Plant Cell Reports最新文献

Key message: Through the study of a point mutation of AtECB2, it is reconfirmed that AtECB2 plays an important role in the early development of chloroplast. AtECB2(EARLY CHLOROPLAST BIOGENESIS 2, At1g15510), a member of the pentatricopeptide repeat motif proteins (PPR) superfamily, and its loss of function mutation ecb2-1causes seedling lethal, while a point mutation ecb2-2 causes delayed chloroplast development. Finding more AtECB2 weak alleles helps to understand the molecular mechanisms of AtECB2. In this study, a leaf virescence mutant was identified from ethylmethane sulfonate (EMS) treated Arabidopsis Col-0 M2 mutants library. The mutation of this mutant was first confirmed as a recessive mutation of one gene through the phenotype of F1 and its F2 phenotype segregation of this mutant crossed with Col-0. The mutation of G1931A of AtECB2 is identified as the cause of this leaf virescence phenotype sequentially through positional cloning, whole genome resequencing, Sanger sequencing and complementation. Therefore, we named this weak allele of AtECB2 as ecb2-3. The chlorophyll content and photosystem II maximum photochemical efficiency of ecb2-3 are obviously lower than that of Col-0 and its complementation lines, respectively. The chloroplast development of ecb2-3 is also inferior to that of Col-0 and its complementation line at the observed time points using the transmission electron microscope. The RNA editing efficiency of three chloroplast gene sites (accD C794 and C1568, ndhF C290) was observed much lower compared with that of Col-0 and its complementation line. In summary, AtECB2 plays an important role in early chloroplast biogenesis through related chloroplast gene editing regulation, and this weak mutant ecb2-3 may be useful material in dissecting the function of AtECB2 in the near future.

Key message: A long tracrRNA (tracr-L), which naturally act as single guide RNA, and its truncated version, Δtracr-L, from S. pyogenes, efficiently induce Cas9-mediated double-strand breaks (DSBs) in plant genomic loci, as demonstrated by in vitro cleavage assay and protoplast transfection. CRISPR-Cas system provides a form of immune memory in prokaryotes and archaea, protecting them against viruses and foreign genetic elements. In Streptococcus pyogenes, this system includes the pre-crRNA along with another non-coding RNA, tracrRNA, which aids in CRISPR-based immunity. In S. pyogenes, two distinct tracrRNAs are produced: a long form (tracr-L) and a short form (tracr-S). The tracr-S regulates crRNA biogenesis and Cas9 cleavage, while tracr-L suppresses CRISPR-Cas expression by targeting the Cas9 promoter to prevent autoimmunity. Deleting 79 nucleotides from tracr-L results in Δtracr-L, which retains similar functionality in gene repression. This study investigates, for the first time, the effectiveness of tracr-L, and Δtracr-L in genome editing within plant systems. In vitro cleavage assays using purified Cas9 and synthesized sgRNAs targeting the Cas9 gene, OsPDS, and the OsSWEET11 promoter revealed that across all target sites, tracr-S demonstrated the highest cleavage efficiency compared to tracr-L and Δtracr-L. For in vivo genome editing, we transfected rice protoplasts with tracr-L, Δtracr-L, and tracr-S, targeting three rice genes: OsPDS, OsSPL14, and the promoter of OsSWEET14. Amplicon deep sequencing revealed various types of indels at the target regions across all three tracrRNA versions, indicating comparable levels of efficiency. This study establishes the utility of both the long-form tracrRNA (tracr-L) and its truncated variant (Δtracr-L) in eukaryote genome editing. These two new forms of tracrRNA provide proof of concept and expand the CRISPR-Cas toolkit for plant genome editing applications, and for eukaryotes more broadly.

