Molecular Plant

The red/far-red sensing photoreceptor phytochrome B (phyB) governs multifaceted plant development and responses to light and temperature stimuli. PhyB photoconversion between red-absorbing, inactive Pr and far red-absorbing, active Pfr states, imparted by its covalently bound bilin chromophore, enables rapid switching and plasticity of phyB signaling activities. The phyBY276H variant (YHB) is photochemically inert but adopts a constitutively active Pfr-like structure regardless of light conditions, which becomes a versatile model to dissect phyB signaling mechanisms. Here, we conducted a large-scale EMS mutagenesis screen on YHB-expressing transgenic lines, mining intragenic suppressor mutations that would unveil critical residues for phyB structure-function relationships. Comparative analyses of 26 nonsense variants suggested modular organization of phyB overall structure and dispensability of the C-terminal HKRD domain for phyB signaling. Amongst fourteen novel and nine known loss-of-function missense variants identified herein, G284E was of particular interest for its fully suppressed constitutive activity in darkness and its restored photochemistry and light responsiveness. The G284E mutation was further tested to also nullify another constitutively active phyBY303V allele by eliminating chromophore attachment. P309L was the sole variant identified which fully suppressed YHB in both dark and light conditions. C402Y profoundly elicited YHB protein instability. Three variants G118R, C402Y and G538D markedly reduced chromophorylation levels of YHB. Although the chromophore binding site variant C357Y was a strong loss-of-function allele, it retained residual signaling activity with respect to PIF3 protein turnover in dark-grown seedlings, presumably due to its ability to noncovalently bind chromophore. Two tandem prolines (P799, P800) proved critical to YHB structural integrity/stability as well as signaling activity. In summary, these diverse variants shed new insights into multiple levels by which the YHB (and thereby phyB) signaling is initiated, tuned, and disseminated.

Salicylic acid (SA), a central hormone in plant immunity, is biosynthesized via a recently elucidated phenylalanine-derived pathway in most seed plants. This pathway requires benzyl alcohol as a key substrate for the formation of the SA precursor benzyl benzoate. However, how benzyl alcohol is produced in plants was unclear. Here, we identify a two-step conversion of benzoyl-CoA to benzyl alcohol via benzaldehyde in Nicotiana (N.) benthamiana. From a forward genetic screen for SA-deficient mutants, the and {beta} subunits of heterodimeric benzaldehyde synthase (BalS) involved in the conversion of benzoyl-CoA to benzaldehyde were found to be required for SA biosynthesis in N. benthamiana. Further reverse genetic analysis revealed that the NADPH-dependent benzaldehyde reductase (BalR1) acts downstream of BalS to convert benzaldehyde to benzyl alcohol. Interestingly, OsBalR1, but not OsBalS or OsBalS{beta}, is required for maintaining high basal SA levels in rice, suggesting the presence of redundant benzoyl-CoA-reducing activities or alternative biosynthesis routes for benzyl alcohol production. Together, this work defines the missing enzymatic steps in phenylalanine-derived SA pathway and provides insights into the evolutionary diversification of SA production strategies in plants.

Carotenoid accumulation underlies fruit color and nutritional quality in squash (Cucurbita pepo). One pair of dominant genes, B and L-2, have been long known to interact epistatically, substantially boosting carotenoid accumulation and producing intensely orange-fleshed fruit. However, their molecular identities and regulatory mechanism are unknown. Here, we show that B encodes a truncated H subunit of magnesium chelatase (CpCHLHB) and L-2 encodes a homolog of Arabidopsis Pseudo-Response Regulator 2 (CpAPRR2-A). Significantly, expression of phytoene synthase (CpPSY-A), which encodes the major rate-limiting enzyme in carotenoid biosynthesis, was dramatically upregulated in fruit of B/B L-2/L-2 plants compared with b/b L-2/L-2 or B/B l-2/l-2, showing that the B and L-2 interaction affects CpPSY-A transcription. A similar upregulation was also observed in Arabidopsis gun5 L-2 transgenic plants, where gun5 is a genetic mimic of the C. pepo B gene. The wild-type CpCHLHb physically interacted with CpAPRR2-A, attenuating the CpAPRR2-A-mediated activation of CpPSY-A. In contrast, the truncated CpCHLHB lost its ability to interact with CpAPRR2-A, enabling CpAPRR2-A to activate CpPSY-A and produce intensely orange fruit. These findings uncover the mechanism underlying the epistatic interaction through which B and L-2 act synergistically to boost carotenoid production, offering novel mechanistic insights and key targets for improving crop quality. One-sentence summarySynergistic epistasis between B and L-2 arises from loss of interaction between their encoded proteins, resulting in dramatically upregulating the key rate-limiting enzyme in carotenoid biosynthesis pathway to produce intensely orange-fleshed fruit in squash.

