Plant Direct

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.

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.

The fungal pathogen Colletotrichum sublineola is a major constraint to sorghum production. The sorghum genotype IS18760 is resistant to multiple C. sublineola strains, but the genetic basis of this resistance is unknown. Bulk segregant analysis using whole-genome resequencing of a biparental mapping population identified a major resistance locus, designated ANTHRACNOSE RESISTANCE GENE LOCUS 3 (ARG3), associated with extensive cell death. Fine mapping delimited the ARG3 locus to a 30 kb interval containing three predicted protein-coding genes and one unannotated non-coding RNA gene. Among these, only the non-coding RNA gene showed consistent sequence polymorphisms between resistant and susceptible lines and elevated pathogen-induced expression in the resistant parent, suggesting that it underlies ARG3-mediated resistance. Regions syntenic to ARG3 across grass species contain conserved non-coding sequences enriched for H3K27me3 and H2A.Z chromatin marks, suggesting a conserved regulatory function. The resistance allele is absent from a 111-line sorghum pangenome. The resistant sorghum line harboring ARG3 and the corresponding avirulent pathogen strain originate from the same geographic region, suggesting co-evolution between host resistance and a prevalent pathogen strain. Phylogenetic analysis suggests that loss of resistance in susceptible lines may relate to insertion of a 12.4 kb repetitive sequence. Importantly, silencing of the ARG3 transcript in the resistant parental line abrogated resistance to C. sublineola, confirming that ARG3 non-coding RNA gene underlies the fungal resistance mediated by the ARG3 locus in IS18760. These lines of evidence show that ARG3 is an important target for resistance breeding and provide new insight into non-coding RNA-mediated disease resistance.

Boron toxicity constrains grapevine (Vitis) production in irrigated arid and semi-arid regions, yet most available rootstocks are sensitive to excessive boron. Although underutilized Vitis species may harbor sources of adaptation, the genetic basis of natural variation in boron exclusion remains poorly understood, limiting the development of boron-tolerant rootstocks. We screened 267 wild Vitis accessions representing 16 species for their ability to limit leaf boron accumulation under excess boron conditions. Leaf boron concentration varied 190-fold among accessions and was highly heritable (entry-mean H^2 = 0.71). Its correlation with stomatal conductance was modest (r = 0.34), indicating that transpiration rate explained only a small proportion of the variation in leaf boron accumulation. Leaf boron was correlated with leaf chloride (r = 0.36), but their largely non-overlapping association profiles supported distinct genetic control. Genome-wide association analysis identified a suggestive association on chromosome 11. Independent quantitative trait locus mapping in a full-sib F1 population (n = 158) derived from Longii-9018 (V. acerifolia) x GRN3 (commercial rootstock) identified qBEx11.1 in the same syntenic region on chromosome 11, which explained 19.5% of the phenotypic variance. Positional candidate genes near the association peak included a pectinesterase and a MATE efflux transporter, suggesting roles for cell wall modification and membrane transport. Leaf spectral reflectance was associated with leaf boron concentration in both populations, particularly at visible and red-edge wavelengths, indicating potential for high-throughput phenotyping. Together, these findings identify qBEx11.1 as a priority locus for boron exclusion and support integrated genetic and spectral approaches for breeding boron-tolerant grapevine rootstocks.

Cannabis sativa L. cultivation in tropical regions or under indoor conditions requires supplemental lighting to maintain plant yields. Night-break (NB) photoperiods offer a cost-effective alternative to conventional day extension, although genotypic responses have not been clearly described. This study evaluated the effects of continuous and pulsed NB treatments on the growth, phenology, physiology, productivity, and cannabinoid accumulation of three Cannabis sativa genotypes representing two chemotypes. Plants were exposed to a commercial 18-h photoperiod (T1), a continuous 1-h NB (T2), or four 15-min NB pulses (T3) with equal total duration. Continuous NB produced responses comparable to the commercial photoperiod. In contrast, pulsed NB accelerated phenological progression by two to three weeks and reduced final plant height by 15-28%, depending on genotype, without impairing photosynthetic performance or decreasing floral dry biomass. Harvest index increased under both NB treatments, indicating enhanced biomass allocation to reproductive structures, while cannabinoid accumulation remained primarily genotype-dependent with only minor treatment-specific modulation. These findings demonstrate that NB efficacy depends not only on the duration of nocturnal light but also on its temporal distribution, highlighting temporal organization of nocturnal light as a key regulator of photoperiodic responses and a promising strategy for optimizing cannabis cultivation.