Key message: This study describes an optimized plastid genetic engineering platform to produce full marker-free transplastomic plants with transgene integrated at homoplasmy in one step in tissue culture. Plastid engineering is attractive for both biotechnology and crop improvement due to natural bio-confinement from maternal inheritance, the absence of transgene positional effects and silencing, the ability to express transgenes in operons, and unparalleled production of heterologous proteins. While plastid engineering has had numerous successes in the production of high-value compounds, no transplastomic plants have been approved for use in agriculture. In order for transplastomic plants to be used in agriculture, the removal of antibiotic selection genes is required. In this work, we developed an optimized strategy to generate homoplasmic marker-free lines of potato (Solanum tuberosum) in a single transformation event. To achieve marker-free transplastomic lines, vectors were redesigned to enable integration of the transgene cassette into the plastid genome, while maintaining the selection cassette on the vector backbone. After an initial round of tissue culture with selection, the selective pressure was removed, leading to the elimination of the vector backbone, while retaining the integrated transgene cassette at homoplasmy. Marker-free transplastomic lines produced using this strategy had a normal phenotype, and transgene integration was stable across generations. The new vectors developed in this work for the generation of marker-free transplastomics will represent a valuable alternative platform for routine plastid genetic engineering in higher plants. It is also anticipated that this approach will contribute to speed the path to commercialization of these novel transplastomic plant varieties.

Key message: Constitutive expression of cucumber CsACS2 in Arabidopsis disrupts anther dehiscence and male fertility via ethylene signaling and DNA methylation, revealing new avenues for enhancing crop reproductive traits. The cucumber gene CsACS2, encoding ACC (1-aminocyclopropane-1-carboxylic acid) synthase, plays a pivotal role in ethylene biosynthesis and sex determination. This study investigates the effects of constitutive CsACS2 expression in Arabidopsis thaliana on anther development and male fertility. Transgenic Arabidopsis plants overexpressing CsACS2 exhibited male sterility due to inhibited anther dehiscence, which was linked to suppressed secondary cell wall thickening. RNA-Seq analysis revealed upregulation of ethylene signaling pathway genes and downregulation of secondary cell wall biosynthesis genes, with gene set enrichment analysis indicating the involvement of DNA methylation. Rescue experiments demonstrated that silver nitrate (AgNO₃) effectively restored fertility, while 5-azacytidine (5-az) partially restored it, highlighting the roles of ethylene signaling and DNA methylation in this process. Constitutive CsACS2 expression in Arabidopsis disrupts anther development through ethylene signaling and DNA methylation pathways, providing new insights into the role of ethylene in plant reproductive development and potential applications in crop improvement.

Seed development is a complex process and co-regulated by genetic and environmental factors, which significantly affects the seed vigor, yield and quality of crops, especially in cereal crops. Abscisic acid (ABA) regulates various biological processes in seed development, including endosperm and embryo development, accumulation of storage materials, achievement of desiccation tolerance and dormancy. Compared to the functional investigation of ABA in germination and stress response, the role of ABA in early seed development and storage product accumulation has not been collectively elucidated. Here, ABA origin in seed was concluded: both maternal source and de novo synthesis of ABA in seed play an important role in seed development. This review also provided an overview of the current knowledge on ABA in early seed development, mainly in endosperm cellularization. ABA promotes endosperm cellularization in Arabidopsis, but this notion has not been spread into cereal crops. Besides, the increasing importance of ABA in seed reserve accumulation was also emphatically described. In the last section, the key problems and challenges (e.g., where dose ABA come from at each stage of seed development? whether same regulators in response to ABA in Arabidopsis apply equally to cereal crops) were addressed.

Key message: PAs varied greatly in leaves of different germplasm accessions in Lotus corniculatus and over-expression of LcMYB5 led to high PA accumulation in L. japonicus hairy roots. Proanthocyanidins (PAs) content in leaves is an important quality trait in forage species. The leaves of most forage crops accumulated no or little PAs, which makes it difficult to discover key genes involved in PA biosynthesis in the leaves. We found PAs content varied greatly in leaves of different germplasm accessions in Lotus corniculatus, which is one of the most agriculturally important forage crops. Through a combination of global transcriptional analysis, GO and KEGG analysis, and phylogenetic analysis, we discovered that LcMYB5 was strongly correlated with PA accumulation in leaves of L. corniculatus. The subcellular localization and transactivation activity assays demonstrated that LcMYB5 localized to the nucleus and acted as a transcriptional activator. Over-expression of the two homologs of LcMYB5 (LcMYB5a and LcMYB5b) in the L. japonicus hairy roots resulted in a particular high level of PAs. Global transcriptional analysis and qRT-PCR assays indicated that LcMYB5a and LcMYB5b up-regulated the transcript levels of many key PA pathway genes in the transgenic hairy roots, including structural genes (eg. CHS, F3H, LAR, ANR, and TT15) and regulatory genes (eg. TT8 and TTG1). Collectively, our data suggests that LcMYB5 independently regulates PA accumulation in the leaves of Lotus as a master regulator, which can be bioengineered for PAs production in the leaves of forage species.

