The establishment of a specific pollen wall pattern is essential for pollen development in flowering plants. This process largely depends on the formation of a temporary wall surrounding microspores in tetrads, known as the primexine, which is primarily composed of Arabinogalactan proteins (AGPs) and polysaccharides. NEF1, a gene identified two decades ago in Arabidopsis thaliana, is required for primexine formation; however, its precise functional mechanism remains unclear. In this study, we generated a nef1 knockout mutant (nef1-2) that exhibits a complete absence of primexine and male sterility. Immuno-fluorescence analysis revealed severe defects in primexine components, including pectin, xylan, and AGPs, in the nef1-2 mutant. Notably, AGPs accumulated abnormally in the tapetum, where material transport was severely disrupted, suggesting that defective AGP accumulation may underlie the primexine deficiency. Fluorescence observations demonstrated that NEF1 is specifically expressed in the tapetum and localizes to the Endoplasmic Reticulum (ER) and Golgi apparatus. Furthermore, when GFP was fused to the N-terminus of NEF1, the protein failed to localize to the Golgi apparatus. Consequently, transgenic plants expressing pNEF1:GFP-NEF1 displayed impaired deposition of both primexine polysaccharides and AGPs, further confirming that proper NEF1 localization is essential for its function. This study improves our understanding of NEF1's role in primexine formation and highlights the contribution of AGPs in pollen wall pattern establishment.

The medicinal plant Andrographis paniculata has garnered attention for its remarkable effectiveness in treating inflammation, which is attributed to its active terpenoid compounds. However, the regulatory mechanisms governing their biosynthesis remain unknown. In this study, we showed that exposing Andrographis paniculata plants to blue light increases the contents of the main active components, andrographolide and 14-deoxyandrographolide, and promotes the expression of key genes encoding enzymes in the diterpene lactone biosynthetic pathway, including ent-copalyl diphosphate synthase (ApCPS2), geranyl(geranyl)diphosphate synthase 1 (ApGGPPS1), and ApGGPPS2. Combined transcriptomic and metabolomic analysis revealed that blue light regulates the light and jasmonic acid (JA) signaling pathways. Furthermore, we demonstrated that the transcription factor ApHY5 directly binds to the promoters of ApCPS2, ApGGPPS1, and ApGGPPS2, thereby activating transcription. In addition, ApHY5 interacted with the transcription factor ApMYC2 to enhance its function, thereby promoting the accumulation of diterpene lactones. Silencing of ApHY5 or ApMYC2 resulted in lower expression levels of genes involved in diterpene lactone biosynthesis and reduced accumulation of these components, while overexpression of either gene had the opposite effect. This study elucidates a mechanism for blue light-regulated terpenoid metabolism that involves the JA signaling pathway, offering strategies to enhance the biosynthesis of effective components in medicinal plants.

Cyanobacteria are photoautotrophic cell factories capable of converting carbon dioxide into valuable chemicals. The rate-limiting step in photosynthesis is the enzymatic dark reaction catalyzed by ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO). The carboxysome is a protein-based organelle that plays a central role in cyanobacterial carbon fixation. It consists of thousands of subunits, including hexameric and pentameric proteins, that form a shell to encapsulate the enzymes RuBisCO and carbonic anhydrase. Here, we enhanced β-carboxysome biogenesis in Synechocystis sp. PCC 6803 by overexpressing a full or partial set of endogenous β-carboxysome components. Overexpression of carboxysome proteins altered protein stoichiometry, morphology, and cellular abundance of β-carboxysomes, as well as improved photosynthetic activities and cell growth. These findings indicate that targeted carboxysome overexpression enhances photosynthetic efficiency and growth of cyanobacteria under favorable conditions. This study provides a framework for engineering cyanobacterial chassis cells as microbial cell factories and carboxysome-based CO2-concentrating mechanisms in heterologous hosts, including crop plants, to improve photosynthetic productivity for sustainable bioproduction.

