Recent developments have shown that the production of recombinant proteins in plants is more useful than in microbial, insect, or mammalian cell-based expression systems in terms of cost-effectiveness, scalability, safety, and sustainability. Furthermore, transient expression systems in plants may be superior to stable transgenic plants in terms of cost, yield, environmental impact, and regulation compliance. Recombinant proteins, such as enzymes, growth factors, scaffolds, and antibodies are in high demand for use in the food and chemical industries, and will be in even greater demand for diagnostic, therapeutic, and pharmaceutical applications that require high-quality proteins. In this review, we summarize the comparison of recombinant protein expression strategies in mammalian cells, microorganisms, insects, and plants. Furthermore, the efficacy of protein expression in plant cultivation environments, the optimal protein extraction, purification methods, and costs and risks are discussed. We should be aware that the production of recombinant proteins has not only scientific challenges, but also economic and political issues that must be overcome.

High-quality pollen grains are essential for artificial cross pollination and grain production. The optimization of culture conditions for in vitro pollen germination is useful for evaluating pollen quality. However, there is limited information on in vitro pollen germination system for rice (Oryza sativa L.). Therefore, this study aimed to develop an efficient pollen germination system for rice and determine the optimal incubation period, incubation temperature, and sucrose concentration. Three rice cultivars were studied: ‘Nanatsuboshi’, ‘Nipponbare’, and ‘Kitaake’ and culture media developed in the previous study were used to optimize the conditions. The highest pollen germination rates for all cultivars were observed in the medium containing 20% (w/v) sucrose. Pollen tube bursting was observed during pollen tube elongation. We discussed the relationship between the incubation period and pollen tube bursting. This study contributes to evaluating rice pollen germination, pollen tube growth, and pollen tube bursting to support grain production.

Flowering time is an important factor in plant fitness and local adaptation. Genome-wide association (GWA) studies have allowed the identification of candidate genes in certain plant species for various traits, including flowering time. Lotus japonicus is widely found throughout the Japanese archipelago. To obtain flowering time data with more prominent difference as more suitable indicator of environmental adaptation, flowering time data were collected for 132 wild accessions originating from various points across this region under shorter day length conditions than in previous studies. The results showed latitudinal variations in flowering time, with southern accessions flowering earlier. Comparing data from four flowering times with varying conditions revealed greater differences under a shorter day length. It is likely that day length significantly affects flowering time in this species. GWA analyses were conducted on flowering time variation measured in this study and the ratios between flowering time under different conditions. Candidate genes different from previous study were detected, including orthologues of known flowering time genes in each analysis. Correlation tests between flowering time and strongly detected single-nucleotide polymorphisms (SNPs) in the GWA analysis suggested that approximately 60% of flowering time variation can be explained by the two main SNPs. This result suggests that the majority of the variation could be explained by a small number of genetic factors. Considering the strong association with flowering time variation, these candidates may be responsible for these differences and therefore can be related to local adaptation in this species.

The pulvinus is a unique motor organ found in leguminous plants. The motor cells surrounding the central vascular bundle of the pulvinus are divided into extensor and flexor halves. The asymmetric change in turgor pressure of the motor cells of the extensor/flexor halves is the driving force behind nyctinastic leaf movement. Omics analysis has recently revealed genes involved in pulvinar development and function, but the molecular mechanism orchestrating the pulvinar movement remains elusive. In this study, we investigated genes predominantly and highly expressed in the pulvinus to find out key genes involved in the regulation of nyctinastic movement. Gene expression in both the pulvinus and stem at dawn and dusk was examined using RNA sequencing analysis. As a result, several genes were identified that preferentially change in expression in the pulvinus at dawn. Among the genes, we first focused on genes that are more highly expressed in the pulvinus than in the stem and validated the results by reverse transcription-polymerase chain reaction (RT-PCR). We further focused on auxin-related genes, as auxin was found to be preferentially expressed in the pulvinus and has been reported to be involved in the regulation of nyctinastic leaf movement. Quantitative real-time PCR and in situ hybridization analyses revealed that at least two auxin-related genes, IAA19/FLS1, are dominantly expressed in the pulvinus. Thus, we provided a new dataset to identify genes involved in the regulation of nyctinastic leaf movement.