In nature, plants are subjected to multiple environmental stress factors simultaneously or sequentially. Recent studies revealed that when three or more stress factors impact a plant simultaneously (termed multifactorial stress combination; MFSC), plant survival declines, even if the intensity of each individual stress involved in the MFSC is low. We previously identified RAP2.3 as a key transcription factor (TF) required for Arabidopsis thaliana survival, specifically under a MFSC of salt+excess light+heat stress (i.e., S+EL+HS). Here we report that RAP2.3 is required for the expression of SIGMA3, a nuclear-encoded factor that directs plastid RNA polymerase to specific plastid promoters, and MYB51, a key stress response TF involved in glucosinolate metabolism and oxidative stress responses, specifically during a MFSC of S+EL+HS. Like rap2.3 mutants, myb51 and sig3 mutants display significantly low survival rate specifically under the MFSC of S+EL+HS. Based on MYB51 gene regulatory network analysis and characterization of jasmonic acid (JA) mutants, we further reveal that suppression of JA signaling could play an important role in promoting plant survival under conditions of S+EL+HS. Our findings uncover an additional layer of the response of plants to MFSC, as well as identify potential targets for breeding crops with enhanced tolerance to climate change.

Chromosome number variation and structural reorganization are key drivers of plant evolution, yet their genomic basis remains unclear due to incomplete representation of repetitive regions in existing assemblies. The Linum genus exhibits exceptional karyotypic diversity (n = 7-43), providing a powerful system to investigate chromosome evolution. Here, we generated near telomere-to-telomere (T2T) genome assemblies for four species, including cultivated flax (L. usitatissimum cv. CDC Bethune; n = 15), its wild progenitor (L. bienne; n = 15), and two related species (L. decumbens and L. grandiflorum; n = 8). Together with published genomes of L. lewisii (n = 9) and L. tenue (n = 10), these enabled reconstruction of chromosome evolution across six lineages. Phylogenomic analyses revealed a shared ancestral whole-genome duplication (WGD) associated with the n = 9 karyotype, followed by lineage-specific WGDs and divergent diploidization. The transition from n = 8 to the derived n = 15 flax lineage not only occurred without chromosome length expansion, but also with genome size reduction, indicating extensive internal restructuring. Comparative analyses showed that this restructuring was associated with lineage-specific expansion of a single DNA transposon family (TE_00003234; hAT), which is highly enriched in expansive pericentromeric regions that are characterized by low gene density and nucleotide diversity, suppressed recombination, segregation distortion, and extensive synteny disruption, unlike the LTR retrotransposon-rich pericentromeres typical of most plant genomes. These findings support a model in which lineage-specific DNA transposon expansion is associated with remodeling of pericentromeric architecture and large-scale chromosome restructuring following polyploidization.

Iron (Fe) is an essential micronutrient for plant growth, and the hormone auxin is a key regulator of developmental processes, including root gravitropism. Here, we investigated the molecular mechanisms underlying the crosstalk between iron nutrition and auxin-mediated root growth in Arabidopsis thaliana. Phenotypic analysis revealed that iron deficiency strongly shaped root system architecture and root gravitropism, and these phenotypes were exacerbated in the iron uptake mutant irt1-1. Genetic analysis revealed that iron deficiency did not aggravate the gravitropic defect of the pin2 mutant, eir1-4, suggesting that iron availability modulates root gravitropism through a PIN2-dependent pathway. Further transcriptomic analysis confirmed that iron deficiency significantly altered the expression of numerous genes related to the auxin pathway, providing molecular evidence for the observed physiological connection. Collectively, this study revealed that iron availability regulates root gravitropic growth by modulating PIN-mediated auxin transport and distribution, providing insights into how plants integrate nutritional cues with developmental programs. Graphical abstract A brief descriptionIron modulates auxin transport and root tip distribution by regulating PIN2 protein, thereby mediating root gravitropism in Arabidopsis. Public summaryO_LIIron nutrition specifically regulates root gravitropism and architecture in Arabidopsis. C_LIO_LIIron deficiency disrupts local auxin homeostasis in root tips and impairs asymmetric distribution in response to gravity. C_LIO_LIIron deficiency stress significantly reduces the abundance of PIN2 protein in root tip cells and disrupts its polar localization pattern on the plasma membrane, thereby precisely modulating polar auxin transport by interfering with the vesicle trafficking and recycling efficiency of PIN2. C_LIO_LIRNA-seq results showed that iron deficiency induced differential expression of multiple auxin-related genes, indicating that iron nutrition affects root development through the auxin pathway. C_LI

