Enhanced Co-culture System Using Escherichia coli and Pichia pastoris (Komagataella phaffii) for Improved Microbial Production of Valuable Plant Alkaloids.

Advancements in synthetic biology have facilitated the microbial production of valuable plant metabolites. However, constructing complete biosynthetic pathways within a single host organism remains challenging. To solve this problem, modular co-culture systems involving host organisms with partial pathways have been developed. We focused on Escherichia coli, a general host for metabolite production, and Pichia pastoris (Komagataella phaffii), a novel synthetic biology host due to its high expression of biosynthetic enzymes. Previously, we reported the co-culture of E. coli cells, which produce reticuline (an important intermediate for various alkaloids) from glycerol, with P. pastoris cells, which produce the valuable alkaloid stylopine from reticuline. However, Pichia cells inhibited E. coli growth and reticuline production. Therefore, we aimed to improve this co-culture system. We investigated the pre-culture time before co-culture to enhance E. coli growth and reticuline production. Additionally, we examined the optimal concentration of Pichia cells inoculated for co-culture and methanol addition during co-culture for the continuous expression of biosynthetic enzymes in Pichia cells. We successfully established an improved co-culture system that exhibited an 80-fold increase in productivity compared to previous methods. This enhanced system holds great potential for the rapid and large-scale production of various valuable plant metabolites.

Enhancing effect of Panax ginseng on Zip4-mediated zinc influx into the cytosol.

Background: Zinc homeostasis is essential for human health and is regulated by several zinc transporters including ZIP and ZnT. ZIP4 is expressed in the small intestine and is important for zinc absorption from the diet. We investigated in the present study the effects of Panax ginseng (P. ginseng) extract on modulating Zip4 expression and cellular zinc levels in mouse Hepa cells. Methods: Hepa cells were transfected with a luciferase reporter plasmid that contains metal-responsive elements, incubated with P. ginseng extract, and luciferase activity was measured. Using 65ZnCl2, zinc uptake in P. ginseng-treated cells was measured. The expression of Zip4 mRNA and protein in Hepa cells was also investigated. Finally, using a luciferase reporter assay system, the effects of several ginsenosides were monitored. Results: The luciferase activity in cells incubated with P. ginseng extract was significantly higher than that of control cells cultured in normal medium. Hepa cells treated with P. ginseng extract exhibited higher zinc uptake. P. ginseng extract induced Zip4 mRNA expression, which resulted in an enhancement of Zip4 protein expression. Furthermore, some ginsenosides, such as ginsenoside Rc and Re, enhanced luciferase activity driven by intracellular zinc levels. Conclusion: P. ginseng extract induced Zip4 expression at the mRNA and protein level and resulted in higher zinc uptake in Hepa cells. Some ginsenosides facilitated zinc influx. On the basis of these results, we suggest a novel effect of P. ginseng on Zip4-mediated zinc influx, which may provide a new strategy for preventing zinc deficiency.

Transport engineering using tobacco transporter NtJAT1 enhances alkaloid production in Escherichia coli.

Transporters have been used in the production of plant metabolites in microorganisms. This study introduced a tobacco multidrug and toxic compound extrusion transporter, NtJAT1, into alkaloid-producing Escherichia coli cells. NtJAT1 expression enhanced alkaloid production secretion into the medium by 14 folds. Our findings further demonstrate the usefulness of the transport-engineering approach.

Establishment of a co-culture system using Escherichia coli and Pichia pastoris (Komagataella phaffii) for valuable alkaloid production.

