both transgenic and non-transgenic rooted plantlets were successfully acclimatised under glasshouse conditions with a survival rate of 90%. This enhanced regeneration system overcomes rooting bottleneck and improves plant survival,enabling faster recovery of transgenic citrus lines within four months. It supports accelerated development for commercial applications and advances in citrus genetic improvement.

Recent studies have demonstrated the presence of kynurenine (KYN) and kynurenic acid (KYNA) in several plant species, but the metabolic function of these metabolites remains undefined. We hypothesized that KYN and KYNA are metabolites of auxin and play a role in plant morphogenesis. To test our hypothesis, we developed a plant tissue-culture-based bioassay using Hypericum perforatum (St. Johns wort; SJW), a model system for auxin and indoleamine metabolism and pharmacological inhibitors (PF-04859989, RO-61-8048, and KMO inhibitor II, JM6) of human kynurenine pathways enzymes. SJW is an interesting model system because explants root in the absence of plant growth regulators but supplementation of the culture media with 10 M IAA induces a callus response without de novo root organogenesis. Supplementation of the culture media with 10 M KYN increased root number and internodal length relative to basal media. We used a previously validated high-resolution mass spectrometry analytical method to quantify KYN, KYNA, and 3-hydroxyanthranilic acid (3-HAA). KYN, KYNA and 3-HAA were quantified in roots and shoots of SJW grown on basal media. Supplementation of the culture media with 10 M KYN increased the concentration of KYN, KYNA and 3-HAA in roots and shoots. Treatment with 10 M IAA increased KYN and 3-HAA concentration in shoots. Three pharmaceutical candidates that are kynurenine pathway inhibitors in humans were taken up into the tissues from the culture media and increased KYN content as compared to basal control. Together, these data propose a role for KYN in IAA metabolism, shoot and root organogenesis. HighlightsO_LIKynurenine metabolites are detected and accumulate in H. perforatum tissue culture C_LIO_LIIAA redirects metabolism towards accumulation of KYN and 3-HAA in shoots C_LIO_LIExogenous KYN promotes KYNA accumulation C_LIO_LIPharmacological inhibition alters kynurenine pathway metabolite profiles in a tissue-specific manner C_LIO_LIKynurenine and IAA differentially regulate root development C_LI

Ovate Family Proteins (OFPs) are a class of plant-specific, negative nuclear transcriptional regulators characterized by conserved C-terminal OVATE domain. This study on comparative functional characterization of two head-to-head arranged OFPs - AtOFP2 (Ovate-OFP with full ovate domain) and AtOFP17 (Ovate-Like OFP with partial ovate domain) provides critical insight into how structural variations in ovate domain leads to functional divergence. Detailed phenotypic analysis of 28 physical and physiological traits of loss- and gain-of-function mutants revealed that both genes act as broad, pleotropic repressors of plant growth and development. Removal of repression in knock-down mutants of both genes exhibited reduced duration of seed dormancy, faster rate of germination and growth, bigger plants and significantly higher seed yield. In contrast, constitutive over-expression showed a generalized repressive nature of both genes, with nuanced differences for fine tuning of specific traits. For example, both genes showed antagonistic behaviours on root hair architecture. AtOFP2 act as a strong repressor of root hair development whereas AtOFP17 is a stronger repressor of hypocotyl and root cell architecture. AtOFP17 owing to partial ovate domain exerts a mild level of repression throughout life span as indicated by smaller plants and lesser yield in knock-down AtOFP17 mutants. On the contrary, AtOFP2 exerted a much stronger repressor effect in which > 90% over-expression mutants died at the juvenile stage ; the survival of remaining 10% is probably owing to activation of dosage-dependent feedback loop mechanism as indicated by normal growth of mature plants, and is also evident by transcriptome data. Transcriptome analysis of roots of 7-day old seedling of knock-down and over-expression mutants of AtOFP2 showed downregulation of OFP2 in over-expressed mutants. However, severely stunted phenotype indicated presence of stable OFP2 protein to exert effects. Analysis of DEGs in OFP2 mutants revealed that it acts as an important regulator working at intersection of hormonal signalling affecting critical genes required for auxin, cytokinin, GA, BR and ABA functioning. Perturbations across hormonal signalling pathways affects cell wall remodelling factors such as EXPANSINS, Xyloglucan hydrolases (XTHs) and cellulose synthases (CSLs) causing overall stunted growth; and epidermal patterning genes such as WER, GL1, EGL3, TTG1 leading to severely reduced root length and root hairs. Significantly, functional analysis of this master regulator highlighted a significant economic potential. Knockdown of both these genes relieves their natural repression on reproductive traits, leading to longer siliques, bigger and heavier seeds, and substantially increased overall seed yield, positioning AtOFP2 and AtOFP17 as highly valuable targets for agricultural crop improvement.

