The Plant Cell

3-Deoxy-D-manno-oct-2-ulosonic acid (KDO) is an essential component of rhamnogalacturonan II (RG-II), a complex pectic polysaccharide required for plant growth and development. While most steps of the KDO biosynthetic pathway have been characterized in plants, KDO-8-phosphatase (KDO8Pase), the phosphatase responsible for converting KDO 8-phosphate (KDO8P) to KDO, remained unidentified. To identify this missing component, we performed gene co-expression analysis and identified At5g57440 (GPP2) as the primary candidate in Arabidopsis (Arabidopsis thaliana L.). Recombinant GPP2 protein exhibited KDO8P-specific phosphohydrolase activity in vitro. A GFP-tagged GPP2 protein was predominantly localized to mitochondria, consistent with the compartmentation of the subsequent step in KDO biosynthesis. Null mutants of GPP2 exhibited significant growth retardation under boron-limited conditions, in which expression of GPP2 and other KDO biosynthetic genes was up-regulated. The growth retardation was also observed in liquid culture in normal media, a condition that induces rapid growth and thus likely increases metabolic demand for KDO. Despite this growth defect, the KDO content per unit cell wall in gpp2 remained equivalent to that in wild-type plants. These results are consistent with the identification of GPP2 as the elusive plant KDO8Pase and suggest a model where KDO availability becomes the rate-limiting factor for cell wall production. Our findings complete the plant KDO biosynthetic pathway and provide new insights into the physiological significance of RG-II in cell wall biosynthesis. Significance statementThis study identifies the previously unknown plant KDO-8-phosphatase, thereby completing the biosynthetic pathway for KDO in rhamnogalacturonan II. Our findings demonstrate that KDO synthesis is up-regulated under boron deficiency, and its supply becomes a rate-limiting factor for cell wall formation.

T-DNA insertion mutants are widely used to disrupt genes and infer their functions, yet the insertions can also trigger unintended genomic changes that confound phenotypic interpretation. Here, we used T-DNA insertion mutants affecting the major mitochondrial malate dehydrogenase (MDH1) and the heterodimeric NAD-dependent malic enzymes (ME1 and ME2) to examine their functional coordination across photoperiods and irradiance regimes. Under short days, especially at low light intensity, mdh1xme2 mutants were markedly smaller than wild type and, unexpectedly, than the mdh1xme1xme2 triple mutant, and they showed a more pronounced reduction in photosynthetic capacity. ME1 was undetectable in mdh1xme2, implying that the double and triple mutants effectively lack heterodimeric ME and should therefore behave similarly, contrary to what we observed. Whole-genome analysis resolved this discrepancy by revealing that the MDH1 T-DNA insertion in mdh1xme2 is accompanied by a major rearrangement, a 137-kbp duplication downstream of the insertion site, which was absent in the mdh1xme1xme2 triple mutant. This duplication increased gene dosage and elevated transcript abundance across the duplicated interval, while proteomics detected 5 of the 38 encoded proteins, including PEPC1. mdh1xme2 accumulated oxaloacetate-derived amino acids and displayed an altered carbon/nitrogen balance, making PEPC1 a plausible contributor to the exacerbated mdh1xme2 phenotype. Together, our data indicate that a T-DNA-linked structural variant can amplify expression of dozens of genes and intensify phenotypes at specific conditions, thereby affecting the interpretation of genotype-phenotype relationships. Because Agrobacterium-mediated DNA transfer also underpins many genome-editing workflows, our findings argue that structural validation around insertion/editing loci should be considered essential when interpreting T-DNA-derived plant lines.

