Plant Science

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.

Drought stress severely limits tree growth and survival, a problem intensifying under climate change due to the rising frequency of extreme weather events. Identifying genetic variants that modulate drought stress responses and enhance resilience is therefore of great interest for developing climate-adapted trees. While the MADS-box transcription factor AGL16 has been implicated in drought responses and stomatal regulation in herbaceous species, the function of its homolog in woody perennials remains largely unexplored. Here, we investigate the role of PtaAGL16 in the poplar hybrid Populus tremula x P. alba. We generated CRISPR/Cas9 knockout lines of PtaAGL16 in the poplar clone INRA 717-1B4. Plants were subjected to a severe drought stress regime followed by a re-watering recovery phase. Morphological and physiological traits, including growth, stomatal conductance, shoot vitality, and stomatal density, together with stress-associated metabolites and drought-responsive gene expression, were analyzed. All agl16 knockout lines exhibited significantly reduced drought-induced damage compared to the wild-type. While the wild-type suffered extensive defoliation and persistent stem necrosis, the knockout lines retained higher shoot vitality and resumed growth after re-watering, including the formation of new leaves. Consequently, vitality in the knockout lines reached up to 86%, compared to only 34% in the wild-type. This improved resilience was accompanied by reduced persistent oxidative membrane damage, as indicated by lower MDA levels after recovery. These findings indicate that the disruption of PtaAGL16 is associated with enhanced drought resilience in a woody perennial species and highlight its potential as a genetic target for improving climate adaptation in fast-growing forest trees.

Amaranthus tricolor is a climate-resilient orphan crop whose betalain pigments contribute to nutritional quality and stress tolerance. However, breeding efforts have traditionally relied on field yield trials due to the limited availability of genomic information and genetic resources. To address this gap, we first used the Illumina and PacBio sequencing platforms to assemble a novel reference genome of A. tricolor, using the Japanese cultivar Bayam. By combining this reference genome with a core collection developed in our previous study, we aimed to identify genomic regions associated with pigmentation-related traits across winter and summer seasonal environments. Betalains are antioxidant phytonutrients that contribute to abiotic stress tolerance, making the regulation of their biosynthesis under heat stress particularly relevant for developing climate-resilient crops such as A. tricolor. As a result, ATR1.0CH03_5131103, which is associated with ATR1.0ch03g000565, was identified under the winter seasonal environment. Meanwhile, transcriptomic profiles in this study revealed that ATR1.0ch03g000565 was differentially expressed under both control and heat stress conditions, suggesting that it may be associated with the regulation of CYP76AD1 expression under optimal conditions. Under the elevated-temperature treatment, transcript levels of CYP76AD1 and 5GT decreased in Am087, coinciding with lower betacyanin-dominated absorbance indices. cDOPA-like homologs were upregulated; however, pigmentation-related absorbance remained reduced. Further molecular experiments, such as CRISPR-Cas9 knockouts or overexpression studies, are necessary to validate the regulatory mechanisms of CYP76AD1 and 5GT under heat stress.

High temperatures during grain filling stage are becoming increasingly frequent, compromising both grain and eating quality and thereby driving demand for heat-resilient cultivars. Such conditions are known to reduce the expression of granule-bound starch synthase I (GBSSI) and starch branching enzyme IIb (BEIIb), which are involved in starch biosynthesis, resulting in a decrease in amylose content and an increase in long-chain amylopectin. Thus, the present study introduced functional mutation in starch synthase IIa (SSIIa) that increases the proportion of short amylopectin chains to genetically compensate for the high-temperature-induced increase in amylopectin long chain. Rice lines carrying the ss2a mutation were grown at two locations with cooler (Akita) and warmer (Okayama) temperatures. Their grain traits, starch structure, and eating quality were compared. The ss2a mutant lines showed an increased proportion of short amylopectin chains as well as an increased apparent amylose content. Furthermore, these alterations in starch structure varied with the grain-filling temperature of the cultivation sites, ultimately affected eating quality. These results suggest that enriching short amylopectin chain via the ss2a mutation can counteract the increase in long amylopectin chain caused by high temperatures during grain filling, thereby maintaining a desirable starch structure and eating quality.

