Frontiers in Plant Science

Background and AimsThe genus Solanum including the wild species S. dulcamara, is rich in specialized metabolites such as steroidal glycoalkaloids (SGAs). Yet, much of this chemical diversity remains poorly characterized. This study aims to provide a comprehensive assessment of intra-specific chemodiversity in S. dulcamara. Using a dataset generated from 42 globally distributed accessions, we tested whether metabolic profiles differ among plant organs. We postulated that metabolic richness and abundance vary across accessions. Additionally, we hypothesized that differences in geographic origin or altitude affect SGA chemodiversity. MethodsAn untargeted metabolomic approach was applied to leaf, flower and root samples of 42 S. dulcamara accessions. Plants were grown in the greenhouse, and the extracted metabolites were analyzed using UHPLC-HRMS/MS in positive and negative ionization modes. Data processing and metabolite annotation were performed with a tailored bioinformatics workflow. Multivariate analyses were performed to evaluate chemical variation across organs and accessions. Key ResultsOur analyses revealed both organ and accession-specific metabolic diversity. Principal component analysis and clustering analyses revealed metabolic differentiation between leaves, flowers and roots. Leaves showed the highest metabolite richness and abundance, while roots showed the lowest. Alkaloids, especially SGAs, dominated positive mode profiles in roots, whereas shikimates and phenylpropanoids were prominent in negative mode profiles. Based on the leaf and flower SGAs profiles, four chemotypes were identified. Analyses of flavonoid and cinnamic acid derivatives, however, did not reveal chemotypes. Feature-based molecular network analyses confirmed that metabolite clusters are associated with plant organs, but not with altitude or geographic origin of the accessions. ConclusionsThe intraspecific chemodiversity within S. dulcamara is mainly driven by organ and accession-specific metabolic differences. We identified four SGA leaf and flower chemotypes, suggesting possible functional and ecological roles of this aboveground chemodiversity. These insights may contribute to applied research in plant resistance breeding and crop production.

The barley yellow mosaic virus disease is one of the most important threats of barley production in Europe and Asia. Transmitted by the soil-borne plasmodiophorid Polymyxa graminis, there are no direct control options against the causal bymoviruses Barley Yellow Mosaic Virus (BaYMV) and Barley Mild Mosaic Virus (BaMMV). Resistance breeding is thus the only viable approach and has been very successful in the past, with the resistance-conferring alleles rym4 and rym5 of the EUKARYOTIC TRANSLATION INITIATION FACTOR 4E being used extensively in European winter barley breeding. However, virus strains have meanwhile overcome this resistance. Therefore, there is an urgent need for new sources of resistance. Genome editing with Cas endonucleases is a timely and promising approach in this respect. However, the small insertions and deletions that frequently arise during site-directed mutagenesis usually lead to the knockout of the target genes. In the case of EIF4E, loss-of-function is accompanied by significant yield reduction. Consequently, more precisely edited alleles with retained function are necessary for crop improvement. The present study represents the first application of base editing in barley plants, using the EIF4E gene as an example. Base exchanges were made at two positions in this gene using an nCas9-cytidine deaminase fusion, resulting in a total of 10 novel EIF4E alleles in addition to the introduction of a single nucleotide polymorphism that is part of rym4. Two of these newly generated alleles led to resistance upon BaMMV inoculation without adverse effects on yield, proving this approach promising to generate new material for resistance breeding.

Although the KINETOCHORE NULL2 (KNL2) protein is an essential inner centromeri c protein that is crucially important for assembly and functioning of kinetochores, our understanding of its organization, dynamics and function of distinct isoforms in the cells of plant species undergoing mitosis/meiosis is far from complete. In this study, we identified and characterized two KNL2.1/KNL2.2 genes in cowpea. GUS reporter constructs and qRT-PCR revealed that the expression profiles of both genes were variable across organs, with the highest expression in leaves and roots. Using an EYFP gene fusion coupled with immunostaining, it was demonstrated that both KNL2 variants colocalized at centromeres in a cell-cycle-dependent manner. The CRISPR/Cas9 technique was used to generate various in-frame deletion and out-of-frame knock-out knl2 mutants. Single- and double-gene knock-out mutants were generated, and the effects of mutations on plant development and seed setting were analyzed. The results are discussed both with respect to the roles of these proteins in kinetochore assembly and in the context of using KNL2 genes for in vivo production of haploids in cowpea. Significance statementThis study identifies two paralogous KNL2 genes in cowpea and reveals their functional redundancy during centromere assembly and essential role in seed development. These findings expand our knowledge of kinetochore dynamics and provide a basis for exploring the evolutionary diversification of centromeric proteins in legumes.