BACKGROUND: Plants produce a variety of specialized metabolites, many of which are used in pharmaceutical industries as raw materials. However, certain metabolites may be produced at markedly low concentrations in plants. This problem has been overcome through metabolic engineering in recent years, and the production of valuable plant compounds using microorganisms such as Escherichia coli or yeast cells has been realized. However, the development of complicated pathways in a single cell remains challenging. Additionally, microbial cells may experience toxicity from the bioactive compounds produced or negative feedback effects exerted on their biosynthetic enzymes. Thus, co-culture systems, such as those of E. coli-E. coli and E. coli-Saccharomyces cerevisiae, have been developed, and increased production of certain compounds has been achieved. Recently, a co-culture system of Pichia pastoris (Komagataella phaffii) has gained considerable attention due to its potential utility in increased production of valuable compounds. However, its co-culture with other organisms such as E. coli, which produce important intermediates at high concentrations, has not been reported. RESULTS: Here, we present a novel co-culture platform for E. coli and P. pastoris. Upstream E. coli cells produced reticuline from a simple carbon source, and the downstream P. pastoris cells produced stylopine from reticuline. We investigated the effect of four media commonly used for growth and production of P. pastoris, and found that buffered methanol-complex medium (BMMY) was suitable for P. pastoris cells. Reticuline-producing E. coli cells also showed better growth and reticuline production in BMMY medium than that in LB medium. De novo production of the final product, stylopine from a simple carbon source, glycerol, was successful upon co-culture of both strains in BMMY medium. Further analysis of the initial inoculation ratio showed that a higher ratio of E. coli cells compared to P. pastoris cells led to higher production of stylopine. CONCLUSIONS: This is the first report of co-culture system established with engineered E. coli and P. pastoris for the de novo production of valuable compounds. The co-culture system established herein would be useful for increased production of heterologous biosynthesis of complex specialized plant metabolites.

Transport engineering for improving the production and secretion of valuable alkaloids in Escherichia coli.

Microorganisms can be metabolically engineered to produce specialized plant metabolites. However, these methods are limited by low productivity and intracellular accumulation of metabolites. We sought to use transport engineering for producing reticuline, an important intermediate in the alkaloid biosynthetic pathway. In this study, we established a reticuline-producing Escherichia coli strain into which the multidrug and toxic compound extrusion transporter Arabidopsis AtDTX1 was introduced. AtDTX1 was selected due to its suitable expression in E. coli and its reticuline-transport activity. Expression of AtDTX1 enhanced reticuline production by 11-fold, and the produced reticuline was secreted into the medium. AtDTX1 expression also conferred high plasmid stability and resulted in upregulation or downregulation of several genes associated with biological processes, including metabolic pathways for reticuline biosynthesis, leading to the production and secretion of high levels of reticuline. The successful employment of a transporter for alkaloid production suggests that the proposed transport engineering approach may improve the biosynthesis of specialized metabolites via metabolic engineering.

Genome-Wide Profiling of WRKY Genes Involved in Benzylisoquinoline Alkaloid Biosynthesis in California Poppy (Eschscholzia californica).

Transcription factors of the WRKY family play pivotal roles in plant defense responses, including the biosynthesis of specialized metabolites. Based on the previous findings of WRKY proteins regulating benzylisoquinoline alkaloid (BIA) biosynthesis, such as CjWRKY1-a regulator of berberine biosynthesis in Coptis japonica-and PsWRKY1-a regulator of morphine biosynthesis in Papaver somniferum-we performed genome-wide characterization of the WRKY gene family in Eschscholzia californica (California poppy), which produces various BIAs. Fifty WRKY genes were identified by homology search and classified into three groups based on phylogenetic, gene structure, and conserved motif analyses. RNA sequencing showed that several EcWRKY genes transiently responded to methyl jasmonate, a known alkaloid inducer, and the expression patterns of these EcWRKY genes were rather similar to those of BIA biosynthetic enzyme genes. Furthermore, tissue expression profiling suggested the involvement of a few subgroup IIc EcWRKYs in the regulation of BIA biosynthesis. Transactivation analysis using luciferase reporter genes harboring the promoters of biosynthetic enzyme genes indicated little activity of subgroup IIc EcWRKYs, suggesting that the transcriptional network of BIA biosynthesis constitutes multiple members. Finally, we investigated the coexpression patterns of EcWRKYs with some transporter genes and discussed the diversified functions of WRKY genes based on a previous finding that CjWRKY1 overexpression in California poppy cells enhanced BIA secretion into the medium.

Comparative analysis using the draft genome sequence of California poppy (Eschscholzia californica) for exploring the candidate genes involved in benzylisoquinoline alkaloid biosynthesis.

