OsNLP4 is a master regulator of nitrate signaling, but its ecological role in the root-associated bacterial communities is unknown. This study investigated how OsNLP4-mediated nitrate signaling influences root-associated microbial composition and root exudate metabolism. We found that the osnlp4 mutants exhibited distinct root-associated bacterial communities compared to the WT under non-nitrogen-fertilized conditions, with a significant enrichment of Bradyrhizobium and other bacterial taxa with potential roles in nitrogen cycling. Isolated Bradyrhizobium strains showed increased colonization in the osnlp4 mutant. Metabolomic analysis revealed increased secretion of sugars and phosphoric acid, along with a lower C:N ratio, in the osnlp4 mutant root exudates. Bacterial growth and chemotactic responses differed between WT- and osnlp4-derived root exudates, and sugars enriched in osnlp4 exudates elicited chemotactic responses in isolated Bradyrhizobium strains. These metabolic changes were associated with differential bacterial responses to WT and osnlp4 root exudates and may contribute to the altered root-associated bacterial community. Taken together, our results suggest that OsNLP4 links nitrate signaling and nitrogen metabolism with root-associated bacterial community assembly. Disruption of OsNLP4 reprograms root exudation and reshapes root-associated microbial communities, highlighting an association between nitrate signaling, nutrient allocation, and root-associated bacterial community assembly in rice.

Improving drought tolerance is crucial for sustainable crop production under climate change. Identifying drought-adaptive variation in diverse genetic resources, including crop wild relatives, requires accurate and temporally resolved assessment of plant responses to water deficit. Here, we developed an indoor, unmanned phenotyping platform that can noninvasively and automatically collect temporal data on plant responses to drought stress. Using this platform, we quantified temporal drought responses in four rice cultivars and five wild rice relatives and integrated these phenotypes with chromosome-scale genome assemblies, gene annotations, and time-course transcriptomes. This integrated phenotyping, genomics, and transcriptomics identified a wild rice accession that maintained shoot growth, leaf temperature, and gene expression more stably under drought stress and exhibited a distinctive constitutive drought-responsive transcriptional state. This state included elevated basal expression of several drought-responsive genes, including ABSCISIC ACID RESPONSIVE ELEMENT-BINDING FACTOR 1 (ABF1), a known regulator of ABA-mediated drought responses. Validation using transgenic and introgression lines suggested that low-level constitutive ABF1 expression, as a component of this drought-responsive regulatory state, can improve drought performance without obvious growth penalties. These findings demonstrate the utility of automated temporal phenotyping combined with accession-specific genomic and transcriptomic resources for uncovering drought-adaptive variation in wild rice, and provide a framework for exploiting natural regulatory variation to improve drought resilience in cultivated rice.

Light regulates the expression of phototropins by decreasing PHOT1 mRNA levels and increasing PHOT2 mRNA levels. In Arabidopsis thaliana, the light-regulated activity of phototropin promoters paralleled the levels of PHOT1 and PHOT2 transcripts. The activity of phototropin promoters in different organs and at various developmental stages reflected the physiological need for those receptors. The PHOT1 promoter was predominantly active in the above-ground parts of etiolated seedlings, which depend on the ability of phototropin1 to sense low light levels. The PHOT2 promoter activity was high in light-grown mature plants, with substantial expression found in inflorescence, rosette, and cauline leaves. The PHOT2 promoter was strongly activated by light also in N. benthamiana leaves. Y1H assay revealed transcription factors that might be responsible for the light regulation of phototropin promoters. Transcription factors from the homeobox (HB) family (ATHB12) were identified as interacting with the PHOT1 promoter, while ANAC102, ARF19, TCP2, and A-PTF-1 were found to interact with the PHOT2 promoter. Using cEMSA, interactions between the DNA-binding domains of TCP2 and ANAC102 with the PHOT2 promoter and the interaction between the ATHB12 domain and the PHOT1 promoter were demonstrated in vitro. The role of ATHB12 in the control of PHOT1 expression was further confirmed at the mRNA level in Arabidopsis seedlings. Our results point to transcription factor families that may be important for the control of phototropin promoter activity.