Although iron (Fe) is abundant in soils, its low solubility, particularly under alkaline conditions, often limits plant Fe acquisition. Under these conditions, coumarin-mediated Fe mobilization becomes increasingly important. Arabidopsis thaliana FE DEFICIENCY-INDUCED KELCH REPEAT PROTEIN 1 (FDK1, AT3G07720) is strongly induced by Fe deficiency and regulated by the central transcription factor FIT, but its function is unknown. We investigated FDK1 using genetic, physiological, metabolite, and transcriptomic analyses. Loss of FDK1 altered root growth, whereas this phenotype was restored in the FDK1-Ox overexpression line. Under alkaline Fe deficiency, growth medium from FDK1-Ox contained strongly increased levels of the Fe-mobilizing coumarins fraxetin and esculetin compared with wild type, whereas their induction was attenuated in fdk1-1. RNA-seq analysis linked FDK1 abundance to coumarin biosynthesis and iron-homeostasis gene expression. FIT and many FIT-associated genes were highly expressed in FDK1-Ox, whereas several upstream iron-homeostasis regulators, including bHLH Ib factors, were highest in fdk1-1. These findings identify FDK1 as a component of the alkaline Fe-deficiency response that promotes iron-mobilizing coumarin accumulation and is associated with coordinated regulation of iron-homeostasis genes.

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.

Background: Plant defensins are small cysteine-rich antimicrobial peptides. In Arabidopsis thaliana, the inducible Class I gene AtPDF1.2a is widely used as a transcriptional marker of jasmonate/ethylene immunity, whereas the constitutively expressed Class II members have received less functional attention. AtPDF2.2 was compared with AtPDF1.2a during infection of Arabidopsis by the vascular fungal pathogen Verticillium longisporum, the necrotrophic fungus Sclerotinia sclerotiorum and the bacterium Pseudomonas syringae pv. tomato DC3000. Results: Profiling of the AtPDF family showed that AtPDF2.2, together with the Class II genes AtPDF2.3 and AtPDF2.5, was significantly reduced during V. longisporum infection, whereas AtPDF1.2a was induced. A promoter-reporter line showed a corresponding decrease in AtPDF2.2 promoter activity during V. longisporum infection, strongest in infected roots. KD-pdf2.2 plants contained more V. longisporum DNA, whereas OE-PDF2.2 plants contained less. The same reciprocal disease pattern was observed during S. sclerotiorum and Pst DC3000 infection. Histochemical Prom::AtPDF2.2::GUS reporter signal decreased during S. sclerotiorum infection but increased following Pst DC3000 infection. Basal AtPDF2.2 expression was lower in JA and ET-signaling mutants and higher in the SA-deficient sid2 background, while the analyzed promoter lacked the GCC-box that mediates inducible AtPDF1.2a regulation. AtPDF1.2a overexpression reduced symptoms across the three pathosystems, but loss of AtPDF1.2a increased disease only during V. longisporum infection under the conditions tested and did not alter Pst DC3000 DNA abundance. AtPDF2.2 overexpression was not associated with reduced vegetative growth under the conditions tested, whereas AtPDF1.2a overexpression reduced rosette size and leaf number. Conclusions: AtPDF2.2 is a constitutively expressed Class II defensin whose expression level is associated with disease outcome across two fungal pathogens and a bacterium. Its promoter shows tissue-dependent regulation during V. longisporum infection, while histochemical reporter signal decreases during S. sclerotiorum infection and increases during Pst DC3000 infection. This regulatory behavior differs from that of the inducible reference gene AtPDF1.2a. The reciprocal disease phenotypes of the AtPDF2.2 transgenic backgrounds support a protective association that may involve direct antimicrobial activity, modulation of host responses or both. The absence of reduced vegetative growth in OE-PDF2.2 under the conditions tested supports further evaluation of PDF2-type genes in Brassica.

