Plant and Cell Physiology最新文献

The Type I-E CRISPR-Cas3 derived from Escherichia coli (Eco CRISPR-Cas3) can introduce large deletions in target sites and is available for mammalian genome editing. The use of Eco CRISPR-Cas3 in plants is challenging because seven CRISPR-Cas3 components (six Cas proteins and CRISPR RNA) must be expressed simultaneously in plant cells. To date, application has been limited to maize protoplasts, and no mutant plants have been produced. In this study, we developed a genome editing system in rice using Eco CRISPR-Cas3 via Agrobacterium-mediated transformation. Deletions in the target gene were detected in 39%-71% of transformed calli by polymerase chain reaction (PCR) analysis, and the frequency of alleles lacking a region 7.0 kb upstream of the protospacer adjacent motif sequence was estimated as 21%-61% by quantifying copy number by droplet digital PCR, suggesting that mutant plants could be obtained with reasonably high frequency. Deletions were determined in plants regenerated from transformed calli, and stably inherited to the progenies. Sequencing analysis showed that deletions of 0.1-7.2 kb were obtained, as reported previously in mammals. Interestingly, deletions separated by intervening fragments or with short insertion and inversion were also determined, suggesting the creation of novel alleles. Moreover, we demonstrated C to T base editing based on Type I-E CRISPR-Cas3 in rice, whereas base editing based on Type I-C and Type I-F2 CRISPR-Cas3 has been reported previously only in human cells. Overall, Eco CRISPR-Cas3 could be a promising genome editing tool for gene knockout, gene deletion, base editing, and genome rearrangement in plants.

During Arabidopsis thaliana photomorphogenesis, light promotes cotyledon expansion and inhibits hypocotyl elongation. This process involves transcriptional reprogramming controlled by various factors, including the Elongator complex which regulates gene expression at the level of transcription and translation via epigenetic and tRNA modifications, respectively. The elo3-6 mutant, lacking Elongator activity, exhibited photomorphogenic defects: less open, hyponastic cotyledons and an elongated hypocotyl. RNA-Seq and miRNA-Seq revealed distinct dysregulated gene sets in elo3-6 hypocotyl and cotyledons. In hypocotyl, the elo3-6 defect affected expression of genes involved in chloroplast physiology, circadian regulation, and auxin responses. Impaired chloroplast biogenesis apparently triggered retrograde signaling and a hypoxia-like state, preventing full inhibition of hypocotyl elongation. The defective elo3-6 cotyledon development is likely due to compromised translation. This was supported by the presence of similar morphological defects in urm11 urm12, defective in the same type of tRNA modification as in elo3-6, and the synergism observed in the elo3-6 urm11 urm12 (euu) triple mutant showing seedling lethality. Moreover, elo3-6 and urm11 urm12 showed increased tolerance to translation inhibitors, including hygromycin B which prevented the narrower cotyledon opening in elo3-6, suggesting that strong Elongator-dependent codon-anticodon interactions are required for proper cotyledon development. Interestingly, the genes enriched in codons recognized by tRNA anticodons modified by Elongator showed decreased mRNA abundance in elo3-6, suggesting a feedback mechanism downregulating the abundance of inefficiently translated mRNAs. Our results suggest that Elongator's transcriptional role is more important in hypocotyl growth, while its translational role is more prominent in cotyledon development.

Stomata in the plant epidermis open in response to light, but the molecular mechanisms underlying this process remain incompletely understood. In this study, we screened for compounds that affect light-induced stomatal opening and identified two stomatal closing compounds (SCLs), SCL10 and SCL11 (clorgyline). Both compounds completely suppressed light-induced stomatal opening in Commelina benghalensis but did not affect fungal toxin fusicoccin (FC)-induced opening. The half-maximal inhibitory concentrations (IC50) of SCL10 and SCL11 on light-induced opening were 1.40 and 38.25 μM, respectively. Consistently, both compounds fully inhibited blue-light-induced phosphorylation of the penultimate residue, a threonine (Thr948; numbering according to Arabidopsis AHA1) of plasma membrane (PM) H+-ATPase, which is essential for stomatal opening, but did not interfere with FC-induced phosphorylation in Arabidopsis thaliana guard cells. Notably, red- and blue-light-induced phosphorylation of Thr881 in PM H+-ATPase, another key step in stomatal opening, was inhibited by SCL11 but not by SCL10 in guard cells. Additionally, stomatal opening induced by PP242, a compound that promotes stomatal opening partly by suppressing steady-state abscisic acid signaling, was inhibited by SCL11 but not by SCL10. These results imply that SCL10 inhibits blue-light-induced phosphorylation of Thr948 and stomatal opening upstream of the site of action of PP242, whereas SCL11 inhibits the phosphorylation of both Thr948 and Thr881, and acts downstream of the site of action of PP242. Finally, the compounds were sprayed onto rose leaves, and we observed that wilting was suppressed only in leaves sprayed with SCL10, for up to 5 h.

