Plant Molecular Biology

O_LIShoot branching is critical not only in breeding for yield but also for ornamentals architecture. In the ornamental plant poinsettia (Euphorbia pulcherrima), shoot branching has traditionally been induced by phytoplasma (Candidatus Phytoplasma pruni) inoculation. This study aimed to identify regulatory genes that could be leveraged in future breeding-by-genetic engineering efforts to develop phytoplasma-free, branching poinsettia plants. C_LIO_LITo elucidate mechanisms of phytoplasma-induced shoot branching, we performed RNA-sequencing and assembled an axillary bud-specific transcriptome for expression analyses in phytoplasma-infected and -free poinsettia. Phenotyping and RNA-sequencing were also conducted on Arabidopsis mutants and wild-type lines to investigate the transcriptional regulatory effects of candidate genes. C_LIO_LIThe transcription factors EpMAF3 and EpbZIP67 were highly de-regulated in phytoplasma-infected poinsettia. We also found a two-fold increase in primary-stem branching levels of the Arabidopsis maf3 and bzip67 mutants, suggesting the two transcription factors as potential shoot branching suppressors. AtTcp1, a CYC-clade TCP transcription factor, was up-regulated (78x) in leaves of the maf3 mutants. Analyzing previously reported protein-level interactions for the differentially expressed genes (e.g., AtClamt, AtGh3.9/3.15, AtSaur32/36, AtAbi3, AtGamt2, AtTcp3, and AtDwf4) in bzip67 mutants shed light on other shoot branching regulators such as TCPs, PINs, ABIs, DWARF14, and BES1, highlighting two regulatory sub-networks including membrane transport and hormonal signaling. C_LIO_LIThe results open the way to rational engineering of shoot branching in poinsettia by targeted mutagenesis of MAF3 and bZIP67. In that way, the tedious and viral-infection prone process of phytoplasma inoculation can be avoided and poinsettia plants would have more homogenous branching. C_LI One-sentence summaryPhytoplasma infection in poinsettia induces bud-specific repression of the transcription factors EpMaf3 and EpbZip67, consistent with the enhanced stem branching observed in Arabidopsis maf3 and bzip67 mutant lines.

Here we reported the 409.0 Mb assembly of the Tainung 67 (TNG67) genome, an early hybrid of japonica and indica cultivars. We used different platforms to sequence the TNG67 genome with a total coverage of 209.8x, from a combination of Illumina paired-end reads, Illumina mate-pair reads, and Oxford Nanopore Technology long reads. The assembly has an N50 of 32.3 Mb with the longest scaffold = 45.2 Mb. We have annotated 38,938 genes where 28,376 (72.9%) finished with the Blast2GO annotation stage. There were 4,821 genes, (98.4%) of the total gene count that have complete BUSCOs. 48.33% (197.5Mb) of the TNG67 genome is composed of repeats. We predicted a total of 527 blast-resistant genes in TNG67. We also analyzed a total of 20 grain size genes and 38 photoperiod-related genes of TNG67, Nipponbare and TN1, and grouped them in terms of whether the TNG67 genes are more similar to Nipponbare, more similar to TN1, hybrid, same, or unique. We also determined whether the sequences of the TNG67 genome were derived from its japonica or indica ancestors. This TNG67 genome may help rice researchers improve yield, develop resistance against biotic and abiotic stress, and understand the evolution of a hybrid cultivar of japonica and indica.