Advances in NASAs astrobiology program have demonstrated the feasibility of cultivating plants in space and in analog extraterrestrial habitats. In addition to abiotic stressors, plants grown in terrestrial and space-like environments are challenged by both phytopathogens and opportunistic human pathogens, with implications for plant productivity and human health. The persistence of human-associated pathogens in spacecraft and space stations raises significant concerns regarding food safety. The molecular, biochemical, and signaling mechanisms governing stomatal development and function under microgravity remain poorly understood. We employed an experimental system incorporating human pathogen Salmonella enterica and lettuce microgreens exposed to simulated microgravity through two-dimensional clinorotation to investigate plant innate immunity and stomatal development and function. We further evaluated four lettuce cultivars to determine whether genetic variation impacts these factors under simulated microgravity conditions. Our findings indicate that simulated microgravity significantly influences stomatal development and function, as evidenced by an increase in stomatal density and variable changes to stomatal aperture. Notably, cultivar-dependent variation in stomatal traits and responses to Salmonella enterica was observed under microgravity conditions. Although increased stomatal density was hypothesized to enhance pathogen ingression, internalization was more strongly predicted by cultivar selection and simulated microgravity; simulated microgravity increased ingression, with red pigmented cultivars having less pathogen than green cultivars. These results suggest that targeted selection of cultivars with favorable physiological traits may improve food safety and the viability of crop production systems in space environments. They also suggest that development and function of stomata may change in spaceflight conditions.

Background and AimsThe development of unisexual flowers relies on the tight coordination of flower organ identity and sex determination. The genus Quercus is typically considered strictly monoecious, bearing fully segregated male and female flowers within the same individual tree. However, several reports of atypical flowering across the genus challenge this canonical view, suggesting that flowering in oaks may be more flexible than traditionally assumed. In this work, the dynamics of flower development in Quercus orocantabrica were examined to correlate contrasting floral morphologies with divergent molecular profiles. MethodsThe flowering phenology of Q. orocantabrica trees was closely monitored over several individuals and years, together with a detailed floral morphological analysis of male, female and atypical flowers. Key floral homeotic gene homologues were identified, and their expression assayed in the development of different flowers. Key ResultsRecurrent and widespread hermaphroditic flowering was detected in several Q. orocantabrica trees, frequently associated with unseasonal flowering events. Gene expression analysis of male, female and hermaphroditic flowers revealed a sex-biased expression of Q. orocantabrica B- and C-class genes, with the B-class gene QoPI in particular being tightly associated with the presence of fully-developed stamens. In addition, the expression of the C-class gene QoSHP contrasted with reports in other Fagaceae, highlighting a potential functional divergence of the C/D-class lineage within the family. ConclusionsThe results here depicted indicate that the dynamics of floral sex identity in oaks are more plastic than traditionally assumed, supporting a reinterpretation of oak reproductive biology based on a versatile and resilient framework responsive to different developmental contexts.

Salinity stress is one of the major abiotic factors limiting cotton productivity worldwide by inducing osmotic imbalance, oxidative stress, and metabolic disturbances in plant tissues. The present study aimed to evaluate the physiological and biochemical responses of different cotton (Gossypium hirsutum L.) genotypes under NaCl-induced salinity stress through analysis of proline accumulation, antioxidant enzyme activities, and lipid peroxidation intensity. The experiment was conducted under controlled conditions using several cotton genotypes exposed to different NaCl concentrations. Proline content, superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) levels were analyzed as major biochemical indicators associated with salinity tolerance and oxidative stress responses. In addition, modern bubble heatmap visualization was applied for comparative assessment of genotype-specific stress response patterns under saline treatments. The obtained results demonstrated that increasing NaCl concentrations generally stimulated proline accumulation and enhanced antioxidant enzyme activities in most investigated cotton genotypes. Increased SOD and CAT activities indicated activation of enzymatic antioxidant defense mechanisms under salinity stress conditions. Simultaneously, elevated MDA accumulation reflected enhanced oxidative membrane damage caused by excessive reactive oxygen species (ROS) production under saline environments. Considerable genotype-dependent variability was observed among the investigated cotton varieties. Genotypes such as "Nasaf", "Gulbahor-2", "Ravnaq-1", "Buxoro-6", "Afsona", "Baraka", "Namangan-77", "Porloq-1", and "C-4727" demonstrated comparatively stronger physiological and antioxidant responses under salinity stress conditions, suggesting relatively higher adaptive capacity to NaCl-induced stress. The heatmap visualization additionally confirmed substantial heterogeneity among cotton genotypes in biochemical stress responses and allowed comprehensive comparative interpretation of salinity-induced physiological variability. Overall, the present findings suggest that proline accumulation, antioxidant enzyme activities (SOD and CAT), and MDA content may serve as important biochemical markers for evaluation of salinity tolerance in cotton. The identified stress-tolerant genotypes may therefore represent valuable genetic resources for future breeding programs aimed at improving cotton productivity under saline environmental conditions.