Key message: Astragalus membranaceus hairy roots induced by direct injection of Rhizobium rhizogenes with AmUGT15 overexpressing genes into the stem explants demonstrate enhanced astragaloside biosynthesis Astragalus membranaceus is a widely used medicinal plant, which has important economic, ecological, medicinal, and ornamental values for accumulating various triterpene saponins named astragalosides in roots. Although the hairy root culture technique has been established in A. membranaceus, the molecular regulation of metabolic pathways for improving astragaloside contents was not reported. In this study, an efficient hairy root induction method was established in A.membranaceus by directly injecting Rhizobium rhizogenes into the stem, with an induction rate of up to 80.1%. We improved the production of astragaloside in hairy roots by overexpressing AmUGT15, a 3-O-glucosyltransferase catalyzed xylosylation at C3-OH. The fluorescence microscopy observation revealed that the AmUGT15 fused with DsRed report gene constructed in T-DNA region was overexpressed in hairy roots, and the maximum biomass of hairy roots was measured on the 28th day of cultivation. HPLC analysis confirmed the total amount of astragalosides produced by AmUGT15 overexpressing hairy roots is 4.2 times higher than the non-transgenic control group. Our study proposed an effective method for astragalosides production in A. membranaceus hairy roots via metabolic engineering.

Key message: The histone variant H2A.Z is crucial for the expression of genes involved in cell elongation under elevated temperatures and shade. Its removal facilitates the activation of these genes, particularly through the activities of PHYTOCHROME INTERACTING FACTORs (PIFs) and the SWR1-related INOSITOL REQUIRING 80 (INO80) complex. Arabidopsis seedlings exhibit rapid elongation of hypocotyls and cotyledon petioles in response to environmental stresses, namely elevated temperatures and shade. These phenotypic alterations are regulated by various phytohormones, notably auxin. Under these stress conditions, auxin biosynthesis is swiftly induced in the cotyledons and transported to the hypocotyls, where it stimulates cell elongation. The histone variant H2A.Z plays a pivotal role in this regulatory mechanism. H2A.Z affects the transcription of numerous genes, particularly those activated by the mentioned environmental stresses. Recent studies highlighted that the eviction of H2A.Z from gene bodies is crucial for the activation of genes, especially auxin biosynthetic and responsive genes, under conditions of elevated temperature and shade. Additionally, experimental evidence suggests that PHYTOCHROME INTERACTING FACTORs (PIFs) can recruit the SWR1-related INOSITOL REQUIRING 80 (INO80) complex to remove H2A.Z from targeted loci, thereby activating gene transcription in response to these environmental stresses. This review provides a comprehensive overview of the regulatory role of H2A.Z, emphasizing how its eviction from gene loci is instrumental in the activation of stress-responsive genes under elevated temperature and shade conditions.

Key message: Glycosylphosphatidylinositol-anchored protein (GPI-AP) Aa049 works as a key pathogenic factor to assist A. alternata in infecting plants, which is associated with the reactive oxygen species (ROS) pathway. Chrysanthemum black spot disease is a common fungal disease caused by A. alternata, which has severely hindered the development of the chrysanthemum industry. However, there are few reports on pathogenic factors in A. alternata, especially regarding GPI-APs. In this study, we identified a GPI-AP, Aa049, from A. alternata. Bioinformatics predictions suggest the presence of GPI-anchored modification sites at the C-terminus of its amino acid sequence, which is relatively conserved among different Alternaria Nees. Transient overexpression of Aa049 in Nicotiana benthamiana can induce programmed cell death (PCD), and the appearance of necrosis depends on its native signal peptide and GPI-anchored sites. Compared with the wild-type strain, the morphology and growth rate of the colony and mycelia of the ΔAa049-deletion mutants do not change. Still the integrity of the cell wall is damaged, and the virulence of the strain is significantly reduced, indicating that Aa049 plays an essential role as a pathogenic factor in the infection process of A. alternata. Furthermore, the results of quantitative real-time PCR (qRT-PCR) and physiological indicators suggested that the virulence of Aa049 may be exerted through the synthesis and clearance pathways of ROS. This study reveals that GPI-APs in A. alternata can act as virulence factors to aid pathogen invasion, providing a potential target for the development of future biopesticides.