Achieving high-throughput and precise phenotypic quantification and imaging modalities of stomatal and epidermal cells across diverse species remains a primary bottleneck in elucidating the mechanisms of stomatal dynamics, epidermal patterning, and environmental adaptation of plants. Here, we developed EpiReasoner, an artificial intelligence framework comprising a vision module, EpiVision, and a knowledge-based reasoning module, EpiBrain, for the quantitative phenotypic analysis and domain-specific knowledge reasoning of stomatal complexes and pavement cells in plants. Operating across bright-field, scanning electron microscopy, and differential interference contrast modalities, EpiVision achieves precise instance segmentation in various monocotyledonous, dicotyledonous, and fern species. Its performance significantly surpasses current state-of-the-art models. Moreover, we defined 23 quantitative indices describing stomatal cell morphology and spatial distribution. For domain-specific tasks such as phenotype prediction, genotype deduction, and molecular mechanism reasoning, EpiBrain demonstrates a human preference rate significantly higher than that of general-purpose large language models, including GPT-5 and Claude Sonnet 4. The application of EpiReasoner to phenotypic data of stomatal density derived from a tomato natural population of 170 accessions successfully identified a major quantitative trait locus on chromosome 8. The candidate gene, SKP1-interaction partner 19L (SKIP19L), encoding an F-box family protein, exhibited severe allele frequency drift during tomato domestication, which is highly consistent with the adaptive trend of reduced stomatal density under artificial selection. EpiReasoner provides a novel paradigm that unifies visual phenomics and knowledge-driven reasoning for the biology of stomata and pavement cells, thereby significantly accelerating scientific discovery in plant science.

Polyploid genome assembly presents unique challenges due to extensive heterozygosity and complex haplotype structure. We report a haplotype-resolved, chromosome-scale assembly of Regen-SY27x, a genotype of autotetraploid alfalfa (Medicago sativa), which is widely used for genetic modification because of its excellent regenerative capacity in tissue culture. Using PacBio HiFi long reads, Omni-C scaffolding, and linkage map guided phasing, we generated a 3.2 GB assembly comprising four haplotypes with high contiguity and completeness. Kmer-based validation confirmed accurate haplotype separation, while linkage map integration and dotplot analysis identified and corrected chimeric scaffolds. Gene annotation yielded 221,688 protein-coding genes, with more than 99% assigned to pseudochromosomes. Repetitive elements accounted for 62.7% of the genome, dominated by long terminal repeat retrotransposons and a high fraction of Helitrons. The spatial enrichment of Helitrons within gene-dense distal chromosome arms underscores their pivotal role as key drivers of genomic innovation and gene family expansion. We identified 3,696 nucleotide-binding leucine-rich repeat R genes, with Toll/interleukin-1 receptor-like and Rx-type subclasses forming large tandem clusters across haplotypes. Comparative analyses revealed strong macrosyntenic conservation among Regen-SY27x and the publicly available Chinese alfalfa genomes but extensive structural variation both within Regen-SY27x haplotypes and between Regen-SY27x and the Chinese genotypes with tens of thousands of duplications, inversions, and translocations detected. These results demonstrate that a single autotetraploid individual captures extensive structural diversity, but individuals from different populations vary greatly. The Regen-SY27x assembly provides a foundational genomic resource for investigating polyploid genome evolution and identifying genetic variation relevant to biological and agronomic improvement in alfalfa. Article SummaryThis study presents the first chromosome-scale, haplotype-resolved genome assembly of the US alfalfa germplasm, Regen-SY27x, a key alfalfa genotype used widely for genetic engineering. We integrated HiFi long reads, Omni-CTM scaffolding, and linkage map-guided phasing to reconstruct all four haplotypes of this complex autotetraploid. Our results identified 221,688 protein-coding genes and reveal immense intra-individual structural variations dominated by small duplications. This high-quality reference serves as a foundational tool for the alfalfa community, enabling researchers to link complex structural diversity with agronomic traits and further enhance the biotechnological potential of this essential forage crop.