PmgA and Ssr1600, homologs of the anti-sigma factor and the anti-sigma antagonist of the bacterial partner-switching system, respectively, are involved in coordination of the repression of photosystem I content and chlorophyll biosynthesis under prolonged high-light (HL) conditions in the cyanobacterium Synechocystis sp. PCC 6803. To characterize the transcriptional responses associated with PmgA and Ssr1600 during HL acclimation, we compared global gene expression profiles of the wild-type (WT) and knockout strains of pmgA (ΔpmgA) and ssr1600 (Δ1600) under low-light conditions and at 15 min, 1 h and 12 h after the shift to HL. The knockout of pmgA or ssr1600 induced temporally distinct transcriptomic alterations between short-term and prolonged HL exposure. At 15 min, Δ1600, but not ΔpmgA, showed enhanced induction of stress-responsive genes, including sigB and heat-shock genes, together with derepression of pmgA. At 12 h, both mutants showed significant upregulation of genes related to chlorophyll biosynthesis, photosynthetic electron transport, nitrogen assimilation, and amino acid biosynthesis. Considering that Ssr1600 does not accumulate in ΔpmgA, these observations can be explained by a shift in the role of Ssr1600, from the suppressor of the amplification of the stress response by PmgA under short-term HL conditions to a factor mediating signal output from the PmgA-Ssr1600 module under prolonged HL conditions, although the contribution of secondary effects cannot be excluded. Our transcriptomic data do not support the hypothesis that PmgA acts as a canonical anti-sigma factor for any of the sigma factors examined in this study, SigB, SigD and SigE, under either condition.

Isoflavonoids are a specialized subclass of flavonoids predominantly distributed in the Fabaceae, where they fulfill essential ecophysiological roles as antimicrobial phytoalexins and symbiotic signal molecules. During their biosynthesis, 2-hydroxyisoflavanone synthase [also called isoflavonoid synthase (IFS)], a cytochrome P450 (P450) of the CYP93C subfamily, catalyzes the oxidative aryl migration of (2S)-flavanones. While various species in the genus Iris (Iridaceae, monocots) are also prolific isoflavonoid producers, the biosynthetic mechanisms in these non-leguminous plants have remained elusive. To identify the Iris IFS, we utilized an established adventitious-root culture system for Iris germanica and employed functional expression-fractionation screening of its cDNA library in yeast cells. This sequence-independent strategy successfully identified CYP736D1 as the enzyme mediating the characteristic skeletal rearrangement of naringenin to form 2-hydroxyisoflavanone. CYP736D1 showed higher in vitro and in vivo catalytic activities than CYP93C2, owing to its higher substrate affinity, kinetic efficiency, and protein stability. The IFS activity was highly specific to naringenin; reactions with other flavanones (liquiritigenin, hesperetin, and pinocembrin) primarily yielded corresponding flavone or 3-hydroxyflavanones. The reaction mechanism of CYP736D1 is considered to be essentially identical to that of CYP93C, but the amino acid residues of CYP93C critical for the IFS activity are not conserved in CYP736D1. Thus, IFS proteins in the Fabaceae and Iridaceae likely evolved from different ancestral P450s and independently acquired the ability to construct the isoflavonoid skeleton through parallel evolution. Bioconversion and transgenic experiments demonstrated that CYP736D1 is a promising tool for biotechnological isoflavonoid production in heterologous systems.