The chloroplast is a semi-autonomous organelle with a compact genome encoding ~100 expressed genes. Numerous and widespread promoters coupled with inefficient transcription termination leads to a highly complex primary transcriptome that undergoes extensive RNA quality control orchestrated by RNA binding proteins and ribonucleases. Among the ribonucleases, only RNase J (RNJ) is absolutely required for plant germination and survival, likely because its deficiency leads to massive accumulation of antisense RNA which inhibits translation. As one approach to understanding the selectivity of RNJ for antisense RNA and noncoding regions, we created Arabidopsis lines overexpressing catalytically inactive versions of the enzyme designed to trap its substrates. Initially, transgenic plants appeared phenotypically normal. However, crosses used to generate homozygous transgenic lines yielded progeny that developed varying degrees of chlorosis within 2-3 weeks. Concomitantly, these plants exhibited chloroplast transcriptome alterations similar to RNJ-silenced lines, along with mRNA changes consistent with retrograde signaling. This chlorosis could be correlated to the likely decay of RNJ protein, but not its endogenous transcript. The result could be replicated by attempting to overexpress a wild-type version of RNJ, in which case even one transgene copy was sufficient to induce chlorosis. Our results suggest that RNJ is under protein expression level surveillance to prevent wholesale destruction of the plastid transcriptome.

Sudden Death Syndrome (SDS), caused by Fusarium virguliforme, can significantly impact soybean yields. To advance the understanding of SDS resistance in plant introductions, we conducted genome-wide association studies (GWAS) on a set of 351 accessions from the USDA Soybean Germplasm Collection. The analysis revealed seven single nucleotide polymorphisms (SNPs) significantly associated with foliar SDS resistance; three significant QTL (on chromosomes 3, 9 and 11) were detected with Fixed and Random Model Circulating Probability Unification (FarmCPU) and four (on chromosomes 5, 6 and two on chromosome 7) with Bayesian-information and Linkage disequilibrium Iterative Nested Keyway (BLINK). Localized linkage disequilibrium blocks were estimated for these regions. Notably, all of them overlapped with regions where QTL associated with SDS resistance had been previously reported and where within, or close to, sequences annotated as implicated in plant defense mechanisms, such as toxin transporters and transcription factors. The architecture of SDS resistance appears quantitative and polygenic, with most loci explaining modest phenotypic variance. Six plant introductions in this study--PI 175188, PI 307889A, PI 323559, PI 578307C, PI 445683, and PI 323558--harbor favorable alleles at all seven loci, providing high-value resources for resistance introgression. This study underscores the value of leveraging the USDA Soybean Germplasm Collection for the discovery of resistance loci and provides a foundation for marker-assisted breeding to enhance SDS resistance in elite soybean lines.

Understanding how grafts form has fascinated scientists for over a century, and numerous transcription regulators involved in wound healing, callus formation, and grafting have been identified in model plants. However, the genetic architecture underlying grafting traits remains poorly characterized. To address this gap, we aimed to identify quantitative trait loci (QTLs) associated with grafting success rates and grafting-related traits (including callus formation, mechanical strength of the graft union, and post-grafting growth) in a grapevine rootstock mapping population, Vitis berlandieri x V. rupestris. Although no QTLs were identified for grafting success rate, QTLs were identified for component traits revealing a genetic architecture that would have remained hidden when considering grafting success as a single composite phenotype. By integrating QTL and transcriptome data, we identified regulators potentially mediating grafting-related traits. We then showed that one transcription factor that regulates callus formation and grafting in Arabidopsis potentially has a similar role in grapevine by silencing with exogenous double-stranded RNAs and that exogenous application of methyl jasmonate increases callus development at the graft interface. Candidate transcriptional regulators located within QTLs and evidence from gene silencing and hormone treatments suggest the conservation of regulatory mechanisms underlying wound-induced callus formation across plant species.