The apoplast is a primary location for interactions between plants and invasive pathogens and is the site where many pathogen-secreted effectors are perceived by cell surface immune receptors to activate defence responses. However, our understanding of apoplastic interactions remains limited because protein interactions are difficult to investigate in this harsh extracellular environment. TurboID-based proximity labelling (PL) has emerged as a powerful approach for studying protein interactions in plants, though its application has to date been restricted to intracellular proteins. Here, we designed and validated a TurboID-based PL strategy for investigating protein interactions in the leaf apoplast using the well-characterised interaction between the Phytophthora infestans elicitor INF1 and the receptor-like protein (RLP) REL in Nicotiana benthamiana. Transient expression of SP-INF1-TurboID (INF1-T) induced a cell death (CD) response comparable to that triggered by native INF1, demonstrating that fusion of the TurboID tag did not impair INF1 recognition by REL. Apoplastic localisation of both INF1-T and the control construct SP-eGFP-TurboID (eGFP-T) was confirmed, validating their suitability for PL experiments. Efficient TurboID-mediated biotinylation was achieved in the apoplast using co-infiltration of biotin, ATP and magnesium acetate. Streptavidin-HRP immunoblotting revealed distinct biotinylation profiles for INF1-T and eGFP-T. Furthermore, co-immunoprecipitation demonstrated specific biotinylation of REL by INF1-T, but not by eGFP-T, in wild-type, bak1 and sobir1/sobir1-like N. benthamiana. These findings demonstrate that TurboID-based PL is functional in the apoplast and provides a proof-of-concept for investigating elicitor-receptor interactions in this compartment.

BackgroundCymbidium tortisepalum var. longibracteatum is a Class II nationally protected endangered plant in China with significant economic value. AimThis study aimed to establish an efficient in vitro flowering system and identify key hormonal factors regulating flower bud differentiation. MethodsOrthogonal experiments and hormone treatments were performed to evaluate the effects of TDZ, 6-BA, NAA, IBA, PP333, ABA, and GA3 on flower bud induction in tissue-cultured plantlets. ResultsTDZ was identified as the key initiator of flower bud differentiation, as no flower buds were induced in TDZ-free treatments. The optimal hormone combination was 1.0 mg{middle dot}L-1 6-BA + 0.4 mg{middle dot}L-1 TDZ + 0.8 mg{middle dot}L-1 NAA + 1.6 mg{middle dot}L-1 IBA, achieving a flower bud induction rate of 23.33% and a normal flower bud rate of 20.00%. PP333 inhibited flower bud differentiation but reduced malformation; at 0.2 mg{middle dot}L-1 PP333, the normal flower bud rate reached 20.00%. GA3 pretreatment resulted in a flower bud induction rate of 12.04%, whereas ABA pretreatment showed no significant promoting effect on flower bud induction. ConclusionsThis study provides an efficient in vitro flowering system and key hormonal parameters for shortening the breeding cycle and elucidating orchid flowering mechanisms.

Background: Weed control is one of the main challenges in agriculture today, particularly due to the increasing occurrence of herbicide-resistant populations. Among the most problematic species are Lolium multiflorum (L.) and Echinochloa crus-galli (L.) Beauv., for which glyphosate-resistant populations have been reported. In this context, glufosinate ammonium has emerged as an alternative for their control; however, its efficacy may vary depending on species and photosynthetic metabolism. Objective: The objective of this study was to evaluate the differential sensitivity of ryegrass (C3) and barnyardgrass (C4) to ammonium glufosinate by analyzing physiological responses associated with leaf senescence and photosystem II activity. Methods: Visual injury, chlorophyll fluorescence, and ammonium accumulation were assessed. Results: Results revealed a differential response between species. Barnyardgrass exhibited earlier symptom onset and a greater reduction in the quantum yield of photosystem II ({Phi}PSII), whereas ryegrass showed a slower senescence process. These differences indicate a higher sensitivity of barnyardgrass to glufosinate ammonium, possibly associated with its C4 photosynthetic metabolism. Conclusions: It is concluded that the effectiveness of glufosinate ammonium depends on the type of photosynthetic metabolism and on the ability of each species to cope with herbicide-induced oxidative stress. This information contributes to optimizing glufosinate ammonium use and to the development of management strategies aimed at delaying the evolution of herbicide resistance.