Heat stress negatively impacts plant growth and development and reduces yield in agricultural crops. Plants respond to elevated temperatures through the heat stress response (HSR) in which Ca2+ signals trigger changes in cellular activity and gene expression that help limit cellular damage. Calmodulin-binding transcription factors (CAMTAs) are a family of transcriptional regulators that participate in various stress-responsive signalling pathways. However, roles for CAMTAs during HSR are unclear. Here, using a combination of genetics and physiological assays, we tested the hypothesis that CAMTAs function as regulatory proteins during the Arabidopsis HSR. Among the six-member CAMTA family, camta6 loss-of-function lines exhibited enhanced tolerance to HS relative to wild-type plants at the germination and seedling stages but not at later developmental stages. Qualitative promoter-reporter assays and quantitative gene transcription analyses indicate that CAMTA6 expression is repressed by HS in wild-type plants. Under control and HS conditions, we investigated the expression of various genes with promoters containing putative CAMTA-binding CGCG-box cis-elements and known to function in HSR. Notably, we found that in response to HS, several key HSR genes such as HEAT-SHOCK FACTOR-A1e (HSFA1e), HSFA2, HSFB1, HSFB2b, and DEHYDRATION-RESPONSIVE ELEMENT-BINDING 2A (DREB2A) are significantly up-regulated in camta6 knockout mutants in comparison to wild-type plants. In addition, we found that expression of ELONGATED HYPOCOTYL 5 (HY5) and HY5 HOMOLOGUE (HYH) were suppressed in camta6 mutants. Collectively, our study indicates that CAMTA6 functions as an important negative regulator of the HSR in Arabidopsis and highlights the expanding roles of CAMTAs in abiotic stress signalling pathways.

Phytopathogenic outer membrane vesicles (OMVs) can elicit protective immune responses and can be utilised for crop protection. Xanthomonas oryzae pv. oryzae (Xoo), a Gram-negative bacterium that causes Bacterial Blight of rice, releases OMVs. However, the role of Xoo-OMVs in the Xoo-rice interaction remains unexplored. We investigated the early signalling events underlying the plant's response to Xoo-OMV to gain insight into plant-pathogen interactions. We observed that Xoo-OMVs trigger a rapid, unique Ca2+ signal in rice that is essential for evoking an immune response. By imaging and proteomic analysis of the rice plasma membrane (PM), we found that this Ca2+ signal is required for Xoo-OMV-induced nanodomain enrichment and aggregation of defence response-associated proteins in rice PM. The specific protein assembly in the PM helps the plant defend itself against Xoo. In the absence of the early Ca2+ signal, rice cannot respond effectively to Xoo-OMVs, resulting in a compromised immune response. Collectively, our study shows that rice responds to Xoo-OMVs via Ca2+-signal-induced nanodomain assembly and protein reorganisation at the PM, preparing the plant for an imminent pathogen invasion.

High-temperature stress driven by global warming and phosphorus (P) deficiency resulting from the depletion of phosphate rock reserves pose serious threats to modern agriculture. However, our understanding of the combined effects of these stresses on plants remains limited. In this study, we employed a hydroponic culture system to investigate the physiological and transcriptomic responses of the model legume Lotus japonicus to simultaneous moderate high-temperature and low-P conditions. Both elevated temperature and low-P individually reduced shoot biomass. Under low-P conditions, shoot biomass remained significantly lower than in P sufficient plants. However, the relative reduction was smaller under elevated temperature than under favorable temperature. L. japonicus exhibited enhanced root-secreted acid phosphatase activity in under low-P alone and displayed an increased organic acid (e.g. malate and citrate) exudation rates under elevated temperature, irrespective of the P supply. Root transcriptome analysis identified up-regulation of the PEPC1 gene, encoding phosphoenolpyruvate carboxylase, suggesting its potential involvement in organic acid root exudation. Additionally, elevated temperatures promoted metal ion translocation and uptake, increased anthocyanin accumulation, and enhanced carbohydrate transport in shoots. Transcriptomic analysis further revealed that low-P stress altered heat shock protein expression, potentially associated with reduced ATP availability. These findings demonstrate that L. japonicus employs distinct and overlapping strategies to adapt to the combined stresses of elevated temperatures and P deficiency. These strategies include the adjustment of root exudation patterns, nutrient transport, and stress-responsive gene expression.

