Plant Biotechnology最新文献

Triterpenoids, a group of specialized plant metabolites with substantial structural diversity, are promising for healthcare applications. Ursolic acid, a pentacyclic triterpenoid with therapeutic potential, is also important as a precursor of corosolic acid, which is known as a "phyto-insulin" for its insulin-like properties. Ursolic acid is synthesized from a linear 30-carbon precursor 2,3-oxidosqualene via cyclization to produce triterpene scaffold α-amyrin, followed by a series of oxidation steps at the C-28 position mediated by cytochrome P450 monooxygenases (CYPs) in the CYP716A subfamily. The Tsukuba system was developed for the high-level transient expression of foreign proteins in plant cells based on the use of a binary vector equipped with geminiviral replication system and a double terminator. In this study, we used the Tsukuba system to produce ursolic acid in Nicotiana benthamiana leaves via transient pathway reconstruction. We used an oxidosqualene cyclase identified from the medicinal legume Bauhinia forficata, exhibiting a preponderant α-amyrin-producing activity. Wild-type Medicago truncatula CYP716A12 and its mutants were assessed in terms of ursolic acid production. We improved the performance of MtCYP716A12 by co-expressing it with the appropriate cytochrome P450 reductase (CPR) isozyme as an electron-transfer partner and tested different Agrobacterium infiltration ratios to optimize the CPR : CYP ratio to maximize ursolic acid production. We also achieved high yield of corosolic acid by co-expressing Avicennia marina CYP716C53 with ursolic acid biosynthetic enzymes. Moreover, engineering of AmCYP716C53 significantly improved corosolic acid yield, resulting in a yield exceeding the content found in banaba leaves, a well-known rich source of corosolic acid.

Structurally diverse benzylisoquinoline alkaloids (BIAs) are found in specific plant families, some of which are desirable for their efficient production because of their strong biological activities. Corydalis plants (e.g., Corydalis yanhusuo) of the family Papaveraceae also produce various BIAs; thus, they have been used in traditional Chinese medicine. Because metabolic engineering and synthetic biology using microorganisms are promising technologies for the effective production of useful metabolites, elucidation of the biosynthetic pathway of each BIA is indispensable. Although several enzyme genes involved in the biosynthesis of Corydalis BIAs have recently been isolated, many remain unknown, such as the protoberberine alkaloid C-methyltransferase involved in the biosynthesis of corydaline, one of the main BIAs found in the tubers of Corydalis plants. In this study, we performed transcriptome analysis combined with metabolite profiling of different tissues of Corydalis solida. Based on the high accumulation of several BIAs, including protopine, allocryptopine, and corydaline, genes encoding putative biosynthetic enzymes, including cytochrome P450, methyltransferase, and oxidase proteins, that were highly expressed in the tubers were screened. Two OMT genes, CsOMT1 and CsOMT2, were highly expressed in the tuber, and further characterization using crude enzyme preparations demonstrated that CsOMT1 showed 7-O-methylation activity against reticuline, whereas CsOMT2 catalyzed 9-O-methylation of scoulerine, followed by 2-O-methylation of tetrahydrocolumbamine. Our findings provide valuable information for the isolation of novel biosynthetic enzyme genes in Corydalis species.

Ditylenchus destructor is a plant-parasitic nematode that severely damages garlic (Allium sativum L.) in Japan. D. destructor is detected in roots, bulbs, and outer bulb skins of garlic at harvest; however, the resistance of garlic to D. destructor infection is not well understood. Here, we investigated the propagation of D. destructor in storage organs and roots using in vitro plantlets of six Japanese garlic varieties to exclude the effects of microbes and to uniform growing conditions. In vitro inoculation can proceed simultaneously with vegetative growth, storage organ formation of garlic plantlets, and D. destructor infection. In 'Fukuchi-white', a variety susceptible to D. destructor, nematodes successfully propagated in storage organs and roots. Furthermore, the nematodes invaded and propagated in the newly formed storage organs. By contrast, 'Kirishima', 'Hirado', and 'Shishimaru' substantially suppressed more the propagation of the nematodes in storage organs and roots than 'Fukuchi-white'. Additionally, the propagation of nematodes in newly formed storage organs was inhibited in these three varieties. 'Shishimaru' showed unique responses to D. destructor infection: nematode propagation was the lowest among six varieties in inoculation tests and the nematode-inoculated cloves turned brown. Our results suggest that several garlic varieties have resistance mechanisms that suppress the propagation of D. destructor in storage organs and roots, and that in vitro inoculation methods are useful for selecting resistant garlic varieties. These findings will help developing novel D. destructor-resistant garlic varieties and our further understanding of garlic-nematode interactions.