Understanding the genetic architecture of ecophysiological traits related to carbon gain and water loss is crucial to develop cultivars tolerant to abiotic stresses and that maintain productivity in the context of climate change. This can be achieved both through experiments in the greenhouse on potted plants under controlled conditions and in the field. However, the stability of genetic determinism of ecophysiological traits across environmental conditions has rarely been evaluated. We studied a grapevine diversity panel grown both in a greenhouse and in the field, to investigate the impact of growing conditions on leaf ecophysiological traits related to leaf morphology, fluorescence, gas exchange and water use efficiency. The correlation structure between traits was conserved within each environment even though, for most traits, cultivars ranking was not conserved between both environments. However, a set of cultivars, sharing the same functioning pattern in terms of gas exchange and fluorescence in both environments, was identified. Single- and multi-environment Genome-Wide Association Studies (GWAS), based on a dense genotyping matrix, were used to dissect the genetic architecture of leaf traits. The Quantitative Trait Loci (QTLs) identified through single-environment analyses differed substantially from those detected using multi-environment ones. Multi-environment approaches highlighted shared QTLs across environments for gas exchange and fluorescence traits. Candidate genes associated with carbon- and water-related functions were further identified in these genomic regions. Despite the weak correlation between greenhouse and field trait values, combining both environments enabled the identification of robust genetic regions associated with ecophysiological traits of interest.

Plants frequently experience recurrent drought events separated by periods of rehydration. Although drought imposes strong constraints on plant physiology, prior exposure may alter subsequent stress responsiveness through memory-based mechanisms. Here, we investigated whether recurrent drought at distinct developmental stages establishes stress memory in sugarcane and whether this response persists across vegetative propagation. Two genotypes contrasting in drought tolerance and productivity (IACCTC07-8008 and IACSP95-5000, respectively) were grown under greenhouse conditions and subjected to three drought cycles imposed either at tillering or maturation stage. Gas exchange, photochemical performance, leaf water status, primary metabolite profile, and growth traits were assessed across cycles, and vegetative propagules were subsequently evaluated under renewed drought. The first drought cycle imposed strong limitations on carbon assimilation and photochemistry in both genotypes and developmental stages. However, subsequent cycles resulted in attenuated reductions in A and g, improved intrinsic water use efficiency, and partial stabilization of PSII performance, indicating a modified stress-response trajectory. Young plants displayed earlier improvements (from the second cycle), whereas in mature plants this shift was evident mainly during the third cycle. Recurrent drought promoted sustained reorganization of amino acid, carbohydrate, organic acid and polyol metabolism alongside increased root biomass and higher relative water content during later cycles. Importantly, propagules derived from drought-conditioned plants exhibited faster recovery of photosynthetic performance and reduced cumulative physiological impairment under renewed drought, despite showing similar stress sensitivity at maximum water deficit. This persistence of enhanced recovery capacity across vegetative propagation indicates that drought-induced memory was maintained beyond the initially stressed plants. Together, our findings demonstrate that recurrent drought establishes a metabolically imprinted state in clonal sugarcane, integrating physiological adjustment, metabolic reprogramming, and whole-plant acclimation. These results highlight the potential of stress memory as a mechanism supporting resilience in perennial crops exposed to increasingly recurrent drought events.

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.