RAP2.6, an AP2/ERF transcription factor (TF), regulates plant stress responses; however, its role in floral transition remains unexplored. Here, we evaluated RAP2.6s role in flowering and the associated transcriptional changes in Arabidopsis thaliana under long-day conditions. RAP2.6-overexpressing line showed early flowering with fewer rosette leaves, whereas rap2.6-1 mutant flowered later, had more rosette leaves, and higher expression of the floral repressor FLOWERING LOCUS C (FLC). Early flowering in the overexpressing line was accompanied by transcriptional activation of the floral integrators GIGANTEA (GI), FLOWERING LOCUS T (FT), and COSTANS (CO), potentially through RAP2.6 interaction with GCC/DRE cis-regulatory elements. RAP2.6-mediated floral transition depended on nitric oxide (NO), with flowering time largely varying based on NO bioactivity. RAP2.6 was found to be a downstream regulator of Arabidopsis S-NITROSOGLUTATHIONE REDUCTASE 1 (GSNOR1) in controlling S-nitrosothiol (SNO) levels, flowering time, and silique formation. The NITRIC OXIDE-ASSOCIATED 1 (NOA1)-dependent reduction in NO levels abolished early flowering in 35S::RAP2.6 plants without affecting silique formation. Furthermore, enhanced cytokinin sensitivity and upregulation of cytokinin biosynthetic genes suggest cytokinin involvement in RAP2.6-mediated flowering. Together, these findings highlight the crucial role of RAP2.6 in regulating flowering time by integrating redox and hormonal signaling to coordinate reproductive development in A. thaliana.

Background and AimsPaspalum notatum reproduces through either sexuality or apomixis, two pathways that may coexist within the same individual and are regulated by interconnected molecular networks responsive to environmental cues. Here, we characterized the transcript structure and expression of BUD SITE SELECTION PROTEIN 13 (BUD13), a component of the RES spliceosomal complex previously reported as differentially expressed in florets of sexual and apomictic plants, as a first step toward testing its involvement in the molecular regulation of the apomixis-sexuality switch. MethodsPreviously generated floral and leaf transcriptomes from sexual and apomictic Paspalum notatum plants, including Oxford Nanopore long-read data, were mined to characterize BUD13 transcript structure and expression. Phylogenetic analyses and in silico mapping were conducted to infer evolutionary relationships and determine the origin of the transcripts. Differential expression was validated by RT-qPCR, while in situ hybridization was used to reveal cell-specific ovule expression patterns. Key resultsBUD13 is expressed in Paspalum notatum florets as a truncated isoform (SHORT) encoding a small protein lacking part of the herpes simplex virus regulatory protein (ICP4) domain. Two SHORT transcripts, SHORT1 and SHORT2, with different 5' untranslated region (UTR) regions, were identified in flowers. SHORT1 was consistently upregulated in apomictic ovules from premeiosis to anthesis. Both transcripts originated from a single genomic locus located in the subtelomeric region of the short arm of chromosome 6. SHORT isoforms with variable structures were detected in other monocots. In situ hybridization showed that, whereas BUD13 was expressed throughout sexual ovules, expression was absent from the female germline of apomictic ovules. A consistent expression was observed in somatic proembryos of aposporous embryo sacs. ConclusionsOur findings reveal structural, spatial and temporal divergence in BUD13 expression between sexual and apomictic reproductive programs, providing new insights into the molecular regulation of asexual seed formation.