In tomato plants, the potassium (K) transporter SlHAK5 is integral to root K uptake and overall plant fertility. Under K deficiency, SlHAK5 expression is induced in roots and the encoded transporter is activated via the Ca{superscript 2}-sensing CIPK/CBL complex SlCIPK23/SlCBL1-9. In Arabidopsis, multiple CIPK/CBL complexes can activate AtHAK5, providing alternative regulatory pathways that enhance K uptake. However, the architecture of CIPK/CBL signaling networks has diverged among plant species, necessitating species-specific identification of novel regulatory components. Accordingly, we screened additional tomato CIPK proteins for their capacity to modulate SlHAK5 activity in yeast. SlCIPK15 and SlCIPK26 emerged as potent activators of SlHAK5, acting in concert with SlCBL9. Functional characterization of slcipk15 and slcipk26 mutants revealed that neither contributed significantly to SlHAK5-mediated K uptake in roots. Conversely, both mutants exhibited impaired pollen tube elongation, correlating with reduced K content in pollen relative to wild type. Notably, slcipk26 mutants displayed more severe pollen defects, phenocopying the slhak5 mutant. Further analyses demonstrated that slcipk26 plants suffered compromised seed set and pistil function, paralleling the reproductive deficiencies observed in slhak5 mutants. These findings implicate SlCIPK26 as the principal regulator of SlHAK5 in reproductive tissues. Collectively, our data underscore the role of CIPK paralogs in orchestrating tissue-specific regulation of target proteins, thereby enabling fine-tuned modulation of K transport essential for both vegetative and reproductive development.

Grain legumes such as pea (Pisum sativum L.) accumulate large amounts of seed storage proteins without nitrogen fertilization due to their symbiosis with nitrogen-fixing bacteria, making them a key source of plant-based proteins. Seed growth and the accumulation of seed storage proteins are tightly regulated by complex gene networks; however, the mechanisms governing these processes in pea remain poorly understood. In this study, we generated a comprehensive seed expression atlas covering six developmental stages in pea (cv Cameor), including the key transition stage from embryogenesis to early seed filling, providing a detailed temporal resolution of transcriptional dynamics throughout seed development in this species. Co-expression network analysis highlighted several candidate transcription factors potentially involved in the transition towards seed filling. Among them, we characterized the seed-specific NF-YB transcription factor PsLEC1-like (PsL1L), the major LEC1-type factor expressed during early pea seed development. Functional analyses using TILLING mutants demonstrated that loss of PsL1L function reduces seed size and seed nitrogen content and impairs early embryo growth from the end of embryogenesis. Finally, we show that the expression of the B3-domain transcription factor PsFUS3, but not that of PsLEC2 or PsABI3, is reduced in the loss-of-function l1l mutant, suggesting that PsL1L acts upstream of PsFUS3 to control seed size.

C4 photosynthesis improves light, water and nitrogen-use efficiencies and can raise yield by 50% compared with the ancestral C3 pathway. Engineering C4 traits into C3 crops could substantially boost food production but requires coordinated modifications to leaf anatomy and cell-specific photosynthetic function. For example, C4 leaves contain more numerous, shorter bundle sheath cells that are photosynthetically active. In searching for transcriptional regulators of bundle sheath development in C3 rice, we unexpectedly found OSA3, a plasma membrane H+-ATPase that is expressed in bundle sheath cells as they elongate, and when knocked out reduces their length due to reduced apoplastic acidification. Bundle sheath cell number and chloroplast occupancy are increased. Thus, switching between C3 and C4 bundle sheath identity is controlled by acid growth, and OSA3 represents a simple tool for C4 engineering.

