Plant Direct

Cannabis is well known for its pungent, skunk-like aroma. Recent chemical studies have identified prenylated and C6 volatile sulfur compounds as contributors to its skunky and citrus-like aromas, but the pathways that produce these compounds remain unknown. This gap limits efforts to explain variation in sulfur-aroma traits and to selectively enhance or reduce those traits. To address this gap, we used the known chemistry of sulfur-containing volatiles in Cannabis and characterized sulfur and volatile biosynthetic pathways in other plant species to select candidate enzyme groups. Because the GMO cultivar is anecdotally associated with a pronounced sulfurous aroma, reference protein sequences and profile hidden Markov models were used to search its version 1 (v1) primary high-confidence protein set of 55,790 sequences. These searches recovered 975 unique proteins. Sequence screening retained 941 candidates across 20 reporting categories; 939 contained all expected domains, while the two candidates assigned to the methionine gamma-lyase (MGL)-nearest category had no category-specific expected-domain rule. The largest reporting category comprised 359 proteins containing a cytochrome P450 domain, recovered through a search motivated by cytochrome P450 family 74 (CYP74) enzymes involved in oxylipin and plant volatile formation. Thirteen of these proteins were also recovered by at least one full-length CYP74 reference search. Other large reporting categories included 218 sugar-transferase, 83 glutathione-transferase, and 61 alcohol dehydrogenase candidates. Comparison with the Cannabis Expression Atlas linked 168 candidates to 128 annotated genes through 100%-identity amino-acid matches spanning at least 80% of each GMO v1 candidate protein. Twenty-nine genes were tissue-specific, including 13 root-specific and 6 trichome-specific genes. These results define candidates for biochemical testing and direct searches for additional enzymes acting upstream and downstream in Cannabis sulfur-volatile pathways.

Research ConductedTo elucidate the functions of glycosylceramides, we generated and characterized mutants deficient in multiple steps contributing to their assembly in the model moss Physcomitrium patens. We mutagenized SPHINGOLIPID {Delta}8-DESATURASE, whose products are preferentially incorporated into glycosylceramides, and a suite of higher-order mutants combining sphingolipid {Delta}4-desaturase and glycosyl ceramide synthase. MethodsWe used targeted lipidomics to describe the chemotypes of all mutants. We used quantitative phenotype analysis, transcriptomics, and phytohormone profiling to understand the effects of these chemotypes on development and physiology. Key ResultsThese mutants present a range of phenotypes that collectively indicate that in P. patens (1) glycosylceramide deficiency impairs development, largely due to imbalance in free ceramide homeostasis (2) the synthesis of glycosylceramides is dependent upon the presence of a specific free ceramide profile (3) the {Delta}4-, but not the {Delta}8-desaturation, is strictly required for glycosylceramide synthesis, (4) cell division and differentiation, but not cell expansion, are affected by sphingolipid imbalance, and (5) sphingolipid imbalance results in oxylipin accumulation. ConclusionCollectively, our results elucidate the assembly and functions of glycosylceramides in a model bryophyte, and highlight conserved and specialized aspects of sphingolipid metabolism among plants.

Plants make diverse metabolites to outcompete neighboring organisms for space and resources. Some of these toxic metabolites broadly disrupt conserved molecular mechanisms, such as protein biosynthesis. Nonproteogenic amino acids (NPAAs) are a structurally diverse class of metabolites that interfere with protein biosynthesis. The proline (Pro) analog azetidine-2-carboxylic acid (Aze) inhibits plant growth through misincorporation during protein biosynthesis. However, it is unknown if a cascade of downstream stress responses is triggered following Aze misincorporation. Here, we investigate the morphological and stress responses in Arabidopsis grown on Aze. Investigation of root morphological responses show not only reduced root growth, but increased root branching following growth on Aze. Altered root morphology is coupled with a reduced gravitropic response. Aboveground organs were also affected by Aze, including reduced chlorophyll content, reduced photosynthetic efficiency, and increased anthocyanin content. We then tested whether Aze induces reactive oxygen species (ROS) accumulation using multiple approaches and observed both immediate and sustained accumulation of general ROS and H2O2 following treatment with Aze. When plants were grown on Aze supplemented with Pro, ROS levels were restored to normal levels, suggesting that reducing misincorporation events results in less downstream stress responses. In summary, we find that following Aze treatment a cascade of downstream stress responses is induced that exacerbates the effects of toxic NPAAs. This study sheds light on the mechanism of action of NPAAs and provides information on the downstream consequences of translational errors.