Cassava produces starch-filled tubers which are a crucial food source for many people, especially in Africa. This plant species is famous for drought tolerance and resilience, but changing conditions due to climate change require genetic improvements to ensure sustainable cassava cultivation. In this context, a comprehensive understanding of the genetic basis responsible for a variety of traits is beneficial. Here, we present insights into the anthocyanin metabolism, responsible for coloration and protection of cassava structures during stress. Intraspecific variation in the transcriptional control of the anthocyanin biosynthesis was characterized through comparative genomics and phylogenetic analyses. A member of the R2R3-MYB transcription factor family, orthologous to crucial activators of anthocyanin biosynthesis in other plant species, was identified as a genetic hotspot for pigment accumulation capacity between cultivars. Orthologs of this gene appeared absent from three cassava cultivars without pronounced anthocyanin pigmentation, while being present in cultivars known for anthocyanin formation. This discovery corroborates previous reports about the importance of transcriptional regulators in the intraspecific diversity of anthocyanin pigmentation.

Climate change is increasing the frequency and intensity of heatwaves and droughts, threatening medicinal and aromatic plants (MAP) production. Temporary shading during the hottest part of the day may reduce heat and radiative stress while limiting the reduction in light availability. We evaluated this strategy in a field experiment using three MAP species with contrasting ecological requirements and harvested organs: lavandin (Lavandula x intermedia), curly parsley (Petroselinum crispum) and valerian (Valeriana officinalis). Vertical shade nets provided midday shading and effects on crop microclimate, leaf temperature, water status, PSII photochemical efficiency, plant growth and biomass and essential oil production were assessed over two growing seasons. Midday shading reduced daily photosynthetically active radiation by 19.9-55.9 %, with weak effects on air temperature and vapor pressure deficit. In contrast, leaf temperature at solar noon decreased by 6.9 degrees C, 5.6 degrees C and 3.0 degrees C in lavandin, valerian and parsley, respectively. Midday shading improved light-adapted and maximum PSII photochemical efficiencies in all three species and reduced branch dehydration in lavandin during heatwave periods. However, agronomic responses were species-dependent. Floral biomass decreased in lavandin, whereas parsley leaf biomass and valerian root biomass increased. Essential oil concentration was unaffected in lavandin and valerian. Overall, our results show that midday shading primarily reduced leaf-level thermal and radiative stress rather than modifying the surrounding air microclimate, and suggest that its agronomic effects may reflect a balance between stress alleviation and reduced carbon acquisition, that varies with species ecology and production objectives.

Enhancing grain carotenoid concentration to increase yellowness and provitamin A content in sorghum (Sorghum bicolor [L.] Moench) is a major breeding objective across sub-Saharan Africa. Carotenoid breeding is constrained by costly post-harvest phenotyping and can be accelerated through genetic markers that capture functional variation in key carotenoid biosynthesis genes. Zeaxanthin epoxidase (ZEP) is a major gene controlling sorghum grain carotenoid accumulation, and SNP-based KASP markers facilitate selection of high-carotenoid grain, although quantitative variation remains among lines carrying the favorable allele. We hypothesized that additional functional variation within ZEP and other carotenoid biosynthesis pathway genes contributes to this variation and can be revealed by a pangenome-informed analysis. Using a 33-member pangenome reference, we characterized sequence and structural variation at ZEP and other key carotenoid biosynthesis genes and applied pangenome-derived genotyping to study marker diversity associated with carotenoid accumulation. Evaluation of ZEP in the pangenome reference revealed previously uncharacterized structural variation that is absent from the BTx623 primary reference genome. A pangenome-based association analysis identified genetic markers associated with the accumulation of carotenoids which were specific to particular pangenome reference members, including in RTx430 and SRN39, both yellow endosperm lines. Machine learning identified markers in ZEP, {beta}-OH, ZDS, and Z-ISO as the most predictive for all carotenoid traits, suggesting a multi-locus genetic architecture underlies the accumulation of carotenoids in sorghum grain. By integrating sorghum pangenomic resources with machine learning, this study establishes a framework for pangenome-accelerated trait discovery and identifies new genetic targets for carotenoid biofortification in sorghum.

Chlorophyll fluorescence imaging is a powerful tool used to characterize abiotic and biotic stress response of photosynthesis in plants. However, extracting metrics such as Fv/Fm (maximum quantum yield of photosystem II) from chlorophyll fluorescence images remains a substantial bottleneck in large measurement campaigns, often requiring significant manual effort. To resolve this issue, we developed fvfmPy, a user-friendly program which enables rapid analysis of chlorophyll fluorescence images of arrays of leaf discs. fvfmPy uses algorithmic image segmentation to identify regions of interest and extract fluorescence metrics from tens or hundreds of leaf samples per image with little to no manual input. fvfmPy is developed in Python and includes a graphical user interface enabling intuitive parameter adjustment and real-time quality control checking. We additionally developed fvfmR, an R wrapper package for fvfmPy. Fv/Fm values extracted with fvfmPy were indistinguishable from those obtained using manual methods (r2 > 0.99) but required 94% less time to complete (13 s vs. 200 s per 100 measurements). fvfmPy and fvfmR were built to directly integrate with existing fluorescence software packages and can greatly increase throughput of fluorescence measurements to help address key challenges in agriculture and ecology.