Petal coloration is a key ornamental trait in Cyclamen persicum Mill. and is closely associated with anthocyanin composition and relative accumulation, yet the major anthocyanins and candidate molecular factors associated with color variation remain unclear. In this study, anthocyanin profiling and transcriptome analysis were integrated to investigate three Cyclamen materials with distinct petal colors, BXK, FXK, and SHXK. Anthocyanin profiling showed clear separation among the three materials, indicating that petal color divergence was associated with differences in anthocyanin composition and relative abundance. Petunidin-, peonidin-, delphinidin-, malvidin-, and cyanidin-related derivatives exhibited distinct accumulation patterns. Transcriptome analysis revealed marked transcriptional divergence, with phenylpropanoid and flavonoid biosynthesis repeatedly enriched across comparisons, whereas isoflavonoid and flavone and flavonol biosynthesis were enriched in selected comparisons. Mapping differentially expressed genes onto the anthocyanin biosynthetic pathway revealed branch-specific expression patterns of structural genes, including CHS, F3H/F3'H, F3'5'H, DFR, and ANS. Notably, Cluster-26256.0, annotated as a putative anthocyanidin synthase gene, showed a progressive increase in expression from BXK to FXK and SHXK, consistent with increasing petal pigmentation. Several DFR-related transcripts also showed relatively high expression in SHXK. Co-expression analysis linked key anthocyanins, structural genes, and transcription factors, while qRT-PCR generally confirmed RNA-seq trends. These results suggest that late anthocyanin biosynthesis, particularly ANS-related transcriptional variation, may contribute to dark red petal coloration. This study provides a metabolite-transcript framework for Cyclamen petal coloration and identifies ANS-related genes as priority candidates for future functional studies and flower-color breeding.

We performed a screening to evaluate the responses to water deficit before the flowering stage in a panel of 31 genotypes composed of one commercial hybrid, one wild species, multiple ex-PVP (vintage) lines, several commercial heirlooms from the Northeast USA, Mexican landraces from hot and humid environments and genome edited lines with altered flowering and inflorescence traits. We characterized 18 different morphological, physiological and biochemical traits to assess the response to 40% water deficit. We observed diverse morphological, physiological and biochemical responses across the 31 genotypes screened, suggesting the presence of useful variation for breeding for drought tolerance in cultivated tomato. Interestingly, some heirlooms exhibited superior performance under drought, suggesting they can be used as donors to discover novel genes involved in drought responses. Unexpectedly, we found mutants in SlCLE9, a paralog of the stem cell regulator SlCLV3 known to co-regulate meristem proliferation in tomato, exhibited enhanced drought tolerance. Notably, the slcle9 mutant also conferred drought tolerance when used as rootstock with grafted scions from our reference cultivar M82, suggesting SlCLE9 influence root architecture and drought response. Consistent with this, we found SlCLE9 is transcriptionally upregulated in roots and leaves during drought stress, and exogenous application of SlCLE9 modifies root growth and response to drought. Altogether, our screening uncovered new potential trait donors for breeding against drought stress and describes a novel role for SlCLE9 in drought tolerance.

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.

Plant encounters with saline conditions trigger a phase of osmotic stress, followed by ionic stress as salt accumulates in plant tissue. Jasmonate (JA) signalling plays a vital role in both phases, but its role for adaptation depends on timing. Using wild-type rice (Oryza sativa L. cv. Kitaake) and a JA-deficient allene oxide cyclase (aoc) mutant, we followed whole-plant physiology (growth, biomass, photosynthesis, ion distribution) with expression analysis for genes of jasmonate signalling, ion transporters and redox balance to separate adaptive from maladaptive JA effects. The aoc mutant sustained shoot and root growth more effectively than the wild type, maintaining cell expansion in the shoot and cell proliferation in the root under saline conditions. Likewise, stomatal conductance in the mutant was more persistent under salinity, accompanied by improved shoot K/Na homeostasis and reduced sodium in young leaves. In the wild-type, prolonged saline conditions were linked with induction of OsJAZ13 and OsNHX1, stomatal closure and chlorophyll degradation. We discuss these findings in the context of a model, where the function of JA signalling does not merely depend on its accumulation, but on its temporal dynamics. Here, OsHAK16 emerges as a marker of early adaptive responses, whereas OsNHX1 and OsJAZ13 characterize later maladaptive stress states, emphasising the potential of modulating JA dynamics to improve salt resilience in rice breeding.