Production of polyhydroxybutyrate (PHB), a kind of biodegradable polymer, was attempted using transformant rice, in which the genes involved in PHB biosynthesis in Cupriavidus necator were introduced. Accumulation of PHB was observed in the transformants containing the genes for β-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB) and PHB synthase (phaC) (PhaABC lines) and those containing phaB and phaC (PhaBC lines). However, they immediately withered after regeneration due to severe growth inhibition, whereas no growth inhibition occurred in the PhaAB lines containing phaA and phaB, and the PhaC lines containing phaC, which did not produce PHB. Crossing between them generated sufficient quantities of F1 seeds. Many of them germinated and produced PHB, but they died at an early stage of growth. This suggests that the accumulation of PHB in the cells caused a strong growth inhibition. Microarray analysis using the PhaBC and PhaC lines revealed very similar expression profiles, suggesting that most of the changes in gene expression were mainly caused by phaC gene expression. PhaC transformants exhibited increased expression of genes involved in the stress response, certain biological processes and cellular components. These results strongly suggest that phaC gene expression results in perturbation of gene expression levels in various cell functions. It was concluded that the disturbance of cell function caused by phaC gene expression is enhanced by the intracellular production of PHB, leading to cell death.

Glyceollins, which are prenylated pterocarpan phytoalexins found in soybean, play important roles in plant–microbe interactions. During biosynthesis, the formation of the cyclic ether ring from the C-5 prenyl side chain provides structural diversity to the glyceollin isomers. This reaction has been attributed to cytochrome P450 (P450); however, it is unclear whether a single enzyme or multiple enzymes are involved in glyceollin isomer formation. In this study, we searched a co-expressed gene network database for soybean. Known genes involved in glyceollin biosynthesis were used as queries, and eight P450s (CYP71D8, CYP81E24, CYP82A2, CYP82A3, CYP82A4, CYP93A2, CYP93A3, and CYP736A33) were selected as candidates. In vitro enzyme assays using recombinant yeast microsomes expressing P450s revealed that CYP71D8 produced glyceollin I from 4-dimethylallylglycinol, and CYP82A2 yielded glyceollin III from 2-dimethylallylglycinol. Real-time PCR analysis showed that transcripts of CYP71D8 and CYP82A2 were transiently induced in soybean cells upon elicitation, prior to the accumulation of glyceollins. Thus, CYP71D8 and CYP82A2 were identified as glyceollin I and glyceollin III synthases, respectively, indicating that distinct P450s catalyze the final steps in the biosynthesis of glyceollin isomers.

Oxygen depletion due to submergence causes cellular energy starvation and severely restricts the growth of most plant species. To survive hypoxic and anoxic environments under submergence, rice (Oryza sativa L.) possesses various adaptive mechanisms including energy production from seed storage starch via anaerobic respiration and coleoptile elongation during early post-germinative growth. However, further investigation of the submergence tolerance mechanism is important for understanding its effect on plant physiology and agricultural production. Here, we found that pretreatment of rice seeds with organic acids, such as citrate and lactate, improved subsequent seedling growth under submergence. Citrate pretreatment promoted coleoptile elongation under submergence. Moreover, the expression of genes related to anaerobic respiration and phenylpropanoid biosynthesis was activated in the embryo of citrate treated seeds during submergence while the expression of genes encoding starch degradation enzymes and signaling factors was not significantly influenced. Accordingly, starch and soluble sugar amounts in the endosperm were not altered by citrate pretreatment. These results suggest that citrate pretreatment promotes coleoptile elongation in rice seeds under submergence via the transcriptional regulation of genes related to anaerobic energy production, possibly through an unknown mechanism related to phenylpropanoid metabolism.