Eggplant (Solanum melongena L.) displays extensive fruit color diversity, in which chlorophyll-related pigmentation contributes to both external appearance and market value. Previous genetic studies identified SmAPRR2 and SmGLK2 as major candidate genes controlling uniform green pigmentation and green netting in fruit, respectively, but their individual and combined functional contributions had not been validated through targeted mutagenesis in a common genetic background. Here, we established a multiplex CRISPR/Cas12a system in eggplant accession MEL3, representing, to our knowledge, the first application of this nuclease for genome editing in eggplant. Transformation efficiency was 2.0%, but all 15 genotyped regenerants were edited, yielding four SmAPRR2 and six SmGLK2 alleles. Segregation and crossing enabled the recovery of six transgene-free lines carrying single or combined edited alleles. Disruption of SmGLK2 abolished the reticulated green netting pattern while preserving a uniformly green peel and the internal green ring. Conversely, disruption of SmAPRR2 reduced the background uniform peel pigmentation and eliminated the green ring while retaining green netting. Double mutants carrying disruptive alleles at both loci produced white fruits lacking internal green pigmentation, whereas putatively hypomorphic SmAPRR2 and SmGLK2 alleles generated intermediate phenotypes. Whole-genome resequencing identified only two predicted off-target sites under a canonical TTTV PAM search allowing up to four mismatches. Both were fully covered, and no edited-line-specific candidate variants were detected. These findings establish complementary and partially separable roles for SmAPRR2 and SmGLK2 in fruit peel and flesh chlorophyll pigmentation and demonstrate the potential of Cas12a for functional genomics, allele engineering, and precision breeding in eggplant.

Tillering plasticity is a key adaptive trait in sorghum influencing resource use efficiency via a plants ability to adjust branching to neighbour density. Neighbour detection through red:far-red (R:FR) light sensing regulates this plasticity. While molecular pathways regulating tiller outgrowth are partly known, the genetic architecture underlying density-responsive tillering has not been resolved in any grass species. A sorghum diversity panel (n = 895) was evaluated over two growing seasons (2023 and 2024) with plant spacing ranging from 5 to 60 cm. A linear mixed model incorporating neighbour distance and tiller counts estimated genotype-specific response. GWAS was conducted on isolated plants (no neighbours within 60 cm) and on estimated responsiveness to neighbours. GWAS identified 52 baseline tillering QTLs and 50 for spacing responsiveness, with 10 overlapping, suggesting shared genetic control. Comparison with 41 R:FR pathway candidate genes revealed enrichment in responsiveness QTLs (5/50, 10%) versus baseline (0/52, 0%) (Fishers exact test, P = 0.025). Our model identified 40 unique density-responsive tillering QTL regions. Reducing genotype response to neighbour absence could be a selection target to develop water-efficient sorghum varieties where controlled architecture may be more valuable than natural plasticity.

Tomato production, yield and fruit quality face major challenges due to several factors, including the complex polygenic inheritance of agronomically relevant traits, biotic and abiotic stresses, and increasingly stringent regulations limiting the use of phytosanitary products. In this context, bioinoculants have emerged as a sustainable strategy capable of enhancing yield without compromising fruit quality, conferring protection against different stresses and exerting a minimal or no impact on environment and human health. In this study, we evaluated the effects and the underlying mechanisms by which Streptomyces sp. N2A, an actinobacteria isolated from soybean rhizosphere, promotes seed germination, vegetative growth and yield in tomato, without modifying fruit quality. The obtained results demonstrated that the bacterial treatment significantly improved seedlin[g]s emergence and growth and development in vegetative stage. At harvest, yield was also significantly enhanced, mainly driven by increased individual fruit weight, which was positively correlated with a thicker pericarp in fruits from N2A-treated plants. Transcriptional analysis during fruit development revealed a coordinated induction of auxin and cytokinin signaling pathways before and after anthesis, providing a hormonal framework that underlies the promotion of pericarp growth. This study provides evidence of the beneficial effect of inoculation with Streptomyces sp. N2A on tomato yield and constitutes the first report describing the modification of fruit morphology and expression of genes involved in phytohormonal modulation during early growth and development, induced by a plant growth-promoting Streptomyces.

Apple scar skin disease (ASSD), caused by Apple scar skin viroid (ASSVd), is characterized by peel scarring, cracking, dappling, and fruit deformation, resulting in reduced fruit quality and marketability. Despite its economic importance, the physicochemical and metabolic alterations underlying disease progression remain poorly understood. To address this knowledge gap, apple fruits representing four stages of ASSD (healthy, lightly infected, moderately infected, and highly infected) were comprehensively characterized. ASSVd infection was confirmed by RT-PCR, amplicon sequencing, and phylogenetic analysis. Fruit morphology and quality attributes, including firmness, total soluble solids (TSS), pH, titratable acidity (TA), and total phenolic content (TPC), were evaluated. ASSVd infection significantly reduced fruit weight and firmness and altered TSS and TA, indicating progressive deterioration of fruit quality. To investigate the underlying metabolic changes, peel and pulp tissues were analysed separately using gas chromatography-mass spectrometry (GC-MS), while major soluble sugars were quantified by 1H nuclear magnetic resonance (1H NMR) spectroscopy. Integrated metabolomic analyses revealed distinct tissue-specific metabolic reprogramming during disease progression. Major soluble sugars declined significantly during early infection, followed by tissue-dependent recovery at later stages, whereas organic acids, amino acids, phenolics, lipids, polyols, and pentacyclic triterpenoids exhibited dynamic stage-dependent changes. Notably, lupeol accumulated progressively, whereas ursolic acid and oleanolic acid declined, indicating disease-associated alterations in host triterpenoid metabolism. Multivariate analyses demonstrated clear metabolic separation among disease stages. Lupeol, ursolic acid, and chlorogenic acid were identified as candidate discriminatory metabolites in the peel, whereas myo-inositol, chlorogenic acid, and aspartic acid were identified in the pulp. Collectively, these findings demonstrate that ASSD induces coordinated, tissue-specific physicochemical and metabolic reprogramming that compromises postharvest fruit quality and reshapes defence-associated metabolism. This study provides the first integrated metabolomic characterization of ASSD progression and identifies potential metabolic biomarkers for disease diagnosis and severity assessment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/741181v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@92e453org.highwire.dtl.DTLVardef@1cc2436org.highwire.dtl.DTLVardef@15d7e27org.highwire.dtl.DTLVardef@1056568_HPS_FORMAT_FIGEXP M_FIG C_FIG