A strategy to improve agricultural water productivity is to increase water use efficiency (WUE) at the level of plant transpiration through genetic selection. This requires detectable genetic variability in WUE and the ability to phenotype and select plants with higher WUE within a population. A proxy for phenotyping leaf level WUE by measuring carbon isotope signature ({delta}13Cleaf) has been supported by theory and data in C4 species. However, the functional relationship of {delta}13Cleaf and WUE in C4 species can be driven by genetics and environment. Therefore, a wide survey of existing natural variation is needed to quantify the heritability and identify various genetic factors that influence {delta}13Cleaf and WUE. In this study a genome-wide association panel was used to quantify the heritability of {delta}13Cleaf. We measured {delta}13Cleaf across a population of 360 genetically diverse lines of the C4 species Sorghum bicolor with single nucleotide polymorphic (SNP) markers determined from whole-genome resequencing. This analysis was conducted on two independent field environments where heritability of {delta}13Cleaf was evident and was driven by small genetic effects from loci that were consistently identified across environments. Candidate genes are presented that offer insights on future targets to manipulate and explore the functional relationship between {delta}13Cleaf and WUEi in C4 plants.

Salinity severely impairs plant growth and development. Increasing evidence suggests that the cell wall (CW) plays a central role in salt stress acclimation, not only as a structural barrier but also as a dynamic sensor that activates downstream stress signaling pathways. To identify extracellular factors involved in cell wall remodeling and integrity signaling during salt stress, we performed a comparative proteomic analysis of the Arabidopsis seedling apoplast. Among the proteins that accumulated under salt stress, we focused on three members of the Subtilisin-Like Protease family (SBT1.1, SBT1.6, and SBT5.3), which have previously been implicated in the processing of signaling peptides and CW-associated proteins. Functional analyses revealed that sbt1.1 and sbt5.3 mutants exhibit enhanced lignin deposition under salt stress, suggesting altered CW remodeling during stress application. Given the established role of SBT1.1 in processing PHYTOSULFOKINE (PSK) peptides, we investigated the involvement of PSK signaling in salt stress responses. Mutants lacking the two PSK RECEPTORS (pskr1 pskr2) displayed reduced growth and increased lignification under salt treatment. Exogenous application of PSK attenuated salt-induced responses, including MITOGEN ACTIVATED PROTEIN KINASE 6 (MPK6) phosphorylation, salt stress marker gene expression, and lignin accumulation, ultimately promoting root elongation under salt stress. Furthermore, PSK treatment mitigated salt-induced changes in CW composition. In cell wall integrity (CWI) mutants, PSK treatment failed to restore wild-type root growth under salt stress and induced a pronounced root bending phenotype in fer-4, indicating that CWI signaling influences PSK-mediated root growth responses. Together, these results support a role for PSK signaling in modulating CW-associated responses to salinity stress in Arabidopsis.

