Journal of Integrative Plant Biology

Canonical ribonucleases such as Argonaute (AGO) participate in RNA-based gene regulatory processes. They are important for small RNA (sRNA) association and the formation of RNA-induced RNA silencing complexes. Potato (Solanum tuberosum) has 14 AGO-encoding genes, including the Solanum-specific StAGO15, which is activated upon infection by filamentous pathogens. The resistant phenotype conferred by ectopic expression of StAGO15 was abolished in plants in which a 21-nt sequence in the PAZ-encoding region of StAGO15 was targeted using an artificial miRNA. We then analyzed the sRNAs associated with StAGO15 in Phytophthora infestans leaf infection. Small RNAs were coimmunoprecipitated, subjected to deep sequencing, and analyzed against the P. infestans and potato genomes, in which cleavage sites were predicted by applying the R package, smartPARE. The most notable difference between pathogen- and water-inoculated StAGO15 samples was observed in the transposon (TE) category. Here, two categories of nonautonomous TE dominated: the miniature inverted-repeat transposable elements (MITEs) and short interspersed nuclear elements. The StAGO15-associated sRNAs bind and target complementary endogenous potato RNAs. One example is siRNA122, which acts on a cleavage site in the resistance gene PGSC0003DMG400015681 (StRES1). siRNA122 is derived from a MITE; upon deletion of this source sequence, resistance is restored. Our findings provide new insights into the plant immune surveillance network and demonstrate that deleting a 21 nt RNA sequence from a MITE transposon released a defense response, against two important filamentous pathogens.

Plants perceive pathogenic threats through receptors localized at the plasma membrane and inside cells to initiate pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), respectively. PTI and ETI pathways are interconnected and partially converge on the EDS1-PAD4-ADR1 branch which stimulates anti-pathogen defence. Besides these canonical immune pathways, proteolysis plays a pivotal role in immunity by regulating the abundance and activities of diverse components. We previously identified Arabidopsis papain-like cysteine protease RD19 as a PAD4 interactor and showed that RD19 promotes resistance to host-adapted pathogens. Here, we investigated the contribution of RD19 to bacterial flg22 and nlp20 elicited PTI responses which exhibit, respectively, partial and high PAD4 dependence. We show that although PAD4 and RD19 both promote PTI, they have separate functions. In resting (non-triggered) and nlp20-triggered tissues, RD19 limits RBOHD protein accumulation and consequently ROS production. Using N-terminomic enrichment and protease cleavage site identification methods we identify candidate RD19 substrates in resting and nlp20-responding tissues. Our analysis shows that two immune-related aspartyl proteases AED3 and AED2/ASPG1 are likely direct RD19 targets. This study reveals a role of RD19 in ROS regulation and advances our understanding of proteolysis and dynamic RD19 substrate targeting in plant immunity.

Aphids deplete their host plants of amino acids and other essential nutrients by feeding on phloem sap. Autophagy, a conserved process across eukaryotes, maintains cellular homeostasis during starvation and environmental stress by recycling damaged or excess cellular components. Here, we identified and characterized a previously uninvestigated aphid salivary protein, termed Aphid Autophagy Related Factor 1 (AAF1), that is secreted into plant cells and manipulates plant physiology in a manner that benefits the aphid. We show that AAF1 interacts with four known or putative host autophagy related proteins: RABD1 and RABD2a (plant homologs of Ypt1/RAB1), ATG8, a ubiquitin like protein required for autophagosome formation, and PI3PT, a previously uncharacterized ER localized putative lipid binding or lipid transfer protein annotated as a phosphoinositide 3 phosphate transfer protein. We further demonstrate that the RAB binding domain of AAF1 and its interaction with RABD2a may be specifically required for AAF1 function. In addition, we identified unique, highly mobile compartments containing interacting AAF1 and RABD2a that are associated with the endoplasmic reticulum (ER) and dynamically traffic with autophagosomes. We propose that these compartments may be derived from endosomes or endosome to ER contact sites (EERCSs) and serve as platforms for autophagosome trafficking and/or biogenesis. We further propose that AAF1, RABD2a, and PI3PT function together to regulate the formation or function of these compartments, thereby supporting autophagosome formation and trafficking. Finally, we show that stable expression of AAF1 in plants enhances growth and biomass in an autophagy dependent manner.

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.

