AbstractLaticifers, the coenocytic cells responsible for latex production, represent one of the most extreme examples of continuous and polarized growth in plants. Their unique architecture, extending over large distances, requires tight coordination between wall extensibility, cytoplasmic organization, and hormonal control. Here, we characterized the poor in latex 13 (pil13) mutant of Euphorbia lathyris which, similar to the previously described pil1 and pil6 mutants, is defective in laticifer expansion during post-embryonic phases of plant growth. Through comparative transcriptomic analyses of these three pil mutants, we identified a core set of 266 differentially expressed genes (DEGs) that define a transcriptomic signature associated with laticifers. We further identified a regulatory network linking two HD-Zip I transcription factors, HAT7 and ATHB13, with brassinosteroid (BR) signaling in the mediation of laticifer growth. This HAT7/ATHB13–BR module regulates the expression of laticifer DEGs involved in cell wall remodeling. Consequently, we found that laticifer cell walls are highly enriched in low-esterified homogalacturonan (HG) and branched rhamnogalacturonan-I (RG-I) pectins. Whole-mount immunolocalization revealed that laticifer cell walls exhibit a peculiar spatial segregation of pectic polysaccharides. These pectins follow a characteristic complementary, non-overlapping deposition pattern along the laticifer longitudinal axis, presumably conferring both elasticity and mechanical integrity. This modular pectin assembly appears compatible with the high flexibility and mechanical sinuosity required for growing of laticifers through host tissues, while accommodating internal turgor pressure changes derived from latex accumulation. Our findings define the laticifer as a cellular model in which wall remodeling, cell growth, turgor pressure, and hormonal signaling are mechanistically coupled.Read more

AbstractIn eukaryotes, autophagy is an evolutionarily conserved cellular degradation and recycling process that is tightly associated with cellular nutrient status. The energy sensor SNF1-RELATED KINASE 1 (SnRK1) is thought to sense nutrient starvation and induce autophagy, but how it regulates autophagosome formation in plant cells remains largely unknown. Here, we show that the SnRK1 α-catalytic subunit KIN10 regulates autophagosome formation in Arabidopsis (Arabidopsis thaliana) by interacting with and phosphorylating AUTOPHAGY-RELATED PROTEIN 13a (ATG13a), a key component of the ATG1a–ATG13a protein kinase complex involved in autophagy initiation. KIN10 directly phosphorylates ATG13a in vitro and in vivo, inhibiting its ubiquitination and increasing its abundance. Moreover, we identified ten serine residues in ATG13a that are important for KIN10-mediated phosphorylation. Transgenic plants accumulating a nonphosphorylatable ATG13a variant with mutations of these ten residues were less tolerant of starvation than ATG13a-overexpressing plants and displayed accelerated leaf senescence and lower autophagic flux. The KIN10-mediated phosphorylation of ATG13a did not affect ATG1a–ATG13a complex formation; instead, it facilitated ATG1a phosphorylation, thereby promoting autophagosome formation. Therefore, our findings suggest that the KIN10–ATG13a module modulates SnRK1-mediated autophagosome formation, likely representing a key step in integrating energy sensing and autophagy initiation in plant cells.Read more

AbstractStilbenes are inducible plant specialized metabolites, best exemplified by resveratrol, a phytoalexin that links environmental perception, defense responses, and phenylpropanoid resource allocation. Stilbene accumulation may be controlled at three mechanistically distinct levels: signaling cascades that connect environmental and hormonal cues to kinase-dependent activation; combinatorial transcriptional control, which governs the response amplitude and specificity of stilbene synthase (STS) genes; and branch selection together with product disposition, which shapes the final metabolic output. Within this framework, MYB and WRKY proteins serve as major transcriptional nodes as activators or repressors, yet their regulatory effects depend on combinatorial interactions, phosphorylation events, competitive promoter occupancy, and metabolic context. We explicitly distinguish strongly supported mechanisms, defined by genetic perturbation, direct target validation, and metabolite quantification from emerging models built primarily on transient expression assays or co-expression correlations. This distinction avoids equating regulators supported by disparate levels of experimental evidence, identifies the most robust principles governing pathway control, and highlights experiments required to turn descriptive associations into causal mechanistic models. We further highlight conceptual insights extending beyond stilbene metabolism to inducible specialized metabolism in general, and outline research priorities for comparative genetics, cell-resolved analysis, and predictive pathway engineering.Read more

AbstractRIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE (RUBISCO) and the Calvin-Benson-Bassham (CBB) cycle assimilate most of the inorganic carbon on earth. Conserved CP12 proteins control diurnal C-assimilation of the CBB cycle. Their function originated in cyanobacteria that deactivate GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE (GAPDH) and PHOSPHORIBULOKINASE (PRK) during darkness by redox-regulated complex-formation with CP12. This mechanism indicates metabolic regulation of C-assimilation. We measured C-assimilation in vivo at the day/night transition by dynamic 13CO2 labeling and 1-C-positional monitoring of 13C-incorporation into 3-phosphoglycerate (3PGA) and aspartate of Synechocystis sp. PCC 6803 grown with high (5%) CO2 supply. Unlike wild type, a Δcp12 mutant accumulated ribulose-1,5-bisphosphate (RuBP) and assimilated 13CO2 in darkness. We validated these observations by proteome measurements and orthogonal quantifications of RUBISCO abundances and specific activities. Complementation of Δcp12 with native CP12 and point-mutants of its GAPDH- and PRK-binding domains demonstrated that defective complex formation is causal for aberrant C-assimilation during darkness and that RuBP accumulation correlates significantly with the time-shifted 13C-incorporation into 3PGA. RUBISCO carboxylase activity is present throughout the diurnal cycle and depends on CP12-controlled substrate availability. 13C-positional analyses suggest dysregulated RuBP regeneration in Δcp12 during darkness via futile metabolic cycling through gluconeogenesis and the oxidative pentose phosphate path. We propose that blocked regeneration and rapid depletion of RuBP contributes to the regulation of RUBISCO activity at day-night transitions.Read more

AbstractThe plant cell wall, composed primarily of polysaccharides, is essential for normal plant growth and development. Many of these polysaccharides are synthesized within and subsequently secreted via the endomembrane system. Mutations in genes including MURUS3 (MUR3), YPT/RAB GTPase-interacting protein 4 homologs (YIP4a/YIP4b), and ECHIDNA (ECH) result in dwarf plants, which is believed to be a consequence of impaired cell wall polysaccharide secretion. Here, we identify a LMBR1 (Limb Development Membrane Protein 1)-like membrane protein (LMBD2) and find that loss of LMBD2 suppresses the secretion and growth defects in the mur3, yip4a/yip4b, and ech mutants. LMBD2 localizes to the trans-Golgi apparatus and trans-Golgi network (TGN)/early endosome (EE), where it interacts with YIP4b and orchestrates TGN/EE-mediated trafficking. Furthermore, LMBD2 can competitively attenuate the interaction of YIP4b and ECH. Our findings establish LMBD2 as a component of endomembrane trafficking homeostasis in plant cells and offer new insights into endomembrane dynamics and their influence on plant development.Read more

AbstractRNA silencing is a conserved defense mechanism that protects plants against viruses, bacteria, fungi, and oomycetes. Although diverse pathogens have evolved effectors that suppress host RNA silencing, whether powdery mildew fungi exploit this strategy and the underlying molecular mechanisms remain unknown. Here, we identify CSEP0004, a secreted effector from the barley powdery mildew fungus Blumeria hordei, as an RNA silencing suppressor that promotes fungal virulence. CSEP0004 physically interacts with the evolutionarily conserved DEAD-box RNA helicases Hordeum vulgare RNA helicase 37 (HvRH37) and HvRH52. Genetic analyses demonstrate that HvRH37 and HvRH52 positively regulate chitin-triggered reactive oxygen species production, resistance to B. hordei, and small RNA accumulation in barley, while their Arabidopsis homologs AtRH11/37/52 perform conserved functions in small RNA accumulation. Mechanistically, HvRH37 and HvRH52 associate with HvAGO1, a core component of the RNA-induced silencing complex, and function as biochemically active RNA helicases capable of binding and unwinding double-stranded RNA. CSEP0004 disrupts the HvRH37/52–AGO1 association and inhibits HvRH37/52 helicase activity in a dose-dependent manner. Together, our findings uncover a previously unrecognized mechanism by which a fungal effector suppresses host RNA silencing through targeting conserved DDX3-like DEAD-box RNA helicases, establishing these helicases as key regulators of RNAi-mediated immunity and identifying RNA helicase-dependent RNA remodeling as a critical regulatory node exploited by fungal pathogens.Read more