The rapid spread of herbicide-resistant Amaranthus palmeri S. Watson across the Mediterranean region poses a severe threat to crop production. This study characterizes the resistance profile of an A. palmeri population from Gonen, Israel, which survived multiple post-emergence herbicide applications in maize. Greenhouse dose-response and screening trials were conducted across four modes of action (MOAs). The Gonen population exhibited three-way multiple resistance to acetolactate synthase (ALS), photosystem II (PSII) and 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors, marking the first confirmed case outside the Americas. High-level resistance was demonstrated for the ALS inhibitor foramsulfuron and the PSII inhibitor metribuzin, maintaining 40-70% and 80-100% survival, respectively, at double the labelled field rate. Sequencing of the ALS gene revealed a Trp574 to Leu substitution in resistant plants. The population showed differential PSII sensitivity, as atrazine provided complete control despite high metribuzin survival. Sequencing revealed no known psbA mutations, and malathion pre-treatment caused only a slight reduction in metribuzin survival. Partial resistance was observed for the HPPD inhibitor tembotrione (30-50% survival), where malathion pre-treatment reduced survival by 20-30%. In contrast, pre-mergence applications of S-metolachlor (very long chain fatty acid inhibitor), isoxaflutole (HPPD inhibitor), and a fomesafen + terbutryn mixture [protoporphyrinogen oxidase (PPO) + PSII inhibitors] significantly reduced emergence. These findings confirm multiple-resistant A. palmeri in the region and highlight the urgent need for integrated weed management incorporating effective pre-emergence herbicides rather than relying solely on post-emergence chemical control in summer cereal rotations.

Genetic transformation of citrus is essential for functional genomics and trait improvement, yet remains limited by prolonged regeneration cycles, genotype-dependent responses, and inefficient screening of transformed tissues. Here, we established an Agrobacterium-mediated indirect organogenesis-based transformation system for Carrizo citrange and evaluated two visible reporter systems, the anthocyanin regulator ROSEA1 and the betalain biosynthetic cassette RUBY, for monitoring transformed tissues throughout regeneration. An optimized regeneration protocol enabled visible callus ini-tiation within 7 days following a 5-day pre-culture treatment and recovery of PCR-confirmed transgenic plantlets within 5 months. Both visible reporters were compatible with callus development and shoot regeneration throughout the indirect organogenesis workflow. During callus proliferation, visible pigmentation closely corresponded with GFP fluorescence, enabling rapid, non-destructive identification of transformed tissues. However, reporter performance diverged during shoot regeneration: RUBY maintained stable pigmentation throughout regeneration, whereas ROSEA1-associated pigmentation progressively declined despite continued GFP fluorescence. Consequently, RUBY exhibited a 4.7-fold higher pigmenta-tion-based detection rate than ROSEA1 at the shoot stage and showed closer agreement with GFP-based detection. Putative transgenic events were confirmed by PCR in independent lines of both constructs, together with GFP fluorescence in leaves and root tips. Together, these findings establish an efficient indirect organogenesis-based transformation platform for citrus and demonstrate that both ROSEA1 and RUBY are effective visual reporters during callus proliferation, whereas RUBY provides more reliable visual identification during shoot regeneration and plant recovery.

