Plant Molecular Biology最新文献

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.

Tiller and spikelet numbers are important agronomic traits affecting wheat grain yield, but the molecular mechanisms controlling these traits are largely unknown. We have identified a gene (Wheat Tiller-1, WT-1) that regulates numbers of these two very important agronomic traits. While trying to understand the early events of tiller development in wheat, cross section analysis of the crown region showed that differentiation of the tiller buds and apical meristem into spikelets occurs during early seedling stages. The gene was identified by VIGS silencing using sequence around the VHIID motif of the LS gene of tomato that controls branching. VIGS gene silencing, first using the tomato sequence and then from the gene identified from wheat resulted in uniculm and reduce tiller number phenotype. Overall, the WT-1 showed only 37.6% predicted protein similarity to the LS gene although the VHIID motif was conserved. The gene has three structural copies one each on the three wheat homoeologous group 7 chromosomes. Although share > 98% sequence similarity, the three gene copies showed different expression pattern in various tissues and growth stages. Silencing of the gene via stable RNAi showed reduction in both tiller and spikelet number. SEM analysis of the RNAi plants showed that silencing of WT-1 reduced the tiller bud initiation. Among the progeny of independent RNAi events, variation in both spikelet and tiller numbers correlated with the level of reduction in the gene expression, showing role of the gene in controlling tiller number and spikelet number per spike.

Sugarcane holds significant economic importance in sugar and biofuel production. Despite extensive research, understanding highly quantitative traits remains challenging due to its complex genomic landscape. We conducted a multiomic investigation to elucidate the genetic architecture and molecular mechanisms governing sugarcane sucrose accumulation. Using a biparental cross and a genetically diverse collection of sugarcane genotypes, we evaluated the soluble solids (Brix) and sucrose content (POL) across various years. Both populations were genotyped using a genotyping-by-sequencing approach. Genotype‒phenotype associations were established using a combination of traditional linear mixed-effect models and machine learning algorithms. Furthermore, we conducted an RNA sequencing experiment on genotypes exhibiting distinct Brix and POL profiles across different developmental stages. Differentially expressed genes (DEGs) potentially associated with variations in sucrose accumulation were identified. All findings were integrated through gene coexpression network analyses. Strong correlations among the evaluated characteristics were observed, with estimates of modest to high heritabilities. By leveraging a broad set of single-nucleotide polymorphisms (SNPs) identified for both populations, we identified several SNPs potentially linked to phenotypic variance. Our examination of genes close to these markers facilitated the association of such SNPs with DEGs for contrasting sucrose levels. Through the integration of these results with a gene coexpression network, we delineated a set of genes potentially involved in the regulatory mechanisms of sucrose accumulation. Our findings constitute a significant resource for biotechnology and plant breeding initiatives. Furthermore, our genotype‒phenotype association models hold promise for application in genomic selection, offering valuable insights into the molecular underpinnings governing sucrose accumulation in sugarcane.

Perilla frutescens (L.) Britt., a traditional Chinese herb used for both medicinal and culinary purposes, contained various bioactive compounds such as volatile oils, flavonoids, and phenolic acids, which contribute to its diverse pharmacological activities. BBXs (B-box zinc finger genes), a subfamily of zinc finger proteins, play critical regulatory roles in plant growth and development, abiotic stress responses, and pigment accumulation. However, research on the PfBBXs in P. frutescens remains limited. In this study, 31 PfBBXs were identified from the P. frutescens genome. Their protein physicochemical properties, phylogeny, conserved domains, motifs, cis-acting elements, and expression patterns were systematically analyzed. Phylogenetic analysis classified PfBBXs into five subfamilies, with similar conserved motifs and gene structures within each subfamily but notable divergence among them. Promoter regions of PfBBXs were enriched in cis-regulatory elements related to light responsiveness, stress responses, and phytohormone signaling. Different light intensities significantly affected the leaf area and the accumulation of anthocyanins and flavonoids. Integrated metabolomic and transcriptomic analyses revealed that light intensity modulated the biosynthesis of flavonoids and anthocyanins. Transcriptomic screening identified five highly light-responsive PfBBXs (PfBBX10, 12, 13, 17, and 18), whose light-induced expression patterns were further validated by qRT-PCR. Among them, PfBBX10, 12, and 17 exhibited significant positive correlations with anthocyanin and flavonoid contents, suggesting their pivotal roles in light signaling and secondary metabolism regulation. This study lays a foundation for functional characterization of PfBBXs in P. frutescens particularly in light signal transduction and anthocyanin accumulation.