Insect herbivory triggers cytosolic proteome reprogramming by activating defense pathways and modulating key metabolic processes. We found that simulated herbivory in pigeon pea (Cajanus cajan) induced reactive oxygen species (ROS) production and molecular alterations within 12 hours (h) of post treatment. We compared the leaf proteome profiles of two cultivated genotypes, ICPL 332 (moderately resistant) and ICPL 87 (susceptible), using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) coupled with mass spectrometry (MS). More than 220 protein spots were detected in ICPL 332 and over 200 in ICPL 87. Comparative analysis revealed 75 differentially accumulated proteins (DAPs), of which 40 were consistently reproducible across biological replicates. These included 11 unique to ICPL 87, 9 unique to ICPL 332, and 10 common to both genotypes. Among the shared DAPs, ICPL 332 showed five upregulated and five downregulated, whereas ICPL 87 exhibited only two upregulated and eight downregulated. Functional categorization grouped DAPs into primary metabolism, stress response, and growth and development. Proteins related to primary metabolism were largely downregulated in both genotypes, while stress-associated proteins exhibited substantial downregulation in ICPL 87 compared to ICPL 332. Overall, the results demonstrate proteomic adjustments underlying defense responses in pigeon pea genotypes.

Flowering time and monocarpic senescence are tightly environmentally and genetically controlled. Typically, early flowering and staygreen traits are associated with opposing life-history strategies; stress avoidance versus adaptation; with flowering time an overarching regulator of crop cycle length. We developed RIL populations segregating for Ppd-1 and NAM-1 variation, which are otherwise isogenic. Multi-year field experiments enabled exploration and uncoupling of the relationship between heading and staygreen traits. Heading date manipulation enabled introduction of staygreen traits to their target breeding environments, characterised by a hot-finish. Under moderate stress, we report a 2.9% and 1.9% increase in grain width (P<0.0001), and 5.8% and 3.7% increase in TGW (P<0.0001), plus significantly greater yield (P<0.1) for late heading staygreen RILs homozygous for NAM-A1, and NAM-D1 missense variants, respectively. Grain yield increases were proportionate to the delay in senescence, being greater for the NAM-A1 than the NAM-D1 variant. For RIL populations segregating for both traits, senescence variation was observed relative to heading-date. Regarding grain yield, the staygreen trait-associated increase in source size could not compensate for the Ppd-1a associated pleiotropic reduction in sink size, even under hypothesised continental target breeding environments, with trait competition identified. Therefore, to maximise the benefits associated with staygreen traits, especially in early-heading favouring environments required targeted manipulation of source-sink dynamics, and we propose multiple strategies. HighlightStaygreen traits were associated with extending grain fill duration, increasing grain width, TGW and grain yield. There appears an antagonist relationship between earlier heading and staygreen traits.

Heavy metals act as essential metalloprotein cofactors in numerous physiological processes but can become toxic when non-essential metals accumulate or when essential metals are in excess. As plants continuously encounter heavy metals through their roots, they have evolved complex homeostatic mechanisms to regulate metal uptake and distribution. The Heavy Metal ATPase (HMA) gene family encodes a group of heavy metal transporting P-type ATPases that have been linked to stress resistance and nutrient supply. Here, we used a bioinformatics approach to identify and characterise 13 HMA genes containing characteristic P1B-type ATPase domains and motifs in the barley Morex V3 reference genome. The genes are located on five of the seven barley chromosomes. Phylogenetic analysis revealed that they cluster into five sub-clades, including one clade unique to barley. Expression profiling across multiple datasets showed distinct temporal and tissue-specific expression patterns among HvHMAs, with several members exhibiting significant transcriptional responses to specific biotic and abiotic stresses. By utilising recently available pan-transcriptomic and pan-genomic resources, we have identified substantial allelic diversity and inter-accession variation in HvHMAs. Our findings suggest that HvHMAs have functions extending beyond canonical heavy metal homeostasis and warrant further investigation for their potential roles in broader physiological and stress-related processes.

