Plant Biotechnology最新文献

The unfolded protein response (UPR) is a central regulatory pathway that ensures the proper function of the endoplasmic reticulum (ER) through efficient protein folding and quality control. In Arabidopsis, bZIP60 mRNA is activated by an IRE1-mediated unconventional splicing that excises a 23-nucleotide intron, resulting in the spliced form (bZIP60s mRNA) that encodes the active bZIP60 transcription factor lacking a transmembrane domain. In this study, we investigated the functional role of the spliced form-specific C-terminal extension, hereafter referred to as ORF2. Transient expression assays in Arabidopsis mesophyll protoplasts demonstrated that full-length bZIP60s potently activates the BiP3 promoter compared to a truncated variant lacking ORF2. Fusion of ORF2 to transcription factors unrelated to the UPR did not enhance their transcriptional potency, underscoring its specialized role in the context of bZIP60s. Furthermore, mutation in a conserved nuclear localization signal within ORF2 decreased promoter activation by bZIP60s. Fusion of ORF2 to GFP enhanced the nuclear localization of GFP. Our results suggest that ORF2 is critical for the full transcriptional activity of bZIP60s to ensure an efficient UPR.

Commercial tomatoes have been increasingly criticized for their declining aroma, driving a search for tomato cultivars with more robust aroma profiles. Volatile organic compounds (VOCs) in tomato fruits play a crucial role in determining their aroma potential. This study investigates tomato cultivars with desirable aroma profiles by examining the levels of free- and glycoside-derived VOCs across 13 cultivars over a three-year period using non-targeted VOC profiling. The analysis detected 41 free VOCs and 35 VOCs released from the glycoside-derived precursors (glycoside-derived VOCs). Principal component analysis of the annotated free and glycoside-derived VOCs revealed that year-to-year differences were more pronounced than cultivar-to-cultivar variations. Among the annotated VOCs, 18 compounds were classified as unique free VOCs, while 12 were unique glycoside-derived VOCs. In 2020, the cultivar Livingstone's stone exhibited significantly higher levels of several key aroma compounds compared to other cultivars and the control, Ailsa Craig. These compounds are known for their low odor thresholds and positive contributions to the overall aroma. These findings suggested that the growth year may substantially influence VOC production in tomato fruits, particularly for specific cultivars or VOCs, likely due to variations in climatic conditions. Consequently, optimizing the release of glycoside-derived VOCs offers a promising strategy to enhance tomato fruit aroma. This approach would be most effective by focusing on key VOCs from specific cultivars, while also accounting for the significant influence of growing year.

In the present study, an efficient regeneration protocol via somatic embryogenesis and organogenesis has been developed for cassava var. Vamas 1 utilizing leaf and node explants, respectively. Leaves were inoculated on Murashige and Skoog (MS) medium containing different concentrations (4, 8, and 12 mg l^-1) of Picloram or 2,4-dichlorophenoxyacetic acid (2,4-D) with 6 mg l^-1 1-naphthaleneacetic acid (NAA). The maximum callus formation (100%) was recorded in medium containing 4 mg l^-1 Picloram or 8 mg l^-1 2,4-D. However, the callus fresh weight (0.11 g) was higher in presence of 4 mg l^-1 Picloram with 2.72 scoring of callus proliferation after 3 weeks. After subculture, 12 mg l^-1 Picloram with 6 mg l^-1 NAA proved optimum medium that formed maximum 10.25±3.49 embryos (44.00±0.04% response) under dark conditions after 6 weeks. The green cotyledons were produced after 2 weeks of light incubation on 0.2 mg l^-1 6-benzyladenin (BA). which further formed shoots within 5 weeks. Simultaneously, nodal explants were placed in MS media augmented with BA (2, 4, 8, and 10 mg l^-1) individually and in combinations with 0.02 mg l^-1 NAA. Results revealed that maximum 4.13±0.56 shoots/explant were formed with 11.07±2.79 number of leaves and 3.61±0.17 cm shoot length at 2 mg l^-1 BA. These shoots induced 7.33±0.58 number of roots after 2 weeks in basal MS medium. At last, the plantlets derived via both the pathways were transferred to soil : rice husk (1 : 1 w/w), and they were successfuly acclimatized with 80% survival in greenhouse. Since the cassava plant regeneration is genotype-dependent, the developed protocol can be applied for mass-propagation of this recently released Indonesian superior variety Vamas 1. This will generate large number of plantlets for the farmers and also the protocol will be utilized for genetic improvement studies.