O_LIViroids are small, non-coding RNAs that rely on host factors for replication, intracellular trafficking and systemic movement. VIRP1, a Bromodomain and Extra-terminal domain (BET) protein, has previously been implicated in Potato spindle tuber viroid (PSTVd) infection, yet its precise role and mode of action remain unresolved. C_LIO_LIIn this work, we highlight VIRP1 as the only Solanaceae BET protein containing a proline-rich domain, which overlaps with the PSTVd-binding site. VIRP1-deficient plants exhibit delayed flowering and increased ABA sensitivity, with differentially expressed genes enriched in stress-related pathways. C_LIO_LIVIRP1 forms condensates in planta and in vitro, consistent with phase-separation behaviour. Condensate morphology is altered by PSTVd RNA, deletion of the intrinsically disordered CTD and bromodomain mutations. C_LIO_LIVIRP1 is particularly important for the early establishment of PSTVd infection, while nuclear localization and bromodomain integrity are required for efficient viroid accumulation. By contrast, the disordered CTD region is dispensable for complementation of PSTVd accumulation. C_LIO_LIOur results support a model in which VIRP1 acts as a host nuclear factor that links stress-related functions, nuclear condensate formation and early viroid infection. C_LI

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 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.

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.

Drought is a major abiotic stress threatening plant productivity and agricultural sustainability, yet the molecular mechanisms underlying adaptive root responses to water deficit in the water use strategies continuum remain insufficiently understood, particularly in perennial crops. In this study, we explored drought responses in nine accessions belonging to three wild Vitis species (V. acerifolia, V. candicans, and V. doaniana) displaying varying drought-response strategies. Plants were subjected to moderate drought stress (40% soil water content) for three weeks under greenhouse conditions. By integrating physiological, metabolic, and transcriptomic analyses, we aimed to identify both conserved and species-specific mechanisms associated with drought adaptation. Differential expression analyses revealed a conserved core set of drought-responsive genes shared among species, including genes involved in abscisic acid signaling, reactive oxygen species detoxification, solute transport, and plant defense. In parallel, each species exhibited distinct transcriptional and metabolic signatures reflecting alternative adaptive strategies related to osmoregulation, and oxidative stress mitigation. Weighted gene co-expression network analysis (WGCNA) further revealed significant associations between constitutive, non-plastic gene expression and root phenotypic traits. Overall, our findings demonstrate that wild Vitis species rely on both conserved stress-responsive pathways and species-specific constitutive regulation to cope with drought stress. These results highlight the importance of root-associated traits and intrinsic regulatory networks in shaping drought adaptation and provide new targets for the development of drought-resilient grapevine rootstocks.

Grain yield is a highly complex quantitative trait in rice, resulting from the interaction of multiple genetic, physiological and environmental factors. Although genome-wide association studies (GWAS) have successfully identified loci associated with grain yield, translating statistical associations into biologically meaningful candidate variants remains a major challenge, particularly for variants located in regulatory regions. This study aimed to identify genomic variants associated with grain yield in upland rice and to develop an integrative framework for functionally prioritizing candidate variants through the combination of genome-wide association analysis, functional annotation and regulatory genomics. A panel of 252 accessions from the Brazilian Rice Core Collection was phenotyped for grain yield and genotyped with 35,763 single nucleotide polymorphism (SNP) markers. GWAS identified 29 SNPs significantly associated with grain yield, including 16 variants located within or near annotated genes and 13 located in intergenic regions. The identified candidate genes were involved in signal perception, metabolite transport, amino acid and energy metabolism, hormone biosynthesis, protein turnover, RNA processing and disease resistance, highlighting the polygenic architecture of grain yield. Functional characterization of the intergenic regions revealed enrichment of cis-regulatory elements recognized by transcription factors associated with hormonal signaling, drought response, carbon metabolism, photosynthesis and reproductive development, indicating that regulatory variation represents an important component of grain yield determination. By integrating GWAS signals, candidate gene annotation, cis-regulatory element characterization and the physical proximity between SNPs and cis-regulatory elements, an integrative prioritization strategy identified seven intergenic SNPs as the most promising candidates for functional validation. Together, these findings establish a robust framework for discovering, prioritizing and functionally validating regulatory variants, bridging the gap between statistical associations and biological function while providing a rational strategy for translating GWAS discoveries into molecular breeding of complex traits.

