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BMC Plant Biology Jun 2024Angelica Gigas (Purple parsnip) is an important medicinal plant that is cultivated and utilized in Korea, Japan, and China. It contains bioactive substances especially...
Response surface methodology mediated optimization of phytosulfokine and plant growth regulators for enhanced protoplast division, callus induction, and somatic embryogenesis in Angelica Gigas Nakai.
BACKGROUND
Angelica Gigas (Purple parsnip) is an important medicinal plant that is cultivated and utilized in Korea, Japan, and China. It contains bioactive substances especially coumarins with anti-inflammatory, anti-platelet aggregation, anti-cancer, anti-diabetic, antimicrobial, anti-obesity, anti-oxidant, immunomodulatory, and neuroprotective properties. This medicinal crop can be genetically improved, and the metabolites can be obtained by embryonic stem cells. In this context, we established the protoplast-to-plant regeneration methodology in Angelica gigas.
RESULTS
In the present investigation, we isolated the protoplast from the embryogenic callus by applying methods that we have developed earlier and established protoplast cultures using Murashige and Skoog (MS) liquid medium and by embedding the protoplast in thin alginate layer (TAL) methods. We supplemented the culture medium with growth regulators namely 2,4-dichlorophenoxyaceticacid (2,4-D, 0, 0.75, 1.5 mg L), kinetin (KN, 0, 0.5, and 1.0 mg L) and phytosulfokine (PSK, 0, 50, 100 nM) to induce protoplast division, microcolony formation, and embryogenic callus regeneration. We applied central composite design (CCD) and response surface methodology (RSM) for the optimization of 2,4-D, KN, and PSK levels during protoplast division, micro-callus formation, and induction of embryogenic callus stages. The results revealed that 0.04 mg L 2,4-D + 0.5 mg L KN + 2 nM PSK, 0.5 mg L 2,4-D + 0.9 mg L KN and 90 nM PSK, and 1.5 mg L 2,4-D and 1 mg L KN were optimum for protoplast division, micro-callus formation and induction embryogenic callus. MS basal semi-solid medium without growth regulators was good for the development of embryos and plant regeneration.
CONCLUSIONS
This study demonstrated successful protoplast culture, protoplast division, micro-callus formation, induction embryogenic callus, somatic embryogenesis, and plant regeneration in A. gigas. The methodologies developed here are quite useful for the genetic improvement of this important medicinal plant.
Topics: Angelica; Plant Growth Regulators; Plant Somatic Embryogenesis Techniques; Protoplasts; Cell Division
PubMed: 38858674
DOI: 10.1186/s12870-024-05243-w -
The Plant Genome Jun 2024Seven in absentia proteins, which contain a conserved SINA domain, are involved in regulating various aspects of wheat (Triticum aestivum L.) growth and development,...
Seven in absentia proteins, which contain a conserved SINA domain, are involved in regulating various aspects of wheat (Triticum aestivum L.) growth and development, especially in response to environmental stresses. However, it is unclear whether TaSINA family members are involved in regulating grain development until now. In this study, the expression pattern, genomic polymorphism, and relationship with grain-related traits were analyzed for all TaSINA members. Most of the TaSINA genes identified showed higher expression levels in young wheat spikes or grains than other organs. The genomic polymorphism analysis revealed that at least 62 TaSINA genes had different haplotypes, where the haplotypes of five genes were significantly correlated with grain-related traits. Kompetitive allele-specific PCR markers were developed to confirm the single nucleotide polymorphisms in TaSINA101 and TaSINA109 among the five selected genes in a set of 292 wheat accessions. The TaSINA101-Hap II and TaSINA109-Hap II haplotypes had higher grain weight and width compared to TaSINA101-Hap I and TaSINA109-Hap I in at least three environments, respectively. The qRT-PCR assays revealed that TaSINA101 was highly expressed in the palea shell, seed coat, and embryo in young wheat grains. The TaSINA101 protein was unevenly distributed in the nucleus when transiently expressed in the protoplast of wheat. Three homozygous TaSINA101 transgenic lines in rice (Oryza sativa L.) showed higher grain weight and size compared to the wild type. These findings provide valuable insight into the biological function and elite haplotype of TaSINA family genes in wheat grain development at a genomic-wide level.