Rhamnogalacturonan I (RG-I) is a major pectin domain and an important wall component for cell-cell adhesion. Arabidopsis seed mucilage serves as a powerful model to identify and characterize pectin-related enzymes, since this gelatinous capsule is composed predominantly of RG-I. Although multiple glycosyltransferase (GT) families participate in pectin biosynthesis, only a limited number of their members have been functionally characterized in vivo or biochemically characterized in vitro. In this study, we characterized the biological functions of PEELING WALLS1 (PEEL1), a Golgi-localized GT106 protein that is related to known RG-I rhamnosyltransferases (RRTs). Knocking out PEEL1, but not its close paralog PEEL1-LIKE (PEEL1L), led to patchy mucilage release from seeds upon hydration. However, RG-I content was not compromised in mucilage extracted from peel1 or peel1 peel1L mutant seeds. Treatment of seeds with a cation chelator restored mucilage expansion but failed to rescue the underlying defect in epidermal cell adhesion. Histological experiments and transgene complementation demonstrated that optimal expression of PEEL1 is required for the adhesion of primary walls to the seed surface. Collectively, these findings identify PEEL1 as a previously unrecognized contributor to primary cell wall adhesion and suggest that different RRT-related proteins contribute to unique pectic structures or localizations in vivo, rather than total mucilage RG-I abundance.

Considering the increasing water and labor constraints in rice production, direct-seeded rice is becoming increasingly important. However, direct-seeded rice is vulnerable to water stress and inefficient nutrient use. Nitrogen use efficiency in rice is generally low, and improving nitrogen acquisition and assimilation is important for sustainable productivity. In this study, 48 recombinant inbred lines were evaluated under ammonium and nitrate nutrition in irrigated and rainfed environments across two cropping seasons. Agronomic, physiological, biochemical and molecular responses were assessed. Significant effects of genotype, nitrogen form, environments and their interactions were observed for several traits. Rainfed conditions reduced grain and biological yield compared with irrigated conditions. Ammonium produced a higher mean grain yield than nitrate by approximately 26.2 g m-2 under irrigated conditions and 154.2 g m-2 under rainfed conditions, although the responses varied among genotypes. Biological yield and SPAD values were generally higher with ammonium, whereas total chlorophyll showed season and environment-dependent responses. Glutamine synthetase activity was highest under ammonium treatment, while glutamate synthase activity was higher under nitrate treatment. Grain yield showed strong positive correlations with biological yield, but correlations between grain yield and GS or GOGAT activity were not significant. Expression of OsGln1.2, OsGln2 and OsGlt1 varied according to genotype, tissue and nitrogen treatment. Overall, the results demonstrate that responses to nitrogen forms are genotype- and environment-dependent, and that ammonium may provide a yield advantage under rainfed conditions. Integrating field performance with biochemical and molecular traits may help identify rice genotypes suited to water-limited systems.

Bulk phloem flow is driven by pressure gradients generated by water drawn from the xylem, coupling source-sink transport with water status. Huanglongbing (HLB), a phloem-affecting bacterial disease of citrus, disrupts phloem transport, and causes canopy water deficit. Although improved water management can enhance HLB-affected tree health, the effects of HLB on water-driven source-sink relations are unknown. Fruits function as both carbon sink and water reservoir, but the role of fruit in regulating whole-tree water status remain poorly understood. We examined leaf-fruit water relations in HLB-affected citrus and evaluated how HLB exposure duration, fruit developmental stage, and source-sink manipulation shape these dynamics. Source-sink relations were manipulated in branches of symptomatic trees with long-term (four years) and recent (six months) HLB exposure. We girdled branches with a single fruit as the sink and defoliated at (0%, 50%, or 75%), and an ungirdled, non-defoliated branch served as control. We measured stomatal conductance, leaf hydraulic traits, leaf and fruit soluble sugars, diurnal fruit shrinkage (to estimate xylem backflow) and fruit water loss. Source-sink manipulation did not affect shoot total soluble sugar. HLB-exposure resulted in less negative osmotic potential, more negative turgor potential and reduced modulus of elasticity and relative water content at turgor loss point. HLB exposure also increased diurnal fruit shrinkage, with shrinkage responses to source-sink manipulation varying across fruit development stages. Collectively, these findings suggest that long-term HLB exposure promotes leaf acclimation to water deficits, however, high sink demand may diminish the leaf water status regulation at Stage II, when fruit sink carbon demand is highest.