In plants, the functional characterization of essential genes is often hindered by the lethality associated with complete loss-of-function alleles. Here, we present a genome editing-based strategy to generate viable hypomorphic alleles through selective removal of the translation start codon. Using Arabidopsis thaliana NON-SMC ELEMENT 1, which encodes a conserved component of the Structural Maintenance of Chromosomes (SMC) 5/6 complex involved in DNA repair and genome stability, as a model, we generated CRISPR-Cas9-edited alleles lacking the start codon. These homozygous mutants exhibited severe developmental defects, including stunted growth and failure to form true leaves. Moreover, they displayed molecular hallmarks of genome instability, such as increased DNA fragmentation, upregulation of DNA repair and cell cycle checkpoint genes, and root meristem cell death. Complementation assays using wild-type and mutated NSE1 genomic constructs confirmed that these alleles retained partial gene function. Overall, the use of start codon removal as an editing strategy is a robust and broadly applicable approach for generating hypomorphic alleles without relying on transcript-level manipulations, such as RNAi. This work therefore provides a practical demonstration of a novel editing strategy for dissecting the functions of essential genes that are otherwise genetically intractable. Consequently, this approach expands the functional genomics toolkit and opens new avenues for basic plant biology and advanced biotechnological applications.

Variability in environmental conditions and farming practices often leads to discrepancies between experimental results and outcomes in farmers' fields. This gap poses a challenge for understanding the effects of agricultural inputs and methods under real-world conditions, particularly in fruit cultivation systems, where large-scale experimental data are limited. In this study, we applied a cohort study approach leveraging data from farmers' fields to investigate the effects of pesticide and fertilizer application methods on fruit quality and soil properties in mandarin orange orchards. Biases arising from differences in covariates among cultivation methods were controlled using the inverse probability weighting (IPW) based on propensity scores. Consequently, compared to local-scale analysis between adjacent fields, the nationwide cohort analysis detected a greater number of significant effects of cultivation methods by utilizing its larger sample size. Through this analysis, we found important insights into the effects of pesticide and fertilizer application methods on plant pathogens, nutritional quality, and soil properties in sustainable cultivation systems of mandarin orange. This study demonstrates that cohort analyses using real-world data have great potential to advance agricultural biotechnology by providing effective feedback from farmers' fields and bridging the gap between scientific research and real-world agriculture.

Suspension-cultured cells of a temperate bamboo species (Phyllostachys nigra) accumulate substantial amounts of hydroxycinnamic acid derivatives and lignin under culture conditions that promote xylogenesis. In our previous study, we found a metabolite specifically produced in bamboo cells cultured under lignification-inducing conditions in a medium containing N ⁶-benzyladenine (BA), but the chemical structure was not elucidated. In this study, we purified and identified this compound as BA N ⁹-β-D-glucopyranoside (BA-9G). Despite the presence of three nitrogen positions (N-3, N-7, and N-9) that may be glucosylated in the adenine moiety of BA, bamboo cells specifically produced BA-9G (i.e., without other glucoside types) when cells were cultured in the presence of BA. This finding suggests that bamboo cells possess a regio-specific N-glucosyltransferase for catalyzing cytokinin glucoside formation. The biological activity of BA-9G as a cytokinin was compared with that of BA on the basis of adventitious shoot formation on internodal segments of ipecac (Carapichea ipecacuanha) plants grown under in vitro conditions. The activity of BA-9G was more moderate than that of BA, but BA-9G was less cytotoxic than BA at a high concentration, suggesting that BA-9G may be useful as a plant growth regulator. The development of a viable system for the regio-specific bioproduction of BA-9G in bamboo cells may increase the availability of this highly expensive and rare cytokinin derivative.

