Plant Biotechnology最新文献

The long 5' untranslated region (5'UTR) exhibits enhancer activity in translation of rice OsMac3 mRNA. In this report, we describe elements of OsMac3 5'UTR that may be responsible for its enhancer activity, including a long uORF and several secondary structure elements. OsMac3 5'UTR can be dissected into three stem-loop structures SL1, small SL and SL2, where the uORF starts within SL1 and ends within SL2. As expected, uORF inhibits translation of downstream ORF since deletion of the uORF AUG or the SL1 stem-loop increases translation by approximately two-fold. Thus, the 158 nt 3' region of the 5'UTR lacking SL1 together with the AUG uORF, which has significant enhancer activity, was named dMac3. We investigated two critical regions within dMac3 mRNA that influence its translation: SL2, which destabilization potentially decreases translation activity, and another 13 nt located downstream of SL2. We further confirmed that dMac3 promotes mRNA translation initiation in an in vitro translation system and during transient expression in either cultured cells or Nicotiana benthamiana leaves. Thus, the dMac3 5'UTR is a useful tool for efficient protein production in various in vitro and in vivo translation systems.

Japanese cedar or sugi (Cryptomeria japonica D. Don) is among the most important plantation conifers in Japan, occupying 12% of the total land area in the country. We have successfully established a CRISPR/Cas9-based genome editing system in C. japonica. However, in practical use, we encountered problems of low efficiency when generating biallelic mutations, i.e., target gene knockouts. As part of our efforts to improve efficiency, we codon-optimized the Cas9 gene, evaluated by the genome editing efficiency of CjChl I, a gene encoding a chlorophyll biosynthesis enzyme. As a result, our codon-optimized SpCas9, named ^CjSpCas9, performed the highest genome editing efficiency of two targets (t4, t1+t2). Specifically, the biallelic disruption efficiency of the CjChl I with ^CjSpCas9 was 1.8-fold higher than that of the SpCas9 gene optimized for Arabidopsis thaliana (^AtSpCas9) and 2.0-fold higher than that of the SpCas9 gene optimized for Orysa sativa (^OsSpCas9) for t4, respectively. For t1+t2, the efficiency was 4.9-fold higher than that of ^AtSpCas9 and 1.4-fold higher than that of ^OsSpCas9, respectively. Our western blotting analysis proved that the Cas9 protein accumulation increased upon codon frequency optimization. We concluded that the observed efficiency improvement was due to the increased Cas9 protein quantity. The efficient genome editing system we report here would accelerate molecular breeding in conifers.

Recently a cytidine deaminase-based method for highly efficient C-to-T targeted base editing was developed and has been used with CRISPR-mediated systems. It is a powerful method for genome engineering, although it is prone to off-target effects and has a limited targeting scope. Transcription activator-like effector (TALE)-based tools which allow longer recognition sequences than do CRISPR/Cas9 systems, can also be used for targeted C-to-T base editing. Here, we describe a method that efficiently achieved targeted C-to-T substitutions in Arabidopsis nuclear genes using cytidine deaminase fused to a TALE DNA-binding domain. We used a single pair of TALEs with a novel TALE-repeat unit that can recognize all four DNA bases, especially to allow for variations in the third base of codons in homologous genes. This targeting strategy makes it possible to simultaneously base edit almost identical sites in multiple isoforms of a gene while suppressing off-target substitutions.