Quercetin glycosides are abundant flavonoids in apple (Malus domestica) leaves; however, how their structural variation shapes plant-insect interactions remain unclear. Here, we show that quercetin glycosylation and hydrolysis underlie a biochemical arms race between apple and multiple herbivorous insects. Herbivory by Helicoverpa armigera and Spodoptera litura activated a jasmonic acid (JA)-regulated transcriptional response that selectively induced the quercetin-glycoside pathway, resulting in elevated accumulation of 5 major quercetin glycosides. Methyl jasmonate treatment and JA content-boosting transgenic plants increased quercetin glycosides, whereas altering quercetin glycoside content did not feedback to JA signaling. Functional analyses revealed strong structure-dependent variation in anti-insect activity. Quercetin 3-O-glucoside (Q3Glc), but not its isomer quercetin 3-O-galactoside (Q3Gal), deterred larval feeding. During herbivory, apple leaves hydrolyzed Q3Glc to release toxic quercetin, while insects displayed species-specific detoxification strategies. H. armigera and S. exigua re-glycosylated quercetin to regenerate Q3Glc, neutralizing toxicity, whereas S. litura and S. frugiperda lacked this ability and suffered reduced growth. These findings reveal a reciprocal metabolic interplay in which apple activates glycoside hydrolysis to deploy toxic aglycones, while insects counter-evolve re-glycosylation to attenuate toxicity. Quercetin glycosylation patterns, therefore, serve as dynamic determinants of apple defense and herbivore susceptibility, offering opportunities for breeding insect-resistant cultivars.

Salinity poses a major global threat to agriculture, severely damaging crop production and productivity. Potato (Solanum tuberosum L.) is an important food crop for ensuring food security and, as a moderately salt-sensitive crop, is extremely stressed by salinity. Melatonin (MT) is involved in various abiotic stress responses in plants. However, the detailed regulatory mechanisms by which melatonin contributes to salt stress tolerance in potatoes remain unclear. Here, we determined that an appropriate MT concentration of 10 µM significantly alleviates salt stress damage in potatoes. Transcription-metabolite association analysis identified the core transcription factor genes StMYB55 and StWRKY28, which exhibit both salt- and MT-responsive expression. Overexpressing StMYB55 enhanced salt tolerance in transgenic potato plants by increasing flavonoid accumulation. Importantly, StMYB55 promoted StF3H, StFLS, and StUGT78D2 transcription, while StWRKY28 directly enhanced StCHI expression. Additionally, StMYB55 interacted with StWRKY28 to promote StWRKY28-regulated StCHI expression and flavonoid biosynthesis in potato. Our findings reveal that the MT-mediated StMYB55-StWRKY28 transcriptional complex module regulates flavonoid accumulation, thereby improving salt stress tolerance in potatoes. These results shed light on the molecular mechanism of potato response to salinity stress, providing the basis for the genetic breeding of a melatonin-enriched salt-resistant potato variety.

Circadian clocks provide plants with an adaptive advantage by enabling them to anticipate daily environmental changes. The periodicity of circadian clocks is regulated at multiple levels of gene expression, including transcription, mRNA processing, translation, and protein modification. Numerous mRNA splicing factors have been implicated in maintaining circadian period length. However, these factors often play additional roles in transcription, making it difficult to determine whether they affect the clock through splicing-dependent or -independent mechanisms. We and others have shown that XAP5 CIRCADIAN TIMEKEEPER (XCT) and components of the PRE-MRNA-PROCESSING FACTOR 19 (PRP19) complex, including the functionally redundant PRP19A and PRP19B, physically associate and regulate both splicing and circadian rhythms. Here, our transcriptome analyses reveal that the antagonistic regulation of circadian period length by XCT and PRP19 likely occurs through splicing-independent mechanisms. Interestingly, both factors co-regulate expression of a substantial set of shared target genes involved in RNA metabolism, photosynthesis, and stress responses despite having largely distinct targets for splicing. Gene co-expression analysis followed by functional characterization identified anthocyanin biosynthesis as another process antagonistically regulated by XCT and PRP19. Nonetheless, we found genetic perturbation of anthocyanin production does not affect circadian period, suggesting that the observed correlation between anthocyanin levels and circadian period may instead reflect disruption of a shared upstream regulatory pathway. Together, our findings suggest involvement of XCT and PRP19 in the transcriptional coordination of anthocyanin biosynthesis and biological timing, expanding their known roles beyond mRNA splicing.

