Plant Molecular Biology

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.

O_LIResearch on the functions and molecular mechanisms of long non-coding RNAs (lncRNAs) involved in regulating plant freezing tolerance is still in its infancy. Our previous research work identified that lncRNA DROUGHT INDUCED LNCRNA (DRIR) regulates gene expressions in Arabidopsis. However, the underlying molecular mechanism is still unknown. C_LIO_LIThis study demonstrates that lncRNA DRIR regulates plant freezing tolerance by affecting alternative splicing patterns of pre-mRNAs. C_LIO_LIThrough chromatin isolation by RNA purification followed by mass spectrometry (ChIRP-MS), we identified two DRIR interacting proteins: CP29A and CP29B. We showed that the drirD mutant, which exhibits elevated DRIR expression and DRIR overexpression lines showed increased sensitivity to freezing stress, whereas DRIR RNAi lines were more tolerant to the stress. CP29A and CP29B bind to nuclear transcripts and, together with DRIR, regulate pre-mRNA alternative splicing under freezing stress. Notably, DRIR induces the relocalization of CP29A and CP29B to autophagosomes, leading to autophagy-mediated protein degradation. C_LIO_LICollectively, our findings elucidate the molecular mechanism by which DRIR influences the autophagy-based degradation of its binding proteins CP29A and CP29B, thereby regulating plant freezing tolerance by altering the alternative splicing patterns of pre-mRNAs, providing novel insights into the functions and mechanisms of lncRNAs in plants adapting to freezing environments. C_LI

Long non-coding RNAs (lncRNAs) have emerged as critical players in plant development and stress responses, yet their involvement in pollination responses is largely unknown. To address this gap, we identified and characterized lncRNAs and their cis-acting, trans-acting, and miRNA-mediated regulatory interactions during both compatible and incompatible pollination in Arabidopsis thaliana. Leveraging publicly available datasets, we analyzed expression profiles at 10 and 60 minutes post-pollination. We identified 1,073 novel and 3,422 annotated lncRNAs, with 1,002 novel and 985 annotated, respectively, showing detectable expression after filtering. Differential expression analysis identified 12 lncRNAs at 10 min and 32 lncRNAs at 60 min post-pollination. Further investigation revealed 9 cis-targets, 112 trans-targets, and 144 miRNA-mediated regulatory interactions, many of which were enriched in pathways related to stress, defense, and self-incompatibility. Notably, the regulatory landscape is more active at 60 minutes than at 10 minutes post-pollination. These findings provide a robust framework and resource to facilitate future functional studies of lncRNAs during pollination.

In nature, plants are subjected to multiple environmental stress factors simultaneously or sequentially. Recent studies revealed that when three or more stress factors impact a plant simultaneously (termed multifactorial stress combination; MFSC), plant survival declines, even if the intensity of each individual stress involved in the MFSC is low. We previously identified RAP2.3 as a key transcription factor (TF) required for Arabidopsis thaliana survival, specifically under a MFSC of salt+excess light+heat stress (i.e., S+EL+HS). Here we report that RAP2.3 is required for the expression of SIGMA3, a nuclear-encoded factor that directs plastid RNA polymerase to specific plastid promoters, and MYB51, a key stress response TF involved in glucosinolate metabolism and oxidative stress responses, specifically during a MFSC of S+EL+HS. Like rap2.3 mutants, myb51 and sig3 mutants display significantly low survival rate specifically under the MFSC of S+EL+HS. Based on MYB51 gene regulatory network analysis and characterization of jasmonic acid (JA) mutants, we further reveal that suppression of JA signaling could play an important role in promoting plant survival under conditions of S+EL+HS. Our findings uncover an additional layer of the response of plants to MFSC, as well as identify potential targets for breeding crops with enhanced tolerance to climate change.

We recently reported the identification of endogenous viral elements (EVEs) originating from the Caulimoviridae family within the alfalfa (Medicago sativa L.) genome. Our subsequent identification of ubiquitous rhabdoviral elements in infected and healthy alfalfa tissues by high throughput sequencing prompted us to suggest that the alfalfa genome might be populated with integrated rhabdoviruses as well. Bioinformatics analysis using 26 publicly available alfalfa genomes proved the suggestion accurate. We found multiple non-retroviral segments of the Rhabdoviridae family belonging to the genera Betanucleorhabdovirus and Betacytorhabdovirus that appeared to be stable constituents of the host genome. In that capacity they could potentially acquire functional roles in alfalfas development and response to environmental stresses. We believe this study reveals the first documented case of rhabdoviruses integrated into the alfalfa genome.

