Proteins in the importin α (IMPA) family play pivotal roles in intracellular nucleocytoplasmic transport. Arabidopsis thaliana possesses nine IMPA members, with diverse tissue-specific expression patterns. Among these nine IMPAs, IMPA1, IMPA2, and IMPA4 cluster together phylogenetically, suggesting potential functional redundancy. To explore this redundancy, we analyzed single and multiple T-DNA mutants for these genes and discovered severe growth defects in the impa1 impa2 impa4 triple knockout mutant but not in the single or double mutants. Complementation with IMPA1, IMPA2, or IMPA4 fused to green fluorescent protein (GFP) rescued the growth defects observed in the impa1 impa2 impa4 mutant, indicating the functional redundancy of these three IMPAs. The IMPA-GFP fusion proteins were localized in the nucleus and nuclear envelope, suggesting their involvement in nucleocytoplasmic transport processes. Comparative transcriptomics revealed that salicylic acid (SA)-responsive genes were significantly upregulated in the impa1 impa2 impa4 triple mutant. Consistent with this observation, impa1 impa2 impa4 mutant plants accumulated SA and reactive oxygen species to high levels compared with wild-type plants. We also found enhanced resistance to the anthracnose pathogen Colletotrichum higginsianum in the impa1 impa2 impa4 mutants, suggesting that defense responses were constitutively activated in the impa1 impa2 impa4 mutant. Our findings shed light on the redundant roles of IMPA1, IMPA2, and IMPA4 in suppressing the autoimmune responses and suggest avenues of research to clarify their potentially unique roles.
Efficient transformation systems are highly desirable for plant genetic research and biotechnology product development efforts. Tissue culture-free transformation (TCFT) and minimal tissue culture transformation (MTCT) systems have great potential in addressing genotype-dependency challenge, shortening transformation timeline, and improving operational efficiency by greatly reducing personnel and supply costs. The development of Arabidopsis floral dip transformation method almost 3 decades ago has greatly expedited plant genomic research. However, development of efficient TCFT or MTCT systems in non-Brassica species had limited success until recently despite the demonstration of successful in planta transformation in many plant species. In the last few years, there have been some major advances in the development of such systems in several crops using novel approaches. This article will review these new advances and discuss potential areas for further development.
Many quantitative traits are controlled by multiple genetic variations with minor effects, making it challenging to resolve the underlying genetic network and to apply functional markers in breeding. Affected by up to a hundred quantitative trait loci (QTLs), fruit-soluble sugar content is one of the most complex quantitative traits in apple (Malus sp.). Here, QTLs for apple fruit sucrose and fructose content were identified via QTL mapping and bulked-segregant analysis sequencing (BSA-seq) using a population derived from a ‘Jonathan’ × ‘Golden Delicious’ cross. Allelic variations and non-allelic interactions were validated in the candidate genes within these defined QTL regions. Three single-nucleotide polymorphisms (SNPs) (SNP −326 C/T, SNP −705 A/G, and SNP −706 G/T) in the MdMYB109 promoter region affected the binding ability of the repressive transcription factor MdWRKY33, leading to increased MdMYB109 expression. MdMYB109 bound directly to the promoter of the sucrose transporter gene MdSUT2.2 and activated its expression, raising fruit sucrose content. A SNP (SNP1060 A/G) in the hexokinase gene MdHXK1 affected the phosphorylation of the transcription factor MdbHLH3, and phosphorylated MdbHLH3 interacted with MdMYB109 to co-activate MdSUT2.2 expression and increase fruit sucrose content. Adding the joint effects of the genotype combinations at the SNP markers based on the SNPs in MdMYB109 and MdHXK1 increased the prediction accuracy of a genomics-assisted prediction (GAP) model for total soluble solid content from 0.3758 to 0.5531. These results uncovered functional variations in MdMYB109 and MdHXK1 regulating apple fruit sucrose content. The updated GAP model with improved predictability can be used efficiently in apple breeding.
Environmental stresses such as salt and drought severely affect plant growth and development. SQUAMOSA-promoter binding protein-like (SPL) transcription factors (TFs) play critical roles in the regulation of diverse processes; however, reports describing the SPL regulation of plant responses to abiotic stress are relatively few. In this study, two stress-responsive TFs from Codonopsis pilosula (CpSPL5 and CpSPL8) are reported, which confer salt stress sensitivity. CpSPL5 and CpSPL8 are expressed in almost all tissues and localized in the nucleus, where the CpSPL5 transcript level is relatively higher than that of CpSPL8. Their expression levels are significantly suppressed in hairy roots treated with ABA, NaCl, PEG-6000, and under high temperature stress. Compared with the control, CpSPL5, or CpSPL8-overexpressed hairy roots increased salt stress sensitivity, and exhibited higher levels of O2− and MDA, as well as lower superoxide dismutase and peroxidase activities. Further, the CpSPL5 or CpSPL8 interference transgenic hairy roots enhanced salt tolerance and exhibited contrasting phenotype and antioxidant indices. Although all genotypes revealed significantly increased Na+ and decreased K+ contents under salt stress, the physiological indicators of CpSPL5 or CpSPL8-interference transgenic hairy roots could be partially restored, where CpSPL5 was more sensitive to salt stress than CpSPL8. A yeast one-hybrid and dual-luciferase assay revealed that CpSPL5 and CpSPL8 directly targeted and inhibited the expression of CpSOS2 in the salt overly sensitive (SOS) pathway, which promoted salt stress sensitivity. Our findings suggest that CpSPL5 and CpSPL8 served as negative regulators of salt tolerance, which indicate that members of the SPL family participate in the plant SOS pathway.
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.
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