The Plant Cell

Metabolite movement into chloroplasts is essential for sustaining chloroplast anabolic metabolism and cellular growth, and yet how the specific factors/transporters that enable import and support metabolism under heterotrophic conditions (in the dark in the presence of fixed carbon) remain poorly understood. Here, we identify CreTPT10, a chloroplast envelope-localized transporter in the unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas throughout) that, of the substrates tested, has the highest specificity for xylulose 5-phosphate (X5P). We also demonstrated that the loss of CreTPT10 caused a pronounced reduction in growth and respiratory activity in the dark, whereas no growth defects were observed in the tpt10 mutants when they were maintained in the light or under nutrient-limiting conditions. Furthermore, dark-grown tpt10 mutants exhibited markedly reduced levels of lipids, nucleotides, isoprenoids, and aromatic secondary metabolites, accompanied by coordinated repression of genes encoding enzymes associated with chloroplast-localized biosynthetic pathways. This metabolic suppression extended beyond the chloroplast, as genes associated with the mitochondrial respiratory chain and cell cycle progression were markedly downregulated in darkness. Together, these findings indicate that X5P import via CreTPT10 is critical for sustaining chloroplast anabolic metabolism and functionally coordinates chloroplast and mitochondrial energy metabolism to support heterotrophic growth.

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.

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.

Histone acetylation shapes transcriptional programs during environmental stress. The Arabidopsis histone acetyltransferase GCN5 (HAG1), a catalytic subunit of the SAGA complex, has been implicated in salt stress responses and cell wall integrity. Here, we show that loss of GCN5 enhances reactive oxygen species (ROS) accumulation and is accompanied by altered expression of class III peroxidase genes, with strong salt-induced upregulation of PRX71 and elevated basal PRX33 transcript abundance. Consistent with a role for these peroxidases in stress-associated cell wall remodeling, overexpression of PRX71 or PRX33 in the wild type is sufficient to promote ectopic lignin deposition in roots. Conversely, prx71 and prx33 mutants show improved growth on salt and on the cellulose biosynthesis inhibitor isoxaben, and they lack the pronounced ectopic root lignification observed in gcn5 under salt stress. Chromatin immunoprecipitation followed by qPCR (ChIP-qPCR) reveals reduced H3K9 acetylation at PRX71 and PRX33 promoter regions in gcn5 compared with the wild type, and reduced transcript abundance of candidate transcription factors (TFs), including MYBS2 and GATA21, accompanied by reduced H3K9ac at their loci. Together, our results support a model in which GCN5 constrains PRX71/PRX33-mediated lignification during stress, likely through an indirect regulatory route that integrates chromatin state and transcription factor activity to limit stress-associated lignification while maintaining root growth under salt stress.

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.

Translational regulation mediated by upstream open reading frames (uORFs) is an important mechanism controlling gene expression in eukaryotes, yet the functional characterization of uORFs in plant transporter genes remains limited. ZINC-INDUCED FACILITATOR 2 (ZIF2) is an Arabidopsis thaliana tonoplast-localized zinc transporter whose expression is regulated at the translational level via alternative splicing of its 5 untranslated region (UTR), but the role of additional cis-regulatory elements within this region has remained unexplored. Here, we characterize the functional significance of three uORFs present in the ZIF2 5UTR and elucidate their contribution to translational control and stress responses. We demonstrate that two of the three uORFs (uORF2 and uORF3) act as negative regulators of ZIF2 translation. uORF2 is constitutively translated and plays the primary role in uORF-mediated translational inhibition, whereas uORF3 functions as a fail-safe mechanism that represses translation only when uORF2 is inactive. We further show that uORF2-mediated repression depends on the amino acid sequence of the encoded peptide and identify a short motif critical for its function. Importantly, uORF-mediated regulation of ZIF2 is physiologically relevant in planta: plants carrying mutations that abolish uORF2 translation accumulate higher levels of ZIF2 protein and display increased tolerance to endoplasmic reticulum stress. Together, our findings reveal a uORF-based fail-safe regulatory mechanism that modulates ZIF2 expression, contributes to plant stress adaptation, and is consistent with translational inhibition mediated by peptide-dependent ribosome stalling.