Drought and heat are key abiotic stresses that severely affect crop production. Sorghum (Sorghum bicolor L. Moench), a drought-resilient cereal, serves as a model for studying abiotic stress responses in cereals. To elucidate the molecular responses to individual drought and heat stress, we conducted a transcriptome analysis of sorghum seedlings subjected to individual drought and heat treatment for 1h and 6h. Our analysis revealed distinct as well as overlapping patterns of gene expression between the two stresses at two time points, with a larger transcriptional shift observed at 6h of drought and heat treatment. We identified 410 and 4,136 differentially expressed genes (DEGs) in response to 1 and 6 h of drought treatments, respectively, whereas 1,807 and 2,776 DEGs were identified under 1 and 6 h of heat treatments. Among all four stress conditions, 32 common DEGs were identified. Genes encoding ion transporters were enriched among DEGs common in both 1h stress treatments. Genes involved in ribosome biogenesis were enriched among the common DEGs in both 6 h stress treatments. Specifically, drought-regulated genes were involved in ribosome biogenesis, whereas heat-induced genes were involved in protein folding and histone modifications. Enrichment of ribosome biogenesis in different sets of DEGs suggests that maintaining a balance between growth and survival by regulating protein synthesis may play a role in defining early stress response in sorghum. 6h of drought resulted in strong upregulation of abscisic acid and jasmonate-associated genes. Genes encoding bZIP, MYB, and HSF transcription factors displayed both stress-specific and common temporal regulation, suggesting vital regulatory roles of these transcription factors in mediating responses to drought and heat treatments. Extensive downregulation of genes encoding core histone proteins, in response to both 6h of drought and heat stress, was detected, indicating possible roles of chromatin structure and accessibility in mediating early responses to drought and heat treatments in sorghum.

Functional validation of genetic components in plants often requires cloning them separately into both plant and bacterial expression vectors, a process that is both time-consuming and laborious. This study aimed to simplify this workflow by developing plant-bacteria dual-host promoter systems that drive high-level constitutive expression in both environments. To achieve this, two variants of the chloramphenicol acetyltransferase promoter (PCAT), a bacterial {sigma} factor-dependent promoter, were integrated into the cauliflower mosaic virus 35S promoter (P35S), and their performance was evaluated using a hygromycin phosphotransferase (HPT)-GFP fusion reporter. One of these variants, PCAT1, conferred hygromycin resistance to Escherichia coli (DH5 and BL21 (DE3)) and maintained high-level expression comparable to the original P35S in onion epidermal cells. A hybrid P35S enhancer-PNOS system also conferred hygromycin resistance to E. coli, but its activity in inducing GFP signals in onion cells remained lower than that of P35S. Due to its compact size (89 bp) and efficiency, PCAT1 can serve as a module for converting standard plant vectors into dual-host systems, accelerating gene characterization and the development of new gene-based tools.

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.

Magnaporthe oryzae, the rice blast fungus, plays a role as a model organism for molecular plant-microbe interaction research. Studies on the pathogenic mechanism of this fungus revealed many genes involved in signaling pathways. As multi-omics data are being available, genomic-level researches have been conducted to uncover the underlying biological processes during the pathogenesis of M. oryzae. Identifying the genome-wide protein-protein interaction (PPI) network is one of the omics-level approaches, which helps to understand signaling and regulatory pathways. However, existing biological network resources of M. oryzae are not sufficient to decipher pathogenesis mechanisms due to the abundance of false positives/negatives. In this study, a reliable PPI network database of M. oryzae, MagNet, was constructed with three methods, including homology-based Interolog search, co-expression network construction, and domain-domain interaction (DDI)-based prediction. With three approaches altogether, the pan-network with 5,600,976 interactions was generated, including 217,531 highly confident interactions supported by all three methods. Experimental data on M. oryzae PPIs supported that our PPI network can predict PPIs with higher accuracy compared to the previously constructed databases. MagNet would provide integrated biological network data, which can help to understand the molecular mechanisms of the rice blast fungus. The PPI data can be accessed via https:/magnet.scnu.ac.kr.