The Unfolded Protein Response (UPR) maintains endoplasmic reticulum (ER) homeostasis during proteotoxic stress while also contributing to plant immunity. However, the functional relationship between UPR signaling, pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and salicylic acid (SA) remains poorly understood. Here, we investigated this interplay using Arabidopsis mutants impaired in the two major UPR branches. Mutants defective in the INOSITOL-REQUIRING ENZYME 1 (IRE1)-bZIP60 branch displayed enhanced resistance to Pseudomonas syringae pv. tomato DC3000 and to the type III secretion-deficient strain hrcC-, indicating potentiation of PTI. Consistently, the ire1a ire1b double mutant exhibited increased flg22-induced callose deposition, whereas reactive oxygen species production was unaffected. Despite enhanced basal resistance, all UPR mutants retained full responsiveness to benzothiadiazole, demonstrating that downstream SA signaling remains functional independently of UPR integrity. Conversely, acute ER stress induced by tunicamycin promoted SA accumulation and extensive leaf necrosis. Reverse genetics revealed that SA-deficient mutants were largely protected from ER stress-induced cell death, whereas mutants over-accumulating SA exhibited enhanced susceptibility, demonstrating that endogenous SA is required for the full execution of this response. Analysis of an independent transcriptomic dataset further showed that loss of IRE1 de-represses genes involved in SA biosynthesis, PTI and defense under ER stress. Together, our results identify IRE1 as a key regulator coordinating ER homeostasis with immune signaling by limiting SA-dependent defense activation during ER stress, while simultaneously preventing excessive cell death.

- The ascomycete fungal pathogen Blumeria hordei (Bh) is predicted to secrete over 530 effector proteins to infect barley and cause powdery mildew disease, yet their individual and collective functions are largely unexplored. We used yeast two-hybrid next-generation interaction screening (Y2H-NGIS) to probe the host targets of 48 Bh candidate secreted effectors (CSEPs), including multiple AVRA effectors that are recognized by MLA nucleotide-binding leucine-rich repeat (NLR) receptors. - Y2H-NGIS and subsequent binary retesting identified 356 high-confidence protein-protein interactions (PPI) among Bh CSEPs, MLA NLR fragments, and barley proteins. We uncovered unique and shared host targets, including numerous proteins involved in ubiquitin-associated processes, gene expression, immune and kinase signaling, metabolism, and vesicle trafficking. - We identified a host protein (HvCAP2) that interacts with MLA and at least five Bh effectors, as well as additional effector-host PPI that correlate with loss of recognition of AVRA13 and AVRA6 by MLA13 and MLA6, respectively. - Results were integrated with an interolog-based barley interactome (HvInt) to assemble a host-pathogen network of 1248 proteins and 1701 interactions. This PPI resource can serve as a platform for functional investigations into the molecular basis of powdery mildew pathogenesis and host recognition.

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.

Cellulose synthase complexes build the load-bearing cellulose microfibrils of plant cell walls, yet how the abundance of their catalytic CELLULOSE SYNTHASE A (CESA) subunits is maintained remains unclear. Here, we used multi-bait TurboID proximity labelling with ten cellulose-synthesis-associated baits and six subcellular controls to define a high-confidence cellulose synthase neighbourhood. Stringent spatial and recurrence-based filtering yielded a core network of 119 interactions among 44 proteins and identified three members of the conserved Bcl-2-associated athanogene (BAG) family as previously unrecognised regulators of cellulose synthase homeostasis. BAG1-3 associated with primary-wall CESAs in reciprocal proximity-labelling experiments. Arabidopsis bag mutants showed reduced cellulose accumulation, hypersensitivity to cellulose-synthesis inhibitors, and markedly decreased CESA protein abundance without corresponding changes in CESA transcript levels. Loss of BAG function also increased the accumulation of CESA6 in vacuolar compartments. These findings identify BAG proteins as previously unrecognised regulators of cellulose synthase homeostasis and link a conserved proteostasis-associated protein family to plant cell wall biosynthesis. More broadly, the study establishes multi-bait proximity labelling, combined with cell location-specific controls, as a strategy for resolving dynamic protein networks whose components traffic through multiple subcellular compartments.

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.

