Plant Direct最新文献

One class of enzymes that plant pathogens employ to manipulate innate immunity and physiology of the infected cells is host-targeted ADP-ribosyltransferases. The bacterial pathogen Pseudomonas syringae uses its Type III secretion system to inject several effector proteins with ADP-ribosyltransferase activity into plant cells. One of them, AvrRpm1, ADP-ribosylates the plasma membrane-associated RPM1-INTERACTING PROTEIN 4 (RIN4) in Glycine max and Arabidopsis thaliana to attenuate targeted secretion of defense-promoting compounds. Substrate identification of host-targeted ADP-ribosyltransferases is complicated by the biochemical lability of the protein modification during plant protein extraction and in several cases requires prior knowledge of plant immune signaling pathways that are impaired by the ADP-ribosylating Type III effector. Using the AvrRpm1-RIN4 pair as a proof of concept, we present an untargeted proteomics workflow for enrichment and detection of ADP-ribosylated proteins and peptides from plant cell extracts that in several cases provides site resolution for the modification.

The reproductive cycle of interior lodgepole pine spans approximately 26 months, with seed cones initiating in late summer, followed by pollen conelets emerging the next spring when pollination occurs. Fertilization and cone maturation occur in the subsequent spring and fall. Successful pollination, critical for seed conelet retention, requires an ~80% success rate. In June 2020, 30 pine clones (genotypes), with contrasting conelet abortion rates, were selected from a production seed orchard in Alberta, Canada. Sixty conelets per clone (30 aborted and 30 healthy) were collected for synchrotron radiation-based microcomputed tomography (SR-μCT) analysis. A subset of two contrasting clones (1401 and 596) was selected for detailed study. Conelets were transported to the Canadian Light Source in Saskatoon, Saskatchewan, for SR-μCT Phase Contrast Imaging (PCI), where samples were mounted and scanned using a PCO Edge 5.5 sCMOS camera and analyzed using Avizo software to visualize internal structures. Results showed that healthy conelets in clones 1401 and 596 had greater tissue volume (248.8 and 251.9 mm³, respectively) and more organized cavity spaces than aborted conelets (64.7 and 102.1 mm³, respectively), with differential internal air space volume (clone 1401: 8.7 mm³ in aborted vs. 17.5 mm³ in healthy; clone 596: 6.9 mm³ in aborted vs. 11.5 mm³ in healthy). Overall, SR-μCT imaging demonstrated significant advantages over traditional methods of visualization by providing a nondestructive, high-resolution analysis of internal structures. In addition, this technology enhances our understanding of pine reproductive processes, improving management practices in seed orchards-the delivery system for tree improvement programs.

This beginner's guide is intended for plant biologists new to network analysis. Here, we introduce key concepts and resources for researchers interested in incorporating network analysis into research, either as a stand-alone component for generating hypotheses or as a framework for examining and visualizing experimental results. Network analysis provides a powerful tool to predict gene functions. Advances in and reduced costs for systems biology techniques, such as genomics, transcriptomics, and proteomics, have generated abundant omics data for plants; however, the functional annotation of plant genes lags. Therefore, predictions from network analysis can be a starting point to annotate genes and ultimately elucidate genotype-phenotype relationships. In this paper, we introduce networks and compare network-building resources available for plant biologists, including databases and software for network analysis. We then compare four databases available for plant biologists in more detail: AraNet, GeneMANIA, ATTED-II, and STRING. AraNet and GeneMANIA are functional association networks, ATTED-II is a gene coexpression database, and STRING is a protein-protein interaction database. AraNet and ATTED-II are plant-specific databases that can analyze multiple plant species, whereas GeneMANIA builds networks for Arabidopsis thaliana and nonplant species and STRING for multiple species. Finally, we compare the performance of the four databases in predicting known and probable gene functions of the A. thaliana Nuclear Factor-Y (NF-Y) genes. We conclude that plant biologists have an invaluable resource in these databases and discuss how users can decide which type of database to use depending on their research question.

[This corrects the article DOI: 10.1002/pld3.70081.].