Senescence enables plants to remobilize and recycle nutrients from aging organs to support growth, reproduction, and survival. In annual crops like maize, nitrogen remobilization from leaves to grain is incomplete, with 30-50% of nitrogen stranded in aboveground tissues and subject to environmental loss. Mitigating nitrogen loss in annual crops could be achieved by leveraging the physiological strategies of perennial grasses, which remobilize nitrogen and other nutrients into underground organs at the end of the growing season, thereby preventing environmental leakage. To uncover the molecular basis of perennial nitrogen recycling to underground organs, we built a transcriptomic atlas from field-grown plants, comprising 2,685 RNA-seq libraries from 14 grass species within the Panicoideae (Poaceae), utilizing maize and sorghum as annual references for comparative analyses. The atlas spans leaves, roots, stalks, and rhizomes across two seasons, from mid-growing season to senescence. Using a photosynthetic index to align the leafs transition from nitrogen sink to source across species, co-expression network analysis revealed that the subnetworks driving leaf nitrogen recycling are preserved across annuals and perennials. However, we discovered that the subnetworks associated with underground sink establishment, specifically those associated with seed-like dormancy and desiccation tolerance pathways, have diverged among annual crop accessions. Our work identifies conserved gene candidates and networks that could be used to reintroduce perennial-like nutrient recycling into annual crops to enhance long-term nutrient retention in the field.

Plants continuously develop shoot branches derived from axillary meristems. In rice (Oryza sativa), TILLERS ABSENT1 (TAB1), an ortholog of Arabidopsis WUSCHEL, plays an essential role in axillary meristem formation by promoting stem cell proliferation. Although several genes associated with TAB1 function have been identified, the molecular mechanisms underlying stem cell proliferation during axillary meristem formation remain poorly understood. Here we identify ABERRANT SPIKELET AND PANICLE1 (ASP1), a TOPLESS-like transcriptional corepressor, as a novel regulator of axillary meristem formation, and investigate downstream mechanisms regulated by TAB1 and ASP1. In asp1, the stem cell region was expanded, indicating that ASP1 negatively regulates stem cell proliferation. Notably, WOX4, a paralog of TAB1, was precociously expressed in asp1, possibly in association with expansion of the stem cell region. Genetic analysis further revealed that asp1 mutation rescued the loss of axillary meristems in tab1. Transcriptome analysis showed that several type-A RESPONSE REGULATOR (OsRR) genes, encoding negative regulators of cytokinin signaling, were upregulated in tab1 relative to wild type, asp1, and the tab1 asp1 double mutant. Consistently, fluorescence of the synthetic cytokinin reporter was absent during axillary meristem formation in tab1 but was detected in wild type and tab1 asp1. Moreover, overexpression of OsRR10 inhibited axillary meristem formation, phenocopying tab1. Collectively, these findings suggest that TAB1 activates cytokinin signaling by repressing type-A OsRR expression, whereas ASP1 negatively regulates cytokinin signaling by promoting the expression of these genes. Thus, rescue of the tab1 phenotype by asp1 mutation probably reflects restoration of cytokinin signaling.

Lipopolysaccharides (LPSs) are pathogen-associated molecular patterns (PAMPs) of Gram-negative pathogenic bacteria recognized by plants, triggering typical pattern-triggered immunity (PTI) responses. However, a LPS sensing receptor for the recognition of plants remains largely undefined. A plant receptor for lipopolysaccharide (LPS) has not yet been identified. Here, we identify a plant protein, OsML1, with homologies to animal MD-2, which is capable of binding LPS. Furthermore, it may act as a molecular chaperone to assist CK1 in perceiving LPS signals. Our results show that OsML1 functions as an LPS-binding protein recognizing LPS and participates in downstream rice immune response activation. Structural modeling and sequence analysis revealed that OsML1 contains both a typical ML domain and a conserved three-dimensional {beta}-barrel structure as mammalian MD-2 proteins. Microscale thermophoresis assays confirmed that OsML1 binds LPS with high affinity. Functional analyses further demonstrated that OsML1 knockout plants show reduced resistance to the rice bacterial blight pathogen, as well as attenuated ROS bursts upon LPS treatments, whereas overexpression plants show enhanced immune responses. Metabolomic profiling indicated significant metabolic changes in OsML1 knockout plants, particularly in immune-related pathways involving lipids, amino acids, and antimicrobial compounds. OsML1 is consequently a structurally conserved and functional LPS-binding protein linking lipid metabolism, LPS perception, immune activation, and metabolic regulation. Phylogenetic and structural analyses revealed that OsML1 likely arose from a duplication of OsML2, forming an independently functional subgroup within the PITP family. Our study identifies OsML1 as a LPS recognition factor involved in LPS sensing and downstream ROS bursts activation, callose deposition, and broad-spectrum gene expression of resistance. These findings expand our knowledge of bacterial LPS perception and immune regulation in plants, offering novel targets and strategies for disease-resistant breeding.