Azetidine-2-carboxylic acid (Aze) is a toxic, non-proteogenic amino acid produced in restricted plants. It can enter the human food chain through direct or indirect dietary exposure and has been linked to neurological disorders due to its ability to misincorporate for its structural analog proline (Pro) during translation and lead to protein misfolding. Although several microorganisms have evolved mechanisms to detoxify Aze, these pathways have not previously been exploited to engineer Aze tolerance in plants. Here, we heterologously expressed a bacterial Aze hydrolase (HAS) in Arabidopsis thaliana to establish a metabolic strategy for Aze detoxification in plants. Transgenic plants exhibited markedly enhanced tolerance to Aze, as demonstrated by improved root growth, cotyledon development, and survival compared with wild-type plants under Aze stress. We further showed that HAS localized to the cytosol, where it converted Aze into the non-toxic metabolite 2-hydroxy-4-aminobutyrate (HAB). HAB formation was confirmed both in planta and in vitro, demonstrating that HAS remained catalytically active in plant cells and effectively detoxified Aze. Enzyme kinetics revealed that HAS has a high affinity for Aze and is stereospecific for L-Aze, with no activity toward D-Aze. Multi-omics analysis including transcriptomics and misincorporation proteomics show that the transgenic lines misincorporate Aze significantly less compared to wild-type leading to enhanced Aze tolerance. Our findings establish bacterial HAS as an effective metabolic detoxification system that confers enhanced Aze tolerance in plants. This study provides a promising strategy for engineering plant resistance to toxic non-proteogenic amino acids.

RNA-guided nucleases (RGNs), such as Cas9 from Streptococcus pyogenes (SpCas9), are widely used for plant genome editing. Previous surveys for off-targeting, the modification of unintended targets with similarity to the intended target, indicate high specificity of SpCas9 in plant cells. However, off-targeting has not been assessed for efficiency-optimized editing systems combined with extensive multiplexing, which might increase the likelihood of cleavage at unintended sites. We therefore analyzed Arabidopsis thaliana lines that had been extensively mutagenized using zCas9i and up to 29 gRNAs addressing >45 target sites over several rounds of editing. Genomes were sequenced by short- and long-read technologies, and genome-wide variants were catalogued. Our pipeline for variant calling reliably detected RGN-induced mutations at on-targets. When excluding these on-target modifications, variants were detected in edited lines at frequencies similar to those previously reported for spontaneous mutations. In further analyses, we did not find any evidence for an origin of these variants from RGN activity. Our data are thus consistent with high specificity of SpCas9. In contrast, we detected genomic reorganization events upon editing at two complex loci, RPP1 and RPP7, encompassing multiple homologous genes, and also identified an allele by WGS that had escaped detection by amplicon sequencing. We conclude that, while off-targets may efficiently be avoided by selection of specific gRNAs, on-target modifications may be more extensive than intended, especially at complex loci and/or during multiplexing.

Local metabolic programs can determine whether developing organs maintain growth, differentiate, or degenerate. However, the spatial establishment of such programs during reproductive development remains poorly understood. In cereal inflorescences, this question is closely tied to grain number because the fate of initiated floral primordia is shaped by the developmental position, metabolic support, genotype, and environmental stress. To understand the metabolic logic underlying floral fate in barley (Hordeum vulgare L.), we mapped the spatial distribution of metabolites across the inflorescence in wild-type Bowman and in hvcmf4, a mutant that undergoes premature apical inflorescence degeneration. Amino acid-, carbohydrate-, and chlorophyll-associated metabolites form clear developmental gradients along the inflorescence axis. These gradients are progressively remodeled during developmental pre-anthesis tip degeneration but are both accelerated and spatially distorted in hvcmf4. Within this broader metabolic landscape, two discrete domains stand out. First, spermidine-associated domains specifically marked reproductive meristems, a pattern corroborated by the meristem-enriched expression of polyamine biosynthetic genes. This polyamine-rich meristem state declined as developmental tip degeneration proceeded and was prematurely lost in hvcmf4, where polyamine metabolism appeared to shift toward oxidative breakdown via the polyamine oxidase pathway. Second, lysophosphatidylcholine accumulated specifically at floral attachment and vascular supply zones. The developmental reduction of this bioactive lysophospholipid-associated domain in the apical regions, and its stronger disruption in hvcmf4, coincided with the reprogramming of lipid-remodeling and auxin transport-associated genes, suggesting impaired coordination of lipid signaling, auxin canalization, and vascular support. Together, our study reveals spatial polyamine and lysophospholipid domains that couple meristem maintenance with vascular support, providing a metabolic framework for the fate of developing cereal inflorescences.