Abstract: Picrotoxane-type sesquiterpene alkaloids, including dendrobine, are characteristic constituents of Dendrobium nobile, but the enzymes that produce their proposed skeletal precursors remain in completely characterized. Here, we combined tissue metabolite profiling with genome-guided screening and biochemical characterization of terpene synthases (TPSs). GC-MS analysis revealed variation in sesquiterpenoid profiles and dendrobine signals among tissues and developmental stages. Manual curation of the chromosome-level genome yielded 16 full-length TPS-a candidates. Functional screening in an FPP-supplying Escherichia coli system showed that three closely related genes on chromosome 1, DnTPS1~DnTPS3, produced copaborneol as their dominant product. The principal product of DnTPS1 was purified and identified by NMR spectroscopy, and a purified-enzyme assay confirmed its direct conversion of FPP to copaborneol. Fluorescence imaging in rice protoplasts and Nicotiana benthamiana leaves indicated predominantly cytosolic localization of DnTPS1. Guided by sequence comparisons and structural modeling, we tested 12 DnTPS1 variants and found that substitutions at W377 and I295 altered the relative distribution of sesquiterpene products. Phylogenetic and regional gene-order analyses placed DnTPS1~DnTPS3 in the same clade and identified related TPS candidates within corresponding genomic regions of six Dendrobium species. These findings establish that three D. nobile TPSs can form copaborneol and identify DnTPS1 residues that influence product selectivity, providing a basis for testing the proposed connection to dendrobine biosynthesis.

Cassava (Manihot esculenta Crantz) is a major staple crop across tropical and subtropical regions. Despite advances in genomic selection, delayed, non-flowering, and asynchronous flowering remain key bottlenecks in breeding programs. To better understand the molecular basis of flowering-time variation, we performed RNA sequencing across three genotypes with contrasting flowering phenotypes (early, late, and non-flowering) sampled at three developmental stages under contrasting light regimes in field conditions (natural light and three-hour night-break with white light). Comparative transcriptomic analysis revealed distinct gene expression patterns associated with flowering responses. Genotype comparisons with no light supplementation revealed stage-specific enrichment of biological processes. Light supplementation was associated with changes in the expression of key components of photoperiodic and circadian regulation, as well as pathways involved in flowering-time integration and hormone and sugar-related signaling. These findings suggest that coordinated changes across multiple biological pathways regulate flowering behavior in cassava. The candidate genes and expression patterns reported provide a foundation for functional studies and advance our understanding of molecular mechanisms governing flowering-time regulation in cassava.

In nature, plants experience complex combinations of environmental stresses. Prior studies using Arabidopsis thaliana show that transcriptional responses to combinatorial stress are unique and cannot be predicted from responses to individual treatments, making it imperative to study combinatorial stress in the monocotyledonous cereals that comprise the majority of our food supply. However, crop species are not easily amenable for such studies because of their large size, long generation time, and organismal complexity. In contrast, the monocotyledonous aquatic duckweeds are small, grow rapidly, and show reduced organismal and genomic complexity. Here, we explore the physiological responses to combinatorial stress in three duckweed species, S. polyrhiza, L. minor, and W. australiana and examine their potential to serve as multiplexable models. Focusing on S. polyrhiza, the most complex and best annotated species, we investigated transcriptional response to individual and combinatorial stress treatments. While most individual treatments elicit few transcriptional changes, combinatorial stress elicited a non-additive transcriptional response unique to each combination. Exploring the duckweed regulatory landscape, we found that regulatory regions near genes that were differentially expressed in combinatorial stress in S. polyrhiza resemble environmentally responsive regulatory elements found in terrestrial plants. Examined duckweed species show numbers and genomic distributions of regulatory elements similar to those of A. thaliana and maize, with subtle effects of genome size but none of organismal complexity. Taken together, our results establish that duckweeds respond to combinatorial stress in a manner similar to terrestrial plants and can therefore serve as high-throughput, multiplexable models for studying its molecular underpinnings.

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.