Genome characterization of California poppy (Eschscholzia californica cv. "Hitoezaki"), which produces pharmaceutically important benzylisoquinoline alkaloids (BIAs), was carried out using the draft genome sequence. The numbers of tRNA and rRNA genes were close to those of the other plant species tested, whereas the frequency of repetitive sequences was distinct from those species. Comparison of the predicted genes with those of Amborella trichopoda, Nelumbo nucifera, Solanum lycopersicum, and Arabidopsis thaliana, and analyses of gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway indicated that the enzyme genes involved in BIA biosynthesis were highly enriched in the California poppy genome. Further comparative analysis using the genome information of Papaver somniferum and Aquilegia coerulea, both BIA-producing plants, revealed that many genes encoding BIA biosynthetic enzymes, transcription factors, transporters, and candidate proteins, possibly related to BIA biosynthesis, were specifically distributed in these plant species.

Genome-wide identification of AP2/ERF transcription factor-encoding genes in California poppy (Eschscholzia californica) and their expression profiles in response to methyl jasmonate.

With respect to the biosynthesis of plant alkaloids, that of benzylisoquinoline alkaloids (BIAs) has been the most investigated at the molecular level. Previous investigations have shown that the biosynthesis of BIAs is comprehensively regulated by WRKY and bHLH transcription factors, while promoter analyses of biosynthesis enzyme-encoding genes have also implicated the involvement of members of the APETALA2/ethylene responsive factor (AP2/ERF) superfamily. To investigate the physiological roles of AP2/ERF transcription factors in BIA biosynthesis, 134 AP2/ERF genes were annotated using the draft genome sequence data of Eschscholzia californica (California poppy) together with transcriptomic data. Phylogenetic analysis revealed that these genes could be classified into 20 AP2, 5 RAV, 47 DREB, 60 ERF and 2 Soloist family members. Gene structure, conserved motif and orthologous analyses were also carried out. Gene expression profiling via RNA sequencing in response to methyl jasmonate (MeJA) indicated that approximately 20 EcAP2/ERF genes, including 10 group IX genes, were upregulated by MeJA, with an increase in the expression of the transcription factor-encoding gene EcbHLH1 and the biosynthesis enzyme-encoding genes Ec6OMT and EcCYP719A5. Further quantitative RT-PCR confirmed the MeJA responsiveness of the EcAP2/ERF genes, i.e., the increased expression of 9 group IX, 2 group X and 2 group III ERF subfamily genes. Transactivation activity of group IX EcAP2/ERFs was also confirmed by a luciferase reporter assay in conjunction with the promoters of the Ec6OMT and EcCYP719A5 genes. The physiological roles of AP2/ERF genes in BIA biosynthesis and their evolution in the regulation of alkaloid biosynthesis are discussed.

Starvation-induced autophagy via calcium-dependent TFEB dephosphorylation is suppressed by Shigyakusan.

Kampo, a system of traditional Japanese therapy utilizing mixtures of herbal medicine, is widely accepted in the Japanese medical system. Kampo originated from traditional Chinese medicine, and was gradually adopted into a Japanese style. Although its effects on a variety of diseases are appreciated, the underlying mechanisms remain mostly unclear. Using a quantitative tf-LC3 system, we conducted a high-throughput screen of 128 kinds of Kampo to evaluate the effects on autophagy. The results revealed a suppressive effect of Shigyakusan/TJ-35 on autophagic activity. TJ-35 specifically suppressed dephosphorylation of ULK1 and TFEB, among several TORC1 substrates, in response to nutrient deprivation. TFEB was dephosphorylated by calcineurin in a Ca2+ dependent manner. Cytosolic Ca2+ concentration was increased in response to nutrient starvation, and TJ-35 suppressed this increase. Thus, TJ-35 prevents the starvation-induced Ca2+ increase, thereby suppressing induction of autophagy.

Dynamism of vacuoles toward survival strategy in plants.

Vacuole is a prominent organelle that often occupies most of the plant cell volume. The vacuolar accumulation of secondary metabolites, also called specialized metabolites, plays important roles in environmental responses such as protecting against insect herbivores and attracting pollinators. The compartmentation of xenobiotics in the vacuole is also essential for adaptation to environmental stresses. These accumulations involve several transport systems, for which some responsible transporter proteins have been reported. Furthermore, studies on biosynthetic enzymes and transporters of secondary metabolites have revealed that vacuoles, which have been recognized for many years as a site for accumulation, also function as a site for biosynthesis of secondary metabolites and are thus actively involved in the entire biosynthetic process. In this review, we briefly summarize recent findings on vacuolar transporters involved in secondary metabolites and xenobiotics, and discuss their roles in plant adaptation to biotic and abiotic stresses, through vacuolar dynamism.