Plant regeneration is a major determinant of transformation and genome-editing efficiency, yet the endogenous regulatory networks controlling regenerative competence in horticultural crops remain incompletely understood. The miR319-TCP module regulates multiple developmental processes in plants, but its function in lettuce regeneration has not been defined. Here, we performed a genome-wide analysis of the TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) gene family in lettuce (Lactuca sativa). Thirty-three LsTCP genes were identified and classified into Class I/PCF, Class II/CIN, and Class II/CYC/TB1 groups. Five CIN-class genes, LsTCP2, LsTCP3, LsTCP4, LsTCP10, and LsTCP24, were predicted as high-confidence miR319 targets and supported by degradome-based cleavage evidence. MIR319-overexpression (OX319) explants showed enhanced de novo shoot regeneration, with 94.5% regeneration efficiency and 1.92 shoots per explant, whereas STTM-miR319 suppression (S319) explants showed reduced regeneration, with 28.5% regeneration efficiency and 0.36 shoots per explant. These phenotypes were associated with altered expression of several miR319-targeted CIN-TCP genes, particularly LsTCP4, LsTCP10, and LsTCP24. Disruption of LsTCP4 increased regeneration efficiency to 91.4% and shoot production to 2.05 shoots per explant, resembling the regeneration-enhancing effect of miR319 overexpression. In contrast, disruption of the non-target CIN gene LsTCP17 did not significantly affect regeneration under the tested conditions. Together, these results identify LsTCP4 as a key miR319-responsive negative regulator of de novo shoot regeneration and highlight miR319-mediated repression of LsTCP4 as a potential endogenous strategy for improving lettuce regeneration.

The TETRATRICOPEPTIDE THIOREDOXIN-LIKE (TTL) proteins are a plant-specific family proposed to function as peripheral membrane proteins that contribute to abiotic stress tolerance in Arabidopsis, likely by maintaining cell wall integrity through brassinosteroid signaling. Previously, we identified a TTL gene that was differentially regulated at the translational level in nitrogen-fixing soybean plants under water deficit (WD) conditions. This finding prompted the characterization of the soybean TTL gene family. Using the Glycine max v4.0 proteome, we identified ten TTL homologs (GmTTL1-GmTTL10), which are unevenly distributed across five chromosomes. Phylogenetic and structural analyses grouped these genes into three clades and revealed a highly conserved exon-intron organization. Likewise, GmTTL proteins display a conserved number and arrangement of TPR and TRXL motifs. To gain insights into their potential biological functions, we integrated co-expression and differential expression analyses. This approach identified a co-expression module enriched for translationally downregulated genes related to the Gene Ontology terms "cellular anatomical entity", "membrane", "cell periphery", "cell wall modification", "nitrate assimilation", and "cell wall organization or biogenesis". Protein-protein interaction network analysis of this specific subset of genes uncovered a novel GmTTL connection with two nitrate reductase enzymes in nitrogen-fixing plants subjected to WD, potentially linking the TTL gene family to new functions or roles. This study provides a framework for future functional studies of GmTTL proteins and their contribution to abiotic stress adaptation in soybean. Key MessageThis work presents the first functional characterization of TTLs proteins in legume species and highlights key processes that may link the TTL gene family to new functions or roles.

O_LISymbiosis between legumes and rhizobia is beneficial on nutrient-poor soils, as it enables the fixation of atmospheric N2. To establish this symbiosis, gene expression in both the host plant and the symbiont has to be regulated. To understand the underlying RNA-mediated regulation of host gene expression, we designed experiments to identify competing endogenous networks involving circular RNA, microRNA, and linear transcripts during symbiosis, using wt and symbiosis-deficient Lotus japonicus mutants with the rhizobium Mesorhizobium loti (M. loti). C_LIO_LICircRNA, miRNA, and linear transcripts were identified from Lotus japonicus wildtype and CCamK mutant (ccamk-13; snf-1) seedlings without inoculation or with M. loti inoculation using deep short-read sequencing with rRNA-depletion and random primers. C_LIO_LIDifferentially expressed miRNAs showed negative correlations to predicted target genes and may regulate symbiotic processes. The symbiosis essential iron-sensor LjnsRING/BRUTUS expresses a circRNA which was upregulated in symbiotic treatments. This circRNA may act as a target mimic and contribute to nodule longevity. CircRNAs are predicted to act predominantly as trans-regulatory molecules with similar frequencies in Arabidopsis thaliania, Oryza sativa, and Lotus japonicus. C_LIO_LIWe identified novel miRNAs, long noncoding RNAs, and circRNAs, and nominated several as potential new regulatory non-coding RNAs that may act as target mimics to stabilize genes and support symbiosis. C_LI SummarySymbiosis between Lotus japonicus and Mesorhizobium loti involves treatment-specific regulation of competing endogenous RNA networks involving circular RNA, miRNA, and linear transcripts.