Dodders (Cuscuta spp.) are obligate parasitic plants that have lost a large portion of photosynthetic genes but gained host genes through parasitism-mediated horizontal gene transfer. Their genetic complexity of speciation is partly clarified in the genome level. Here, we report the de novo genome assemblies of two phylogenetically distinct dodders: C. campestris (2n = 4x = 60) and C. chinensis (2n = 4x = 60), which are classified into distinct section of subgenus Grammica. Relatively low completeness of eudicot Benchmarking Universal Single-Copy Orthologs genes (ca. 87%) indicated progressive gene loss after evolution of the parasitic lifestyle due to release from functional constraints. Comparative genomics analyses revealed that the genome size of each species differs significantly, despite having the same chromosome numbers and allopolyploidy via independent hybridization involving different ancient parents. Various genomic rearrangements have likely contributed to the genomic diversity of the two lineages, which partly share habitats, including (1) gene gain and loss events, (2) homoeologous recombination between two subgenomes, and (3) lineage-specific transposable elements dynamics. Our findings not only provide a genomic basis for surveying parental species for allopolyploidization but also enhance understanding of the unique speciation of parasitic dodders through these chromosomal events.

Lysophosphatidylcholine acyltransferase (LPCAT) is a key enzyme of the Lands cycle that remodels phosphatidylcholine (PC). Proper balance between lysophosphatidylcholine (LPC) and PC is essential for phospholipid turnover and membrane homeostasis. Here, we show that the Arabidopsis lpcat double mutant exhibits an altered LPC/PC ratio in root tissues, reflecting a compromised Lands cycle. Using a chemical phenocopying strategy, we found that root growth of the lpcat mutant was more sensitive than that of wild type to lysoPAF, a structural analog of LPC. This defect was largely rescued when lysoPAF treatment was combined with ONO-RS-082, a phospholipase A2 (PLA2) inhibitor. Subcellular localization experiments with GFP fusions demonstrated that both LPCAT1 and LPCAT2 are targeted to the endoplasmic reticulum (ER) membrane. Furthermore, RNA-seq analysis of root tissues revealed transcriptional changes indicative of ER stress and altered endomembrane trafficking. Together, our results demonstrate that ER-localized LPCAT plays a pivotal role in maintaining root growth integrity when plants encounter conditions that require adjustments in lipid homeostasis. The likely role of ER localized LPCAT in modulating ER stress and root growth will be discussed.

Salt stress causes ion toxicity and oxidation in plants, and severely restricts agricultural production, the regulatory network and detailed mechanisms of salt tolerance remain incompletely known. Many lncRNAs involved in stress responses have been identified in plants, most of which are transcribed by RNA polymerase II and respond to a single stress factor. Arabidopsis AtR8 is transcribed by RNA polymerase III, it responds to SA treatment and interacts with AtWRKY53/AtWRKY70 to affect root elongation. AtR8 also responds to light and ABA and together with AtABI3 and crucial genes for light signal affects hypocotyl elongation. In the present study, AtR8 expression was significantly inhibited under salt stress. Loss-of-function of AtR8 significantly reduced seed germination and seedling root growth under salt stress, the ability to maintain ROS homeostasis was weakened, and H2O2 tolerance was reduced. The AtWRKY53 and AtABI3 expression levels were simultaneously significantly increased in the atr8 mutants. The abi3 mutant showed strong salt tolerance. The AtWRKY53 directly binds to the W-box in the promoter region of AtABI3, and promotes AtABI3 expression. These results suggest that AtR8 affects the salt tolerance of Arabidopsis by regulating ROS homeostasis and AtWRKY53-mediated AtABI3 transcription. Identification of the AtR8/AtWRKY53/AtABI3 regulatory module contributes to elucidation of the molecular regulatory network of salt stress in plants. Elucidation of the functional diversity of AtR8 improves our understanding of the evolution and diversity of lncRNA mechanisms and molecular pathways involved in adaptation to different environments, which is important for the cultivation of stress-resistant cultivars.