The agroinfiltration technique using sprouts as a host is one of the most cost-effective, efficient, and rapid methods for producing recombinant proteins. We previously reported that radish sprouts were the best host for this purpose. To find suitable alternative sprouts comparable to radish sprouts, we investigated rye sprouts using a wheat dwarf virus (a geminivirus) DNA-containing expression vector. Various rye cultivars were tested, and Raitaro and Ryokuhiyo sprouts exhibited the highest enhanced green fluorescent protein (EGFP) productivity. When agroinfiltrated after a 5-d cultivation period, including 1 day of seed imbibition, approximately 1.8 mg of EGFP was produced per gram fresh weight of leaf in areas exhibiting EGFP fluorescence. This yield is comparable to that of mature leaves from Nicotiana benthamiana and radish sprouts. However, only a limited number of leaves produced the protein, and production was confined to areas near the leaf tips. Elevated production levels were observed in the guard cells of stomata and at wounded sites via microneedling, suggesting that the limiting factors for protein production may involve the entry of Agrobacterium into the leaves and/or the subsequent transfer of T-DNA into the plant cells.

A plant-based expression system provides a cost-effective, scalable, and safe alternative to traditional cell culture platforms. In this study, recombinant human interleukin-15 (IL-15) was transiently expressed in Nicotiana benthamiana plants using agroinfiltration. IL-15 is a cytokine with significant potential in cell engineering, immunotherapy, and cancer therapy. A codon-optimized IL-15 gene was cloned into a binary vector designed for plant expression and introduced into Rhizobium radiobacter (formerly Agrobacterium tumefaciens). The R. radiobacter for human IL-15 expression was infiltrated into N. benthamiana leaves. Following purification, receptor-binding assays confirmed that the plant-derived IL-15 could bind to the IL-15 receptor comparably to its mammalian-produced counterpart. This first report of IL-15 expression in plants highlights the promise of plant-based systems for biopharmaceutical production and lays the groundwork for further development of IL-15 for applications in cell engineering, clinical therapies, and the cultured meat industry.

Bioproduction of high-value natural compounds in cultured plant cells represents a favorable strategy compatible with the framework of green sustainable chemistry. Raspberry ketone, a phenylpropanoid-derived compound utilized as a food additive and cosmetics constituent, is a distinctive aromatic component that is accumulated in the ripe fruit of raspberry (Rubus idaeus). Its natural abundance is extremely limited. Consequently, it is imperative to develop methodologies for efficient bioproduction of raspberry ketone. In the present study, we examined the effect of expressing the raspberry ketone biosynthetic genes RpBAS and RiRZS1 in bamboo (Phyllostachys nigra) cells, which had been demonstrated previously to be an appropriate host for production of phenylpropanoid-derived compounds, on the bioconversion of precursor compounds into raspberry ketone. The maximal production yield of raspberry ketone, primarily accumulated in the glycosylated form, reached 293 µg g^-1 fresh weight when the RpBAS-RiRZS1-transgenic bamboo cells were cultured in medium supplemented with 4-hydroxybenzalacetone, the immediate precursor of raspberry ketone. These findings underscore the potential utility of bamboo cells for the bioproduction of raspberry ketone from accessible precursors.

Catechins includ galloylated catechins and non-galloylated catechins, among which galloylated catechins exhibit stronger antioxidant, anti-inflammatory and anti-cancer activities. Section Chrysantha Chang, the only group of yellow Camellia with rich catechins in their flowers, is a common health drink in southern China. To date, few studies have examined galloylated catechins biosynthesis in flowers of this group. To enrich the genetic information of the galloylated catechins biosynthesis, the ONT sequencing platform was used to perform full-length transcriptome sequencing of C. perpetua flowers and 7,972,574 transcripts was identified, including 42,883 simple sequence repeats (SSRs), 41,961 coding sequences (CDSs) and 2,602 long non-coding RNAs (lncRNAs). 36,516 transcripts were successfully annotated, and 147 critical enzyme-encoding genes were identified as involved in the galloylated catechins biosynthesis pathway, including 17 CpSCPL1A genes. Bioinformatics analysis revealed that each CpSCPL1A protein consisted of 427-506 amino acids, and all CpSCPL1A proteins were divided into 5 groups with conserved motifs 1, 4, 5, 6 and 8. Based on the correlation analysis between the gene expression of 17 CpSCPL1A genes and the content of galloylated catechins, 11 candidate CpSCPL1A genes were identified to be involved in the biosynthesis of 4 types of galloylated catechins in C. perpetua flowers. The results enrich the transcriptome data for C. perpetua and provide valuable insights into the importance of the CpSCPL1A gene family members in the galloylated catechins biosynthesis.