Uptake of adenine by purine permeases of Coffea canephora.

Purine permeases (PUPs) mediate the proton-coupled uptake of nucleotide bases and their derivatives into cytosol. PUPs facilitate uptake of adenine, cytokinins and nicotine. Caffeine, a purine alkaloid derived from xanthosine, occurs in only a few eudicot species, including coffee, cacao, and tea. Although caffeine is not an endogenous metabolite in Arabidopsis and rice, AtPUP1 and OsPUP7 were suggested to transport caffeine. In this study, we identified 15 PUPs in the genome of Coffea canephora. Direct uptake measurements in yeast demonstrated that CcPUP1 and CcPUP5 facilitate adenine - but not caffeine - transport. Adenine uptake was pH-dependent, with increased activity at pH 3 and 4, and inhibited by nigericin, a potassium-proton ionophore, suggesting that CcPUP1 and CcPUP5 function as proton-symporters. Furthermore, adenine uptake was not competitively inhibited by an excess amount of caffeine, which implies that PUPs of C. canephora have evolved to become caffeine-insensitive to promote efficient uptake of adenine into cytosol.

Structure of constituents isolated from the bark of Cassipourea malosana and their cytotoxicity against a human ovarian cell line.

Three aromatic compounds, 2α,3α-epoxyflavan-5,7,4'-triol-(4ß â†’ 8)-afzelechin (1), 2ß,3ß-epoxyflavan-5,7,4'-triol-(4α → 8)-epiafzelechin (2), and methyl 4-ethoxy-2-hydroxy-6-propylbenzoate (3), as well as eight known compounds (4-11) were isolated from the bark of Cassipourea malosana (Rhizophoraceae). Their structures were determined on the basis of an analysis of spectroscopic data. The in vitro cytotoxic activities of these compounds against human ovarian cancer cell line TOV21G were evaluated. Most compounds showed little activity; however, the methyl derivatives of flavan dimers (1a and 2a) showed higher activity (IC50 value of 30.3 and 75.4 µM) than parent compounds 1 and 2.

Transcriptome analysis of Petunia axillaris flowers reveals genes involved in morphological differentiation and metabolite transport.

The biosynthesis of plant secondary metabolites is associated with morphological and metabolic differentiation. As a consequence, gene expression profiles can change drastically, and primary and secondary metabolites, including intermediate and end-products, move dynamically within and between cells. However, little is known about the molecular mechanisms underlying differentiation and transport mechanisms. In this study, we performed a transcriptome analysis of Petunia axillaris subsp. parodii, which produces various volatiles in its corolla limbs and emits metabolites to attract pollinators. RNA-sequencing from leaves, buds, and limbs identified 53,243 unigenes. Analysis of differentially expressed genes, combined with gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses, showed that many biological processes were highly enriched in limbs. These included catabolic processes and signaling pathways of hormones, such as gibberellins, and metabolic pathways, including phenylpropanoids and fatty acids. Moreover, we identified five transporter genes that showed high expression in limbs, and we performed spatiotemporal expression analyses and homology searches to infer their putative functions. Our systematic analysis provides comprehensive transcriptomic information regarding morphological differentiation and metabolite transport in the Petunia flower and lays the foundation for establishing the specific mechanisms that control secondary metabolite biosynthesis in plants.

The Crotalaria juncea metal transporter CjNRAMP1 has a high Fe uptake activity, even in an environment with high Cd contamination.

Large quantities of Fe and Cd accumulate in the leaves of the metal-accumulating leguminous plant, Crotalaria juncea. A member of the metal transporter NRAMP family was cloned from C. juncea. The amino acid sequence of this clone, designated CjNRAMP1, was similar to the sequence of Arabidopsis AtNRAMP1, which is involved in Fe and Cd transport. Organ-specific analysis showed that CjNRAMP1 mRNA was expressed mainly in the leaves of C. juncea plants, as well as in stems and roots. Use of green fluorescent protein fused to CjNRAMP1 suggested its localization to the plasma membranes of plant cells. Complementation experiments using yeast strains with impaired metal transport systems showed that CjNRAMP1 transported both Fe and Cd in an inward direction within the cells. Transgenic Arabidopsis plants overexpressing CjNRAMP1 showed high tolerance to Cd, with Cd translocation from roots to leaves being substantially greater in transgenic than in wild-type plants. Overexpression of CjNRAMP1 resulted in a greater accumulation of Fe in shoots and roots, suggesting that CjNRAMP1 recognizes Fe and Cd as substrates and that the high Cd tolerance of CjNRAMP1 is due to its strong Fe uptake activity, even in the presence of high Cd concentrations in the rhizosphere.

