The Plant Journal最新文献

The implementation of genome editing strategies in grapevine is the easiest way to improve sustainability and resilience while preserving the original genotype. Among others, the Mildew Locus-O (MLO) genes have already been reported as good candidates to develop powdery mildew-immune plants. A never-explored grapevine target is NPR3, a negative regulator of the systemic acquired resistance. We report the exploitation of a cisgenic approach with the Cre-lox recombinase technology to generate grapevine-edited plants with the potential to be transgene-free while preserving their original genetic background. The characterization of three edited lines for each target demonstrated immunity development against Erysiphe necator in MLO6-7-edited plants. Concomitantly, a significant improvement of resilience, associated with increased leaf thickness and specific biochemical responses, was observed in defective NPR3 lines against E. necator and Plasmopara viticola. Transcriptomic analysis revealed that both MLO6-7 and NPR3 defective lines modulated their gene expression profiles, pointing to distinct though partially overlapping responses. Furthermore, targeted metabolite analysis highlighted an overaccumulation of stilbenes coupled with an improved oxidative scavenging potential in both editing targets, likely protecting the MLO6-7 mutants from detrimental pleiotropic effects. Finally, the Cre-loxP approach allowed the recovery of one MLO6-7 edited plant with the complete removal of transgene. Taken together, our achievements provide a comprehensive understanding of the molecular and biochemical adjustments occurring in double MLO-defective grape plants. In parallel, the potential of NPR3 mutants for multiple purposes has been demonstrated, raising new questions on its wide role in orchestrating biotic stress responses.

Proanthocyanidin, synthesized in the endoplasmic reticulum and stored in vacuoles, is key to grape and wine quality. Glutathione S-transferase (GST) plays a crucial role in proanthocyanidin accumulation. However, little is known about the mechanisms of GSTs in the process. Here, we found that a TAU-type GST VvGSTU60 is required for proanthocyanidin accumulation in Vitis vinifera. Gene expression analysis revealed a favorable correlation between the expression pattern of VvGSTU60 and proanthocyanidin accumulation in the seed of V. vinifera. We discovered that the overexpression of VvGSTU60 in grapes resulted in a significant increase in proanthocyanidin content, whereas the opposite effect occurred when VvGSTU60 was interfered with. Biochemical analysis indicates that VvGSTU60 forms homodimers and heterodimers with VvGST1. Interestingly, we also found that VvGSTU60 interacts with VvDTX41B, a MATE transporter protein localized on the tonoplast. Heterologous expression of VvDTX41B in the Arabidopsis tt12 mutant rescues the proanthocyanidin deficiency, and interfering with VvDTX41B expression in grapes remarkably reduces the accumulation of proanthocyanidin. In addition, compared with the VvGSTU60-OE callus, the content of proanthocyanidin in VvDTX41B-RNAi + VvGSTU60-OE callus was significantly decreased but higher than that in VvDTX41B-RNAi callus. The results suggest that VvGSTU60 and VvDTX41B are coordinated in proanthocyanidin accumulation. These findings offer new insights into the accumulation mechanisms of proanthocyanidin in plants and provide the molecular basis for optimizing grape quality and wine production.

Establishing reciprocal symbiosis with arbuscular mycorrhizal (AM) fungi is an important evolutionary strategy of most terrestrial plants to adapt to environmental stresses, especially phosphate (Pi) deficiencies. Identifying the key genes essential for AM symbiosis in plants and dissecting their functional mechanisms will be helpful for the breeding of new crop varieties with enhanced nutrient uptake efficiency. Here, we report a nuclear factor YC subunit-encoding gene, OsNF-YC3, whose expression is specifically induced in arbuscule-containing cells, plays an essential role in AM symbiosis. Knockout of OsNF-YC3 resulted in stunted arbuscule morphology and substantially decreased P accumulation, while overexpressing OsNF-YC3 enhanced mycorrhization and Pi uptake efficiency. OsNF-YC3 is directly regulated by OsPHRs, the major regulators of Pi starvation responses. Chromatin immunoprecipitation sequencing analysis uncovered multiple genes with crucial roles in arbuscule development as its potential downstream targets, including the AM-specific Pi transporter gene OsPT11. OsNF-YC3 can form a heterotrimer with the other two NF-Y subunits, OsNF-YA11 and OsNF-YB11, in yeast. Loss of OsNF-YA11 function also severely impaired arbuscule development in its mutants. Overall, our results highlight an essential role of OsNF-YC3 and its potential interacting NF-Y subunit, OsNF-YA11, in regulating AM symbiosis and arbuscule development.

Xanthanolides, also described as seco-guaianolides, are unique sesquiterpene lactones (STLs) with diverse bioactivities. Most of xanthanolides are 12,8-olides based on the position of their lactone ring. The biosynthetic pathway leading to xanthanolides has hitherto been elusive, especially how nature creates the xanthane skeleton is a long-standing question. This study reports the elucidation of a complete biosynthetic pathway to the important 12,8-xanthanolide 8-epi-xanthatin. The xanthane-type backbone is directly derived from the central precursor germacrene-type sesquiterpene, germacrene A acid, via oxidative rearrangement, catalyzed by an unusual cytochrome P450. Subsequently, a 12,8-lactone ring is formed within this xanthane-type backbone resulting in xanthanolides. The biosynthetic pathway for xanthanolides contrasts with the previously unified biosynthetic route for diverse 12,6-guaianolides, in which a 12,6-lactone ring formation precedes the transformation of a germacrene-type skeleton into a guaiane-type structure. The discovery of the full biosynthetic pathway of 8-epi-xanthantin opens new opportunities for producing xanthanolides in microbial organisms using synthetic biology strategies.