The COP1/SPA ubiquitin ligase is a key repressor of photomorphogenesis that is inactivated by photoreceptors to initiate light signalling. The four SPA proteins (SPA1-SPA4) confer functional specificity to COP1 during plant growth, yet the underlying molecular mechanisms remain unclear. Here, we used a domain-swap approach in transgenic seedlings to address the functional divergence of SPA2 and SPA3. We show that the respective N-terminal kinase domain determines the contrasting protein stabilities of SPA2 and SPA3 in light-grown seedlings. The instability of SPA2 correlates with a specific ability of the SPA2 N-terminal domain to bind phytochrome A in the light, suggesting that phytochrome A promotes the CUL4DET1/COP1-dependent degradation of SPA2 but not of SPA3. We uncover that the coiled-coiled and WD-repeat domains of SPA2 and SPA3 substantially differ in their activity in repression of photomorphogenesis, with those of SPA2 being more active repressors than those of SPA3. Thus, SPA2 combines a potent repressor activity with light-induced instability. We conclude that the evolution of SPA2 instability in response to light counterbalances its inherent strong repressor activity, thereby allowing seedling etiolation in darkness followed by rapid reduction in COP1 activity through SPA2 degradation upon light-exposure as seedlings emerge from soil to initiate photosynthetic growth. HighlightThe repressor of light signaling SPA2 combines a phytochrome A-interacting instability domain with a potent repressor domain to allow greatly contrasting activities of COP1 in skoto- and photomorphogenesis.

Polyamines (PAs) are ubiquitous metabolites that, despite their simple structure, profoundly influence plant growth, development, and stress adaptation. Their cellular levels are largely determined by arginine decarboxylase (ADC), a key rate-limiting enzyme in their biosynthesis. We previously identified a [~]50 bp GC-rich sequence in the 5' untranslated region (UTR) of plant ADC genes, termed the ADC-box, that is conserved across land plants. Transient reporter assays in tomato, in which ADC upstream regions were decoupled from their native coding sequences and fused to reporter genes, suggested that this element represses translation. However, its function in the native genomic context and its impact on PA homeostasis remain unclear. Here, we combined CRISPR-Cas9 genome editing, metabolite profiling, enzymatic assays, and RNA structure probing to define ADC-box function in tomato and in the seedless land plant Marchantia polymorpha, which retains a conserved [~]20 bp core region. Mutation of the M. polymorpha ADC-box increased ADC activity and altered PA levels, indicating that the ADC-box functions as a conserved translational repressor. In tomato, disruption of the ADC-boxes in SlADC1 and SlADC2 increased ADC activity, demonstrating that the ADC-box acts as a translational repressor in its native context. These ehects were most pronounced under cold stress, when ADC transcript levels increase, suggesting that the ADC-box buhers stress-induced translation. Metabolically, ADC-box disruption led to agmatine accumulation and alterations in upstream intermediates, while downstream PA pools remained largely unchanged. SHAPE analysis revealed that the tomato ADC-box folds into a three-stem RNA structure, with a central stem representing the major inhibitory module. ADC-box mutants displayed altered plant-microbe interactions, with enhanced resistance to Pseudomonas syringae and Tobacco rattle virus, but increased susceptibility to Ralstonia solanacearum and Tomato yellow leaf curl virus. Together, these findings establish the ADC-box as an evolutionarily conserved cis-regulatory element that stabilizes PA homeostasis and modulates plant-microbe interactions.