Secondary metabolites in plants: transport and self-tolerance mechanisms.

Plants produce a host of secondary metabolites with a wide range of biological activities, including potential toxicity to eukaryotic cells. Plants generally manage these compounds by transport to the apoplast or specific organelles such as the vacuole, or other self-tolerance mechanisms. For efficient production of such bioactive compounds in plants or microbes, transport and self-tolerance mechanisms should function cooperatively with the corresponding biosynthetic enzymes. Intensive studies have identified and characterized the proteins responsible for transport and self-tolerance. In particular, many transporters have been isolated and their physiological functions have been proposed. This review describes recent progress in studies of transport and self-tolerance and provides an updated inventory of transporters according to their substrates. Application of such knowledge to synthetic biology might enable efficient production of valuable secondary metabolites in the future.

Molecular Characterization of LjABCG1, an ATP-Binding Cassette Protein in Lotus japonicus.

LjABCG1, a full-size ABCG subfamily of ATP-binding cassette proteins of a model legume, Lotus japonicus, was reported as a gene highly expressed during the early stages of nodulation, but have not been characterized in detail. In this study we showed that the induction of LjABCG1 expression was remarkable by methyl jasmonate treatment, and reporter gene experiments indicated that LjABCG1 was strongly expressed in the nodule parenchyma and cell layers adjacent to the root vascular tissue toward the nodule. LjABCG1 was suggested to be localized at the plasma membrane based on the fractionation of microsomal membranes as well as separation via aqueous two-phase partitioning. The physiological functions of LjABCG1 in symbiosis and pathogenesis were analyzed in homologous and heterologous systems. LjABCG1 knock-down L. japonicus plants did not show clear phenotypic differences in nodule formation, and not in defense against Pseudomonas syringae, either. In contrast, when LjABCG1 was expressed in the Arabidopsis pdr8-1 mutant, the penetration frequency of Phytophthora infestans, a potato late blight pathogen, was significantly reduced in LjABCG1/pdr8-1 than in pdr8-1 plants. This finding indicated that LjABCG1, at least partially, complemented the phenotype of pdr8 in Arabidopsis, suggesting the multiple roles of this protein in plant-microbe interactions.

Translocation and accumulation of nicotine via distinct spatio-temporal regulation of nicotine transporters in Nicotiana tabacum.

In plants, secondary metabolites play important roles in adaptation to the environment. Nicotine, a pyridine alkaloid in Nicotiana tabacum, functions as chemical barrier against herbivores. Nicotine produced in the root undergoes long-distance transport and accumulates mainly in the leaves. Since production of such defensive compounds is costly, plants must regulate the allocation of the products to their tissues; however, the molecular mechanism of nicotine translocation remains unclear. Our recent studies identified a novel multidrug and toxic compound extrusion (MATE)-type nicotine transporter, JAT2 (jasmonate-inducible alkaloid transporter 2). This transporter is specifically expressed in leaves, localizes to the tonoplast, and transports nicotine as its substrate. The specific induction of JAT2 expression in leaves by methyl jasmonate (MeJA) treatment suggests that this transporter plays an important role in nicotine distribution to leaves, especially under herbivore attack, by transporting nicotine into the vacuole. Considering JAT2, together with the previously identified MATE transporters JAT1, MATE1, and MATE2, and the PUP (purine permease) transporter NUP1 (nicotine uptake permease1), we show a model of nicotine translocation and accumulation via distinct spatio-temporal regulation of nicotine transporter expression. Furthermore, we discuss the possible role of nicotine transporters in determining outcrossing rates and seed production.

Tobacco NUP1 transports both tobacco alkaloids and vitamin B6.