'Fengtang' plum (Prunus salicina Lindl. cv. 'Fengtang'), a high-sugar cultivar, suffers from severe yield losses due to early physiological fruit drop. We identified 24 days after flowering (DAF) as the critical abscission period, characterized by ovule abortion and abscission zone formation. Hormonal profiling revealed a significant reduction in gibberellin (GA) and jasmonic acid (JA) levels, along with elevated abscisic acid (ABA) content in abscised fruits. Integrated transcriptomic and metabolomic analyses highlighted the central role of hormone signaling pathways. Among differentially expressed genes, PsGA2ox1, encoding a GA-inactivating enzyme, was highly up-regulated in abscised fruits and exhibited strong negative correlations with bioactive GA and JA levels. Functional validation demonstrated that PsGA2ox1 overexpression reduced GA3 level and induced the expression of key abscission-related genes, whereas its silencing had the opposite effects. Our findings indicate that PsGA2ox1-mediated GA inactivation plays a pivotal role in modulating hormonal balance and transcriptional reprogramming, which involves cell wall remodeling, during early fruit abscission in plum. This study provides molecular insights into a key regulatory node for fruit retention and identifies a potential target for improving fruit set.

Fructokinase participates in carbohydrate metabolism via phosphorylation of fructose and plays an important role in plant growth and development. In this study, we revealed the role of fructokinase OsFRK1 and OsFRK2 as positive regulators of seed germination and seedling growth by influencing abscisic acid (ABA) content and energy metabolism. Compared with the wild type (WT), the seed germination, seedling growth and seed vigor of osfrk1 and osfrk2 mutants were suppressed, with the osfrk1 mutants showing a larger inhibitory impact than osfrk2 mutants. Energy metabolomics analysis indicated that OsFRK1 and OsFRK2 might involve in the biosynthesis of secondary metabolites and carbohydrate metabolism to affect the energy required for seed germination. Furthermore, UPLC-MS/MS analysis demonstrated that the ABA content in osfrk1 and osfrk2 mutants was significantly higher than in the WT. RT-qPCR results indicated that the expression of genes associated with ABA metabolism in the mutants was down-regulated. Additionally, exogenous ABA treatment showed that the osfrk1 and osfrk2 mutants exhibited heightened sensitivity to ABA during seed germination and seedling growth. Collectively, this work provides significant insights into the regulation of OsFRK1 and OsFRK2 on seed germination and vigor in rice, as well as theoretical references for practical problems such as direct seeding of rice.

The AAA+ ATPase CDC48A is a central regulator of proteostasis in plants, functioning through interactions with a diverse set of cofactors. Among these, the plant-specific ubiquitin regulatory X (UBX) domain-containing proteins (PUX) are key adaptors that direct CDC48A to specific substrates and pathways. The molecular basis of PUX-CDC48A interactions remains incompletely understood. Here, we combine structural, biophysical, and computational approaches to dissect the binding modes of representative PUX proteins from different subfamilies in Arabidopsis thaliana. Although all PUX proteins tested exhibit low micromolar affinities for CDC48A, they form unexpectedly stable complexes, suggesting additional mechanisms of interaction. We identify two distinct strategies for complex stabilisation, producing different dynamic features. One relies on combining two weak associations: PUX5 employs a SHP-UBX module that engages the CDC48A N domain at two proximal sites, whereas PUX2 uses a SHP motif and a distant PUB domain to engage the N- and C-termini of CDC48A. In contrast, PUX6, PUX7, and PUX9 allosterically stabilise the association between their UBX domain and the CDC48A N domain. These multi-pronged strategies likely enable durable yet reversible associations, facilitating fine-tuned competitive regulation of CDC48A activity across diverse cellular contexts. Our findings provide a mechanistic framework for understanding how PUX proteins achieve specificity, stability, and regulatory flexibility in directing CDC48A function.