PubMed: 38840306
DOI: 10.1002/tpg2.20480 -
Genomic deletions in Aureobasidium pullulans by an AMA1 plasmid for gRNA and CRISPR/Cas9 expression.Fungal Biology and Biotechnology Jun 2024Aureobasidium pullulans is a generalist polyextremotolerant black yeast fungus. It tolerates temperatures below 0 °C or salt concentrations up to 18%, among other...
BACKGROUND
Aureobasidium pullulans is a generalist polyextremotolerant black yeast fungus. It tolerates temperatures below 0 °C or salt concentrations up to 18%, among other stresses. A. pullulans genome sequencing revealed a high potential for producing bioactive metabolites. Only few molecular tools exist to edit the genome of A. pullulans, hence it is important to make full use of its potential. Two CRISPR/Cas9 methods have been proposed for the protoplast-based transformation of A. pullulans. These methods require the integration of a marker gene into the locus of the gene to be deleted, when the deletion of this gene does not yield a selectable phenotype. We present the adaptation of a plasmid-based CRISPR/Cas9 system developed in Aspergillus niger for A. pullulans to create deletion strains.
RESULTS
The A. niger CRISPR/Cas9 plasmid led to efficient genomic deletions in A. pullulans. In this study, strains with deletions ranging from 30 to 862 bp were obtained by using an AMA1 plasmid-based genome editing strategy.
CONCLUSION
The CRISPR/Cas9 transformation system presented in this study provides new opportunities for strain engineering of A. pullulans. This system allows expression of Cas9 and antibiotic resistance while being easy to adapt. This strategy could open the path to intensive genomic engineering in A. pullulans.
PubMed: 38824542
DOI: 10.1186/s40694-024-00175-4 -
Frontiers in Plant Science 2024Environmental stressors disrupt secretory protein folding and proteostasis in the endoplasmic reticulum (ER), leading to ER stress. The unfolded protein response (UPR)...
Protein disulfide isomerase-9 interacts with the lumenal region of the transmembrane endoplasmic reticulum stress sensor kinase, IRE1, to modulate the unfolded protein response in .
Environmental stressors disrupt secretory protein folding and proteostasis in the endoplasmic reticulum (ER), leading to ER stress. The unfolded protein response (UPR) senses ER stress and restores proteostasis by increasing the expression of ER-resident protein folding chaperones, such as protein disulfide isomerases (PDIs). In plants, the transmembrane ER stress sensor kinase, IRE1, activates the UPR by unconventionally splicing the mRNA encoding the bZIP60 transcription factor, triggering UPR gene transcription. The induced PDIs catalyze disulfide-based polypeptide folding to restore the folding capacity in the ER; however, the substrates with which PDIs interact are largely unknown. Here, we demonstrate that the PDI-M subfamily member, PDI9, modulates the UPR through interaction with IRE1. This PDI9-IRE1 interaction was largely dependent on Cys63 in the first dithiol redox active domain of PDI9, and Cys233 and Cys107 in the ER lumenal domain of IRE1A and IRE1B, respectively. and , PDI9 coimmunoprecipitated with IRE1A and IRE1B. Moreover, the PDI9:RFP and Green Fluorescence Protein (GFP):IRE1 fusions exhibited strong interactions as measured by fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) when coexpressed in mesophyll protoplasts. The UPR-responsive PDI9 promoter:mCherry reporter and the UPR-dependent splicing of the bZIP60 intron from the mRNA of the 35S::bZIP60-intron:GFP reporter were both significantly induced in the mutants, indicating a derepression and hyperactivation of UPR. The inductions of both reporters were substantially attenuated in the mutant. We propose a model in which PDI9 modulates the UPR through two competing activities: secretory protein folding and via interaction with IRE1 to maintain proteostasis in plants.