Plant survival and growth depend partly on the ability to manage energy resources in response to changing environmental conditions. SnRK1 plays a central role in this process by restricting growth under energy-limiting conditions while promoting stress adaptation and survival. When activated, SnRK1 triggers transcriptional reprogramming that prioritizes energy-producing pathways. A key mediator of this response is the transcription factor bZIP63, whose activity is regulated by SnRK1-dependent phosphorylation. Given its roles in energy homeostasis and its interaction with the circadian clock, bZIP63 influences growth and is therefore a candidate component of the Metabolic Daylength Measurement (MDLM) system, which integrates starch and sucrose metabolism with circadian timing and photosynthetic duration to regulate vegetative growth under contrasting photoperiods. We show that 39 bZIP63 direct targets regulated by SnRK1 correspond to a subset of short-day-induced genes associated with the MDLM system and are downregulated in a bZIP63 T-DNA mutant (bzip63-2) and/or in an RNAi-induced silencing line (RNAiWs_L9). Downregulation of these genes was more extensive in RNAiWs_L9 than in bzip63-2, possibly due to the unexplained silencing of BAM4, a {beta}-amylase that promotes starch degradation. Under short-day conditions, the frameshift mutant bzip63-5 (Col-0), bzip63-2 (Ws), and the bzip1-1/bzip53-1/bzip63-5 (Col-0) triple mutant, which disrupts bZIP63 heterodimerization partners, showed similar deregulation of a subset of these genes and comparable growth inhibition, whereas both growth and gene deregulation were more strongly affected in RNAiWs_L9. We further show in two partially complemented bzip63-2 lines that bZIP63 protein levels increase toward the end of the night and decline toward the end of the day, in synchrony with the diel oscillation of its transcript. Additional analyses of these lines, together with bzip63-2 line overexpressing bZIP63, suggest that the timing and amplitude of bZIP63 accumulation contribute to shaping the expression profiles of a subset of the 39 MDLM-associated genes. Together, these findings indicate that bZIP63 participates in a regulatory network linking SnRK1 signaling, photoperiod-changes, and growth within the MDLM system.

RD21-like proteases are papain-like cysteine proteases with a C-terminal granulin domain that are abundant and ubiquitous in angiosperms and have often been implicated in immunity. We previously found that the activity of RD21 in Nicotiana benthamiana (NbRD21) is suppressed during infection with Pseudomonas syringae. Here, we studied the role of NbRD21 in immunity and proteome processing. NbRD21 was disrupted by genome editing and rd21 mutants were subjected to disease assays and shot-gun proteomics. Dipeptide substrate zLR-AMC was used in protease assays and agroinfiltration was used to transiently express NbRD21 and candidate substrates. Genome edited lines lacking NbRD21 develop normally but have drastically reduced zLRase activity and are significantly more susceptible to P. syringae. Shot gun proteomics revealed an increased accumulation of [~]20 diverse receptor-like kinases (RLKs) in untreated rd21 knockout lines, but their transcript levels are unaltered when compared to wild-type plants. 35S-driven GFP-tagged RLKs accumulate more upon transient expression in rd21 plants than in wild- type plants. These data indicate that NbRD21 post-translationally controls RLK homeostasis, either by directly degrading RLKs, or indirectly by regulating endocytic RLK recycling.

Higher plants possess two classes of myosin molecular motors, class XI and class VIII, both unique to the plant lineage. The diverse cellular functions of class XI myosins, including organelle transport and nuclear positioning, have been elucidated largely through systematic identification of cargo adaptor proteins that bind to their globular tail domains (GTDs). In contrast, no proteome-wide screen for class VIII myosin tail-binding proteins has been reported; the few known interacting proteins were each discovered through studies focused on the binding partner rather than on the myosin itself, leaving the full repertoire of class VIII myosin-associated proteins largely unknown. Here, we employed TurboID-based proximity labeling to systematically identify proteins associated with the GTD of the class VIII myosin ATM1 in Arabidopsis thaliana, as this approach covalently biotinylates neighboring proteins in vivo, enabling their identification even after proteolytic degradation during cell lysis. We identified 233 non-redundant candidate ATM1-proximal proteins. Candidates were prioritized by AlphaFold3-based protein complex structure prediction and validated by co-immunoprecipitation. We identified two ATM1-associated proteins: C3H61/AtTZF5, a tandem zinc finger protein involved in mRNA turnover at processing bodies and stress granules; and SFH7, a Sec14-nodulin domain protein that mediates phosphatidic acid transfer from the endoplasmic reticulum to chloroplasts. These findings provide initial evidence linking ATM1 to proteins involved in post-transcriptional gene regulation and interorganellar lipid transport, raising the possibility of previously unrecognized connections between class VIII myosins and these cellular processes.