Chinese chestnut (Castanea mollissima Blume) is distinguished by its remarkable nut quality and robustness against disease and environmental stressor. However, its somatic embryogenesis process is notably slow, presenting a significant bottleneck in its cultivation. This study focuses on the WUSCHEL (WUS)-related homeobox 2 gene (WOX2), a member of WOX transcription factors gene family, known for its critical role in the somatic embryo development of Arabidopsis. We have identified and explored the function of a WOX2 homolog in Chinese chestnut, termed CmWOX2, in the context of somatic embryogenesis. Our analysis revealed seven WUS gene family members in the species, with CmWOX2 being uniquely upregulated in callus. Our experiments demonstrated that suppression of CmWOX2 expression diminishes somatic embryo production, whereas its overexpression enlarges the embryonic callus diameter. Notably, CmWOX2 expression levels are threefold higher in varieties with high embryogenic competence, such as 'Jingshuhong' and 'Huaihuang', compared to those with lower competence, including 'Jiujiazhong' and 'Shandonghongli'. These findings underscored the pivotal role of CmWOX2 in the initial stages of Chinese chestnut somatic embryogenesis, highlighting its potential as a target for enhancing somatic embryogenesis in this species.

Strawberry, a member of the Fragaria genus within the Rosaceae family, is one of the most cherished fruits worldwide. This perennial herbaceous plant also serves as a model for studying the Rosaceae family. Despite the complex polyploidy of strawberries, extensive efforts in traditional breeding over the years have resulted in improvements in yield, fruit size and shape, berry quality, and various other aspects of strawberry production. However, in addition to these conventional methods, advanced genetic technologies such as genetic modification and gene editing in intricate polyploidy varieties of strawberry are also required. Here, we present the Rhizobium rhizogenes-mediated hairy-root transformation of daughter plants from the model strawberry Fragaria vesca's stolons (also called runners), which exhibit diploid genomes. As a case study, new daughter plants were cut from the stolons, infected with R. rhizogenes harboring the mVENUS gene under Cauliflower mosaic virus 35S promoter, and then transferred on vermiculite-filled pots. After a couple of months of growth, fluorescence was observed in a few adventurous roots of the daughter plants. The hairy root transformation of daughter plants isolated from its vegetative propagation circumvents the need for seed production or callus formation and subsequent plant regeneration, which are often problematic for maintaining preferred genetic traits in complex ploidy levels. This method, which excludes genetic modification of the above-ground parts, especially the edible fruits, will open new avenues for strawberry breeding, particularly in the areas of plant nutrient absorption and fostering growth through interactions with microorganisms.

The development of cell-free protein synthesis technology has made it possible to easily and quickly synthesize recombinant proteins. Among cell-free protein synthesis systems, wheat germ cell-free protein synthesis using eukaryotic ribosomes is an efficient approach to synthesize proteins with diverse and complex structures and functions. However, to date, cell-free protein synthesis systems, including wheat germ cell-free systems, have not been widely used in plant research, and little is known about their applications. Here, I first introduce a basic overview of the cell-free protein synthesis system of wheat germ. Next, I will focus on our previous research examples on plants and present the applications in which the wheat germ cell-free system is used. We provide protein expression and protein function screening methods at the semi-genomic level and also introduce new approaches to enhance study of chemical biology by adapting the cell-free system of wheat germ. With this review, I would like to highlight the potential of the wheat germ cell-free system and position it as a widely used tool for the previously difficult task of recombinant protein preparation and functional analysis.

Wheat, the second most produced cereal globally, is primarily cultivated in cooler regions. Unexpected freezing temperatures can severely impact wheat production. Wheat and other temperate plants have a cold acclimation mechanism that enhances freezing tolerance, but reduces growth under low, non-freezing temperatures. During cold acclimation, plants break down storage polysaccharides like starch and fructan to accumulate soluble sugars such as glucose and fructose. These soluble sugars aid freezing tolerance through osmotic adjustments, membrane stabilization, and freezing point depression. However, plant cell walls, composed of insoluble polysaccharides, are the first line of defense against extracellular freezing. We analyzed the contributions of soluble sugars, storage polysaccharides, and cell wall polysaccharides to freezing tolerance and growth under cold acclimation in wheat. The study involved two Japanese winter cultivars (Yumechikara and Norin-61) and one Japanese spring cultivar (Haruyokoi). While Yumechikara showed poor growth after four weeks of cold acclimation, it exhibited higher freezing tolerance than the other cultivars. Our analysis revealed that Yumechikara accumulated higher levels of glucose, fructose, starch, and fructan than Norin-61 and Haruyokoi, whereas no significant differences in cell wall composition among the cultivars were observed. Gene expression patterns related to soluble sugar metabolism supported these findings. Additionally, the distribution of sugar changes between leaves (source) and crown (sink) correlated with the relationship between growth and freezing tolerance. These results suggest that freezing tolerance in wheat involves a balance between sugar accumulation and growth regulation during cold acclimation.