PubMed: 38817940
DOI: 10.3389/fpls.2024.1389658 -
Frontiers in Bioscience (Landmark... May 2024Oliver is a unique high-quality natural rubber tree species and rare medicinal tree species in China. The rapid characterization of gene function has been severely...
BACKGROUND
Oliver is a unique high-quality natural rubber tree species and rare medicinal tree species in China. The rapid characterization of gene function has been severely hampered by the limitations of genetic transformation methods and breeding cycles. The polyethylene glycol (PEG)-mediated protoplast transformation system is a multifunctional and rapid tool for the analysis of functional genes , but it has not been established in .
METHODS
In this study, a large number of highly active protoplasts were isolated from the stems of seedlings by enzymatic digestion, and green fluorescent protein expression was facilitated using a PEG-mediated method.
RESULTS
Optimal enzymatic digestion occurred when the enzyme was digested for 10 h in an enzymatic solution containing 2.5% Cellulase R-10 (w/v), 0.6% Macerozyme R-10 (w/v), 2.5% pectinase (w/v), 0.5% hemicellulase (w/v), and 0.6 mol/L mannitol. The active protoplast yield under this condition was 1.13 × 106 protoplasts/g fresh weight, and the protoplast activity was as high as 94.84%.
CONCLUSIONS
This study established the first protoplasm isolation and transient transformation system in hard rubber wood, which lays the foundation for subsequent functional studies of genes to achieve high-throughput analysis, and provides a reference for future gene function studies of medicinal and woody plants.
Topics: Protoplasts; Eucommiaceae; Transfection; Green Fluorescent Proteins; Polyethylene Glycols
PubMed: 38812327
DOI: 10.31083/j.fbl2905187 -
The Plant Genome Jun 2024The clustered regularly interspaced short palindromic repeats (CRISPR) systems have been demonstrated to be the foremost compelling genetic tools for manipulating...
The clustered regularly interspaced short palindromic repeats (CRISPR) systems have been demonstrated to be the foremost compelling genetic tools for manipulating prokaryotic and eukaryotic genomes. Despite the robustness and versatility of Cas9 and Cas12a/b nucleases in mammalian cells and plants, their large protein sizes may hinder downstream applications. Therefore, investigating compact CRISPR nucleases will unlock numerous genome editing and delivery challenges that constrain genetic engineering and crop development. In this study, we assessed the archaeal miniature Un1Cas12f1 type-V CRISPR nuclease for genome editing in rice and tomato protoplasts. By adopting the reengineered guide RNA modifications ge4.1 and comparing polymerase II (Pol II) and polymerase III (Pol III) promoters, we demonstrated uncultured archaeon Cas12f1 (Un1Cas12f1) genome editing efficacy in rice and tomato protoplasts. We characterized the protospacer adjacent motif (PAM) requirements and mutation profiles of Un1Cas12f1 in both plant species. Interestingly, we found that Pol III promoters, not Pol II promoters, led to higher genome editing efficiency when they were used to drive guide RNA expression. Unlike in mammalian cells, the engineered Un1Cas12f1-RRA variant did not perform better than the wild-type Un1Cas12f1 nuclease, suggesting continued protein engineering and other innovative approaches are needed to further improve Un1Cas12f1 genome editing in plants.
Topics: Oryza; Solanum lycopersicum; Gene Editing; CRISPR-Cas Systems; Protoplasts; Genome, Plant
PubMed: 38807445
DOI: 10.1002/tpg2.20465 -
International Journal of Molecular... May 2024A homeobox transcription factor is a conserved transcription factor, ubiquitous in eukaryotes, that regulates the tissue formation of structure, cell differentiation,...