In flowering plants, pollen tubes communicate with ovular cells to achieve precise one-to-one pollen tube reception. The final step of this communication between the pollen tube and synergid cells has been extensively investigated and visualized by calcium imaging. Synergid cells exhibit characteristic cytoplasmic calcium concentration oscillations, which are thought to play a critical role in pollen tube reception. However, their significance and relationship with calcium dynamics in the entire ovule remain unclear. Here, we show, using the calcium sensor GCaMP6s, that proteins involved in asparagine-linked glycosylation (N-linked glycosylation) are required for normal calcium oscillations in synergid cells but are not essential for pollen tube reception. Using a semi-in vivo assay in Arabidopsis thaliana, we found that the amplitude of these oscillations prior to rapid pollen tube growth across the filiform apparatus was reduced in mutants lacking the oligosaccharyltransferase (OST) 3/6 subunit or alpha1,2-glucosyltransferase (ALG) 10, both of which are involved in N-linked glycosylation. Notably, these mutants did not exhibit reduced fertility attributable to defects in the female gametophyte but instead showed a polytubey phenotype due to a sporophytic defect. These findings suggest that N-linked glycans mediate communication between synergid cells and the pollen tube and indicate that the typical pattern of calcium oscillations in synergid cells is not essential for triggering pollen tube rupture. Furthermore, we show that sporophytic tissues of the ovule exhibit calcium waves that propagate toward the funiculus in correlation with pollen tube contact and rupture, implying that ovular tissues can potentially transmit these signals distantly beyond the ovule. Together, these findings reveal previously unrecognized intercellular calcium signaling and its significance in pollen tube reception by the ovule.

In plant cells, the multi-domain proteins FRIENDLY and REC regulate the cellular organization, distribution and proliferation of mitochondria and plastids, respectively. Both proteins share a similar overall domain architecture and belong to the larger CLUSTERED MITOCHONDRIA (CLU) superfamily. Domains of CLU proteins have been shown to interact with translation related proteins, tRNA synthetases and even mRNA, but their exact modes of operation remain cryptic and how organelle specificity of CLU paralogs in plant cells is achieved unknown. We characterized the single CLU family protein of the liverwort Marchantia polymorpha that we demonstrate to be transcribed either with or without exon 22, which changes the configuration of the TPR domains in the C-terminus. Knockout of MpCLU affects both mitochondria and plastids, and independent rescues show that the splice variant with exon 22 (MpCLU22) serves mitochondrial- and the one lacking exon 22 (MpCLUspl22) plastid biology. The CLU-C domain of the protein is responsible for nuclear localisation and expressed alone induces a phenotype that differs in photosynthesis performance and transcriptome changes from that of the knockout of MpCLU. Our results identify the C-terminal TPR motif to be responsible for organelle specificity in plants and they provide an example of how genome reformatting and gene loss can be compensated for by the alternative splicing of a single exon.