Plant vascular tissues consist of multiple cell types that together coordinate long-distance transport, signaling, and perform diverse metabolic functions. While extensive research uncovered key regulators of vascular patterning and function, the dissection of individual vascular cell types has been hindered by their inaccessibility in planta and the interdependence among the cell types. To overcome these limitations, in vitro transdifferentiation-based approaches have emerged as powerful experimental tools. Here, we provide a comprehensive review of current transdifferentiation strategies used to generate vascular cell types in Arabidopsis thaliana (Arabidopsis), discuss conceptual and technical limitations, and suggest future possibilities and necessity of transdifferentiation-based frameworks to dissect cell-type-specific regulatory programs within complex vascular tissues.

Sexual reproduction in land plants has diversified over more than 500 million years of evolution. Ferns occupy a key phylogenetic position among land plants because they possess a dominant sporophyte, as in seed plants, while retaining a free-living gametophyte that produces gametes through mitotic divisions, as in bryophytes. This combination of life cycle features provides a useful framework for understanding how reproductive and developmental programs have evolved across land plants. In this review, we summarize current knowledge of fern reproduction, with particular emphasis on the model fern Ceratopteris richardii and the related species C. thalictroides. We focus on four interconnected processes: (1) gametophyte development and the meristem, which functions as a signaling center required for prothallus formation and gametangium positioning; (2) sex determination by antheridiogen, including classical genetic models, hormonal regulation, and recent studies linking meristem identity to sexual fate; (3) gametangium development and gametogenesis, covering antheridium and archegonium formation as well as the differentiation of motile sperm and egg cells; and (4) fertilization and embryogenesis, including gamete interaction, zygotic polarity, and embryonic axis formation. Recent advances in genome sequencing, transformation, genome editing, live imaging, and transcriptomic analyses are beginning to connect the rich morphological literature on ferns with molecular mechanisms. These advances make Ceratopteris a key comparative system for uncovering how reproductive structures, stem cell functions, and embryogenic programs have been conserved, modified, and redeployed during land plant evolution.

The circadian clock is an endogenous time-keeping mechanism found in most eukaryotes that integrates external signals to synchronize biological processes with the environment. Circadian and diel rhythms across multiple regulatory layers help coordinate gene expression underlying plant processes such as metabolism, stress responses, and agronomic traits. In the context of a changing environment, a comprehensive understanding of how circadian-mediated regulatory mechanisms can contribute to plant resilient strategies continues to be a priority. In this mini review, we highlight the pleiotropic functions of the rice circadian clock in coordinating diverse processes.

Plasma membrane transporters play an essential role in plant development and all physiological processes. Their activity is tightly controlled by a variety of mechanisms, including the rate of transporter endocytosis. There is evidence that the local membrane environment, in particular the sterol content, influences transporter endocytosis. Here, we analyzed the sucrose transporters StSUT1 (potato) and AtSUC2 (Arabidopsis) transiently expressed in Nicotiana benthamiana epidermal cells. Using live-cell imaging of fluorescent protein fusions combined with extracellular quenching by Trypan Blue, we quantitatively distinguished plasma membrane-localized from internal transporter pools. Pharmacological perturbation of sterols and sphingolipids with methyl-β-cyclodextrin, fenpropimorph and myriocin reduced sucrose transporter abundance at the plasma membrane and decreased uptake of the fluorescent sucrose analogue esculin. In Arabidopsis leaves, methyl-β-cyclodextrin treatment reduced endogenous AtSUC2 protein levels, confirming sterol-dependent regulation in planta. Single-molecule localization microscopy of AtSUC2-reporter constructs revealed its enrichment in ∼50 nm plasma membrane domains, with the localization becoming more homogeneous upon sterol depletion. Together, our results demonstrate that plasma membrane lipid composition modulates sucrose transporter localization and abundance, with likely relevance for the regulation of phloem loading and other membrane proteins.