DNA methylation dynamics regulate diverse plant stress responses, but the mechanisms governing arsenic (As) tolerance through epigenetic pathways remain largely unknown. Here we report that Arabidopsis mutants lacking the DNA demethylase, REPRESSOR OF SILENCING (ROS1), show substantially enhanced As resistance. Integrating transcriptome and methylome data, we found that PIN-FORMED 2 (PIN2), encoding an auxin efflux carrier that also transports As out of cells, represents a key ROS1 target. In ros1, we observed increased methylation in the last exon of PIN2, a region functioning as the promoter for an antisense long non-coding RNA (lncRNA). Hypermethylation of the promoter suppresses the antisense lncRNA, which follows reduction of sense PIN2 transcription. As a result, PIN2 expression increases in ros1 mutants during As exposure, enhancing As tolerance. Analysis of ros1 pin2 double mutants confirmed that PIN2 acts downstream of ROS1 in As tolerance. Our study demonstrates an epigenetic pathway in which ROS1 regulates As tolerance by controlling PIN2 expression through antisense lncRNA-mediated transcriptional interference, offering new epigenetic strategies using plants in heavy metal contaminated environments.

Calcium (Ca2+) is a universal second messenger that orchestrates plant growth, development, and stress responses. Plant calcineurin B-like (CBL) proteins sense and relay Ca2+; signals in response to pathogens to activate downstream defense signalling pathways. Early blight disease, caused by the necrotrophic fungal pathogen Alternaria solani, affects tomato crops and severely limits their productivity. In the present study, we found that the tolerant tomato cultivar (Arka Rakshak) restricts A. solani infection and disease progression compared to the susceptible cultivar (PKM-1). Comparative transcriptome and qRT-PCR analysis of A. solani-infected tomato leaves showed that SlCBL1-2 (Solyc08g007160.3), a Ca2+; sensor, is up-regulated in the tolerant cultivar, both relative to its own uninfected control and the infected susceptible cultivar, whereas it was not significantly induced in the uninfected susceptible cultivar. Protein sequence analysis indicates that SlCBL1-2 contains four EF-hand motifs, an N-terminal N-myristoylation/S-acylation module and a C-terminal FPSF motif, and it is predicted to interact with several CBL-interacting serine/threonine protein kinases (CIPKs). However, the evolutionary analysis, conformational transitions upon binding to Ca2+, and the Ca2+-binding affinity of SlCBL1-2 have not been previously characterized. Molecular Dynamics (MD) simulation of the AlphaFold structure of SlCBL1-2 indicates a well-folded structure and binding of two Ca2+ ions at EF-hands 3 and 4. Further, far-UV circular dichroism and tryptophan fluorescence spectroscopy of recombinant SlCBL1-2 indicate a natively folded structure in the absence of Ca2+ and undergo minimal secondary and tertiary structural changes upon Ca2+-binding, in contrast to several Ca2+-binding proteins. Of note, SlCBL1-2 binds Ca2+ with apparent KD values of 526 {+/-} 113 {micro}M and 28.7 {+/-} 9.47 {micro}M, as evidenced by isothermal titration calorimetry. Together, our study provides an experimental description of the Ca2+-binding properties of tomato CBL protein and defines the molecular basis by which pathogen-induced SlCBL1-2 may act as a Ca2+ sensor in defense signalling.