Diversification of plant development has largely enabled land colonization and establishment of different ecosystems. This diversification relies on the gain/loss of cell-types and tissues, such as stomata, vascular tissue and functional roots. Likewise, diversification of immunity is thought to rely on expansion/contraction, followed by functional specification, of the different components of plant defense mechanisms. Although anatomical changes might result in altering the infection routes of pathogens and the cells and tissues they interact with, very little is known about the co-evolution of plant development and immunity. We have recently described that RNAi-dependent antiviral responses observed in the non-vascular Marchantia polymorpha are confined to leaf vasculature in Nicotiana benthamiana plants, suggesting repatterning of antiviral responses as result of the acquisition of developmental innovations. Here, we explored the genetic basis of that repatterning by establishing the basal immunity toolkit across different cell-types in the non-vascular Marchantia polymorpha and the vascular Arabidopsis thaliana. The results from our comparative transcriptomic studies show that while RNAi is the major antiviral defense across Marchantia cell-types, that configuration is only maintained in phloem companion cells in Arabidopsis leaves, suggesting that plant immunity might co-evolve with developmental diversification. Additionally, differential levels of RNAi expression in different cell-types correlate with their vulnerability to viral countermeasures, with companion cells been the most resilient to the presence of viral silencing suppressors.

Plant receptor-like kinases (RLKs) are involved in diverse processes, ranging from growth and reproduction to interactions with microbes. Variation in the extracellular domains delineates several RLKs subfamilies, including the malectin-like domain leucine-rich repeat receptor-like kinases (MLD-LRR-RLKs). Symbiosis Receptor-like Kinase (SymRK) is the prototypical member of MLD-LRR-RLKs and is required for microbial accommodation in host roots during root endosymbiosis. Yet, comparative phylogenetic analysis of SymRK orthologs in the broader context of MLD-LRR-RLK subfamily evolution remains limited. In this study, we examined the inventory, phylogeny and clade-specific evolutionary and transcriptional characteristics of this receptor group. SymRK and its closest homologs are present in most land plant lineages and group into four major clades and six additional species-specific clades. These clades can be distinguished by their evolutionary characteristics as either conserved with reduced gene copy number changes (including SymRK) or expanded and diversified, as observed in clade IV. Clade IV dynamics are largely driven by tandem gene duplications, which often arise within gene clusters. We further analysed the evolutionary characteristics of MLD-LRR-RLKs at the population level in Arabidopsis thaliana accessions. We found that some genes are conserved across accessions and are therefore likely to be functionally important, whereas a subset of genes, often located within tandem clusters, are highly diverse and likely contribute to accession-specific adaptations. Finally, most MLD-LRR-RLKs in the A. thaliana Col-0 accession are expressed in roots and respond broadly to biotic stimuli at the transcriptional level. Notably, clustered genes frequently exhibited divergent expression profiles, suggesting transcriptional diversification. Together, we revealed two contrasting evolutionary characteristics among members of the MLD-LRR-RLK subfamily, potentially associated with their functions in plants.

Chloroplast HSP70 is an essential component of the plastid proteostasis network, supporting protein folding, complex assembly and disassembly, and stress acclimation. Despite extensive genetic evidence for its essentiality, the cellular consequences of reduced chloroplast HSP70 activity remain poorly defined. Here, we investigated the function of the sole chloroplast HSP70 in Chlamydomonas reinhardtii, HSP70B, using an inducible artificial microRNA approach that reduced HSP70B abundance to below 30% of wild-type levels. HSP70B depletion resulted in cell division arrest and extensive proteome remodeling, characterized by strong upregulation of proteins involved in chloroplast protein quality control and membrane remodeling. Notably, this response was accompanied by increased abundance of protein quality control components in the endoplasmic reticulum, cytosol, and mitochondria, indicating pronounced proteostasis cross-talk between cellular compartments. In contrast, chloroplast and cytosolic ribosomes, photosynthetic and respiratory protein complexes, and central metabolic enzymes were broadly depleted, consistent with a collapse of cellular proteostasis. At the ultrastructural level, HSP70B-depleted cells exhibited lesions at thylakoid membrane conversion zones previously described in VIPP1-depleted cells. Accordingly, higher-order oligomeric forms of VIPP1 accumulated, and cells displayed extreme sensitivity to high-light stress. These findings confirm HSP70B as a key regulator of VIPP1 oligomer dynamics and highlight its central role in coordinating chloroplast membrane remodeling with cellular proteostasis in Chlamydomonas. One-sentence summaryDepletion of chloroplast HSP70B causes cell division arrest, proteostasis collapse, impaired VIPP1 oligomer dynamics with aberrant thylakoid structures, and increased light sensitivity.

