Plant Molecular Biology最新文献

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.

High temperature (HT) is a critical abiotic factor that restricts plant growth and development. The role of abscisic acid (ABA) in stress tolerance is well established, and ABA 8'-hydroxylase (ABA8ox), a key enzyme in ABA degradation, is crucial for plant responses to abiotic stress. In this study, a CsABA8ox1-deficient mutant, yf-343, and its wild-type counterpart, BY, were subjected to continuous HT treatment to assess phenotypic, physiological, and transcriptomic changes. Under HT, ABA accumulation increased in BY and yf-343, with significantly higher levels in the yf-343 mutant. Exogenous ABA application accelerated leaf yellowing in BY and triggered pronounced leaf senescence and cell death in yf-343. HT treatment also increased the activities of superoxide dismutase and peroxidase, elevated ABA and malondialdehyde content, and simultaneously inhibited catalase activity and photosynthetic rate. Comparative RNA sequencing (RNA-seq) revealed that genes associated with plant hormone signaling, secondary metabolite biosynthesis, starch and sucrose metabolism, phenylalanine metabolism, and the mitogen-activated protein kinase signaling pathway were differentially expressed between yf-343 and BY under 42 °C HT treatment. Among these, two genes, heat shock proteins 70 (CsHSP70) and wall-associated receptor kinase 2 (CsWAKL2), were validated through virus-induced gene silencing. Knockdown of CsHSP70 and CsWAKL2 enhanced susceptibility to HT, confirming the reliability and significance of the candidate genes involved in HT stress response identified by RNA-seq. These findings establish a strong foundation for elucidating the role of ABA8ox in cucumber resistance to abiotic stress.

Highly specific, second-generation RNA interference tools are based on artificial small RNAs (art-sRNAs), such as artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs). Recent progress includes the use of minimal-length precursors to express art-sRNAs in plants. These minimal precursors retain the minimal structural elements for recognition and efficient processing by host enzymes. They yield high amounts of art-sRNAs and remain stable when incorporated into potato virus X-based viral vectors for art-sRNA-mediated virus-induced gene silencing (art-sRNA-VIGS). However, further adaptation to new viral vector systems with reduced symptomatology is needed to improve the versatility of art-sRNA-VIGS. Here, we developed a novel platform based on tobacco rattle virus (TRV)-a widely used viral vector inducing minimal or no symptoms-for the delivery of art-sRNAs into plants. TRV was engineered to express authentic amiRNAs and syn-tasiRNAs from minimal precursors in Nicotiana benthamiana, resulting in robust and highly specific silencing of endogenous genes. Notably, the expression of syn-tasiRNAs through TRV conferred strong resistance against tomato spotted wilt virus, an economically important pathogen. Furthermore, we established a transgene-free approach by applying TRV-containing crude extracts through foliar spraying, eliminating the need for stable genetic transformation. In summary, our results highlight the unique advantages of minimal precursors and extend the application of art-sRNA-VIGS beyond previously established viral vector systems, providing a scalable, rapid and highly specific tool for gene silencing.

Legumes are the second most important food crop after cereals for the world population. It is a significant protein source for developing countries and integral to global food security. However, various agroecological constraints and biotic and abiotic factors often compromise the production of pulses. Legumes are long-term neglected crops worldwide and follow traditional breeding, leading to a time-consuming, labor-intensive, less economically feasible program associated with linkage drag. Recent sequencing attempts in the twenty-first century, with the development of an enormous repertoire of genetic and genomic resources, allowed scientists to accelerate the improvement of legumes with modern genome editing tools. One such promising tool is CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), which has revolutionized and transformed the landscape of genetic engineering. The emergence of CRISPR/Cas systems has redefined precision breeding, offering unprecedented control over genome manipulation in legume crops. It has tremendous potential for crop improvement and can precisely make changes at genomic locations with incredible accuracy. Therefore, identifying the desired genes and their precise manipulation has enormous implications for legume crop improvement. This review will give an overview of the genome editing tools available for crop improvement and the efficiency of different transformation methods in legume crops. It will also discuss the current status of genome editing in legume crops, including challenges and future perspectives.