Abstract Seed longevity is a critical agronomic trait underpinning crop productivity, yet its regulation remains less understood due to the complexity of interacting signaling pathways. Here, we identified an aging-resistant rice (Oryza sativa L.) cultivar ZH11 that shows a higher seed germination rate and vigor, higher salicylic acid (SA) content, and lower reactive oxygen species (ROS) level than other cultivars (NJ505 and XF9) under aging treatment. Integrated multi-omics analyses revealed six key pathways—oxidative phosphorylation, plant-pathogen interactions, photosynthesis, carbon fixation, ATP-binding cassette (ABC) transport, and hormone signaling—converging on nine core genes (OsUbOR, OsA10, WRKY42, OsPKD, OsPRCW, OsYGD1-1, OsABCB14, OsPYL3, OsNPR1). Deficiency of each of the nine key genes leads to mutants with a phenotype of markedly reduced seed viability. The differential expression of these key genes across ZH11, NJ505, and XF9, together with the dose-dependent rescue of the shortened seed longevity phenotype in NJ505 and XF9 by SA, confirms that SA acts as a key bridge mediating distinct signaling pathways. The interactions between the SA receptor NPR1 (Nonexpressor of Pathogenesis-Related 1) and bZIP03/bZIP28 (Basic Leucine Zipper 03/28) were identified as potential targets and components for regulating seed longevity. Furthermore, the interactions of NPR1-Protein Phosphatase 2C 30 (PP2C30) and PP2C30-bZIP28 further underscored the role of SA signaling in the aging process of rice seeds. Our findings provide mechanistic insights into seed viability regulation and offer genetic targets for breeding crops with extended seed storage potential and quality for sustainable agriculture.

Chloroplast stress granules (cpSGs) are emerging as dynamic suborganellar condensates that play a crucial role in mediating stress response in plants. In this study, we demonstrate that the chlorophyll biosynthesis enzyme PROTOCHLOROPHYLLIDE OXIDOREDUCTASE C (PORC) localizes to cpSGs in response to acute and prolonged heat stress in Arabidopsis (Arabidopsis thaliana). While PORC promoter activity was developmentally regulated and remained unresponsive to heat, PORC protein re-localized from a diffuse chloroplast distribution into punctate structures under elevated temperatures. This condensation was reversible, translation-dependent, and absent under optimal growth conditions. Genetic disruption of PORC resulted in compromised thermotolerance, whereas overexpression enhanced photosynthetic recovery following both acute (42°C) and prolonged (35°C) heat stress. High-throughput phenotyping and chlorophyll fluorescence imaging confirmed enhanced Photosystem II (PSII) efficiency and increased post-stress growth rate. Proteomic profiling of heat-induced PORC-cpSGs revealed functional enrichment of photosystem I/II components, proteases (e.g., FtsH), and proteins involved in chlorophyll biosynthesis and photoprotection, suggesting a stress-protective role of cpSG under heat. These findings establish PORC as a key player in the chloroplast stress response, implicating cpSGs as protective hubs that facilitate the maintenance of photosynthetic integrity under elevated temperatures. Our study provides insight into chloroplast-specific biomolecular condensates, enhancing our understanding of plant stress resilience and paving the way for future studies on the regulation of their dynamics. Additionally, it highlights how components such as PORC could be utilized to develop heat-tolerant crops.