Plant architecture is a crucial component of maize productivity. Tailoring architectural component traits like leaf area and angle can increase productivity by promoting deeper light penetration into the canopy and better resource utilization. Novel genetic variants can increase the rate of gain for optimized plant architecture. Here, we map a moderate-effect mutation denoted reduced leaf area1 (rdla1) to the RAGGED5 (RGD5) locus and characterize it as a transposon insertion allele. Mutant leaf area reductions were most extreme in mid-upper canopy positions. Photosynthetic gas exchange rates were not significantly impacted in rdla1 relative to wild-type, indicating that mutant leaf structure, but not function, is altered. Functional annotations of RDLA1 were supported by metabolite profiles suggesting a role in cuticular wax biosynthesis. Introgression of the rdla1 allele into 27 commercially relevant genetic backgrounds identified differences in effect size across genotypes, revealing modifier effects that could serve as targets for modulating plant architecture.

Rapid shoot growth (flushing) phenology is a fundamental developmental process in perennial woody plants such as citrus. In a separate study, we identified physiological shifts from photosynthesis to mobilization of nitrogen and carbohydrate to support new shoot growth. However, the underlying molecular and biochemical signals remain largely unknown. Here, we integrated proteomic and metabolomic analyses to investigate carbohydrate and hormone dynamics across three flush stages in Citrus sinensis: quiescent period (stage 1), new shoot initiation (stage 2), and full expansion (stage 3). Sucrose, maltose, and trehalose accumulated in apical leaves during early shoot initiation and declined during subsequent shoot expansion, indicating depletion of carbohydrate reserves and enhanced resource remobilization. These changes were accompanied by coordinated regulation of starch-metabolizing enzymes, including ADP-glucose pyrophosphorylase, -amylase, and isoamylase, supporting a transition from carbon storage to carbon export during active shoot growth. Indole-3-acetic acid increased continuously across stages, while trans-zeatin and gibberellin A{square} showed opposite trends in apical versus basal leaves before jointly increasing at stage 3. Hormone analysis revealed dynamic and coordinated signaling changes during flush development. Abscisic acid declined from stage 1 to 2, whereas jasmonoyl-isoleucine and salicylic acid increased from stage 2 to 3. Some hormone-responsive proteins, including Gretchen Hagen 3 and Gibberellin-insensitive dwarfing 1, exhibited expression patterns consistent with hormonal fluctuations. Together, these results support a stage-specific regulatory framework in which carbohydrate metabolism and hormone signaling are tightly coordinated to regulate rapid source-sink transitions during citrus flush development. HighlightWe reveal how carbohydrate metabolism and hormone signaling are spatiotemporally coordinated during citrus shoot growth phenology, and we develop an integrated metabolic-hormonal model that connects carbon allocation to developmental transitions.