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

Tuber shape and size are important traits in potato. Although several factors governing tuberization have been well studied, the molecular mechanisms regulating the tuber shape and size are still elusive. Here, we demonstrate that suppression of miR166, which targets class III HD-ZIP transcription factors, alters tuber morphology and productivity in potato. Target mimicry of miR166 (MIM166) in Solanum tuberosum ssp. Andigena resulted in elongated, pigmented tubers with reduced yield under short-day conditions. Transcriptome profiling of the tuberizing stolons - swollen head vs. stalk revealed differential expression of auxin-, cytokinin-, and gibberellin-associated genes, consistent with altered hormone levels. The coloured tubers from MIM166 line exhibited differential accumulation of cyanidin 3-glucoside, pelargonidin 3-glucoside, and delphinidin 3-glucoside. Tuber productivity in these lines was reduced possibly due to the decreased expression of SWEET11b and distorted vascular structures. Further, the miR166 target-StREVOLUTA exhibited dynamic expression during stolon-to-tuber transition, and could modulate StYUCCA7 expression, an auxin biosynthesis gene. Increased pigmentation and auxin accumulation in MIM166, reduced expression of auxin biosynthesis genes in REVOLUTA antisense, and low tuber yield collectively suggest miR166-REV as a regulatory module of auxin homeostasis in differentiating stolons that influences tuber morphology. These results reveal a previously unrecognized miRNA-mediated pathway governing storage organ shape, and extends the functional scope of the miR166-HD-ZIP III module beyond organ polarity. Significance statementLimited studies describe the molecular mechanisms regulating tuber shape in storage organ development. Here, we show that the miR166-REVOLUTA-auxin network has significant regulatory roles in potato development, influencing tuber shape, pigmentation, and productivity. These findings provide insights into molecular control of tuber morphology, uncover hitherto unknown functions of the miR166-REVOLUTA module in tuber crops, and broadens our understanding of miR166-mediated developmental regulation in plants.

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.

Self-incompatibility (SI) is the single most important mechanism utilized by flowering plants to avoid self-pollination, thus preventing inbreeding and promoting outcrossing. Many plant SI systems are genetically controlled by a multi-allelic S-locus, containing two tightly linked genes that encode the female and male S-determinants. When pollen lands on a "self" pistil, interaction between cognate female and male S-determinants induces an SI signalling response, resulting in the failure of self-fertilization. Here, we review currently known SI systems that utilize receptor-ligand interactions to control pollen rejection on the stigma surface. Although detailed molecular and cellular information is only known for the SI systems in the Brassicaceae and Papaveraceae, it is apparent that the S-determinants of other SI systems (e.g. in the Poaceae and the Convolvulaceae) are likely to also utilize receptor-ligand interactions to prevent self-fertilization. Strikingly, although most of these systems all appear to utilize cysteine rich proteins (CRPs) as ligands to induce an SI response, only one of these receptors is a receptor-like kinase (RLK); the other "receptors" identified to date are proteins of unknown function, which we propose to be atypical receptors (ATRs). Although many of these receptors were identified some time ago, their atypical nature raises many questions, including how they function mechanistically, how they evolved and whether they are found in other plant cell-cell communication systems. Significance StatementSelf-incompatibility involves the precise recognition and rejection of incompatible pollen, often using a receptor-ligand type of interaction between male and female S-determinants. In this review we compare several S-determinants that appear to function as novel, atypical "receptors" (ATRs), with no kinase- or other distinct domains. We propose that the discovery of these novel "receptors" suggests that further, as yet, unidentified ATRs could be more widely utilized in angiosperms than currently appreciated.

Plant genes can each produce multiple RNA isoforms, but little is known about when and how they are produced, or their functions. We conducted a detailed time course analysis of rapid changes in abundance of RNA isoforms in response to transient water deficit in Arabidopsis thaliana seedlings. We identified 95,104 transcripts in total, 21,935 of which were differentially expressed. Strikingly, 1,258 differentially expressed genes were identified only by means of RNA isoform analysis and would have been missed by conventional analysis. Several hundred genes exhibited a very rapid and reversible switch in the most abundant RNA isoform, with timings corresponding to defined changes in seedling water potential. In most cases it is predicted that RNA isoform switching would generate new protein products. Many of these genes encoded proteins of RNA metabolism and splicing, while others potentially function more directly in the response to water deficit. We propose a model in which very rapid RNA processing mechanisms come into play within minutes of imposition of the stress, changing the RNA isoform population including RNAs that encode components of the RNA processing apparatus itself. These changes together with subsequent changes in transcription facilitate further changes in the RNA isoform population as part of the stress and recovery responses. More broadly we propose that responses to abiotic stress involve substantial changes in gene function through the production of RNA isoforms, and we present a detailed approach to identifying such genome-wide RNA isoform switching.

In cereals such as maize, the kernel accumulates large quantities of storage compounds, including carbohydrates, lipids, and proteins, a process that requires tight regulation of nutrient transport. Seeds are composed of distinct tissues: the embryo, the endosperm, and maternal tissues that are symplastically isolated (not connected through plasmodesmata), necessitating specialized nutrient transfer mechanisms. In maize, nutrient transfer from maternal tissues to the endosperm via specialized basal endosperm transfer layer (BETL) cells is well characterized. However, nutrient transfer at the endosperm/embryo interface remains poorly understood. Consequently, the routes by which maternal carbon-derived sugars support embryo growth are still unclear. Our previous transcriptomic profiling uncovered a novel Endosperm domain Adjacent to the embryo Scutellum (EAS) with strong enrichment for transporter genes. Notably, genes encoding three sugar transporters from the SWEET (Sugars Will Eventually be Exported Transporters) family are highly and preferentially expressed in the EAS, suggesting the existence of a specialized sugar transfer mechanism at this interface. We show that the ZmSWEET proteins encoded by these genes are membrane-localized sucrose transporters and are functionally important for kernel development. A gene-edited triple zmsweet14a/14b/15a knock-out mutant exhibits reduced kernel weight and embryo size, significantly decreased embryo oil accumulation at maturity, and altered carbon partitioning within the kernel. In addition to these defects, mutant kernels display a significant reduction in primary root length during germination, indicating either lasting physiological consequences of disrupted sucrose transport during seed development or an additional role for these SWEET transporters during germination. Together, our findings demonstrate that sucrose transport at the endosperm/embryo interface is critical for proper carbon allocation, embryo development, and seed vigor, and identify the EAS as a key functional domain and potential target for improving seed composition.