Mildew Locus O (MLO) genes, initially identified as powdery mildew susceptibility factors, are increasingly recognized as multifunctional regulators implicated in diverse processes, including plant reproduction, root thigmotropism, and interactions with beneficial microbes. Recent evidence shows that MLO proteins can act as Ca2+-permeable channels in response to Rapid Alkalinization Factors (RALF) peptides in reproductive cells, pointing to broader roles in Ca2+-mediated signalling. In this study, we investigate the symbiotic clade IV member LjMLO4 in the model legume Lotus japonicus, focusing on its role in root development and responsiveness to LjRALF34 peptides. We show that LjMLO4 expression is strongly induced in root cells colonized by arbuscular mycorrhizal (AM) fungi, yet loss-of-function mutants exhibit only subtle AM-associated phenotypes. Instead, we uncover a previously uncharacterized function of LjMLO4 as a regulator of primary root growth and lateral root formation, acting even in the absence of AM fungal colonization and in a Ca2+-dependent manner. Heterologous expression in E. coli confirms that LjMLO4 facilitates Ca2+ transport, while genetic and physiological assays demonstrate its contribution to LjRALF34-triggered root growth responses and Ca2+ signalling. Together, these findings identify LjMLO4 as a molecular hub between peptide signalling, Ca2+ transport and root system architecture, highlighting how MLO proteins integrate developmental, nutritional and symbiotic cues.

Rice blast, caused by the fungus Magnaporthe oryzae, poses a severe threat to global rice production. Although numerous M. oryzae effectors have been identified, the molecular mechanisms remain poorly understood. The effector AvrPii triggers ETI upon recognition by NLR receptor Pii and has been shown to target host exocytosis and oxidative metabolic pathways to suppress immunity. Here, we identify AVIN8 (AvrPii-interacting protein 8) as a new target of AvrPii. AVIN8 is an ankyrin-repeat (ANK) protein localized to the plasma membrane and nuclear envelope, with sequence similarity to known ANK calcium channels. AVIN8 expression is induced during early M. oryzae infection. Overexpression of AVIN8 enhances blast resistance, whereas its silencing increases susceptibility. AVIN8 promotes chitin-triggered ROS production in a Ca2+-dependent manner. In contrast, AvrPii impairs Ca2+ influx and chitin-triggered ROS burst. Our findings reveal a virulence strategy in which M. oryzae effector AvrPii hijacks the Ca2+ influx-associated ANK protein AVIN8 to suppress early calcium and PTI signaling in rice.

Transcriptional regulation is one of the fundamental approaches for young plants to cope with environmental fluctuations and maintain active development. The transposable element (TE) subclass long terminal-repeat retrotransposons (LTR-RTs) can act as additional regulators for genes through enhancer and promoter activity, but their promoters, transcription initiation, and contributions during maize development remain uncharacterized. Here, we developed IsoClassifier to resolve the transcription start site (TSS) and RNA isoforms of LTR-RTs based on long-read transcriptomics, delineating LTR U3 regions as the native promoter and enhancer of LTR-RTs. We reveal conserved motifs associated with core promoter activity in transcribed LTR-RTs that are highly comparable to gene promoters. Further, we found that LTR-RT transcription in maize was dominated by spliced, long non-coding RNA. Finally, a genome-wide coexpression analysis revealed that LTR-RTs are transcribed as hub-like elements in coexpression networks, suggesting important roles in gene regulation. We conclude that LTR-RTs have similar promoter compositions to gene promoters and likely share similar transcription regulation programs.

Photomorphogenesis, the light-driven development of seedlings, is governed by a complex network of transcription factors and circadian regulators. While the TIMING OF CAB EXPRESSION 1 (TOC1) is known to link circadian rhythms with light-responsive growth, the mechanisms fine-tuning its activity remain poorly understood. Here, we identify PHOSPHATE 1 HOMOLOG 2 (PHO1;H2) as a novel negative regulator of seedling photomorphogenesis in Arabidopsis. Loss-of-function pho1;h2 mutants exhibit hypersensitivity to light, characterized by markedly shorter hypocotyls and increased photopigment accumulation, whereas overexpression lines display reduced photomorphogenic response. We demonstrate that the N-terminal SPX domain of PHO1;H2 is both necessary and sufficient to repress seedling photomorphogenic growth. Mechanistically, in vitro and in vivo interaction assays reveal that the SPX domain physically binds and sequesters TOC1, inhibiting its regulatory function. This PHO1;H2-mediated sequestration of TOC1 alleviates the repression of PHYTOCHROME INTERACTING FACTOR 4 (PIF4), thereby promoting the expression of downstream genes involved in cell elongation and hormone signaling. Collectively, our findings reveal a competitive binding mechanism by which PHO1;H2 modulates the TOC1-PIF4 signaling axis, providing a crucial checkpoint for seedling growth in dynamic light environments.