A homeobox transcription factor is a conserved transcription factor, ubiquitous in eukaryotes, that regulates the tissue formation of structure, cell differentiation, proliferation, and cancer. This study identified the homeobox transcription factor family and its distribution in var. at the whole genome level. It elucidated the gene structures and evolutionary characteristics of this family. Additionally, knockout experiments were carried out and the preliminary function of these transcription factors was studied. Through bioinformatics approaches, nine homeobox transcription factors () were identified in var. , and these contained HOX-conserved domains and helix-turn-helix secondary structures. Nine homeobox gene deletion mutants were obtained using the homologous recombinant gene knockout technique. Protoplast transformation was mediated by polyethylene glycol (PEG) and the transformants were identified using PCR. The knockouts of , , , , , , and genes resulted in a smaller growth diameter in var. . In contrast, the knockouts of the , , and genes inhibited the formation of conidia and led to a significant decrease in the pathogenicity. This study's results will provide insights for understanding the growth and development of var. . The pathogenic mechanism of the affected sugarcane will provide an essential theoretical basis for preventing and controlling sugarcane twisted leaf disease.
Topics: Saccharum; Homeodomain Proteins; Plant Diseases; Ascomycota; Transcription Factors; Plant Leaves; Phylogeny
PubMed: 38791383
DOI: 10.3390/ijms25105346 -
Microbial Cell Factories May 2024
PubMed: 38741121
DOI: 10.1186/s12934-024-02377-2 -
Horticulture Research May 2024The seasonal flowering Chinese produce an axillary floral meristem and require a dormancy period during cold conditions for flower development. However, the bud...
The seasonal flowering Chinese produce an axillary floral meristem and require a dormancy period during cold conditions for flower development. However, the bud activation mechanism remains elusive. This study evaluates the multi-omics across six stages of flower development, along with functional analysis of core genes to decipher the innate mechanism of floral bud initiation and outgrowth in the Chinese orchid . Transcriptome and proteome analyses identified 10 modules with essential roles in floral bud dormancy and activation. Gene clusters in the early stages of flower development were mainly related to flowering time regulation and meristem determination, while the late stages were correlated with hormone signaling pathways. The metabolome identified 69 potential hormones in which gibberellin (GA) and abscisic acid (ABA) were the main regulatory hubs, and GA4 and GA53 exhibited a reciprocal loop. Extraneous GA application caused rapid elongation of flower buds and promoted the expression of flower development genes. Contrarily, exogenous ABA application extended the dormancy process and ABA inhibitors induced dormancy release. Moreover, () was identified as the potential target of ABA for floral bud activation. Transformation of in and its transient overexpression in protoplasts not only affected flowering time and floral organ morphogenesis in but also orchestrated the expression of flowering and hormone regulatory genes. The presence of ABA response elements in the promoter, rapid downregulation of after exogenous ABA application, and the activation of the floral bud after ABA inhibitor treatment suggest that ABA can control bud outgrowth through .
PubMed: 38738212
DOI: 10.1093/hr/uhae073 -
Plant Methods May 2024With the accumulating omics data, an efficient and time-saving transient assay to express target genes is desired. Mesophyll protoplasts, maintaining most...
BACKGROUND
With the accumulating omics data, an efficient and time-saving transient assay to express target genes is desired. Mesophyll protoplasts, maintaining most stress-physiological responses and cellular activities as intact plants, offer an alternative transient assay to study target genes' effects on heat and oxidative stress responses.
RESULTS
In this study, a perennial ryegrass (Lolium perenne L.) mesophyll protoplast-based assay was established to effectively over- or down-regulate target genes. The relative expression levels of the target genes could be quantified using RT-qPCR, and the effects of heat and HO-induced oxidative stress on protoplasts' viability could be quantitatively measured. The practicality of the assay was demonstrated by identifying the potential thermos-sensor genes LpTT3.1/LpTT3.2 in ryegrass that over-expressing these genes significantly altered protoplasts' viability rates after heat stress.
CONCLUSION
This protoplast-based rapid stress regulatory gene identification assay was briefed as 'PRIDA' that will complement the stable genetic transformation studies to rapidly identify candidate stress-regulatory genes in perennial ryegrass and other grass species.
PubMed: 38725058
DOI: 10.1186/s13007-024-01192-5