Peptide hormone signaling coordinates plant growth and osmotic stress responses, yet how the transition between these responses is regulated remains poorly understood. Here, we investigated the function of the rice PLANT PEPTIDES CONTAINING SULFATED TYROSINE 8 (OsPSY8) peptide in osmotic stress responses. OsPSY8 was predominantly expressed in root tissues under non-stress conditions, with preferential expression in lateral roots where it promoted root growth. Osmotic stress rapidly reduced OsPSY8 expression in roots through the OsWRKY24 transcription factor. Loss-of-function ospsy8 mutants exhibited enhanced osmotic stress tolerance, whereas OsPSY8 overexpression increased osmotic stress susceptibility. Transcriptomic analyses revealed that disruption of OsPSY8 activated stress-responsive pathways, including those associated with lignin biosynthesis, compatible solute production, cell wall remodeling, and reactive oxygen species (ROS) scavenging, and was accompanied by increased lignin accumulation in roots. In contrast, overexpression of OsPSY8 resulted in maintenance of growth-associated transcriptional programs while suppressing stress-responsive pathways under osmotic stress. Together, these findings identify OsPSY8 as an important regulator of the transition from growth to stress adaptation in rice and suggest that stress-induced repression of PSY signaling is required to disengage growth programs and activate adaptive responses during osmotic stress. Significance StatementCrop survival during drought depends on the ability to transition from growth to stress adaptation. Plant peptide hormones have emerged as important regulators of this critical transition, highlighting the importance of investigating their roles and potential for improving crop resilience. We show that a rice peptide hormone regulates this transition. Under non-stress conditions, this peptide hormone, predominantly expressed in rice roots, promotes root growth while suppressing stress responses. During osmotic stress, expression of the peptide hormone decreases, resulting in activation of stress-responsive pathways, such as lignin biosynthesis and reactive oxygen species scavenging. These findings demonstrate that a peptide hormone coordinates the balance between growth and stress adaptation in rice, with broader implications for understanding and improving crop resilience.

Water-use efficiency (WUE), the ratio of accumulated plant biomass to water lost through transpiration has conventionally been determined using a destructive single-point measurement. Recent advances in high-throughput phenotyping now enable repeated, non-destructive estimation of biomass and WUE. However, these digital measurements must be statistically validated against conventional destructive methods to validate their use as reliable proxies. Therefore, we compared digital biomass determined point clouds produced from multispectral camera scanners with destructive harvests across eight harvests using Samsun tobacco grown under both drought and high-water conditions. WUE efficiency, calculated using the digital biomass estimated from a point cloud and gravimetric water use determinations, were compared to destructive harvest determinations. The coefficient of variation (CV) showed there were no significant differences in digital and destructive measurements for either biomass or WUE. Indicating that digital measurements can be used in place of destructive measurements. Drought plants used significantly less water and were significantly smaller than high-water plants from Harvests 4 through 8. However, there were no significant differences in the ratio of evapotranspiration to leaf area or WUE, indicating that drought plants were simply smaller and used less water than the high-water plants. This work validates that estimating plant biomass from a digital point coupled with continuous gravimetric determination of water use provides a reliable nondestructive measure of WUE in high-throughput measurements across the full plant life cycle.

Arabidopsis Leucine-Rich Repeat-Malectin Receptor Kinase (LRR-MAL RK) genes have been previously implicated in the early stages of pollen-pistil interactions to support compatible pollen. One member, Receptor Kinase in Flowers 1 (RKF1), has been associated with roles in the stigma to support pollen hydration as well as pollen tube growth. To better understand the function of RKF1 in these processes, a yeast two-hybrid screen was conducted with the RKF1 cytosolic kinase domain. Two positive interactors identified from this screen were the Group VII Ethylene Response Factors (ERFVIIs), RELATED TO APETALA 2.12 (RAP2.12) and RAP2.3. Their putative roles in pollen-pistil interactions were investigated using the quintuple erfvii mutant, and novel pistil-mediated pollen tube callose deposition phenotypes were uncovered during the pollen tube growth stage. Loss of seven LRR-MAL RKs including RKF1 in the pistil was previously found to cause an unusual phenotype where shorter callose plugs were deposited in wildtype pollen tubes compared to that seen in wildtype Col-0 pistils. Contrary to this, wildtype pollen tubes growing through the quintuple erfvii mutant pistil deposited callose plugs that were more elongated than that seen in wildtype Col-0 pistils. Further analyses with the proteolysis 6 (prt6) mutant and RAP2.12 rescue constructs were consistent with these phenotypes providing support that RKF1 is a negative regulator of RAP2.12 and RAP2.3 in the pistil during pollen tube growth.