The ability of seeds to survive until dormancy recedes and the germination requirements are met is an adaptive strategy. Proteomics improves our understanding of the mechanisms that control the adaptation to environmental heterogeneity. In this study, we investigated two European beech populations from different habitats that differed in dormancy and germination traits. We found that the populations exhibited different germination strategies, which were reflected in coordinated but quantitatively different proteomic reprogramming. The Miekinia population exhibited stronger accumulation of proteins involved in nucleotide sugar biosynthesis, S-adenosylmethionine metabolism, and flavonoid biosynthesis. Enhanced nucleotide sugar biosynthesis indicates more intensive cell wall remodelling and carbohydrate metabolism, which support embryo growth and faster germination. Increased S-adenosylmethionine metabolism suggests the epigenetic and hormonal regulation of germination differences between populations. Higher flavonoid biosynthesis indicates an enhanced antioxidant capacity associated with environmental protection. In contrast, the Wisa population showed stronger accumulation of proteins involved in RNA processing, suggesting tighter post-transcriptional regulation and proteome reorganization during germination. Consistent with its deeper dormancy and later germination, the Wisa population appears to rely more on RNA-level regulation, whereas the Miekinia population prioritizes metabolic activation. These contrasting proteomic profiles likely reflect population-specific physiological strategies associated with dormancy depth and adaptation to different climatic conditions. HighlightProteomic reprogramming reveals population-specific germination strategies in European beech, linking dormancy depth with contrasting metabolic activation and RNA-level regulation during the transition from dormancy to germination.

Background and aims Plant volatile organic compounds (VOCs) change dynamically with plant development and in response to environmental conditions. However, their potential as non-invasive indicators of phenological progression remains poorly explored. In this study, we developed a framework integrating automated VOC sampling, time-resolved VOC profiling, and machine-learning analysis for the non-invasive assessment of plant phenology. Using soybean (Glycine max (L.) Merr.), we investigated whether development-associated temporal variation in VOC emissions could delineate and predict developmental phases. Methods We collected VOCs daily under controlled environmental conditions from 16 to 43 days after sowing, spanning the transition from vegetative to reproductive stages, using an automated sampling system coupled with thermal desorption-gas chromatograph-mass spectrometer (TD-GC-MS). To characterise temporal changes in VOC profiles associated with phenological progression, we analysed the daily VOC data using a multi-step pipeline combining statistical filtering and similarity-based network analysis. We defined VOC-derived developmental phases from similarity patterns in the VOC profiles, then developed and evaluated machine-learning models to predict these phases. Key results Seven VOCs exhibited distinct phase-dependent dynamics, including green leaf volatiles and monoterpenes showing characteristic temporal changes during phenological progression. Network-based clustering of VOC profiles resolved five developmental phases closely aligned with conventional developmental stages. A machine-learning model predicted these phases from the VOC profiles with high predictive accuracy on independent test data, demonstrating that phenological progression could be quantitatively inferred from VOC emission patterns. Conclusions Our findings support VOC profiling as a reliable and non-invasive approach for assessing phenological progression in soybean. By extracting temporally structured VOC signals, this framework captures developmental information that may be difficult to obtain through visual observation alone, particularly after canopy closure. VOC profiling offers a practical tool for monitoring crop developmental dynamics and has broader potential for plant phenotyping and precision crop management.

O_LISelf-incompatibility can limit the availability of compatible mates in small and isolated populations, eventually reducing average seed set to the point that the long-term persistence of the populations can be impaired. This phenomenon, named the S-Allee effect, is caused by the loss of alleles (S-alleles) at the self-incompatibility locus (S-locus) due to the intense genetic drift experienced by small populations. Quantifying the diversity of S-alleles is therefore of direct interest for biological conservation, but efficient genotyping methods have been lacking so far because of technical challenges associated with the typically extreme levels of polymorphism and complex genomic structure of the S-locus. C_LIO_LIWe used two alternative approaches to genotype the S-locus using NGS sequencing technologies in four natural populations of the endangered Brassica insularis in Corsica. First, we used an NGS amplicon-sequencing approach using generalist primers for each of the two classes of Brassica S-alleles. Second, we obtained whole genome shotgun short-read resequencing data and analyzed them with a recently developed bioinformatic pipeline dedicated to hypervariable loci, which we successfully validated on a public dataset comprising 119 cultivated accessions of B. oleracea. C_LIO_LIBy combining the two approaches in natural populations of B. insularis we identified 31 distinct S-alleles and obtained fully resolved S-locus genotypes for 319 out of 326 sampled individuals. The number of S-alleles varied from four in the smallest population to 18 in the largest one. As a result, the smallest population exhibited very low proportions of compatible individuals, potentially threatening its persistence. We conclude that introducing individuals carrying S-alleles currently absent from the population could help rescue fertility. C_LI