Rational metabolic-flow switching is an effective strategy that we previously proposed to produce exogenous high-value secondary metabolite(s) in cultured plant cells. Specifically, it involves redirecting a highly active inherent metabolic pathway to a pathway producing related exogenous compounds. The success of this strategy depends on the identification of at least one highly active metabolic pathway in host plant cells that can be redirected to produce a target compound following the introduction of exogenous biosynthetic gene(s) via genetic transformation. Active metabolic pathways may be predicted on the basis of the major metabolites that accumulate in cells. In previous proof-of-concept studies, we demonstrated that cultured cells of a temperate bamboo species (Phyllostachys nigra; Pn) are an appropriate host for producing phenylpropanoid-derived compounds. However, developing a series of host plant cells with a variety of metabolic properties is necessary to maximize the utility of rational metabolic-flow switching. In this study, we established cultured cells of two tropical bamboo species (Dendrocalamus giganteus and Dendrocalamus brandisii). By analyzing the metabolites that increased in abundance in response to phytohormone treatments, we determined that exogenous gibberellin A₃ (GA₃) substantially induced the accumulation of an unknown metabolite in D. giganteus (Dg) cells. This compound was isolated and identified as serotonin (5-hydroxytryptamine). After optimizing the culture conditions, the serotonin production titer in Dg suspension cells reached 360 mg l⁻¹. These findings indicate that Dg cells are potentially suitable for the bioproduction of exogenous tryptophan-derived indolic compounds via rational metabolic-flow switching.

α-Ketol octadecadienoic acid (KODA), an oxylipin, has stimulatory effects on flowering, rooting, and resistance to pathogens. It also increases the yield of rice, Oryza sativa L. Here we examined the effects of KODA on the early growth of rice under various temperature conditions. KODA was applied by imbibing seeds in 1 µM KODA solution overnight. KODA treatment did not promote the growth at 25°C or 28°C, which are appropriate temperatures for rice cultivation. At a constant temperature of 15°C, seedling growth was poor, and KODA application did not promote seedling growth. On the other hand, at a night temperature of 15°C and day temperature of 25°C, KODA prominently enhanced the growth. We analyzed the transcript levels of several marker genes associated with chilling signaling and stress tolerance in rice. The expression of the dehydration-responsive-element-binding protein 1/C-repeat binding factor (DREB1/CBF), which regulate the expression of many stress-responsive genes was promoted. The expression of the late embryogenesis abundant (LEA), which has a DRE/CRT cis-element, was also increased by KODA treatment. Additionally, the expression of the b-amylase 4 (OsBMY4), which is important for starch degradation during cold-stress adaptation in rice, and that of the probenazole-induced protein 1 (PBZ1), a molecular marker in the rice immune response, were significantly elevated in KODA-treated rice. Thus, the enhanced growth of KODA-treated rice under chilling stress may be attributed, at least in part, to the enhanced transcriptional regulatory network mediated by DREB1/CBF genes and sugar metabolism, including starch degradation mediated by abscisic acid.

Two types of free N-glycans (FNGs), high mannose type (HMT) and plant complex type (PTC), occur ubiquitously in plants, the former mainly in the cytoplasm and the latter in the vacuole or extracellular fluid. It has been hypothesized that these plant FNGs have auxin-like activity that promotes fruit ripening based on the experimental results of adding FNGs to plant tissues; however, the postulated biological functions have not been proven at this time. In this study, using fluorescence analysis in vitro, we found that Man₃Fuc₁Xyl₁GlcNAc₂ (a PCT-FNG) occurring in plant extracellular fluids, significantly decreased the fluorescence intensity of IAA in a concentration-dependent manner at acidic (extracellular fluid) and neutral pH (cytosol), suggesting that this FNG interacts with IAA. These results suggest a possibility that the interaction of PCT-FNG and IAA may reduce the hydrophobicity of IAA in acidic environments and support the movement of IAA in plant extracellular fluids. The Interactions with IAA bearing the indole ring, appear to be unique to free N-glycans, since they were not for other oligosaccharides such as sucrose, lactose, or chitooligosaccharides. Some other PCT-FNGs and HMT-FNGs found in plants have also been confirmed to interact with IAA, suggesting that the common trimannosyl core structure of FNGs may be a prerequisite for such interactions.