O_LIHerbivory is a major biotic stress for plants, triggering the induction and modulation of diverse specialized metabolites. Such induction responses are well studied for leaves and have been shown to depend on the herbivore feeding mode. Little is known about changes in flower metabolites and chemodiversity due to florivory type. Moreover, we lack an understanding of the intraspecific variation in such responses and whether these are spatially structured. C_LIO_LIThe aromatic plant Tanacetum vulgare, which shows high intraspecific chemodiversity in terpene profiles, was used to examine chemotype-specific metabolic responses of flower heads to infestation by the inflorescence-infesting aphid Macrosiphoniella tanacetaria or the flower-feeding beetle Olibrus spp. under field conditions. At peak flowering, each plant received both florivory treatments on separate stems, leaving one stem herbivore-free as a control. After four days, flower heads were harvested to analyze terpenes (GC-MS) and metabolic fingerprints (LC-MS). C_LIO_LIWe found stem-specific floral metabolic responses, with florivory altering specific chemical families and their chemodiversity. Levels of a few terpenes decreased following infestation, while none increased. Untargeted analyses revealed that aphid infestation had a lower effect on flower chemistry than beetle infestation, with aphid infestation mainly causing decreases and beetle infestation predominantly leading to increases in some metabolite intensities, but little overlap across treatments and chemotypes. C_LIO_LIOur results demonstrate that floral metabolic responses to florivory are spatially structured, florivore type-specific and shaped by plant chemotype. These findings highlight that the interplay between vascular organization, insect feeding mode, and intraspecific chemodiversity governs how flowers adjust their chemical defenses. C_LI One-sentence summaryTanacetum vulgare showed chemotype-specific responses to florivory by aphids (Macrosiphoniella tanacetaria) and beetles (Olibrus spp.), with aphids causing decreased and beetles increased levels of metabolic features within the same plant individuals, with little overlap in significant features across chemotypes.

Banana (Musa spp.) is a vital staple food and cash crop cultivated in over 140 countries, providing nourishment and livelihoods to more than 400 million people worldwide. In this context, Bhimkol (Musa balbisiana, BB genome), a diploid banana variety native to Northeast India holds significant nutritional and commercial value. Its high iron and nutrient content have already been commercially validated through products like Bhimvita and Bhimshakti, which utilize fresh fruit pulp as nutrient-rich food for infants. However, Bhimkol fruits typically contain 100-150 seeds, an undesirable trait for product development. The manual removal of these seeds significantly increases production time and labour costs. Furthermore, because bananas are recalcitrant to traditional breeding, there is a constant need for rapid in vitro transformation protocols. To address these challenges, as a proof of concept, our research aims to knockout the INNER NO OUTER (INO) gene, which is responsible for ovule development. Using CRISPR/Cas12a technology, we established an efficient and reproducible in vitro regeneration and transformation system using Embryogenic Cell Suspensions (ECS). The resulting CRISPR-edited plantlets exhibited various mutations, including insertions and deletions (INDELs) within the targeted INO gene. These INDELs resulted in frameshift mutations that triggered premature stop codons. While these genetic changes are expected to render the banana seedless, phenotypic verification is currently underway to confirm the absence of seeds in mature fruit. Significance StatementDespite its superior nutritional profile, the commercial viability of the Bhimkol banana (Musa balbisiana) is restricted due to abundance of seeds (100-150 per fruit). This study employs CRISPR/Cas12a-mediated knockout the INNER NO OUTER (INO) gene in Bhimkol and expected to develop seedless fruits. The resulting plantlets exhibit targeted indels that trigger frameshift mutations, effectively disrupting ovule developmental INO gene.

Glycosidic nitriles (GN) in barley are precursors to carcinogens formed during distillation, making GN reduction a critical breeding objective for malting and distilling industries. Measurement of GN is time-consuming. Grain must first be malted before GN can be quantified, and generally cannot be completed before selections must be made in a winter barley breeding program. Here, feasibility of same-season genomic selection against GN content was evaluated in elite Virginia Tech winter barley germplasm. In 2023, all 176 elite breeding lines screened for presence of GN were shown to be GN producers. A subset of 95 lines was then quantitatively measured for GN concentration to determine the genetic variability for the trait. Efficacy of genomic selection for GN was first assessed using a divergent selection approach on the remaining 81 predicted lines. The highest 16 and lowest 16 of the predicted lines were chosen for GN quantification. A significant phenotypic difference was found between the predicted high and low group means (0.8 ppm; P = 0.003). An additional 120 lines were quantified the following year to determine repeatability. GN exhibited moderate narrow-sense heritability (h2 = 0.42) and a high genetic correlation (r = 0.79) across years. Moderate predictive ability as was observed in cross-validation (range 0.38 - 0.61), and forward prediction using 2023 to predict 2024 (r = 0.39). A genome-wide scan did not identify any major-effect loci, suggesting GN content is polygenic, thus enabling same-season genomic selection to reduce GN content in this germplasm.