Rising atmospheric CO2 concentrations consistently reduce mineral concentrations in C crops, yet the mechanisms driving this decline - dilution, reduced transpiration-driven mass flow, and altered root nutrient acquisition capacity - have rarely been tested simultaneously under field conditions. Here, we grew two soybean (Glycine max Merr.) varieties with contrasting yield responses to elevated [CO2] (Loda and HS93-4118) at the Soybean Free Air CO2 Enrichment (SoyFACE) facility and quantified the independent contributions of dilution, transpiration, and root uptake to observed changes in tissue mineral concentration and nutrient yield. Elevated [CO2] increased grain yield by 21% and 6% in Loda and HS, respectively, and reduced transpiration by 13-14% across both varieties. A decomposition analysis revealed that while dilution and reduced transpiration each exerted negative effects on tissue nutrient concentrations, with their combined effect representing a 31-40% potential reduction in whole-plant concentration, active root uptake responses were positive for nearly all nutrients, largely offsetting these losses and resulting in observed concentration declines substantially smaller than either mechanism alone would predict. Consequently, whole-plant nutrient yield increased under elevated CO2 for all elements measured, and grain nutrient yield increased for most. The relationship between seasonal transpiration and nutrient yield was steeper under elevated CO2 for most macronutrients, indicating that plants acquired more nutrients per unit water transpired, not less, under elevated CO2. These results demonstrate that root uptake is the dominant compensatory response to elevated [CO2] driven dilution and reduced mass flow in soybean, that nutrient yield is maintained or increased for most elements under elevated [CO2], and that Fe represents a physiologically and nutritionally significant vulnerability in future CO2 environments.

High-phenolic plant genera present a major technical limitation in genomic research. Standard extraction approaches that perform reliably across diverse flora often perform poorly when applied to recalcitrant taxa, producing low DNA yield and integrity incompatible with sequencing requirements. The genus Anigozanthos (Kangaroo paws) from the family Haemodoraceae exemplifies this problem. We identified key physicochemical factors governing extraction failure in this genus and resolved them through targeted modifications to lysis chemistry and contaminant management. The resulting protocol achieved a near threefold improvement in DNA purity, substantially reducing contaminant carry over and consistently yielded high-integrity, long DNA fragments (DIN > 7) across a diverse sample set spanning cultivated and wild material across four diverse genera of Haemodoraceae. We also tested a straightforward purity assessment framework that can be implemented in any standard molecular laboratory, enabling rapid pre-submission quality assessment without the need for specialised equipment. Together these advances open a practical path to genomic characterisation of Anigozanthos that establishes a transferable model for genomic research across Australia ' s chemically complex native flora.

BackgroundN6-Methyladenosine (m6A) regulates RNA fate in flowering plants, but the repertoire and expression patterns of m6A-related genes remain incompletely characterized in liverworts. Here, we characterized these gene families in the liverwort Marchantia polymorpha within a comparative framework spanning diverse plant lineages. ResultsA comparative survey of 119 plant genomes using sequence similarity and conserved-domain criteria identified 18 candidate m6A-related loci in M. polymorpha, comprising seven writer-related genes, five AlkB homolog genes, and six YTH- or CPSF30-related genes. Across the surveyed genomes, canonical writer components generally had low copy numbers, whereas AlkB homolog and reader-related families showed greater copy-number variation. Phylogenetic and domain analyses supported the family assignments of the M. polymorpha candidates. Eight of the 18 candidate loci showed detectable collinearity with Marchantia quadrata. Putative promoter analysis identified motifs annotated as hormone-, light-, and stress-responsive, with hormone-responsive motifs accounting for the largest share of predicted sites. Public transcriptome analysis revealed variation in candidate gene expression across developmental stages and tissues. Quantitative reverse-transcription PCR showed higher transcript abundance for all six YTH- or CPSF30-related genes in thalli than in gemmae or gemma cups. Cold, heat, osmotic, salt, and dark treatments elicited gene-specific expression responses. Darkness increased transcript abundance of several candidates, including MpMTA, MpALKBH1A, and MpECT2. A predicted protein-association network suggested links between subsets of the candidates and RNA-processing proteins. ConclusionsThese findings define a candidate m6A-related gene repertoire in M. polymorpha and identify tissue- and stress-responsive genes for targeted functional studies of RNA methylation in this liverwort.