Plastidial phosphorylase (Pho1) regulates starch metabolism and also modulates photosystem I (PSI) activity. In rice, Pho1 contains an 80 amino acid intrinsically disordered region (IDR), L80, that acts as a negative regulator. While total removal of the IDR boosts seedling growth, flowering time, overall biomass, and grain yield and alters PSI redox properties, the precise underlying regulatory basis remains unclear. To pinpoint IDR regulatory elements, four Pho1 partial-IDR deletion variants ({delta}N, {delta}C, {delta}M, and {delta}N{delta}C) were evaluated. Lines carrying {delta}M and {delta}C exhibited enhanced starch metabolism, yielding larger, heavier grains, and flowered 5 to10 days earlier than wild type. Both d{delta}M and {delta}C share an acidic motif, (E/V)SEEV, identifying it as a primary negative regulator of starch synthesis and floral timing. By contrast, no partial deletion fully mirrored the PSI redox modifications of Pho1{delta}L80. These results demonstrate that starch biosynthesis and flowering are governed by one or more discrete sequence motifs within the middle and C-terminal regions, whereas PSI modulation likely requires the structural integrity of the intact IDR.

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.

Groundnut (Arachis hypogaea) is an important oilseed crop, and its production is severely affected by drought worldwide. Improving the drought-tolerance of groundnut without compromising its agronomic traits is a major challenge for researchers. Here, a meta-transcriptome analysis identified tandemly duplicated G{beta} subunits (AhG{beta}1 and AhG{beta}3) as early signaling genes under water deficit conditions. Heterotrimeric G-protein {beta}-subunit is a major signaling element that can regulate diverse responses in plants. Heterologous complementation studies in Arabidopsis G{beta} mutant (agb1-2) demonstrated evolutionary conservation of AhG{beta} function, while the transgenic overexpression and RNAi knockdown in groundnut established AhG{beta}1 as a positive regulator of plant growth, yield, and drought tolerance. Integrated transcriptomic, transcription factor regulatory network, and metabolomic analyses demonstrated that AhG{beta}1 coordinates drought-responsive transcriptional and metabolic reprogramming characterized by suppression of cell wall-associated processes and activation of phenylpropanoid, flavonoid, and isoflavonoid biosynthesis. This reprogramming resulted in enhanced accumulation of antioxidant flavonoids, including quercetin and rutin, reduced ROS accumulation, and improved drought tolerance. Furthermore, exogenous application of these flavonoids enhanced drought recovery, highlighting their role as protective downstream metabolites. Collectively, our findings establish AhG{beta}1 as a conserved regulator linking early G-protein signaling with transcriptional and metabolic reprogramming to maintain redox homeostasis during drought stress and identify promising molecular and metabolic targets for improving drought resilience in groundnut.

Lipid droplets (LDs) play a central role in cellular carbon and energy homeostasis, in particular through the roles played by numerous LD-associated proteins. A long-standing question concerns how these proteins are molecularly organized at the seemingly overcrowded LD surface. Delayed in TAG Hydrolysis 1 (DTH1) has been identified as a key protein for efficient LD degradation in Chlamydomonas reinhardtii, however the underlying molecular mechanism remains unknown. DTH1 contains several structural features including a coiled-coil domain, a phosphatidylethanolamine (PE) specific lipid-binding domain, intrinsically disordered regions, and an alpha-helical C-terminal domain, that make it a compelling candidate for acting as a molecular scaffold in protein recruitment and/or organelle tethering. In this study, we used the Chlamydomonas knock-out mutant dth1 as an entry point to begin dissecting the molecular organization of proteins at the LD surface. Comparative lipidomic and proteomic analyses of isolated LDs from both WT and dth1 mutants revealed several key features associated with LD degradation during nutrient recovery. First, PE emerged as an important lipid species involved in LD degradation. Second, active communication occurs among multiple subcellular organelles particularly between LDs, peroxisomes and mitochondria. Third, DTH1 was found to play a critical role in remodeling both the LD proteome and lipidome. Furthermore, detailed analyses of LD degradation during nitrogen recovery uncovered the formation of micro-LDs, a phenomenon not yet reported in microalgae. Collectively, these results highlight the tight coordination of metabolic activities across subcellular compartments and emphasize the importance of intracellular carbon trafficking during stress recovery.

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.