RNA editing and maturation are critical regulatory mechanisms in plant organelles, yet their quantification remains technically challenging. Traditional Sanger sequencing lacks sensitivity and reproducibility, whereas advanced next-generation sequencing (NGS) approaches, such as rRNA-depleted long non-coding (lnc) RNA-seq or targeted amplicon-seq, involve high costs, complex workflows, and limited accessibility. To address these limitations, I developed a rapid and cost-effective long-read sequencing approach, termed Target-Indexed-PCR (TIP) sequencing, for digital quantification of RNA editing and intron retention events in targeted chloroplast transcripts. This method combines multiplexed high-fidelity PCR amplification with Oxford Nanopore sequencing and custom in-house Perl and Python scripts for streamlined data processing, including barcode-based demultiplexing, strand reorientation, alignment to a pseudo-genome, manual editing-site inspection, and splicing variant identification. As a proof of concept, TIP sequencing was applied to ndhB and ndhD transcripts, two chloroplast NAD(P)H dehydrogenase genes with the highest number of known editing sites in Arabidopsis thaliana. These transcripts were analyzed both in an inducible CRISPR interference (iCRISPRi) system targeting MORF2, a key RNA-editing factor, and in MORF2-YFP transgenic lines with either overexpression or co-suppression silencing. My findings revealed dose- and development-dependent impacts of MORF2 on C-to-U editing efficiency. Moreover, I identified an accumulation of intron-retaining ndhB transcripts, specifically in Dex-treated iCRISPRi lines and in both MORF2-YFP overexpression and silencing rosette leaves, indicating impaired chloroplast splicing functions when MORF2 expression is perturbed beyond an as-yet-undefined threshold. The platform achieves single-molecule resolution, robust reproducibility, and high read coverage across biological replicates at a fraction of the cost of lncRNA-seq. Collectively, this study establishes TIP sequencing as a versatile, scalable, and affordable tool for targeted post-transcriptional analysis in plant organelles and expands our understanding of MORF2's role in chloroplast RNA maturation. By overcoming key limitations of existing approaches, TIP sequencing enables routine, site-specific quantification of post-transcriptional regulation in organelles, including RNA editing and splicing, making it broadly accessible to researchers studying plastid biology, stress responses, and organelle-nucleus communication.

Programmed cell death (PCD) mediates plant development and environmental interactions. Photosynthesis-derived singlet oxygen (¹O₂) is one of key reactive oxygen species (ROS) implicated in acclimation and PCD responses to environmental stress conditions. Using Arabidopsis thaliana cell suspension culture system, we characterized the PCD induced by Rose Bengal (RB), a photosensitizer generating ¹O₂ upon light exposure. Obtained results reiterated that RB-induced PCD is light and chloroplast dependent. Further, we demonstrate that PCD induced by RB involves calcium signaling and mitochondria, thus sharing common features with other forms of regulated cell death in plants. The PCD induced by RB was associated with early transcriptional reprogramming, involving switching off the primary metabolism and activation of stress response and cell death related pathways (e.g., oxidative stress, hypoxia, immunity, and salicylic acid). The constructed gene regulatory network featured ¹O₂-responsive genes and suggested involvement of transcription factor ANAC102 in retrograde regulation of RB-induced PCD. Interestingly, treatment with RB also induced light independent toxicity, showing features of uncontrolled, necrotic cell death. Presented findings highlight RB as a valuable tool for studying ¹O₂-induced PCD that may advance future work on chloroplast-mediated oxidative stress responses and enhancing plant resilience to climate change-related stressors through targeted modulation of ROS pathways.

Powdery mildew (PM), mainly caused by Podosphaera xanthii, is a severe destructive disease that threatens the production of cucurbit crops globally. Heterologous transformation has shown that the CmSGT1 gene (suppressor of the G2 allele of skp1) improved PM resistance in tobacco. However, the function of the gene in pumpkins (Cucurbita moschata) is largely unknown. Herein, transient CmSGT1 overexpression in pumpkin cotyledons inhibited the spore germination and mycelia growth of P. xanthii by inducing an increase in salicylic acid (SA) content, and exogenous SA intensified the inhibitory effect of the gene on the growth of P. xanthii. The β-glucuronidase activity of cotyledons transformed with the CmSGT1 promoter was induced by PM and signaling molecules (gibberellic acid, ethephon, SA, abscisic acid, and methyl jasmonate). The yeast one-hybrid assay verified that transcription factor CmWRKY21, CmWRKY31, and CmWRKY75 proteins interact with the CmSGT1 promoter. Transactivation analysis revealed that CmWRKY21 and CmWRKY31 significantly triggered the expression of GUS driven by the CmSGT1 promoter under PM. Furthermore, transient co-overexpression of CmWRKY21/CmWRKY31 and CmSGT1 enhanced the inhibitory effect on the growth of P. xanthii. In conclusion, the CmSGT1 gene is a PM resistance gene in pumpkin and is transcriptionally regulated by CmWRKY21 and CmWRKY31. Our study provides a reference for resistance breeding of pumpkins.