Coronaridine, a monoterpenoid indole alkaloid, is present in Tabernanthe iboga and the related species Tabernaemontana divaricata. Recent exhaustive analysis revealed its presence in Catharanthus roseus, though specific details remain unknown. We conducted a detailed analysis of coronaridine in C. roseus, detecting it in seedlings post-germination up to 8 weeks after sowing, with peak abundance at 3-4 weeks. Gradual decrease occurred from the flowering stage, and it was absent during seed formation. The accumulation varied dramatically with the plant's growth phase. LC-MS/MS analysis confirmed (-) coronaridine, consistent with T. iboga. Additionally, cultivating at 35°C increased coronaridine accumulation over 10-fold. These findings hold potential for enhancing the stable production of iboga alkaloids for pharmaceutical use.

Rational metabolic-flow switching is an effective strategy that we previously proposed to produce exogenous high-value natural products in cultured plant cells through redirecting a highly active inherent metabolic pathway to a pathway producing related exogenous compounds. In previous proof-of-concept studies, we demonstrated that bamboo (Phyllostachys nigra; Pn) cells are a suitable host for production of phenylpropanoid-derived compounds, in particular those derived from feruloyl- and p-coumaroyl-CoAs. To expand the utility of Pn cells for production of exogenous metabolites via the rational metabolic-flow switching strategy, it is important to evaluate the metabolic potential of Pn cells under diverse culture conditions. In this study, we examined highly active metabolic pathway(s) in Pn suspension cells cultured under light. The Pn suspension cells strongly accumulated two light-induced compounds. These compounds were isolated and identified as 3-O-caffeoylquinic acid (neochlorogenic acid) and its regioisomer 5-O-caffeoylquinic acid (chlorogenic acid). Through optimization of the culture conditions, production titers of 3-O-caffeoylquinic acid and 5-O-caffeoylquinic acid in the Pn suspension cells reached 121 and 77.9 mg l^-1, respectively. These findings indicate that Pn cells are a suitable host for bioproduction of exogenous metabolites, in particular those derived from caffeoyl-CoA via the rational metabolic-flow switching strategy.

Plant hormones like salicylic acid (SA) and jasmonic acid (JA) play crucial roles in regulating defense gene expression systems. SA mainly regulates defense against biotrophic pathogens, while JA mediates defense against necrotrophic pathogens. Compounds called plant activators including probenazole, acibenzolar-s-methyl and 2,6-dichloroisonicotinic acid (INA) activate plant immune systems, providing protection against pathogens. Unlike conventional pesticides that directly target pathogens, plant activators boost the host's defense mechanisms, potentially reducing the likelihood of drug resistance development. Various high-throughput screening systems (HTS) have been developed with the aim of searching for plant activators. Transgenic Arabidopsis lines expressing luciferase under the control of defense gene promoters allow us to monitor the activity of defense-related gene in vivo. To investigate the influence of nutrients on the HTS system, we conducted luciferase assays using Arabidopsis seedlings and observed the suppression of defense gene expression in response to the treatment of plant activators. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was employed to monitor the expression levels of endogenous genes in response to nutrient-rich conditions and confirmed the suppression effect of defense gene expression as observed in the luciferase reporter assays. The findings highlight the importance of considering nutrient effects when evaluating plant activators and screening for compounds that induce defense gene expression under nutrient-rich conditions.