Plant Direct最新文献

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.

Phototropic bending of plants towards a light source allows them to position their photosynthetic tissues to optimize light capture. In light-grown (de-etiolated) Arabidopsis seedlings, phototropic bending of the hypocotyl is inhibited by light with a high red:far-red ratio (HRFR) and high levels of blue light (HBL). This occurs via activation of the phytochrome B (phyB) and cryptochrome 1 (cry1) photoreceptor signaling pathways. Both phyB and cry1 act upstream of PHYTOCHROME INTERACTING FACTOR (PIF) transcription factors, which are required for hypocotyl bending in light-grown seedlings. Presently, it is not known whether other pathways are involved in the inhibition of PIF-mediated phototropism in light-grown seedlings. To address this, we conducted a screen to identify mutants with increased phototropic bending relative to wild type in HRFR + HBL conditions. Through this screen, we identified EARLY FLOWERING 3 (ELF3), a member of the Evening Complex (EC), as a key inhibitor of phototropic bending in green seedlings. We show that both ELF3 and LUX, another component of the EC, inhibit phototropic bending upstream of PIF4/PIF5. Furthermore, we show that phototropic bending in Arabidopsis seedlings is subject to circadian regulation in an ELF3-dependent manner. Finally, we provide evidence that ELF3 in the grass Brachypodium distachyon also affects phototropism but in an opposite way than in Arabidopsis.

In apple (Malus domestica), flowering is repressed by the phytohormone gibberellin (GA) and high temperatures (> 27°C), but the molecular mechanisms underlying this repression remain unknown. In Arabidopsis thaliana (Arabidopsis), GA and temperature signaling converge on DELLA protein regulation, with both factors promoting DELLA degradation through independent 26S proteasome-mediated pathways. Here, we tested whether high-temperature-induced DELLA degradation is conserved in apple. Using the heterologous systems Arabidopsis and Nicotiana benthamiana, we characterized the function of the apple DELLA protein DELLA REPRESSOR OF ga1-3 (MdRGL1a) and found that high temperatures promote its degradation via a 26S proteasome-dependent mechanism. Additionally, MdRGL1a interacts with apple orthologs of Arabidopsis CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) and SUPPRESSOR OF phyA-105 2 (SPA2), components of an E3 ubiquitin ligase complex that mediates protein ubiquitination and degradation. These findings suggest a conserved mechanism of temperature-induced DELLA degradation between apple and Arabidopsis. The degradation of MdRGL1a may underlie flowering suppression in apple under high temperatures, providing molecular insights that could aid in developing strategies to stabilize apple and other crop production in the face of climate change.

Plant diseases caused by pathogenic microorganisms result in significant damage to agriculture. Lactic acid bacteria (LAB), in particular strains of Lactiplantibacillus plantarum (L. plantarum), are used as one of the biocontrol methods against plant pathogenic bacteria due to high antagonistic activity associated with their metabolic potential. We have investigated the influence of nutrient medium components (various carbon and nitrogen sources) and cultivation conditions (temperature, duration, and pH) of L. plantarum strains on the level of their antagonistic activity against the test strains of plant pathogenic bacteria. The antimicrobial activity of LAB supernatants was maximal in the presence of 3% (30 g/L) sucrose as the main carbon source and 1% (10 g/L) tyrosine as the main nitrogen source in the nutrient medium against all investigated test strains of PPB. However, the use of such a carbon source as galactose or arabinose led to a decrease or even absence of antimicrobial properties of LAB against phytopathogenic bacteria. The optimal conditions for cultivation of lactobacilli were determined: cultivation temperature +30 ± 1°C, pH 7.8, and duration 72 h. Strains of L. plantarum 13c and 21c caused zones of inhibition in test pathogens from 298 to 291 mm. Whereas during 24-h cultivation of LAB strains, their antagonistic activity was significantly lower, and the zones of inhibition decreased by 30%. The duration and temperature had a significant effect on increasing the antagonistic activity of L. plantarum strains, in contrast to the pH of the medium (p ≥ 0.05).

Microalgae are promising sources to sustainably meet the global needs for energy and products. Algae grow under different trophic conditions, where nutritional status regulates biosynthetic pathways, energy production, and growth. The green alga Chromochloris zofingiensis has strong economic potential because it co-produces biofuel precursors and the high-value antioxidant astaxanthin while accumulating biomass when grown mixotrophically. As an emerging reference alga for photosynthesis, metabolism, and bioproduction, C. zofingiensis needs a defined, optimized medium to standardize experiments during fast growth for batch cultivation. Because the interplay of glucose treatment (+Glc) and mineral deficiency influences photosynthesis, growth, and the production of lipids and astaxanthin, we designed a replete nutrient medium tailored to the C. zofingiensis cellular ionome. We combined inductively coupled plasma mass spectrometry (ICP-MS) and +Glc growth curves to determine a medium that is nutrient replete for at least 5 days of +Glc logarithmic growth. We found that there are high nutritional needs for phosphorus and sulfur during mixotrophy. Iron was the only element measured for which the cellular concentration correlated with exogenous concentration and was iteratively adjusted until the cellular ionome was consistent through the logarithmic growth phase. This Chromochloris-Optimized Ratio of Elements (CORE) medium supports fast growth and high biomass and lipid accumulation without causing excess nutrient toxicity. This defined, nutrient-replete standard is important for future C. zofingiensis investigations and can be adapted for other species to support high biomass for batch cultivation. The method used to develop CORE medium shows how ionomics informs replicable media design and may be applied in industrial settings to inform cost-effective biofuel production.