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.

Root depth is a major determinant of plant performance during drought and a key trait for strategies to improve soil carbon sequestration to mitigate climate change. While the model Arabidopsis thaliana offers numerous advantages for studies of root system architecture and root depth, its small and fragile roots severely limit the use of the methods and techniques currently available for such studies in soils. To overcome this, we have developed ClearDepth, a conceptually simple, non-destructive, sensitive, and low-cost method to estimate the root depth of Arabidopsis in relatively small pots that are amenable to mid- and large-scale studies. In our method, the root system develops naturally inside of the soil, without considerable space constraints. The ClearDepth parameter wall root shallowness (WRS) quantifies the shallowness of the root system by measuring the depth of roots that reach the transparent walls of clear pots. We show that WRS is a robust and sensitive parameter that distinguishes deep root systems from shallower ones while also capturing relatively smaller differences in root depth caused by the influence of an environmental factor. In addition, we leveraged ClearDepth to study the relation between lateral root angles measured in non-soil systems and root depth in soil. We found that Arabidopsis genotypes characterized by steep lateral roots in transparent growth media produce deeper root systems in the ClearDepth pots. Finally, we show that ClearDepth can also be used to study root depth in crop species like rice.

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.

Plants have evolved sophisticated defense mechanisms against insect herbivores, including cell wall fortification through lignin biosynthesis. Insect attack primes systemic acquired resistance in plants, preparing them to respond more swiftly and vigorously to subsequent insect assaults. Here, we found that Beauveria bassiana-exposed maize plants can emit phytol upon infestation by Spodoptera frugiperda, inducing plant-to-plant (PTP) communication of alert signals for neighboring plants, and revealed the expansin protein EXP-A20 as a pivotal node mediating maize defense responses in neighboring plants against the destructive pest Ostrinia furnacalis via stimulation of ethylene (ET) synthesis and lignin production. Through virus-induced gene silencing, we showed that EXP-A20 is essential for maize resistance, while downregulating ET and lignin pathways. Critically, protein-protein interactions determined via luciferase complementation and yeast two-hybrid assays demonstrated that EXP-A20 binds to and likely activates the ET-forming enzyme gene ACO31 to initiate defense signaling cascades, representing a novel signaling modality for expansins. Treatment with the plant volatile phytol has known insecticidal/priming activity, but we found that its effectiveness requires EXP-A20. This finding highlights the importance of EXP-A20 upstream of hormone-cell wall crosstalk in defense activation by volatiles. Overall, our multifaceted dissection of EXP-A20 revealed key molecular intersections underlying inducible maize immunity against herbivores. Furthermore, we provide functional evidence that extensive cell growth processes directly stimulate defense programs in plants. Our work opens new avenues for enhancing durable, broad-spectrum pest resistance in maize through the use of volatile organic compounds and PTP interactions.

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.

The endoplasmic reticulum (ER) utilizes ER-associated degradation (ERAD), a highly conserved eukaryotic pathway, to eliminate misfolded or unassembled proteins and maintain protein homeostasis in cells. The clearance of misfolded glycoproteins involves several distinct steps, including the recognition of a specific glycan signal, retrotranslocation to the cytosol, and subsequent degradation of the misfolded protein by the ubiquitin proteasome system. Confocal microscopy was used to track the fate of a well-characterized ERAD substrate via a self-complementing split fluorescent protein assay. The results demonstrate that a misfolded variant of the STRUBBELIG (SUB) extracellular protein domain (SUBEX-C57Y) is retrotranslocated to the cytosol when transiently expressed in Nicotiana benthamiana leaf epidermal cells. Retrotranslocation requires a protein domain with a lesion that is exposed in the lumen of the ER, N-glycan trimming by α-mannosidases, HRD1-mediated ubiquitination, and the ATPase function of CDC48. The retrotranslocated SUBEX-C57Y ERAD substrate undergoes deglycosylation, and proteasomal degradation is blocked by a catalytically inactive cytosolic peptide N-glycanase. These findings define distinct aspects of ERAD that have been elusive until now and may represent the default pathway for degrading misfolded glycoproteins in plants.

Flower color change, a common phenomenon that is important in pollination ecology, has intrigued scientists for decades. While previous flower color studies have mainly focused on color diversity among different plant species, our focus is on unraveling the mechanism of post-anthesis color change (PACC) and the molecular basis for its presence and absence, respectively, in two closely related species of Lotus, Lotus filicaulis and Lotus japonicus MG20. Metabolomic analysis reveals anthocyanins as the key metabolites responsible for the observed PACC. Differential expression of anthocyanin biosynthetic and transport genes causes the variation in PACC between the two Lotus species. Crucially, the significant upregulation of a functionally characterized MYB regulator, LfPAP1, is linked to the accumulation of anthocyanins and visible color alterations in L. filicaulis flowers. Notably, we uncover a nucleotide polymorphism in the initiation codon of LjPAP1. Although this mutation does not affect transcription, we show that it has a major effect in attenuating protein translation, reducing its capacity to activate anthocyanin biosynthesis, and leading to a failure of PACC in L. japonicus MG20. Our study sheds light on mechanisms of PACC phenomenon and highlights the potential for mutations in initiation sequences to generate phenotypic differences between species in evolution.

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.

The transition from vegetative to reproductive growth is essential for the flowering process of plants. In summer chrysanthemum, CmBBX8 exploits prominence function in floral transition by activating the expression of CmFTL1. However, how CmBBX8 induces CmFTL1 during the photoperiod inductive cycles remains unknown. Here, we show that CmBBX8 interacts with the SGS3-like protein CmFDM2, and the CmFDM2 overexpression strains presented early flowering, while knockdown strains delayed flowering. Additionally, CmFDM2 could bind to the CmFTL1 promoter and activate the expression of CmFTL1, and associate with chromatin remodeling factor CmSWI3B, and CmBBX8 induces flowering dependent on CmFDM2 and CmSWI3B. CmFDM2 also partially depends on CmSWI3B. The CmSWI3B knockdown strains exhibited a significant late flowering phenotype. Interestingly, CmBBX8 also interacts with CmSWI3B. Moreover, the level of H3K27me3 at the CmFTL1 locus was reduced when CmBBX8 and CmFDM2/CmSWI3B occupied the locus to promote chrysanthemum flowering during the photoperiod inductive cycles, which was accompanied by the increasing level of CmFTL1 transcripts. Thus, our work provides novel insights into the gradually increasing level of CmFTL1 for the floral transition through CmBBX8 recruiting chromatin modifiers CmFDM2/CmSWI3B.