Jasmonic acid (JA) and its derivatives are lipid-derived phytohormones that coordinate plant growth, development, and stress responses through the bioactive conjugate jasmonoyl-isoleucine (JA-Ile). Their role in reproductive development is well established, particularly in stamen maturation through the R2R3-MYB transcription factor MYB21, which has been considered largely flower-specific. Here, we reveal a previously unrecognized role of MYB21 in vegetative tissues of Arabidopsis thaliana. Although basal MYB21 transcript levels in leaves are extremely low and spatially restricted, wounding and exogenous hormone applications induced MYB21 transcription in a JA- and COI1-dependent manner. Transcriptional GUS reporter analyses showed localized MYB21 promoter activity in specialized epidermal cells, including trichomes, hydathodes, and in the vasculature at wound sites. Functional characterization using the myb21-5 mutant indicated roles in germination and vegetative growth, partially phenocopying JA-insensitive mutants despite unaltered JA biosynthesis and signaling. Transcriptome profiling further revealed changes in expression of genes involved in lignin biosynthesis, light-harvesting complex components, cytokinin pathways, and defense-related responses, consistent with reduced resistance of myb21-5 to insect herbivory and infection by Botrytis cinerea. Together, these findings identify MYB21 as a JA-responsive regulator of growth and defense in seedlings and leaves, extending its function beyond reproductive development. HighlightThe transcription factor MYB21, previously considered flower-specific, is induced by jasmonate signaling in Arabidopsis leaves and seedlings, where it regulates growth and defense responses, thereby expanding its role beyond reproductive development.

Soil salinization is a growing global threat that limits crop productivity. To cope with sodium (Na) stress, plants have evolved tolerance mechanisms, including excluding Na from shoot tissues and tolerating elevated Na within shoots through tissue- and cellular-level mechanisms. Most current knowledge of Na accumulation comes from organ- or whole-plant measurements that lack the spatial resolution needed to resolve cellular tolerance mechanisms. Here, we used histological approaches to map leaf Na distribution in tomato (Solanum) species with contrasting salt-tolerance strategies. In the Na-excluding domesticated tomato (cv. M82), Na was largely confined to the bundle sheath, whereas Na-including wild relatives accumulated Na throughout the blade mesophyll. Consistent with these cell population-specific Na patterns, M82, but not S. pennellii, exhibited reduced symplastic transport and plasmodesmal permeability under salt stress. A genetic screen combined with transcriptome profiling implicated Plasmodesmata-Located Protein 1 (PDLP1), a regulator of callose-mediated plasmodesmal closure, in establishing symplastic domains in M82 that restrict Na movement into the mesophyll. Moreover, PDLP1 expression negatively correlated with mesophyll Na+ levels across wild and domesticated tomatoes. Collectively, these results link cellular Na enrichment patterns to symplastic connectivity and suggest that PDLP1-mediated regulation of plasmodesmata contributes to leaf-level salt-tolerance strategies. HighlightsO_LICell type-specific Na accumulation differs between domesticated tomato (Solanum lycopersicum cv. M82) and its wild relative S. pennellii. C_LIO_LIAdditional salt-tolerant wild tomato relatives exhibit leaf Na enrichment patterns similar to S. pennellii. C_LIO_LISalt stress reduces symplastic transport and plasmodesmal permeability in M82 leaves but not in S. pennellii. C_LIO_LIAn introgression line (IL6-4) between the two tomato species, which carries S. pennellii Plasmodesmata-Located Protein 1 (SpPDLP1), shows S. pennellii-like Na enrichment patterns. C_LIO_LIPDLP1 expression shows a negative correlation with mesophyll Na+ levels across tomato species. C_LI

1.O_LISalt stress triggers overlapping osmotic and ionic effects. In halophytes, rapid acclimation can obscure how early responses are coordinated across organs. The use of facultative halophytes and salt shocks provides a useful framework to resolve this transition. C_LIO_LIWe investigated the first 24 h of salt shock responses in the facultative halophyte Lobularia maritima by integrating physiological, ionomic, transcriptional and phytohormonal analyses to resolve organ- and time-dependent acclimation dynamics. C_LIO_LISalt shock induced a rapid but transient osmotic effect, with shoot turgor recovery after 8 h. This recovery was associated with sustained osmotic adjustment, proline accumulation and increased Na+ levels in shoots. Conversely, photosynthetic impairment persisted beyond osmotic recovery. Salt exposure rapidly reshaped shoot and root ionomes and was associated with dynamic expression of LmSOS1, LmNHX1, and LmHKT1, consistent with coordinated Na+ partitioning. Oxidative responses diverged between organs; shoots maintained a stable oxidative state, while roots exhibited progressive loss of meristem viability. Abscisic acid (ABA) was strongly accumulated at all time points and emerged as the dominant regulator of early responses. C_LIO_LIThese results show that early salt acclimation in L. maritima is rapid but spatially and functionally uncoupled, combining fast shoot osmotic adjustment with persistent photosynthetic constraints and increased root vulnerability. C_LI