The sustainable production and utilization of lignocellulose biomass are indispensable for establishing sustainable societies. Trees and large-sized grasses are the major sources of lignocellulose biomass, while large-sized grasses greatly surpass trees in terms of lignocellulose biomass productivity. With an overall aim to improve lignocellulose usability, it is important to increase the lignin content and simplify lignin structures in biomass plants via lignin metabolic engineering. Rice (Oryza sativa) is not only a representative and important grass crop, but also is a model for large-sized grasses in biotechnology. This review outlines progress in lignin metabolic engineering in grasses, mainly rice, including characterization of the lignocellulose properties, the augmentation of lignin content and the simplification of lignin structures. These findings have broad applicability for the metabolic engineering of lignin in large-sized grass biomass plants.

The members of glutathione S-transferase (GST) belonging to the Phi class of the GST family are known to play a role in anthocyanin transport to the vacuole. We isolated a GST orthologue from the torenia petal cDNA library. Transgenic plants transcribing GST double stranded RNA were generated from a torenia cultivar having blue flowers. These plants exhibited a range of flower colors, from blue to almost white. Quantitative RT-PCR confirmed the downregulation of the GST transcript, accompanied by a decrease in anthocyanin levels in the petals of the transgenic plants, whereas flavone levels remained unchanged. These results suggest that GST is involved in anthocyanin transport in torenia petals, and that anthocyanins and flavones are likely transported to the vacuole through different mechanisms.

Cell division is important for organisms to grow and repair damaged tissues. A mutant screen in rice has identified dwarf korpokkur (kor) mutants that code for a novel protein potentially involved in mitosis including cytokinesis in rice. The KOR gene is expressed during the mitotic phase and a defect in the KOR gene induces cells with two nuclei. Analysis of kor mutants suggests that the KOR gene promotes cell division in the rice leaf primordia for a period after initiation, and maintains proper cell morphology especially in non-meristematic tissues. Additionally, kor mutants showed a delayed transition from juvenile phase to adult phase. Future research will shed light on the relationship between the mitotic defect and other features observed in the kor mutants.

Initial light reception after germination is a dramatic life event when a seedling starts proper morphogenesis. Blue light contains a range of light wavelengths that plants can perceive. A previous report suggested that the chemical compound 3-bromo-7-nitroindazole (3B7N) inhibits blue light-mediated suppression of hypocotyl elongation by physically interacting with the blue light receptor Cryptochrome 1 (CRY1). We previously examined changes of genome-wide gene expression in Arabidopsis seedlings germinated in the dark and then exposed to blue light by RNA-seq and Ribo-seq analyses. The expression of ribosome-related genes was translationally upregulated in response to the initial blue light exposure, depending on signals from both the nucleus and chloroplasts. Here, we re-analyzed our previous data and examined the effect of 3B7N treatment on changes in gene expression upon blue light exposure. The results showed that 3B7N negatively affected translation of ribosome-related genes and, interestingly, the effects were similar to not only those in cry1cry2 mutants but also plants under suppression of photosynthesis. We propose an apparent crosstalk between chloroplast function and blue light signaling.

Aromatic compounds play essential roles in plant physiology and various industries because of their unique fragrances and beneficial properties. In this study, we investigated the transport and biosynthesis of eugenol, a prominent aromatic compound, within the Ocimum genus, using grafting experiments. Grafting sweet basil (Ocimum basilicum) scions onto diverse rootstocks, including tobacco (Nicotiana benthamiana) and thyme (Thymus vulgaris), revealed that eugenol is transported from the shoot to the root across distinct plant species. Furthermore, grafting within the Ocimum genus, which includes O. basilicum, O. tenuiflorum, and O. americanum, resulted in variations in eugenol transport and accumulation. The eugenol content in the shoots remained constant across all combinations, whereas the root eugenol levels varied depending on the scion-rootstock pair. To elucidate the biosynthetic capabilities of eugenol in Ocimum roots, we performed in vitro enzyme assays using crude protein extracts from roots, which revealed that eugenol can be synthesized in roots in addition to being transported. Expression analysis of eugenol synthase (EGSs) genes showed that EGS4 expression was influenced by grafting in O. basilicum roots, suggesting compensation by other EGSs. Our results suggest that eugenol transport and biosynthesis are multifaceted processes influenced by the interactions between different species and tissues. The potential to engineer eugenol levels in rootstocks lacking biosynthetic capacity has potential applications in agriculture and industry. This study reveals the dynamic interplay between eugenol transport and biosynthesis in the Ocimum genus, providing insights into the manipulation of aromatic compound production in plants.

Floral scents play important ecological roles because they attract pollinators and seed-dispersers. Historically, humans have used plant volatiles, including floral scents, as food additives, cosmetic products, and medicines. Floral scent formation and emissions are sometimes considerably affected by environmental and climatic conditions. Both enzymes and genes involved in floral scent biosynthesis have been consistently identified, and have provided insights into the potential of metabolic engineering of floral scents. This review summarizes recent studies on various aspects of floral scent biosynthesis and emission, including biosynthetic enzymes and genetic engineering. The findings ultimately show that the metabolic pathways of floral volatiles may be regulated by a more complex system than previously thought.