The plant cytoskeleton, composed of microtubules and actin filaments, is an essential structural element for plant growth and development; it optimizes cell size and shape along the differentiation trajectories. Thus, visualizing and observing the cytoskeleton’s spatial organization within cells is crucial to better understanding plants’ developmental strategies as sessile organisms. Here, we developed a whole-mount immunostaining method for double-labeling actin filaments and microtubules using Arabidopsis thaliana roots. To enable this, we examined the specificity of the secondary antibody toward the primary antibody raised in different host-species to propose two optimal methods to double-label actin filaments and microtubules, depending on the combinations of the host-species for primary antibodies: “simultaneous immunostaining”, in which two sets of primary and secondary antibodies are applied simultaneously and “sequential immunostaining”, where two rounds of antibody-antigen reactions are conducted sequentially. The sequential reaction aims to avoid cross-species immunoreaction, where the secondary antibody undesirably binds to the primary antibody from a different host species. Our findings can provide valuable information on how to select antibodies not only for the cytoskeletal elements but also for other proteins of interest.

DNA extraction with reliable purity and concentration is essential for most of the molecular genetics studies. Extracting DNA from young leaves in seedling stage is advantageous because it causes less damage to remaining plant which can be further used for phenotypic analysis. DNA extraction from seeds is even more advantageous in terms of saving time, labor, space, and cost for germination. Maize is one of the most important food and feed sources and provides great materials for genetic and breeding studies which are accompanied by genotyping and phenotyping. We present seed DNA extraction method which does not cause damage the seed’s germination ability. DNA was extracted using cetyltrimethyl-ammonium bromide method or a commercial DNA extraction kit from the seed fragment, and the quantity and quality of the DNA were examined. Seed germination was tested for proportional seed cuts at 0, 10, 30, and 50% of the distal end of a seed, proportionally by weight. Extracting DNA from the distal seed fragments resulted in high-quality and sufficient amount of DNA. Germination rates were not significantly reduced when seed cuts were made at 10 or 30% of seed weight. DNA extraction from seeds cut can be an efficient way to obtain samples for genotyping and phenotyping. Moreover, it can be applied for high-throughput DNA extraction in maize and possibly to other smaller seeds.

Plants display diverse resistance responses that are influenced by their age and the timing of pathogen exposure. In Arabidopsis thaliana (Arabidopsis), nonhost resistance (NHR) to Pyricularia oryzae varies with leaf age and the time of inoculation. While the circadian clock and photoperiod have been linked to the time-dependent regulation of NHR in Arabidopsis, the mechanism underlying leaf age-dependent NHR remains unclear. To identify key players in leaf age-dependent NHR to P. oryzae in Arabidopsis, we utilized rice-Full-length cDNA OvereXpressing (FOX) Arabidopsis lines and identified the rice eIF2β (eukaryotic translation initiation factor 2 beta subunit) variant (Os03g0333300-2). Overexpression of the rice eIF2β variant reduced NHR to P. oryzae and modulated host resistance (HR) to Colletotrichum higginsianum in Arabidopsis. The effect of Os03g0333300-2 expression on resistance is dependent on leaf age in Arabidopsis. These results suggest that overexpression of the rice eIF2β variant Os03g0333300-2 could contribute to defense responses in a leaf age-dependent manner in Arabidopsis. Our findings might suggest the involvement of the rice eIF2β variant in eIF2-dependent translation regulation of resistance response to pathogens in plants.

High-quality green tea is produced from developing shoots (apical buds and young leaves) of tea plants (Camellia sinensis (L.) Kuntze) grown under shaded conditions. However, the removal of shade covers causes shaded tea plants to experience a sudden exposure to high light (HL). Since in ordinary tea plantation new shoots are cropped immediately following shade removal, the remaining leaves emerging from the canopy are exposed to HL. In this study, we investigated the HL response of old leaves on shaded tea plants to evaluate possible deleterious effects of HL illumination after shade removal and shoot harvesting in two years (2017 and 2018). Old leaves of both shade-grown and unshaded tea plants suffered from temporal photoinhibition caused by HL exposure after shoot harvesting but were able to recover within two weeks. Moreover, chlorophyll a/b ratios remained unchanged in old leaves experiencing shading treatment, suggesting that old leaves have a weakened capacity to respond to low light conditions. Furthermore, protein carbonyl content was elevated 3–7 days after shade removal in summer 2018. Shoot growth during the subsequent autumn season was inhibited in shaded plants relative to the control group. Taken together these results indicate that old leaves on shaded tea plants suffer from oxidative damage after shade removal in summer, and this may inhibit the growth of autumn shoots.