The JAGGED transcription factor family regulates lateral organ development across angiosperms. In soybean (Glycine max Merr.), a D9H mutation in the EAR repression motif of GmJAG1 causes a narrow leaflet phenotype and explains over 70% of phenotypic variance in leaf shape. Because this mutation does not affect the zinc finger DNA-binding domain, both alleles bind identical targets but differ in repressor recruitment. Previous studies mapped GmJAG1 binding sites, but the functional targets controlling leaf morphology are uncharacterized. Here, we used comparative transcriptomics across four soybean genotypes with contrasting leaf shape, spanning a developmental time series from shoot apex to mature leaf, and identified 1,567 candidate target genes. GmJAG1 expression was confined to the shoot apex, yet 99.1% of candidate targets maintained differential expression throughout development. We found that neither Kip-Related Protein (KRP) cell cycle inhibitors nor Cyclin-Dependent Kinases (CDKs) showed differential expression despite binding evidence in Arabidopsis. However, D-type cyclins were upregulated in narrow-leaf genotypes suggesting cyclin-mediated rather than KRP-mediated cell cycle regulation in soybean. Pathway analysis revealed enrichment of auxin (1.8-fold, P = 0.02) and salicylic acid (4-fold, P = 0.016) genes among JAG1D9H targets. Filtering by differential expression, binding data, phenotype correlation, and co-expression network membership identified 79 high-confidence targets, including orthologs of NPH3 (phototropin-mediated leaf flattening), MIK2 (cell wall integrity sensing), RD22 (ABA-responsive stress signaling), and SCL23 (GRAS transcription factor in bundle sheath development). These candidates provide targets for functional validation and breeding in legumes.

In plant cytokinesis, the partitioning membrane is made by homotypic fusion of secretory vesicles, progressing in a centre-to-periphery direction. In Arabidopsis, this process is mediated by a cytokinesis-specific fusion machinery involving Qa-SNARE KNOLLE which is made during G2/M phase and degraded at the end of cytokinesis. Here we analyse how the turnover of KNOLLE protein is regulated. KNOLLE is ubiquitinated, which is best detected after combined treatment with inhibitors of endocytosis and de-ubiquitination. Site-directed mutagenesis of three clustered lysine residues prevented ubiquitination and internalisation, resulting in stable accumulation of KNOLLE at the plasma membrane in all cells of the seedling root. This is in stark contrast to the transient accumulation of wild-type KNOLLE in dividing cells only. Partial-substitution mutant lines revealed redundancy of lysine residues in both KNOLLE ubiquitination and turnover. KNOLLE ubiquitination resulted in K63-linked ubiquitin chains known to be involved in endocytosis whereas K48-linked chains were not detected. To explore the spatio-temporal conditions, we analysed KNOLLE ubiquitination in cis-SNARE and trans-SNARE complexes during membrane traffic and cell-plate formation. Our findings suggest that KNOLLE protein turnover is caused by a ubiquitination process that depends on successful membrane fusion generating the cell plate.