The purine permeases (PUPs) constitute a large plasma membrane-localized transporter family in plants that mediates the proton-coupled uptake of nucleotide bases and their derivatives, such as adenine, cytokinins, and caffeine. A Nicotiana tabacum (tobacco) PUP-family transporter, nicotine uptake permease 1 (NtNUP1), was previously shown to transport tobacco alkaloids and to affect both nicotine biosynthesis and root growth in tobacco plants. Since Arabidopsis PUP1, which belongs to the same subclade as NtNUP1, was recently reported to transport pyridoxine and its derivatives (vitamin B6), it was of interest to examine whether NtNUP1 could also transport these substrates. Direct uptake measurements in the yeast Saccharomyces cerevisiae demonstrated that NtNUP1 efficiently promoted the uptake of pyridoxamine, pyridoxine, anatabine, and nicotine. The naturally occurring (S)-isomer of nicotine was preferentially transported over the (R)-isomer. Transport studies using tobacco BY-2 cell lines overexpressing NtNUP1 or PUP1 showed that NtNUP1, similar to PUP1, transported various compounds containing a pyridine ring, but that the two transporters had distinct substrate preferences. Therefore, the previously reported effects of NtNUP1 on tobacco physiology might involve bioactive metabolites other than tobacco alkaloids.

Involvement of the leaf-specific multidrug and toxic compound extrusion (MATE) transporter Nt-JAT2 in vacuolar sequestration of nicotine in Nicotiana tabacum.

Alkaloids play a key role in higher plant defense against pathogens and herbivores. Following its biosynthesis in root tissues, nicotine, the major alkaloid of Nicotiana species, is translocated via xylem transport toward the accumulation sites, leaf vacuoles. Our transcriptome analysis of methyl jasmonate-treated tobacco BY-2 cells identified several multidrug and toxic compound extrusion (MATE) transporter genes. In this study, we characterized a MATE gene, Nicotiana tabacum jasmonate-inducible alkaloid transporter 2 (Nt-JAT2), which encodes a protein that has 32% amino acid identity with Nt-JAT1. Nt-JAT2 mRNA is expressed at a very low steady state level in whole plants, but is rapidly upregulated by methyl jasmonate treatment in a leaf-specific manner. To characterize the function of Nt-JAT2, yeast cells were used as the host organism in a cellular transport assay. Nt-JAT2 was localized at the plasma membrane in yeast cells. When incubated in nicotine-containing medium, the nicotine content in Nt-JAT2-expressing cells was significantly lower than in control yeast. Nt-JAT2-expressing cells also showed lower content of other alkaloids like anabasine and anatabine, but not of flavonoids, suggesting that Nt-JAT2 transports various alkaloids including nicotine. Fluorescence assays in BY-2 cells showed that Nt-JAT2-GFP was localized to the tonoplast. These findings indicate that Nt-JAT2 is involved in nicotine sequestration in leaf vacuoles following the translocation of nicotine from root tissues.

RCN1/OsABCG5, an ATP-binding cassette (ABC) transporter, is required for hypodermal suberization of roots in rice (Oryza sativa).

Suberin is a complex polymer composed of aliphatic and phenolic compounds. It is a constituent of apoplastic plant interfaces. In many plant species, including rice (Oryza sativa), the hypodermis in the outer part of roots forms a suberized cell wall (the Casparian strip and/or suberin lamellae), which inhibits the flow of water and ions and protects against pathogens. To date, there is no genetic evidence that suberin forms an apoplastic transport barrier in the hypodermis. We discovered that a rice reduced culm number1 (rcn1) mutant could not develop roots longer than 100 mm in waterlogged soil. The mutated gene encoded an ATP-binding cassette (ABC) transporter named RCN1/OsABCG5. RCN1/OsABCG5 gene expression in the wild type was increased in most hypodermal and some endodermal roots cells under stagnant deoxygenated conditions. A GFP-RCN1/OsABCG5 fusion protein localized at the plasma membrane of the wild type. Under stagnant deoxygenated conditions, well suberized hypodermis developed in wild types but not in rcn1 mutants. Under stagnant deoxygenated conditions, apoplastic tracers (periodic acid and berberine) were blocked at the hypodermis in the wild type but not in rcn1, indicating that the apoplastic barrier in the mutant was impaired. The amount of the major aliphatic suberin monomers originating from C(28) and C(30) fatty acids or ω-OH fatty acids was much lower in rcn1 than in the wild type. These findings suggest that RCN1/OsABCG5 has a role in the suberization of the hypodermis of rice roots, which contributes to formation of the apoplastic barrier.