Lachrymatory factor, an irritating volatile with tear-inducing property, is produced when onion bulbs are cut or chopped. We aimed to generate onion plants with reduced lachrymatory factor synthase (LFS) activity via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (CRISPR/Cas9) genome editing. Calli induced from primary roots were transformed with Agrobacterium tumefaciens carrying expression cassettes for CRISPR/Cas9, guide RNA, green fluorescent protein (GFP), and hygromycin resistance; callus lines that showed a high-frequency stable GFP expression were selected as "elite callus lines" that were suitable for transformation. Cleaved amplified polymorphic sequence (CAPS), heteroduplex mobility assay (HMA), and Sanger sequencing confirmed mutations introduced into the LFS gene, and plants were regenerated from the confirmed LFS-edited callus lines. The LFS enzyme activity in the leaves and bulbs of the LFS-edited plants was lower than that in control plants, while the LFS-edited plants exhibited severe growth abnormalities and failed to set seed, possibly due to long-term culture to maintain the elite callus line. The present study first demonstrated that onion genome editing, which modified a specific trait of onion, the reduction of LFS activity, was achieved. The results obtained opened the feasible way toward the final goal: the production of tear-free, higher health-functional onions.

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.

Thermal adaptation is critical for organismal viability, with acquired thermotolerance (AT) in Arabidopsis thaliana typically conferred by acclimation temperatures between 34{degrees}C and 37{degrees}C, while acclimation at 40{degrees}C fails to protect against lethal heat stress. To elucidate the transcriptional mechanisms underlying the loss of AT at higher temperatures we performed RNA-seq profiling of Arabidopsis seedlings across a single-degree thermal gradient from 37{degrees}C to 40{degrees}C. Our analysis reveals that all of these temperatures result in a robust heat shock response, characterized by the upregulation of genes involved in protein folding and stress responses. However, each temperature elicits a distinct transcriptional signature. These findings demonstrate that temperature-specific fine-tuning of the heat shock response occurs within this narrow range and that these differences may dictate the successful acquisition of thermotolerance. This dataset provides a valuable resource for understanding the molecular architecture of heat-stress adaptation and the distinct transcriptional states associated with different thermal regimes.

Salinity is a major abiotic stress that severely limits plant growth and productivity, causing substantial yield losses. Despite barleys relative tolerance to salinity, the underlying physiological and molecular mechanisms remain incompletely understood. In this study, we employed an integrative approach combining agronomic, physiological, biochemical, and transcriptomic analyses to investigate salinity responses in the spring barley cultivar Giza 134 under both field and lysimeter-based conditions. Salinity stress significantly reduced growth and yield-related traits, with more pronounced effects observed under lysimeter-imposed salinity, reflecting higher stress intensity. These reductions were associated with impaired water status, altered leaf structural traits, and declines in photosynthetic pigment content. In contrast, proline accumulation increased, indicating activation of osmotic adjustment mechanisms. Salinity also disrupted ionic homeostasis, as evidenced by elevated Na+ levels, reduced K+ content, and an increased Na+/K+ ratio. Enhanced lipid peroxidation and elevated catalase and peroxidase activities suggested increased oxidative stress and activation of antioxidant defenses. Transcriptome profiling identified 4,298 differentially expressed genes, including 1,764 upregulated and 2,534 downregulated genes. Functional enrichment analyses revealed upregulation of pathways related to stress adaptation, redox regulation, and metabolic reprogramming, while genes associated with photosynthesis, ribosome biogenesis, and protein synthesis were strongly suppressed. Several novel stress-responsive genes involved in signaling, osmoprotection, antioxidant defense, and central metabolism were highly induced, supported by coordinated enrichment of cis-regulatory motifs in their promoter regions. Together, these findings provide a comprehensive physiological and molecular framework for salinity tolerance in Giza 134 and highlight candidate genes and pathways for breeding salt-resilient cultivars suited to saline-prone environments.