GAMETE EXPRESSED PROTEIN1 (GEX1) is a gamete nuclear membrane protein essential for nuclear fusion during sexual reproduction in Arabidopsis thaliana. GEX1 orthologs have been identified in several terrestrial plants. The liverwort Marchantia polymorpha, which uses motile gametes (spermatozoids) for fertilization, possesses a GEX1 homolog, M. polymorpha GEX1 (MpGEX1). In the present study, we investigated the function of MpGEX1 in M. polymorpha. MpGEX1 is expressed during both male and female gametogenesis. During male gametogenesis, MpGEX1 was detected in the nuclear envelope of spermatogenous cells and spermatids but not in mature spermatozoids. In female plants, MpGEX1 was detected in the nuclear envelope of mature egg cells. Mpgex1 mutant strains were not defective in egg or spermatozoid formation. However, Mpgex1 mutant female plants were defective in fertilization when crossed with wild-type spermatozoids. In fertilized egg cells, the male pronucleus, formed from the sperm nucleus, fuses with the female nucleus. However, fertilized Mpgex1 mutant egg cells lack an egg cell-derived nuclear envelope in their male pronuclei. The aberrant pronuclei were elongated or oval and remained separated from female nuclei. Fertilized Mpgex1 mutant egg cells do not undergo zygotic cell divisions. Female-derived mCitrine-MpGEX1 was transiently localized to the male pronucleus shortly after fertilization in females. These results demonstrate that maternally supplied MpGEX1 is essential for male pronucleus formation in M. polymorpha. Our findings reveal a novel role for the plant GEX1 ortholog in fertilization, suggesting the functional diversification of this conserved protein family during the evolution of plant fertilization mechanisms.

Photoperiod, the duration of the daily light phase, is a key seasonal signal that shapes vegetative growth in plants. While its role in controlling the transition to flowering has been extensively characterised, the mechanisms through which photoperiod modulates processes such as hypocotyl elongation, leaf expansion, root growth, and carbon allocation during the vegetative phase are only now coming into focus. Understanding these mechanisms is critical, as the vegetative phase establishes the architecture and resources needed for reproduction. Recent work has revealed that plants possess distinct daylength-measurement mechanisms for flowering and vegetative growth, with twilight-length sensing as a complementary mechanism. The circadian clock translates these signals into daily growth rhythms by restricting growth-promoting factors to defined time windows that widen or narrow depending on daylength, contributing to different growth outcomes between short and long photoperiods. Photoperiod further shapes the daily carbon budget, with opposing energy-sensing pathways dynamically adjusting growth to match carbon availability across the day-night cycle. In this review, we examine how photoperiod regulates vegetative growth in Arabidopsis thaliana, from daylength-measurement systems through clock-controlled transcriptional cascades that drive organ elongation and competitive shade avoidance responses, to carbon economy and stress tolerance. We consider how photoperiod modulates responses to temperature extremes, drought, and salinity, and survey emerging evidence from crops and trees. Finally, we discuss chronoculture, propose experimental recommendations, and highlight outstanding questions for the field.

Legumes have developed a symbiotic relationship with soil bacteria known collectively as rhizobia, which when present in root nodules fix atmospheric nitrogen into a form usable by the plant. The functioning unit of symbiotic nitrogen fixation is referred to as the symbiosome and the membrane separating symbionts acts to regulate the metabolite exchange within the root nodule cells. The efflux of fixed nitrogen from the symbiosome is through an unidentified non-selective cation channel, which is inwardly rectified by cytosolic Mg2+ and has a preference for ammonium. Expression of Nodulin 26 (NOD26), a major component of the soybean symbiosome membrane, in Xenopus laevis oocytes reveals monovalent cation-induced currents. These currents display time- and voltage dependent characteristic similar to those previously observed in patch-clamp studies of the symbiosome membrane and exhibited an apparent monovalent cation preference of NH4+ > methylammonium (MeA+) ≥ K+ > Na+ ≥ C5H15NO+. GmNOD26-mediated currents in X. laevis oocytes were inhibited by divalent cations and inhibited by phosphorylation at serine 262. Together, these findings demonstrate that GmNOD26 functions as a non-selective cation channel capable of facilitating ammonium transport in nitrogen-fixing soybean nodules. Furthermore, pronounced inhibition of GmNOD26 by divalent cations provides as likely explanation for why monovalent cation-induced currents have not previously been characterised in GmNOD26-expressing X. laevis oocytes.