Plant Molecular Biology最新文献

Auxin is one of the major driving forces of plant development and requires careful regulation of transporter proteins to establish polar auxin transport. The PIN-FORMED (PIN) family plays a pivotal role in plant development by establishing auxin gradients that govern vascular patterning and organogenesis. However, the PIN family remains severely underexplored in Setaria viridis, a well-established model for C₄ monocots. In this study, we identified and characterized 13 PIN genes in the S. viridis genome. Phylogenetic and collinearity analyses revealed duplication events in the SvPIN1, SvPIN5 and SvPIN10 subfamilies. Structural analysis uncovered unique features, including potential pseudogenization of SvPIN5a. Expression profiling across five developmental stages unveiled the potential developmental roles of SvPINs, with SvPIN1 and SvPIN10 paralogues predominantly expressed in shoots and panicles, SvPIN2 and SvPIN9 in roots, while SvPIN5b showed leaf-enriched expression, suggesting potential involvement in leaf vascular development. Hormonal treatments in callus cultures revealed auxin-mediated upregulation of SvPIN1b, SvPIN2, SvPIN5d, SvPIN8 and SvPIN10a. Our findings provide significant insights into the role of PIN genes in S. viridis and other C₄ monocots, establishing a foundation for future functional studies and offering potential targets for crop improvement through auxin transport manipulation.

Thermal energy has become an increasingly severe environmental stressor to cash crop production worldwide because of global warming. Annexins, proteinaceous protectants against abiotic stress, are multifunctional proteins capable of peroxidase- and Ca²⁺-dependent and Ca²⁺-independent binding to or insertion into membranes. Annexins in plants belong to the annexin D family and are further clustered into six phylogenetic clades on the basis of their structural diversity. A previous study in peanut identified six annexins, but their thermotolerance functions remain unknown. In this study, we report that AhANN6, a peanut annexin, confers heat resistance in Escherichia coli and Arabidopsis when overexpressed. AhANN6 expression led to positive responses to drought stress, ABA supplementation, and heat stress in leaves and was developmentally regulated during germination and pegging. The AhANN6-YFP fusion protein was targeted to the plasma membrane of tobacco cells, suggesting that AhANN6 is localized in the cell membrane. AhANN6-overexpressing E. coli exhibited better growth under heat stress and oxidative stress, validating the molecular function of AhANN6 against abiotic stress. AhANN6-overexpressing Arabidopsis also presented increased heat resistance during vegetative growth. The decreased response of electrolyte leakage in the transgenic Arabidopsis to heat stress indicates potentially improved membrane stability as a result of AhANN6 overexpression. Additionally, the overexpression of AhANN6 in Arabidopsis led to increased expression of AtPOD and AtAPX, key enzyme-encoding genes involved in ROS scavenging, suggesting that AhANN6 is involved in maintaining ROS detoxification. Our findings suggest that AhANN6 plays a crucial role in protecting cell membrane integrity and promoting vegetative growth under adverse environmental stressors.

The gynoecium, a highly specialized structure in flowering plants, ensures their high reproductive success through the control of different crucial steps spanning from ovule protection to fertilization and seed maturation and dispersion. Multiple bpc mutants show reduced vigor, small fruit size and height, a reduced number of seeds and problems in septum fusion and formation. BPCs are known to be involved in the regulation of key factors involved in plant development, and they are thought to function both as activators and repressors of target gene expression. Here we showed that gynoecium development is affected in different multiple mutants of the Basic PentaCysteine (BPC) genes, where the septum fails to develop properly, and that BPCs of class I and II regulate the expression of different genes involved in carpel development and phytohormonal pathways regulation. Considering the fundamental role of the gynoecium, which affects the reproductive success of the plants, we focused on understanding which genes could be putative direct targets of BPCs and thus involved in gynoecium development. We demonstrated that SPATULA and NO TRANSMITTING TRACT (NTT), which play pivotal roles in carpel and transmitting tract development, are downregulated. As a consequence, bpc multiple mutants fail to properly develop the septum and the transmitting tract. Interestingly, among the downregulated genes, we also found PIN-LIKES3, whose promoter can be directly bound by BPCs, which is an auxin efflux carrier that regulates and controls cytoplasmic availability of auxin and could also contribute to various growth processes.