High temperature during grain filling increases rice grain chalkiness and deteriorates grain appearance under climate warming. Although several loci that reduce chalkiness have been identified, breeding strategies that integrate grain level heat tolerance with panicle level heat avoidance remain limited. Here we characterized SL2033, a chromosome segment substitution line carrying a long IR64 derived segment on chromosome 10, and evaluated the combination of the chromosome 10 segment with Appearance quality of brown rice 1 (Apq1), a quantitative trait locus associated with reduced heat induced chalkiness that acts at the grain level. Compared with its recurrent parent Koshihikari, SL2033 had longer flag leaves, altered vertical plant architecture, and lower panicle temperature. Total starch and protein contents were comparable between the two genotypes, whereas RNAseq analysis of the developing endosperm identified specific differences in heat, stress, and cell wall related transcripts. In a two year field trial, a pyramided line combining the SL2033 derived segment with Apq1 had the highest proportion of perfect grains and lowest frequencies of multiple chalky kernel types during the year with hotter grain filling conditions, with no detectable yield penalty. The pyramided line combined longer flag leaves, as in SL2033, with shorter panicle exsertion, as in an Apq1 near isogenic line, and had the lowest panicle temperature among the tested genotypes. Time series unmanned aerial vehicle imaging also detected genotype dependent differences in plant height during early grain filling, supporting distinct temporal patterns of plant development among the lines. These findings demonstrate that pyramiding genetic loci that confer panicle level and grain level heat tolerance is a promising strategy for improving rice grain appearance under high temperature field conditions, which are becoming increasingly prevalent.

Poisonous plants frequently deploy toxic metabolites in an organ- and ontogenetic-specific manner, yet the developmental dynamics of these plant specialised metabolites remain poorly understood. In the Solanaceae, steroidal glycosides (SGs), including steroidal glycoalkaloids (SGAs) and steroidal saponin glycosides (SSGs), are major defence metabolites with potent ecological and pharmacological activities. Here, we investigated how their metabolic profiles are reorganised during flower and fruit development in Solanum dulcamara using untargeted metabolomics and chemometric molecular networking. Non-metric multidimensional scaling (NMDS) using classified metabolite features recovered clear organ-specific and chemotype-specific segregation of samples, which was further strengthened when analyses were restricted to metabolomic features annotated as SGs. In flowers, orthogonal projections to latent structures discriminant analysis (OPLS-DA) separated both floral developmental stage and chemotype, demonstrating that developmental stage and inherited SG polymorphism represent independent axes of chemodiversity in flowers. Chemometric molecular networks showed pronounced diversification of hydroxycinnamate metabolism towards anthesis, including accumulation of caffeoylputrescine in flower buds and progressive acylation of spermidine conjugates across flower development. Upon anthesis, glycosylated hydroxycinnamates and flavonoids, and hydroxycinnamoyl-acylated flavonoid glycosides are accumulated. Fruit ripening followed a contrasting trajectory, in which SGAs were associated with unripe fruit pericarp, whereas ripe pericarp accumulated N- and O-acetylated SGA derivatives, oxidative deamination products, and predicted steroid degradation products, indicating stepwise detoxification of toxic steroidal glycosides during seed maturation. Together, these findings show that the poisonous chemistry in S. dulcamara is highly dynamic and developmentally orchestrated. Flowers accumulate structurally complex metabolites toward anthesis, including conjugates of hydroxycinnamates, whereas fruits metabolise toxic SGAs into less toxic SGs during ripening, linking metabolic remodelling to reproduction and seed dispersal.

Black pod disease is a major threat to global cacao (Theobroma cacao L.) production. It is caused by multiple Phytophthora species with geographic ranges from regionally restricted to pantropical, and developing resistant cultivars requires screening against the diversity of these pathogens. This study evaluated the response of 25 cacao clones to 13 isolates of P. palmivora (eight), P. megakarya (two), and P. theobromicola (three) by nonwounding inoculation of detached pods. Lesion size and lesion expansion status were evaluated 4 days after inoculation. The linear mixed model included 178 combinations among 17 clones and 12 isolates, each represented by at least two biological replicate pods. Lesion size differed among clones (F(16, 449.2) = 25.16) and among isolates (F(11, 416.0) = 32.90), and the clone by isolate interaction was significant (F(150, 465.4) = 1.72; all P < 0.001). PENTAGONA-17 had the smallest estimated marginal lesion size (9.3 mm) and fitted values of 3.8 to 16.9 mm across 11 isolates representing all three species. ARF-31, ICS-47, POUND-7, and PMCT-73 had the next four smallest estimates, whereas NA-246 had the largest (36.7 mm). Isolate estimates varied more than fourfold, from 9.6 to 42.0 mm. Despite a marginal estimate of 12.9 mm, POUND-7 had a fitted estimate of 49.2 mm with P. megakarya GH34. The probability of lesion expansion also differed among clones and isolates (both P < 0.001). Together, these findings identify candidates for further resistance evaluation and show that multi-isolate screening provides a stronger basis for selecting materials for black pod resistance breeding.