Iron (Fe) homeostasis is fundamental to plant growth and development and is centrally regulated by a well-conserved network of basic helix-loop-helix (bHLH) transcription factors. In Arabidopsis thaliana, bHLH121 regulates the expression of FER-like iron deficiency-induced transcription factor (FIT) and clade Ib bHLH38/39/100/101 genes, among others. However, its functional conservation remains unexplored, and some contradictory results have been reported regarding the effects of bHLH121 overexpression. Here, we investigated the evolutionary conservation of bHLH121 through complementary tests in the Arabidopsis bhlh121 mutant created by CRISPR/Cas9 gene editing. We also investigated the effects of ectopically expressing full-length, N-terminal, and C-terminal fragments of bHLH121 on plant responses to Fe deficiency in Arabidopsis. Transgenic plants overexpressing the full-length bHLH121 driven by the cauliflower mosaic virus 35S promoter (bHLH121 ox) resulted in shorter roots and more severe leaf chlorosis compared to wild-type plants under Fe limiting conditions. Moreover, overexpression of the N-terminal fragment (N121 ox) rendered plants highly sensitive to Fe deficiency, whereas overexpression of the C terminal fragment (C121 ox) did not. Consistent with the phenotypic observations, N121 ox plants accumulated lower Fe content, and the ferric chelate reductase activity was significantly reduced. In N121 ox plants, the induction of key Fe regulatory genes, especially FIT, was significantly inhibited under Fe deficiency. Confocal imaging showed that both N121-GFP and C121-GFP fusion proteins could localize to the nucleus. Electrophoretic mobility shift assay showed that both full-length bHLH121 and N121 could effectively bind the probes derived from the promoter of FIT. The present work reveals a conserved function of bHLH121 in Fe homeostasis across several dicot species and demonstrates that the N121 may possess a dominant nature in Fe deficiency responses.

Macaranga tanarius (Euphorbiaceae), a myrmecophilic plant grown in subtropical countries, was identified as a source of Okinawan propolis, a honeybee product, which is glandular trichomes developed on the surface of the fruits. This tissue contains a high amount of characteristic geranylated flavonoids known as nymphaeols (nymphaeol A - C and isonymphaeol B), which exhibit strong antioxidant activity. In this study, we characterized the structure of glandular trichomes of M. tanarius, showing that they are sac-like structures containing drupelet-like aggregates of secretary cells, and a subcuticular cavity was also observed. From a cDNA library, we identified two prenyltransferase (PT) genes involved in biosynthesis of nymphaeols, and a PT gene grouped in a primary metabolic PT. Our biochemical experiments demonstrated that one of the former secondary metabolic PTs (MtPT1) is a B-ring-specific geranyltransferase for eriodictyol to yield nymphaeols B and isonymphaeol B. MtPT1 was unusual for a plant PT, an enzyme that yielded multiple reaction products. The other PT (MtPT3) transferred a geranyl moiety to the A-ring of eriodictyol to form nymphaeol A. Although dimethylallyl diphosphate (DMAPP) was not recognized as its prenyl donor substrate of eriodictyol, giving a B-ring geranylated eriodyctiol as the prenyl acceptor substrate, this enzyme used DMAPP to attach the prenyl moiety to the A-ring and produced nymphaeol C. These data suggest that M. tanarius has evolved its PT function to produce a diverse range of prenylated flavonoids using a limited set of genes.

Palmer amaranth (Amaranthus palmeri S. Watson) is a highly competitive weed that has evolved resistance to several modes of action and has recently become a significant problem in Turkiye. This study was designed to determine the distribution of common target-site resistance (TSR) mechanisms to the acetolactate synthase (ALS)-inhibiting herbicide, nicosulfuron, and the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)-inhibiting herbicide, glyphosate, in A. palmeri accessions collected from two agricultural regions of Turkiye; the Cukurova Region and the Gediz Basin. A total of 96 accessions were analyzed for ALS gene mutations and relative EPSPS gene copy number variation. Whole-plant responses were also evaluated based on relative dry weight following treatment with nicosulfuron and glyphosate applied at twice the field rate. The ALS mutations Pro197Ser, Trp574Leu, and Ser653Asn were far more prevalent in plants from the Cukurova Region than those from the Gediz Basin. Elevated relative EPSPS gene copy numbers were more common and higher in the Gediz Basin. Single rate herbicide screening experiments confirmed that accessions from the Cukurova Region exhibited higher dry weight response to nicosulfuron, whereas those from the Gediz Basin had higher to glyphosate Multivariable regression analyses revealed that nicosulfuron response was primarily associated with the Pro197Ser and Trp574Leu mutations, while glyphosate response was correlated with EPSPS gene copy number. These findings reveal distinct regional patterns of TSR mechanisms in A. palmeri in Turkiye that are consistent with differences in herbicide use and/or introduction history, but the roles of local selection, demographic processes, and gene flow remain unclear.