Most transgene applications require high and sustained expression, particularly in stably transformed plants. Achieving optimal transgene performance, however, depends on the combined influence of multiple genetic and regulatory factors. In previous work, we systematically evaluated the contribution of different genetic elements to transient transgene expression and demonstrated that terminators are key determinants of transgene performance by reducing transcriptional read-through and preventing transgene silencing. Here, we extend these findings by investigating the roles of terminators and introns in the expression of transgenes in stably transformed plants. Our results show that optimal transgene performance arises from the complementary actions of these two elements. Terminator choice was a major determinant of transgene expression levels, whereas introns played a critical role in maintaining expression stability. We further demonstrate a strong relationship between transgene expression levels and small RNA accumulation and show that intron-containing endogenous genes are enriched among highly expressed and stress-responsive genes, suggesting that intron-mediated protection from silencing may facilitate higher levels of gene expression and have contributed to the emergence and evolutionary retention of intron-containing genes.

Phenylacetic acid (PAA) is a naturally occurring auxin whose biosynthesis and function during plant regeneration remain poorly understood. PAA may be synthesized from phenylalanine via the CYP79A2-dependent phenylacetaldoxime pathway, in which benzyl cyanide/phenylacetonitrile (BnCN/PAN) is a potential intermediate and substrate for NITRILASE (NIT) enzymes. Here, we investigated whether BnCN/PAN promotes somatic embryogenesis (SE) through NIT-dependent PAA biosynthesis. Low concentrations of BnCN/PAN stimulated somatic embryo formation in Arabidopsis thaliana, whereas exogenous PAA also promoted embryogenic induction. Transcriptome profiling revealed that BnCN/PAN upregulated genes associated with SE, including key embryogenic regulators and EMBRYO DEFECTIVE genes. RNA-seq data further indicated enhanced auxin signalling, which was independently confirmed using the pDR5::GUS reporter line. Inhibition of NIT activity by heatin reduced the embryogenic competence of BnCN/PAN-treated explants, supporting the involvement of NIT enzymes in this response. Collectively, our findings provide the first evidence that BnCN/PAN promotes embryogenic transition and suggest that NIT functions in SE extend beyond their proposed role in indole-3-acetic acid biosynthesis. Summary statementThis study uncovers a previously unrecognized pathway regulating plant regeneration, providing new insights into how naturally occurring metabolites influence embryo formation.

Heat stress is one of the most damaging abiotic constraints on crop productivity, and its consequences are expected to intensify as extreme temperature events become more frequent and severe. Pepper (Capsicum annuum L.) is particularly vulnerable to sustained high temperatures, which can disrupt photosynthetic performance, cellular homeostasis, and redox regulation. However, the physiological and transcriptional dynamics underlying genotype-dependent responses to prolonged heat exposure remain insufficiently understood. We combined repeated physiological measurements with time-course RNA sequencing to compare GPC003240, previously identified as a candidate heat-tolerant accession, with two non-elite accessions, GPC010350 and GPC014930, which are phenotypically divergent from each other, under 40/30 {degrees}C Day/night temperatures for up to six days. GPC010350 maintained comparatively stable photosystem II performance and higher stomatal conductance, whereas GPC014930 showed progressive photochemical impairment and lower conductance; GPC003240 displayed a distinct, moderately responsive profile. Transcriptomic responses showed partial functional convergence during the early phase of stress exposure but diverged markedly after six days. When gene expression at day 6 was compared with the pre-treatment baseline separately within each genotype, 4,436 differentially expressed genes were detected in GPC010350, compared with 680 in GPC003240 and only 78 in GPC014930. The late response of GPC010350 was associated with enrichment of RNA- and ribosome-related, biosynthetic, DNA-repair, and genome-maintenance functions. By contrast, GPC014930 showed negative enrichment of photosynthesis, plastid organization, redox homeostasis, and translation-related processes. Global co-expression analysis identified a time-decreasing photosynthesis-associated module (ME5) and two time-increasing modules, ME12 and ME19, that were enriched in genes contributing to the late GPC010350 response. Integration of differential expressions, module membership, and functional annotation highlighted a heat shock transcription factor (Caz03g27980), HSP101 (Caz03g07770), and a dual-specificity phosphatase (Caz05g20970) as candidates for further investigation. Overall, the results suggest that genotype-dependent responses to prolonged heat exposure were associated not only with the magnitude of early transcriptional change, but also with differences in the temporal organization of stress-response, maintenance, and metabolic processes. The contrasting responses of the non-elite accessions GPC010350 and GPC014930 further highlight the value of phenotypically diverse germplasm for uncovering mechanisms relevant to future heat-tolerance breeding.