Understanding why sex chromosomes repeatedly evolve recombination suppression, gene loss, and repeat accumulation remains a central challenge in evolutionary genomics. Plant sex chromosomes may be particularly informative, because they have often evolved recently from hermaphroditic ancestors. We studied the sex-linked region of the dioecious annual Mercurialis annua using new long-read genome assemblies of an XX female and a YY male, a published female assembly, linkage maps, and population-genomic data from several Mercurialis species. We identify two discrete nested evolutionary strata on the Y chromosome of diploid M. annua. A young stratum was generated by a large inversion and shows little degeneration, whereas an older stratum nested within it exhibits substantial gene loss, transposable-element accumulation, insertion of paralogous gene copies, and elevated X-Y sequence divergence. These findings indicate that recombination suppression evolved in at least two stages, with a recent inversion expanding an older non-recombining region. Comparative analyses among several Mercurialis species further show that the extent of sex-linked differentiation varies markedly among them. We also identify APRR7 as the only gene showing consistent male-specific inheritance across the genus; this gene is a strong candidate master sex-determination gene. Together, our results refine the structure and gene content of the sex-linked region in M. annua and contribute to our understanding of the diversity of sex chromosomes in plants.

Metal homeostasis in plants relies on coordinated uptake, chelation, and transport mechanisms involving, but not limited to, citrate and nicotianamine (NA). In Arabidopsis (Arabidopsis thaliana), disruption of the citrate exporter FRD3 (FERRIC REDUCTASE DEFECTIVE 3) causes constitutive Fe deficiency responses, altered iron (Fe), manganese (Mn) and zinc (Zn) distribution, with Fe accumulation in the root cell wall. This ultimately results in oxidative and biotic stress responses, and impaired root development, phenotypes that are partially alleviated by Zn excess. In this study, we investigated the consequences of impairing both citrate loading into xylem vessels and NA partitioning within cells. The frd3 zif1 double mutant exhibits enhanced sensitivity to Zn excess, severe defects in root system architecture and meristem maintenance, persistent oxidative stress, and compromised reproductive development. These phenotypes correlate with sustained activation of Fe deficiency signaling and marked defects in root-to-shoot metal translocation. Our findings reveal that coordinated citrate export and NA compartmentation form an integrated buffering strategy required to maintain metal homeostasis and partitioning, as well as redox balance and proper development, including root plasticity and seed yield, under fluctuating metal availability.

Aldoximes are amino acid-derived metabolites that serve as precursors of auxins and modulate phenylpropanoid production in Arabidopsis. However, the enzymes responsible for aldoxime production in Solanaceae remain unknown. Here, we report the identification of aldoxime-producing enzymes in tomato (Solanum lycopersicum) and examine how altered aldoxime production affects auxin production and phenylpropanoid metabolism. Through homology-based analysis, we identified five putative CYP79 homologs in tomato, among which SlCYP79DB32 and SlCYP79DB52 exhibited aldoxime-producing activity toward multiple amino acids, including phenylalanine and tryptophan. SlCYP79DB32 and SlCYP79DB52 converted phenylalanine into phenylacetaldoxime (PAOx), whereas only SlCYP79DB52 converted tryptophan into indole-3-acetaldoxime (IAOx). Stable isotope-labeled feeding experiments revealed that IAOx and PAOx can be converted to the auxins indole-3-acetic acid (IAA) and phenylacetic acid (PAA), respectively. Consistently, tomato plants engineered to overproduce IAOx and PAOx accumulated elevated levels of IAA and PAA. These plants also accumulated lower levels of phenylpropanoids. In Brassicaceae plants such as Arabidopsis and Camelina, aldoxime accumulation represses phenylpropanoid production by promoting degradation of phenylalanine ammonia-lyase (PAL). However, aldoxime accumulation did not reduce PAL activity in tomato, suggesting an alternative mechanism in this species. Transcriptome analysis revealed extensive transcriptional reprogramming in aldoxime-overaccumulating tomato plants, including upregulation of stress- and defense-related genes. Despite the observed reduction in phenylpropanoid levels, transcript levels of most phenylpropanoid biosynthetic genes were not decreased, suggesting possible post-transcriptional regulation of this repression. Together, our findings demonstrate that aldoximes can serve as intermediates in auxin biosynthesis in tomato and reveal that aldoxime-mediated repression of phenylpropanoid metabolism extends beyond Brassicaceae.