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.

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.

Salinity stress affects rice productivity due to reduced growth and sodium ion toxicity. Previously, we identified a splice variant of RADIATION SENSITIVE23a (RAD23a) as the potential basis for variation in salt-tolerance in rice germplasm. RAD23 is a known moonlighting protein associated with protein degradation. To validate the role of RAD23a in salt stress response, we characterized gene edited mutant lines that targeted the UBL and UBA2 domains of this protein. Mutation in either domain promoted shoot growth under saline and control conditions. The mutants also differed from wildtype plants in Na and K accumulation in roots and shoots under salt stress. Transcriptome analysis of mutants versus wildtype showed differential transcript abundance of multiple inorganic phosphate (Pi) starvation related genes, including OsSPX2 and OsPHO2. As a result, mutants accumulate higher Pi compared to wildtype plants. The two allelic groups for RAD23a locus also differ in root and shoot phosphorus (P) content. Further, we show that RAD23a interacts with OsSPX2, a negative post-translational regulator of OsPHR2, the master regulator of Pi starvation response. Mutants have higher shoot growth and Pi levels under low Pi conditions, linking enhanced growth of mutants to increased Pi uptake. The UBA2 domain specific mutants have higher single grain weight and per plant grain weight than wildtype. In summary, we show that the RAD23a regulates differential growth, salt response and Pi uptake in rice in a domain-specific manner supporting the moonlighting roles of RAD23a in salt tolerance and phosphorus-dependent shoot growth.

COBRA-like (COBL) genes encode plant-specific proteins associated with cell-wall organization, cellulose deposition, and developmental processes. Despite their functional relevance in model plants and crops, this gene family remains poorly characterized in Amazonian Malvaceae species. Here, we performed a comparative genomic analysis of COBL genes in two Theobroma grandiflorum clones, two T. cacao cultivars, and Herrania umbratica. Using homology searches, conserved domain validation, phylogenetic reconstruction, gene structure analysis, chromosomal mapping, transcript abundance profiling, molecular modeling, and selection tests, we identified 62 COBL genes across the analyzed genomes. COBL copy number varied among genomes, with T. cacao Matina showing the largest repertoire. All retained proteins contained the conserved COBRA domain and grouped into the two major COBL subgroups previously described in angiosperms, with subgroup-specific exon-intron organization largely conserved across species. Genomic distribution and duplication classification indicated contributions from tandem, segmental, dispersed, and proximal duplications, suggesting that multiple genomic processes shaped COBL family organization in Theobroma and Herrania. Phylogenetic analyses further recovered a putative lineage-specific COBL clade within the analyzed Malvaceae, indicating lineage-specific variation in this gene family. Structural modeling indicated overall conservation between representative proteins from this clade and COBL6-associated proteins, although localized amino acid differences were observed within the COBRA domain. Selection analyses supported predominant purifying selection across the family, with limited evidence of episodic diversifying selection in specific lineages and no significant branch-level signal within the putative lineage-specific clade after correction. Overall, these results provide a comparative framework for COBL gene evolution in Theobroma and Herrania and identify candidate genes for future functional studies on cell-wall-related traits in economically important Malvaceae crops.

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.