Although the biosynthetic pathways of benzylisoquinoline alkaloids (BIAs) have been extensively investigated in several plant species, their transcriptional regulatory mechanisms remain only partially understood. Jasmonate (JA)-responsive group IX APETALA2/Ethylene Responsive Factor (AP2/ERF) transcription factors (TFs) are well-known regulators of specialized plant metabolism, including the biosynthesis of various alkaloids. However, their specific roles in BIA biosynthesis remain largely elusive. Here, we isolated five novel group IX AP2/ERF TFs, designated Benzylisoquinoline alkaloid Jasmonate-responsive AP2/ERF (BJE1-5), from Coptis japonica. Phylogenetic analysis revealed that Benzylisoquinoline alkaloid Jasmonate-responsive AP2/ERF (BJE) proteins belong to subclades distinct from group IXa, which contains well-known AP2/ERF TFs involved in alkaloid biosynthesis. Transient expression analyses in C. japonica protoplasts demonstrated that certain BJEs, particularly CjBJE3 and CjBJE5, positively regulated BIA biosynthetic genes through a mutual regulatory network among BJE members. Moreover, CjBJE3 expression was regulated by CjbHLH1, a unique-type basic helix-loop-helix (bHLH) TF specific to BIA-producing plants. Furthermore, heterologous expression of CjBJE3 and CjBJE5 in cultured Eschscholzia californica cells significantly enhanced the overall BIA production, particularly by increasing end-product benzophenanthridine BIAs, highlighting several uncharacterized biosynthetic genes clustered in the genome. Our findings suggest that BIA-producing species have developed a specific regulatory network comprised of CjbHLH1 and BJE TFs, providing valuable clues for identifying novel biosynthetic enzymes.

To identify novel regulators of plant drought tolerance, we performed a forward genetic screen using thermal infrared imaging to detect alterations in leaf temperature, a proxy for transpiration. This screen identified AT-rich Interacting Domain Protein 4 (ARID4), a protein involved in chromatin remodeling, as a modulator of transpiration. arid4 loss-of-function mutants exhibited cooler leaves, increased water loss from detached leaves, and heightened susceptibility to drought relative to wild-type plants. Stomata of arid4 mutants were hyposensitive to abscisic acid (ABA), displaying reduced ABA-induced reactive oxygen species (ROS) production and impaired stomatal closure. Transcriptome profiling under drought revealed extensive misregulation of genes involved in stress-responses, redox homeostasis, iron acquisition, and photosynthesis. These findings indicate that ARID4 modulates the expression of key regulatory genes to maintain redox homeostasis and photosynthetic efficiency, thereby conferring drought stress tolerance.

The continuous transfer of organellar DNA into the nuclear genome during eukaryotic evolution has resulted in the widespread occurrence of nuclear plastid DNA insertions (NUPTs) and nuclear mitochondrial DNA insertions (NUMTs). However, their functional significance in nuclear gene expression and genome evolution remains largely unresolved. In this study, we employed Oxford Nanopore Direct RNA Sequencing (DRS) to investigate the transcription of NUPTs and NUMTs in the Populus nuclear genome and compared their transcriptional characteristics with their genome-wide insertion patterns. Our analyses revealed that the majority of transcribed NUPTs and NUMTs are enriched within introns and are co-transcribed with their host or adjacent genes in polycistronic-like transcriptional units. In addition, NUPTs and NUMTs frequently generate intronless transcripts, features reminiscent of their prokaryotic ancestry. We further identified a putatively functional NUPT-derived psbH gene that is unique to P. trichocarpa, providing new insights into the evolution of nuclear-encoded organelle-targeted genes. In addition, we identified transcribed NUPT and NUMT insertion polymorphisms among alleles, suggesting that organellar DNA insertions contribute to allelic variation and may participate in environmental adaptation. Collectively, our findings reveal previously unrecognized roles of NUPT and NUMT transcription in gene regulation, allelic variation, genome evolution, and the emergence of novel genes.