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.

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.

Salinity is a major crop production constraint in dry pea (Pisum sativum L.), making the development of salt-tolerant varieties essential to improve crop productivity and land-use efficiency. The genetic mechanisms of salt tolerance in dry pea is largely unknown, and research on salt-tolerant genes is limited. In this study, we established comprehensive genomic and transcriptomic resources, along with a robust screening protocol, to dissect the genetic basis of salinity tolerance using two germplasm sets: the USDA pea diversity panel, consisting of approximately 200 globally sourced accessions, and a set of 300 modern elite lines from the NDSU Pulse Crops Breeding Program. Genetic variation for the salinity response was assessed based on ten phenotypic traits, with root dry weight, shoot dry weight, and specific root length identified as key indicators based on their heritability. Genome-wide association mapping uncovered significant genomic regions and several candidate genes linked to salt stress, with the strongest association found on chromosome 6. Overlapping QTL signals across traits suggest a shared genetic architecture underlying salinity tolerance. Field-based transcriptomic analysis further identified five putative genes involved in salinity response conserved across multiple crop species. Notably, Psat5g000800, encoding a glycosyl hydrolase gene, was markedly upregulated under salinity stress. These findings highlight the complex, multi-gene regulatory nature of salinity tolerance in dry pea and underscore the importance of functional validation of candidate genes. This study provides key insights and practical tools to support breeding efforts aimed at improving salt tolerance in dry pea.

Abscisic acid (ABA) plays a critical role in regulating plant responses to abiotic stress by modulating various physiological processes through complex molecular networks. TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTORS (TCP) transcription factors are known developmental regulators, but their roles in ABA signaling and abiotic stress responses remain poorly understood. We addressed this question by analyzing the closely related Arabidopsis thaliana class I TCPs TCP14 and TCP15 using a variety of genetic and molecular approaches. We found that TCP14 and TCP15 negatively influence ABA and salt stress responses. They function by directly activating genes encoding negative regulators of ABA signaling, such as ABA-INSENSITIVE FIVE-BINDING PROTEIN 2 (AFP2), thereby suppressing the expression of ABA-INSENSITIVE 5 (ABI5) and downstream ABA-responsive genes to inhibit ABA responses under non-stressful conditions. Notably, the TCPs are targets of ABA-mediated regulation, as ABA negatively affects TCP14 and TCP15 protein abundance, providing a mechanism to limit TCP-dependent transcriptional growth responses and de-repress ABA-signaling pathways during times of stress. We propose that the antagonistic interplay between class I TCPs and ABA may serve to fine-tune plant growth and stress responses according to environmental conditions, positioning TCP14 and TCP15 as crucial players in balancing plant developmental progression and stress adaptation.

Understanding how gene families evolve can offer great insight into adaptation at the phenotypic and ecological levels. This is particularly true in plants, where transcription factor gene families are often targeted for breeding programs to improve the agronomic traits of economically important crops. While recent advances in next generation sequencing have accelerated the wealth of genomics data, there remains a lack of accessible and reproducible genome mining pipelines tailored for gene family characterisation. Here, we address this gap by developing GENE-FAM, an automated, scalable and open-source pipeline designed to mine and predict gene families based on conserved domains and motifs. To illustrate its application, we apply GENE-FAM to annotate MADS-box transcription factor genes across multiple Cannabis sativa genomes. A comprehensive set of MADS-box genes was identified across three C. sativa cultivars, including both previously annotated and newly predicted genes. Through phylogenetic analyses, we confirm that all type II MADS-box gene subfamilies represented in flowering plants are present in C. sativa. Comparing our annotations with those of Arabidopsis thaliana and Solanum lycopersicum revealed that while most MADS type II families are highly conserved, SEPALLATA-like genes have undergone diversification in C. sativa. Together, these results demonstrate the application of GENE-FAM for genome-wide identification and characterisation of gene families in non-model species, revealing novel insights into MADS-box gene family evolution in C. sativa.