Palladium (Pd), a technology critical platinum group metal, is increasingly lost to the environment through anthropogenic activity, yet the molecular basis of Pd uptake and tolerance in plants remains poorly understood. Here, we examine the role of the Arabidopsis thaliana (Arabidopsis) COPT2 copper transporter in Pd transport and plant stress responses. Expression of COPT2 in Saccharomyces cerevisiae conferred Pd sensitivity, indicating a capability for Pd transport. Arabidopsis copt2 loss-of-function mutants accumulated less Pd in shoots and exhibited reduced reactive oxygen species (ROS) production, supporting a direct role for COPT2 in Pd distribution and associated oxidative stress. Our subsequent transcriptome profiling revealed extensive changes under Pd exposure, including downregulation of HMA2 and aquaporins, and upregulation of HMA7, glutathione transferases, and glutamine synthetase GLN1;1, consistent with the activation of metal detoxification and redox homeostasis pathways. Together, these findings identify COPT2 as a key transport protein mediating the uptake of Pd in Arabidopsis and suggest that Pd induces similar detoxification mechanisms as zinc (Zn), copper (Cu) and cadmium (Cd), providing a foundation for engineering plants for precious metal recovery.

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.

Abstract Tricyclic coumarins (TCs) exhibit defensive properties against herbivorous insects and possess valuable pharmacological properties for humans. They are mainly distributed in the Apiaceae, Rutaceae, Moraceae, and Fabaceae families. The chemical structural diversity of TCs in these families primarily arises from differences in skeletal variety and attachment position of the third ring, as observed in unsaturated furanocoumarins (FCs) and saturated dihydrofuranocoumarins (DHFCs). In TC biosynthesis, the FC and DHFC pathways diverge through distinct oxidation patterns. The product specificity of the reported cytochrome P450 monooxygenase at the branching point suggests that these TC groups are produced in nearly identical ratios; however, some species of the genus Angelica (Apiaceae) accumulate DHFCs at higher levels than FCs. In this study, we investigated the enzymatic mechanism responsible for forming these two TC groups using A. keiskei, a Japanese native species used as a medicinal and edible herb. Organ-specific transcriptome analysis highlighted a P450 member CYP71AJ62 that exhibited a close correlation with TC accumulation across organs. Enzymatic characterization demonstrated that CYP71AJ62 predominantly forms a hydroxylated DHFC, vaginidiol, which may promote DHFC over FC skeletal formation in TC biosynthesis. Phylogenetic analysis suggested that CYP71AJ62 has been derived from a gene responsible for FC formation by functional shift without gene duplication. Subsequent three-dimensional structural modeling and site-directed mutagenesis revealed that two amino acid residues on substrate recognition sites cooperatively determine the product specificity of this P450 enzyme. This study provides new insights into the molecular evolutionary process underlying ring-structure diversification and, more broadly, the adaptation of plants to their environments.

Progression of leaf senescence is regulated by internal factors like the plant hormone cytokinin (CK) that acts to delay senescence and retain photosynthesis by maintaining chloroplast-functionality. While initial CK-signaling components controlling senescence have been described, downstream targets regulating physiological performance have remained less characterized. Here we present the CHLOROPLAST IMPORT APARATUS 2 (CIA2) and CIA2-like (CIL) transcription factors (TFs) as mediators of CK senescence activity, enabling chloroplast-function. Utilizing loss-of-function mutants, we show CIA2 and CIL negatively regulate senescence progression and alter physiological performance in Arabidopsis thaliana. Integrated transcriptomic-metabolomic analysis shows that CIA2 and CIL action is distinct from other known chloroplast-related TFs (like GLKs/GATAs) and can boost antioxidant response over senescence. We find that CIA2 and CIL are essential in relaying CK activity during chloroplast biogenesis, photosynthesis, rubisco function, and metabolite accumulation. In turn, ARR10/12 CK signaling components were needed for CK induction of CIA2 and CIL themselves - a cascade that induced chloroplast- and photosynthesis-associated nuclear genes, to boost chloroplast-function and metabolism. We also show that CK acts to repress senescence through accumulation of the polyamine, spermidine, in a CIA2/CIL dependent manner. This work presents a CIA2/CIL-cascade as a new anterograde signal for CK antisenescence effects, indicating a mechanism through which CK prolongs leaf function. This fundamental understanding of CK action has implications towards engineering plants with elevated photosynthesis performance and delayed senescence.

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.