SUMOylation is an essential and evolutionarily conserved post-translational modification that enables rapid cellular adaptation to environmental stress. Despite its importance, how the SUMOylation machinery controls its dynamics remains poorly understood. Here, we integrated a mathematical model of the plant SUMO cycle with quantitative imaging and biochemical data to decode the regulatory logic of SUMOylation dynamics. The model reveals a fundamental principle: steady-state SUMOylation levels are determined not by the full complement of SUMO-cycle components, but by the abundance of SUMO modifier proteins and E1 enzymes, together with SUMO protease abundance and activity. Strikingly, when we integrated fluorescence measurements from reporters for SUMO-cycle components in Arabidopsis roots after three hours of abiotic stress, the model predicted decreased SUMOylation levels across several tissues, contrary to prevailing evidence. Motivated by this unexpected prediction, we performed time-resolved imaging to better characterize the dynamics of key SUMO-cycle components after salt and osmotic stress. These data revealed highly dynamic component levels within the first three hours of stress that were not captured in the previous measurements. Incorporating this temporal data into the model uncovered a twin-peaked SUMOylation response to salt stress: an early surge underpinned by protease dynamics, followed by a decline and a second peak driven by elevated SUMO modifier and E2 levels. Western blot analysis revealed a corresponding twin-peaked pattern of SUMOylation. Together, our findings demonstrate that the plant SUMO cycle is a highly dynamic system that achieves tissue- and stress-specific control of SUMOylation through the coordinated interplay of multiple SUMO-cycle components.

Coordinated organogenesis and rhythmic stem development determine shoot morphogenesis, which governs distinct plant architecture and crop yield. The shoot apical meristem (SAM) drives organ initiation and stem growth, with its activity sustained by the spatially defined CLAVATA3 (CLV3)-WUSCHEL (WUS) feedback loop. However, how the temporal CLV3-WUS module modulates SAM activity remains elusive. Here, we demonstrate that both CLV3 and WUS are required for circadian stem elongation. The clock component TIMING OF CAB EXPRESSION1 (TOC1) directly activates and sustains the circadian expression of CLV3, and this regulatory process is controlled by the APETALA1-CIRCADIAN CLOCK-ASSOCIATED1 module. Notably, the rhythmic expression pattern of CLV3, rather than its transcript abundance, plays a more important role in regulating WUS expression and sustaining SAM activity, and CLV3 confers circadian rhythmicity to WUS protein accumulation. Transcriptome and ChIP seq analyses reveal that WUS modulates multiple developmental programs, including meristem homeostasis, phytohormone responses, and cell wall remodeling. Mechanistically, WUS directly activates PECTIN METHYLESTERASE5 expression to promote stem elongation. Collectively, our findings uncover a spatiotemporal regulatory cascade that fine-tunes SAM activity to coordinate rhythmic organogenesis and stem development, thereby establishing optimal phyllotactic patterning in plants.

Jasmonates are essential phytohormones that coordinate defense responses and developmental programs across land plants. In angiosperms, the active jasmonate ligand jasmonoyl-L-isoleucine (JA-Ile), is produced through GH3-mediated conjugation of jasmonic acid to isoleucine and JA-Ile homeostasis is further shaped by ILR1/ILL-family amidohydrolases. In contrast, the primary bioactive jasmonate ligand in bryophytes, dinor-12-oxo-phytodienoic acid (dn-iso-OPDA), is inactivated through conjugation with amino acids, raising the question of whether these conjugates constitute a reversible hormone reservoir or an irreversible catabolic end point. Although the ILR1-like family has been characterized extensively for its role in auxin and jasmonate homeostasis in angiosperms, its function in bryophytes remains basically unexplored. Here we show that MpILR1, the sole Marchantia ortholog of the ILR1/ILL family, hydrolyzes a specific subset of dn-iso-OPDA-amino acid conjugates in vivo. Loss-of-function Mpilr1 mutants exhibit enhanced accumulation of dn-iso-OPDA conjugated to hydrophobic amino acids (Val, Leu and Ile) but not to hydrophilic residues (His, Glu and Gln), demonstrating substrate-selective hydrolysis. MpILR1 hydrolytic activity is required for full dn-iso-OPDA-mediated responses, including transcriptional activation and defense against gastropod herbivory. These findings establish MpILR1 as a key positive regulator of jasmonate signaling in Marchantia polymorpha and suggest that hormone conjugation/deconjugation is an ancient regulatory mechanism evolved during plant terrestrialization.