Rice (Oryza sativa) is highly sensitive to salinity, yet the metabolic mechanisms underlying salt tolerance remains incompletely understood. In this study, we performed leaf tissue-specific untargeted metabolomic profiling of the salt-tolerant introgression line JN100 (JN), its donor parent Nona Bokra (NB), and its recurrent parent Jupiter (JU) to characterize metabolic responses to salt stress. Comparative analysis identified differentially accumulated metabolites (DAMs) spanning diverse chemical classes, including amino acids, sugars and carbohydrates, lipids, organic acids, cofactors, electron carriers, and nucleotides. Under salt stress (SS), 201 DAMs (89 upregulated and 112 downregulated) were detected in JN relative to JU. Notably, metabolites such as allantoin, glycitin, nicotinamide ribotide, D-arabinono-1,4-lactone, violanthin, L-methionine S-oxide, ribitol, lysine, rutin, glutamine, pantothenic acid, and quinic acid, showed significant differential accumulation. Pathway enrichment analysis revealed significant enrichment of arginine biosynthesis, purine metabolism, and alanine, aspartate, and glutamate metabolism, indicating extensive reprogramming of nitrogen and energy-associated metabolic pathways under salinity stress. Integration of transcriptomic and metabolomic datasets from the SS experiments further identified ten differentially expressed genes (DEGs) associated with the metabolite network in the JN vs. JU comparison. Among these, OsDHQDT/SDH, OsFd-GOGAT, phenylalanyl-tRNA synthetase, OsP5CS1, OsP5CS2, and a pyridoxal phosphate-dependent transferase were linked to metabolites involved in shikimate, amino acid, and proline metabolism. Collectively, these results demonstrate that salinity tolerance in rice is associated with coordinated transcriptional and metabolic reprogramming that supports oxidative stress mitigation and adaptive stress responses.

Flower opening is a complex developmental process involving coordinated changes in cell proliferation and cell expansion. Although several regulators of flower opening have been identified, how transcriptional programs are coordinated with the cellular and metabolic changes underlying petal expansion immediately before flower opening remains incompletely understood. Japanese morning glory (Ipomoea nil) is a suitable model for investigating these processes because its flowers open synchronously at a predictable time. This study aimed to identify transcriptional regulators involved in petal development and flower opening in Japanese morning glory. Temporal analyses of petal growth, sugar metabolism, and gene expression revealed that petal development was driven by both cell proliferation and cell expansion until approximately 48 h before flower opening, whereas cell expansion predominated thereafter. Weighted gene co-expression network analysis identified two genes encoding R2R3-MYB subgroup 19 transcription factors, InMYB21A and InMYB21B, as candidate regulators associated with petal development. CRISPR/Cas9-mediated knockout analysis revealed a prominent role for InMYB21B, whose loss markedly impaired petal cell expansion and prevented flower opening. InMYB21B knockout also impaired stamen and pistil development, resulting in male and female sterility. Starch degradation and glucose accumulation were impaired in InMYB21B knockout petals. Transcriptome analysis revealed delayed transcriptomic progression during petal development and reduced expression of genes associated with starch degradation, sucrose metabolism, cell wall remodeling, and water transport. These findings identify InMYB21B as a key regulator of petal cell expansion and flower opening in Japanese morning glory and show that loss of InMYB21B disrupts both metabolic and transcriptomic progression during late petal development.