Rapid alkalinization factors (RALFs) are short-chain polypeptides that regulate methyl-esterified pectin accumulation and reactive oxygen species (ROS) metabolism in pollen tubes across diverse plant species. In pear (Pyrus) self-incompatibility (SI), pollen tube polar growth is inhibited by increased apical methyl-esterified pectin content and disrupted apical ROS gradients, while pear RALF family members show no expression response to SI, indicating they are not inherently involved in the SI regulatory pathway. We investigated pollen tube-highly expressed pear RALFs (PbrRALF2/5/6/7/9/10), among which PbrRALF5/10 interact with pollen tube-expressed PbrLRX7/8/10/11 and negatively regulate apical methyl-esterified pectin content (in contrast to PbrRALF6, which competitively binds PbrLRX8 with PbrRALF10 and exerts opposite pectin-regulatory effects) and positively regulate ROS accumulation via the PbrANX/PbrBUPS receptor kinase pathway. Exogenous application of recombinant PbrRALF5/10 (rPbrRALF5/10) during pear SI responses achieved phenotypic rescue in vitro: it significantly reduced apical methyl-esterified pectin content (not to self-compatible levels), re-established the ROS polarity gradient, alleviated SI-induced nuclear DNA degradation, and alleviated incompatible pollen tube growth inhibition. These findings, based on exclusive in vitro experiments, clarify that PbrRALF5/10, while not participating in the SI pathway, mitigate SI-induced pollen tube defects by regulating pectin and ROS, providing insights into their potential for improving pear reproductive success. Notably, in vivo validation remains critical to fully support these conclusions, as no in vivo evidence was obtained to confirm the function of PbrRALF5/10 in alleviating SI under natural pollination conditions.

Cation/H⁺ exchangers (CAXs) mediate vacuolar Ca²⁺ sequestration and are critical for maintaining cytosolic Ca²⁺ homeostasis in plants. Arabidopsis CAX1, a member of the Ca²⁺/Cation Antiporter (CaCA) superfamily, features a modular architecture comprising two pseudosymmetrical domains separated by a cytosolic loop called the acidic motif. CAX1 is also regulated by a cytosolic N-terminal autoinhibitory domain. To define the structural basis of CAX1 activity, we characterized truncated constructs of the N-terminal half of CAX1, comprising a 6-transmembrane (TM) module lacking the autoinhibitory domain (½N-sCAX1), using yeast complementation, structural modeling, and protein interaction studies. The ½N-sCAX1 monomer folded into a stable topology but it failed to interact with itself or with full-length CAX1, or confer transport activity. Functional reconstitution required tethering two ½N-sCAX1 modules via the acidic motif or removal of TM1, which restored partial Ca²⁺ transport in yeast. Protein interaction assays revealed that the autoinhibitory domain contributes to ½N-CAX1 dimerization, while TM1 interferes with complex assembly. Structural models demonstrated that correct alignment of the conserved GNxxE motif across ½N-sCAX1 monomers, either by artificial tethering or potentially by higher order hexameric oligomerization, is essential to reconstruct a functional Ca²⁺-binding pocket. These findings show that CAX1 functionality depends on specific topological constraints and modular interactions that guide formation of CAX1 halves. Our results highlight how architectural features such as TM1 and the autoinhibitory domain regulate transporter assembly and activity, offering insight into CaCA biogenesis and providing a framework for engineering transporters with tailored functional properties.

Glyoxalase I (GLYI) constitute the first enzyme of the glyoxalase pathway which is a two-step reaction to convert methylglyoxal (MG), an inherent cytotoxin to D-lactate. In plants, multiple members of the glyoxalase pathway genes have been reported. However, not all exhibit glyoxalase activity. OsGLYI-10 is one such member from rice GLYI family which we report here, to lack GLYI enzymatic activity. Instead, OsGLYI-10 shows high structural homology to glutathione-S-transferase (GST) proteins and exhibits GST activity. Further, we found OsGLYI-10 to be highly expressed in seeds, its expression starting at the milk stage, reaching its maximum in the mature seed and finally disappearing after four days of imbibition. Importantly, through molecular docking and site-directed mutagenesis studies, we showed that GLYI activity can be reinstated to some extent via the introduction of a 10 amino acid stretch as well as substitution of certain amino acids in OsGLYI-10. Further increase in GLYI activity could be achieved through the substitution of Met with Tyr at 55th position, restoring 35% activity in OsGLYI-10 relative to a functionally active and highly efficient GLYI, OsGLYI-8 enzyme from rice. Our findings therefore, suggest OsGLYI-10 to be a reminiscent of GLYI enzyme that has diverged in its catalytic function over the course of evolution to adopt newer activities and roles in cellular physiology.