Soybean, a widely cultivated leguminous crop valued for its protein, amino acids, and oil, faces the challenge of maintaining protein levels, which have an inverse correlation with yield. Reducing leaf chlorophyll levels could increase seed protein levels without compromising yield; however, this is yet to be tested. Therefore, to understand the impacts of low chlorophyll mutations on soybean yield and seed composition, we screened and compared 25 low chlorophyll soybean mutants to their 11 dark green parents. PI548210 (Lincoln mutant) demonstrates a higher concentration of protein without affecting yield compared to its dark green parent PI548362 (Lincoln), suggesting it as a good candidate for further large-scale field trials. PI547555 (Y11/y11, Clark mutant) demonstrates a lower concentration of oil without impacting yield, alongside lower gross photosynthesis, but with chlorophyll levels in the pod and seed tissues that are comparable to its dark green parent PI548533 (Clark). These findings are consistent with the oil concentration of the soybean being influenced by pod and seed photosynthesis, which is correlated with pod height and row spacing. Chlorophyll levels in the leaf do not necessarily correlate with those in the pod and seed of low chlorophyll mutants, possibly due to substantially lower expression of chlorophyll synthesis genes in the pod and seed. SIGNIFICANCEO_LIPI548210 (Lincoln mutant), one of twenty-five low chlorophyll soybean mutants, demonstrates a higher concentration of soybean protein without affecting yield compared to its dark green parent (Figure 1 and Table 1). C_LIO_LIPI547555 (Y11/y11, Clark mutant), a low chlorophyll soybean mutant, demonstrates a reduced concentration of soybean oil without impacting yield, alongside lower gross photosynthesis in pod and seed tissues compared to its dark green parent (Figures 3 and Table 2). These findings suggest that the oil concentration of the soybean is influenced by pod and seed photosynthesis, which is in turn influenced by pod height and row spacing (Figure 2). C_LIO_LIChlorophyll levels in the leaf do not necessarily correlate with those in the pod and seed of low chlorophyll mutants, possibly due to substantially lower expression of chlorophyll synthesis genes in the pod and seed (Figure 5-6). C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/744892v1_fig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@4282dcorg.highwire.dtl.DTLVardef@9d565forg.highwire.dtl.DTLVardef@1918292org.highwire.dtl.DTLVardef@1359b1_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Two low chlorophyll mutants are as healthy as their dark green parents. Lincoln and its low chlorophyll mutant, left; Clark and its low chlorophyll mutant, known as Y11/y11, right. It can be seen by eye that the plants have low chlorophyll (light green/yellow leaves) but a similar growth habit to their dark green parents. See Supplemental Figures 1-4 for contrast, where low chlorophyll mutants are stunted in growth compared to their dark green parents. C_FIG O_TBL View this table: org.highwire.dtl.DTLVardef@657ec9org.highwire.dtl.DTLVardef@166e75borg.highwire.dtl.DTLVardef@df23c7org.highwire.dtl.DTLVardef@1a60124org.highwire.dtl.DTLVardef@194ed96_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 1.C_FLOATNO O_TABLECAPTIONComparison of seed yield, weight, seed composition between low chlorophyll mutants and their dark green parents. ANOVA is used with linear mixed model (random effect = block, fixed effect = variety). Least squares mean is used to compare. For yield and seed composition, N=4 blocks. For leaf chlorophyll (SPAD), N=40. Yield is average yield per plant (g). n.s. = not significant. C_TABLECAPTION C_TBL O_FIG O_LINKSMALLFIG WIDTH=179 HEIGHT=200 SRC="FIGDIR/small/744892v1_fig3.gif" ALT="Figure 3"> View larger version (26K): org.highwire.dtl.DTLVardef@7a368aorg.highwire.dtl.DTLVardef@192b8f0org.highwire.dtl.DTLVardef@1abb738org.highwire.dtl.DTLVardef@89e978_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 3.C_FLOATNO Light response curve of low chlorophyll mutant (Y11/y11, PI547555) and its parent (Clark, PI548533). Rates of net and gross photosynthesis of low chlorophyll (white) and dark green parents (black) pods under field conditions. Each dot represents a value (n=4) {+/-}SE. We assumed that the seeds greatly inhibited the transmittance of light through the pod and used photosynthetic photon flux density for a single-side. C_FIG O_TBL View this table: org.highwire.dtl.DTLVardef@3f0528org.highwire.dtl.DTLVardef@16ba712org.highwire.dtl.DTLVardef@a5ab2aorg.highwire.dtl.DTLVardef@889254org.highwire.dtl.DTLVardef@3efa4f_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 2.C_FLOATNO O_TABLECAPTIONPod photosynthetic parameters for low chlorophyll mutant (Y11/y11, PI547555) and its parent (Clark, PI548533). Photosynthesis was measured 1 September through 15 September 2021 at the University of Illinois