Cytochrome P450 monooxygenases (CYP450s) are key oxidative enzymes that diversify plant specialized metabolites and play a central role in the biosynthesis of bioactive withanolides in Withania somnifera (L.) Dunal. Despite their importance, genome-wide information on CYP450s in W. somnifera has remained elusive. Herein, the first high-quality genome assembly (2.2 Gb, scaffold N50: 47.4 kb) of an Indian W. somnifera cultivar was generated using a hybrid Oxford Nanopore-Illumina sequencing strategy. Comparative analysis with the NCBI reference genome revealed moderate SNP and indel variations, reflecting intraspecific genetic diversity. A comprehensive CYP450 catalog was established and analyzed phylogenomically across nine plant genomes, encompassing both withanolide-producing and non-producing Solanaceae and non-Solanaceae species. Unique CYP families (CYP450A, CYP1194, and CYP705A) were detected exclusively in W. somnifera, suggesting lineage-specific metabolic innovations, while Solanaceae-restricted (CYP82E/M) and absent (CYP81B, CYP6) lineages highlight taxonomic divergence. Across all analyzed genomes, 36 conserved CYP450 subfamilies, including triterpenoid-associated members, were identified, suggesting a shared oxidative framework adaptable to specialized metabolism. Moreover, potential candidate genes in the triterpenoid pathway, including CYP72A692_1, CYP72A560_4, CYP716A48, CYP724B2, and CYP51G1, were identified through phylogenetic integration with functionally validated triterpenoid-modifying enzymes from other plant species. Gene family evolution analysis further revealed contraction of monoterpenoid-related subfamilies (CYP76A), implying a metabolic shift toward triterpenoid specialization. The comprehensive genome assembly and CYPome of W. somnifera offer a valuable resource for functional characterization, evolutionary analysis, and the identification of genes underlying its specialized metabolism. Furthermore, the study advances our understanding of CYP450 diversity and evolution, revealing lineage-specific innovations, conserved subfamilies, and key candidate genes involved in triterpenoid biosynthesis. Together, these findings lay a foundation for future functional studies and pathway engineering aimed at optimizing the metabolic potential of this important medicinal plant.

Indica rice, being a tropical crop, is highly sensitive to cold temperature. Cold stress affects vegetative growth, photosynthetic efficiency, along with reproductive features. Genetic resource screening in diverse landraces is an approach for identifying cold-tolerant traits. Here, we have characterised a boro germplasm, CB1, with an efficient germination rate and growth vigour when treated at chilling temperatures. CB1 seedlings show a higher survival rate compared to IR36 when subjected to prolonged chilling stress. Biochemical analyses indicated efficient ROS modulation, higher chlorophyll content, enhanced photosystem II efficiency and unique stomatal traits, leading to higher relative water content in CB1 plants during stress and recovery. Transcriptome analysis supported upregulation of chlorophyll biosynthesis, photosystem, & light harvesting complex and ROS scavenger genes in CB1 seedlings. Interestingly, high D1 protein turnover in CB1 promotes damage-repair of PSII for efficient photosynthesis. Furthermore, key transcription factors for stomatal development and expression of photosynthetic genes were upregulated in CB1 during stress recovery. Notably, higher expression of OsGLK1 and enrichment of GLK1 targets were observed in CB1 plants during chilling stress and recovery. Taken together, our results suggested that CB1 plants exhibit cold tolerance by modulating photosynthesis efficiency and stomatal behavior for better adaptability and survival against chilling temperature. HIGHLIGHTSThe efficient photosynthetic recovery, active ROS scavenging system and maintenance of water content through regulating stomatal traits, enhance the survival of indica germplasm CB1 against chilling stress.

O_LIEngineering nitrogen fixing crops requires not only transferring the nitrogenase structural genes, but also the accessory genes to synthesize its iron-sulphur cofactors. Scaffold protein NifU is a critical element in this system as the starting point of nitrogenase cofactor assembly. NifU has been successfully produced in plants, however, its optimal production required high levels of iron in the medium. This is likely due to a faulty connection with the endogenous iron trafficking network C_LIO_LITo identify specific elements targeting iron to NifU, pull-down assays were performed to identify showing bacterioferritin A (BfrA) as a likely candidate. Co-immunopurification, mutant characterization, iron transfer assays, and co-expression in Nicotiana benthamiana assays were carried out. C_LIO_LIBfrA transfers iron to NifU through protein-protein interactions. When these two proteins were co-expressed in N. benthamiana leaves, there was an increase in NifU production. In turn, it led to doubling NifH synthesis, a nitrogenase structural protein that is also required for the synthesis of the more complex nitrogenase cofactors. C_LIO_LIOur results provide a new element towards engineering nitrogen-fixing crops. They also underscore the importance of transferring the metal delivery systems when expressing metalloproteins in heterologous systems. C_LI