Plant immunity relies on the detection of microbes and the rapid activation of intracellular defense pathways. Catalyzed by protein kinases and E3 ubiquitin ligases, respectively, phosphorylation and ubiquitination are among the most abundant post-translational modifications that regulate immune pathways. It has been well established that members of the receptor-like cytoplasmic kinase (RLCK) and plant U-box E3 ligase (PUB) families are critical components of plant immune signaling. Interestingly, a group of proteins that contain both an RLCK domain and a PUB domain has been conserved throughout plant evolution, referred to as subgroups RLCK-IXb and PUB-VI within their respective families. While very little is known about these proteins, evidence from multiple independent studies indicates that orthologous PUB-VI/RLCK-IXb proteins in potato, tomato, Nicotiana benthamiana, and Arabidopsis thaliana associate with diverse pathogen effectors from the oomycete pathogen Phytophthora infestans, bacterial pathogen Ralstonia pseudosolanacearum, and the mirid bug Apolygus lucorum, suggesting that they may be critical virulence targets or components of the immune response. However, the biochemical activities of these proteins and how they contribute to plant health remain poorly defined. Here, we introduce the PUB-VI/RLCK-IXb clade in Arabidopsis, focusing on PUB32, PUB33, and PUB50. We show that PUB33 exhibits dual kinase and E3 ubiquitin ligase activities that are inversely regulated by autophosphorylation at Thr333. PUB33 forms homomers and heteromers with PUB32 which attenuate PUB33 catalytic activity. Although we did not observe clear defects in innate immune signaling in pub32, pub33, or pub50 mutants, we found that overexpression of PUB33 can suppress cell death triggered by the R. pseudosolanacearum effector RipV1 in N. benthamiana. Moreover, PUB33 directly ubiquitinates RipV1 in vitro and reduces RipV1 accumulation in planta, suggesting that it functions as part of the immune response against R. pseudosolanacearum.

Wood is the greatest reservoir of terrestrial biomass and an essential carbon sink. Formed of xylem, it is derived from the cambium, a meristematic zone within plant stems from which phloem also forms. In Arabidopsis, cell division within the cambium is promoted by three major factors: auxin, cytokinin, and the TDIF-PXY ligand-receptor pair. Meristems and other stem cell populations are typically regulated by a balance between cell division-promoting factors and those that repress cell division to control meristem size, however few factors with cambium-repressing activity are known. Here we combined transcriptomics and transcriptional network analysis, which led to identification of related homeodomain zinc-finger transcription factors, ATHB23, ATHB30, and ATHB34, that repress cambium activity. These factors inhibit cambium activity by directly binding of promoters from a subset of auxin, cytokinin and TDIF-PXY transcriptional target genes, resulting in attenuation of their transcription. Our findings thus reveal a new mechanism underpinning balanced cambium activity.

Malate is a central metabolite in plant energy metabolism and biosynthesis and serves as a major carrier of carbon and reducing equivalents between chloroplasts, the cytosol, and mitochondria. However, how individual malate-converting systems contribute to physiology in specific subcellular compartments remains incompletely understood. Here, we investigated the impact of combined loss of mitochondrial malate dehydrogenase (MDH) and NAD-dependent malic enzyme (NAD-ME) activity in Arabidopsis thaliana by integrating reverse genetics, physiological analyses, transcriptomics, quantitative proteomics, and metabolite profiling. Specifically, we generated triple mutants (mdh1xme1xme2) lacking the predominant mitochondrial isoform MDH1 together with both NAD-ME subunits, thereby reducing overall mitochondrial malate conversion capacity. By growing plants under contrasting photoperiod and irradiance regimes to vary photosynthetic demand on malate-linked fluxes, we uncovered a conditional phenotype that was most pronounced under short-day/low-light conditions. Under these conditions, mdh1xme1xme2 exhibited impaired growth and photosynthetic performance, accompanied by cytosolic redox imbalance and altered chloroplast ultrastructure. Transcriptomic profiling revealed that low light unmasks a dawn-phase bottleneck in establishing photosynthetic and redox homeostasis. Consistent with this, the low-light plastid proteome revealed a reallocation away from chloroplast translation and photosynthetic capacity toward proteome maintenance, photoprotection/repair, and iron/ROS management, consistent with a protective acclimation state that nevertheless constrains carbon gain under energy limitation. Low light also triggered C/N imbalance and ammonium accumulation in the mutants. In contrast, increasing irradiance or extending the photoperiod largely alleviated these defects. Together, our results identified mitochondrial malate conversion capacity as a key control point coupling respiratory energy supply and redox homeostasis to photosynthetic metabolism when photosynthetic energy input is limiting.