Small RNAs are involved in diverse cellular processes, including plant immunity to pathogens. Here, we report that miR158a negatively regulates plant immunity to the oomycete pathogen Phytophthora parasitica in Arabidopsis thaliana. By performing real-time quantitative PCR, transient expression, and RNA ligase-mediated 5' rapid amplification of cDNA ends assays, we demonstrate that miR158a downregulates AtTN7 expression by cleaving its 3'-untranslated region. AtTN7 positively affects plant immunity and encodes a truncated intracellular nucleotide-binding site and leucine-rich repeat receptor containing the Toll/interleukin-1 receptor. AtTN7 can degrade oxidized forms of nicotinamide adenine dinucleotide (NAD+). Further genetic and molecular analyses reveal that the Enhanced Disease Susceptibility 1-Phytoalexin Deficient 4-Activated Disease Resistance 1 complex is required for AtTN7-mediated immunity. ADR1-dependent Ca²⁺ influx is crucial for activating salicylic acid signaling to condition AtTN7-triggered immunity. Our study uncovers the immune roles and regulatory mechanisms of miR158a and its target AtTN7. Both miR158a-downregulation and AtTN7-overexpression lead to enhanced plant resistance to P. parasitica without affecting plant growth phenotypes, suggesting their application potentials and the utilization of miRNAs in identifying novel immune genes for the development of plant germplasm resources with enhanced disease resistance.

Calcium signaling plays an essential role in integrating plant responses to diverse stimuli and regulating growth and development. While some signaling components and their roles are well-established, such as the ubiquitous calmodulin (CaM) sensor, plants possess a broader repertoire of calcium sensors. Notably, CaM-like proteins (CMLs) represent a poorly characterized class for which interacting partners and biological functions remain largely elusive. Our work investigates the role of Arabidopsis thaliana CML8 that exhibits a unique expression profile in seedlings. A reverse genetic approach revealed a function of CML8 in regulating root growth and hypocotyl elongation. RNA-seq analyses highlighted CML8 association with the regulation of numerous genes involved in growth and brassinosteroid (BR) signaling. Using co-immunoprecipitation experiments, we demonstrated that CML8 interacts with the BR receptor, BRI1, in planta in a ligand-dependent manner. This finding suggests the existence of a novel regulatory step in the BR pathway, involving calcium signaling.

Because plants are immobile, they have developed intricate mechanisms to sense and absorb nutrients, adjusting their growth and development accordingly. Sulfur is an essential macroelement, but our understanding of its metabolism and homeostasis is limited. LSU (RESPONSE TO LOW SULFUR) proteins are plant-specific proteins with unknown molecular functions and were first identified during transcriptomic studies on sulfur deficiency in Arabidopsis. These proteins are crucial hubs that integrate environmental signals and are involved in the response to various stressors. Herein, we report the direct involvement of LSU proteins in primary sulfur metabolism. Our findings revealed that the quadruple lsu mutant, q-lsu-KO, which was grown under nonlimiting sulfate conditions, exhibited a molecular response resembling that of sulfur-deficient wild-type plants. This led us to explore the interactions of LSU proteins with sulfate reduction pathway enzymes. We found that all LSU proteins interact with ATPS1 and ATPS3 isoforms of ATP sulfurylase, all three isoforms of adenosine 5´ phosphosulfate reductase (APR), and sulfite reductase (SiR). Additionally, in vitro assays revealed that LSU1 enhances the enzymatic activity of SiR. These results highlight the supportive role of LSU proteins in the sulfate reduction pathway.

Ascorbic acid (AsA) serves as a key antioxidant involved in the various physiological processes and against diverse stresses in plants. Due to the insufficiency of AsA de novo biosynthesis, the AsA regeneration is essential to supplement low AsA synthesis rates. Redox reactions play a crucial role in response to biotic stress in plants; however, how AsA regeneration participates in hydrogen peroxide (H₂O₂) homeostasis and plant defense remains largely unknown. Here, we identified a Golgi vesicle-membrane-localized cytochrome B561 (CytB561) encoding gene, GhB561-11, involved in AsA regeneration and plant resistance to Verticillium dahliae in cotton. GhB561-11 was significantly downregulated upon V. dahliae attack. Knocking down GhB561-11 greatly enhanced cotton resistance to V. dahliae. We found that suppressing GhB561-11 inhibited the AsA regeneration, elevated the basal level of H₂O₂, and enhanced the plant defense against V. dahliae. Further investigation revealed that GhB561-11 interacted with the lipid droplet-associated protein GhLDAP3 to collectively regulate the AsA regeneration. Simultaneously silencing GhB561-11 and GhLDAP3 significantly elevated the H₂O₂ contents and dramatically improved the Verticillium wilt resistance in cotton. The study broadens our insights into the functional roles of CytB561 in regulating AsA regeneration and H₂O₂ homeostasis. It also provides a strategy by downregulating GhB561-11 to enhance Verticillium wilt resistance in cotton breeding programs.