Flowering dogwood (Cornus florida L.) and Asian dogwood (Cornus kousa F. Buerger ex Hance) are popular deciduous ornamental trees native to a wide range of the eastern and southeastern United States and East Asia, respectively. Anthocyanin pigments enhance desirable pink or dark red colored bracts in dogwoods. Although anthocyanin biosynthesis is one of the best-studied biological processes in nature, genomic and genetic resources to understand the molecular regulation of its synthesis in dogwoods are still lacking. Two classes of genes control anthocyanin production; both structural genes and MYB transcription factors may function as positive or negative regulators of anthocyanin biosynthesis. To reveal the molecular mechanisms that govern color production in ornamental dogwoods, mature bracts of three cultivars of C. florida (white bracts: "Cloud Nine"; red bracts: "Cherokee Brave," and "Cherokee Chief") and two cultivars of C. kousa (light green bracts: "Greensleeves" and midtone pink bracts "Rosy Teacups") were sampled when color was maximally visible. Differential gene expression analysis of the RNAseq data identified 1156 differentially expressed genes in C. florida and 1396 in C. kousa. Phylogenetic analysis with functional orthologues in other plants grouped the candidate R2R3-MYB identified in this study into two distinct subgroups. CfMYB2, CfMYB3, and CkMYB2 belong to Subgroup 4, whereas CfMYB1 80 and CkMYB1 clustered in Subgroup 5. Genes in the former group repress anthocyanin and proanthocyanidin synthesis in flowering and Asian dogwoods, whereas genes in the latter increase it. Our study contributes to understanding processes behind anthocyanin production and lays the foundation for the future development of molecular markers for faster development of desirable red-bracted dogwoods.

Davidia involucrata Baill. is a species that thrives in warm, humid climates with consistently moist soil conditions. With rising global temperatures and an increasing frequency of droughts, the natural habitat of Davidia involucrata Baill. is facing severe threats. In-depth investigation of the molecular mechanisms underlying Davidia involucrata Baill.'s response to drought stress is crucial for the conservation of this rare species and the enhancement of its environmental adaptability. This study systematically analyzed the drought stress response of Davidia involucrata Baill. under varying light conditions through transcriptome data analysis. The results showed that under different light conditions, Davidia involucrata Baill. responded to drought stress by regulating its internal osmotic balance via the "response to mannitol" pathway. Notably, the molecular mechanisms by which Davidia involucrata Baill. responds to drought stress vary significantly under different light conditions. Compared with high light intensity, Davidia involucrata Baill. under shaded conditions responded to drought stress by upregulating glycosyltransferase-related pathways. In addition, three soil drought-related pathway genes (SDRPGs) (Dinv08247, Dinv34952, and Dinv00865) involved in the regulation of drought stress in Davidia involucrata Baill. were identified, and both ABA and SA were found to influence their expression. As a key environmental factor, air humidification may enhance the drought stress adaptability of Davidia involucrata Baill. by modulating ABA biosynthesis. The SDRPGs and signaling pathways identified in this study may serve as important candidate targets, providing theoretical guidance and scientific reference for the genetic improvement of drought resistance in Davidia involucrata Baill. and the long-term conservation of rare plant resources.

Anthocyanins are pigments that contribute to plant defense and adaptation to environmental stresses. Given their antioxidant properties and positive impacts on human health, enhancing anthocyanin biosynthesis in plants holds significant economic importance. In potato, several genotypes produce a high amount of anthocyanins, but the molecular mechanisms underlying the genotypic variation of anthocyanin content remain poorly understood. Here, key genes that may determine the genotype-dependent capacity for anthocyanin biosynthesis were analyzed. Anthocyanin content in tubers from five genotypes was measured, and Heimeiren and Desiree, exhibiting high and low anthocyanin content, respectively, were selected. We were unable to identify any evidence of differing activity in anthocyanin biosynthesis enzymes based on single amino acid polymorphism analysis between the two genotypes. However, transcriptome sequencing coupled with prediction of gene function identified 27 candidate genes showing different expression levels in tubers of these genotypes. We additionally verified expression patterns of these genes and found that four genes encoding flavanone 3-hydroxylase, flavonoid 3',5'-hydroxylase, anthocyanin synthase (ANS), and anthocyanin O-methyltransferase (AOMT) were strong candidates for high accumulation of anthocyanins in Heimeiren. Particularly, ANS and AOMT are strong candidates increasing anthocyanin content in the tuber flesh. These results imply that genotype-dependent variations of anthocyanin biosynthesis may be due to difference of gene expression, but not enzymatic activities. Our study suggests key anthocyanin biosynthesis genes showing different expression levels in high- and low-anthocyanin genotypes, offering potential for the metabolic engineering of potatoes to increase anthocyanin content.