Energy Farm in Urbana, IL, USA. The statistical analysis was done using ANOVA with linear mixed model (alpha=0.05). N=4 {+/-} SEM for Clark and N=3 {+/-} SEM for Y11. C_TABLECAPTION C_TBL O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/744892v1_fig2.gif" ALT="Figure 2"> View larger version (23K): org.highwire.dtl.DTLVardef@a36c26org.highwire.dtl.DTLVardef@1116c8forg.highwire.dtl.DTLVardef@ee5e61org.highwire.dtl.DTLVardef@1766712_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 2.C_FLOATNO Low chlorophyll mutant (Y11/y11, PI547555) and its parent (Clark, PI548533) differ in concentration of seed oil, which interacts with height of pod and row spacing. The box plots show the median (central line), the lower and upper quartiles (box) and the minimum and maximum values (whiskers). The statistical analysis was done using ANOVA with linear mixed model (n=3 blocks, alpha=0.05). Least squares mean is used to compare. N.s., non- significant in the analysis. A. Concentration of oil in low chlorophyll mutant seeds from the upper canopy decreased by 4% compared to the dark green parent (18.2% vs 19%) while there was no difference between them in the seeds from the lower canopy (20.2% vs 20.6%). B. Schematic layout of 2013 field setting showing two different row spacings. C. Concentration of oil in low chlorophyll mutant decreased by 2% in 38cm spacing (21.4% vs 22%) while there was no difference in 19cm spacing (21.3% vs 21.7%) in 2013 field. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/744892v1_fig5.gif" ALT="Figure 5"> View larger version (22K): org.highwire.dtl.DTLVardef@68e508org.highwire.dtl.DTLVardef@94a6ccorg.highwire.dtl.DTLVardef@152a187org.highwire.dtl.DTLVardef@1eae137_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 5C_FLOATNO (greenhouse). Correlation between the level of leaf chlorophyll (x-axis: SPAD reading) and the level of immature pod or seed chlorophyll (y-axis, mg/g DW). Line represents the linear regression model. R-squared is a coefficient of determination, the percentage of the response variable variation that is explained by the linear model. Pod is labeled by the fresh weight of seeds it contained. A. Level of chlorophyll of 25-100mg pod (n=18). B. Level of chlorophyll of 100-200mg pod (n=17) . C. Level of chlorophyll of 25-100mg seed (n=17). D. Level of chlorophyll of 100-200mg seed (n=20). C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=180 SRC="FIGDIR/small/744892v1_fig6.gif" ALT="Figure 6"> View larger version (28K): org.highwire.dtl.DTLVardef@167fd88org.highwire.dtl.DTLVardef@361472org.highwire.dtl.DTLVardef@786325org.highwire.dtl.DTLVardef@1b53855_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 6.C_FLOATNO Levels of gene expression in chlorophyll synthesis pathway. A. CHL common pathway genes; Glutamyl-tRNA reductase (GluTR). Glutamate 1- semialdehyde aminotransferase (GSA-AT). ALA dehydratase (ALAD). Uroporphyrinogen III synthase (UROS). Uroporphyrinogen III decarboxylase (UROD). Protoporphyrinogen IX oxidase (PPO). B. Mg branch; Mg-chelatase (Mgch). Magnesium-protoporphyrin IX monomethyl ester cyclase (MPEC). Protochlorophyllide reductase (POR). 3,8-divinyl protochlorophyllide a 8-vinyl-reductase (4VCR). Heme pathway; Ferrochelatase (FECH). Heme oxygenase (HO). Phytochromobilin synthase (HY). Data come from Severin et al (2010). RPKM, reads per kilobase per million mapped reads. DAF, days after flowering. The source seed is experimental line A81-356022 which was generated by introgressing G. soja (PI468916) into G. max (A81-356022). C_FIG

Yield improvement is a main issue in the oil palm industry. Several ways to achieve this goal are possible, like, for instance, improving fruit set. Here we explore a planting strategy to increase two major factors influencing pollination; i.e. (1) the number of individuals of its main pollinator, Elaeidobius kamerunicus Faust, and (2) pollen availability, by mixing oil palm planting material, usually highly feminine, with a new oil palm material, the "super-male" (SM) planting material, with a high ratio of male inflorescences. We design and study a field trial comprising extremely feminine planting material mixed with different SM densities. Using Mark-Release-Recapture experiments we estimate the average dispersal of the pollinators over three different periods of the year, from 2017 till 2022. Second, using phenological data recorded over the same period, we studied the impact of the SM planting density on the population size of the pollinators. Finally, we assess the impact over time of different SM planting densities on the fruit set, the fruit-to-bunch, and the number of trapped wild pollinators. The combination of SM material with a highly feminine planting material provides very promising results in terms of fruit set, linked with increased pollinator population and pollen availability, demonstrating its potential for application in oil palm plantations.