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J Nutr Sci Vitaminol, 62, 19–25, 2016

The Effects of Light and Temperature on Biotin Synthesis in Pea Sprouts

Shin Kamiyama1, Risa Ohnuki1, Aoi Moriki1, Megumi Abe1, Mariko Ishiguro1,2 and Hideyuki Sone1,*

1 Department of Health and Nutrition, Faculty of Human Life Studies, University of Niigata Prefecture, 471 Ebigase, Higashi-ku, Niigata 950–8680, Japan

2 Department of Health and Nutrition, Faculty of Human Life Studies, Jin-ai University, 3–1–1 Ohde-cho, Echizen, Fukui 915–8586, Japan

(Received May 22, 2015)

Summary Biotin is an essential micronutrient, and is a cofactor for several carboxylases that are involved in the metabolism of glucose, fatty acids, and amino acids. Because plant cells can synthesize their own biotin, a wide variety of plant-based foods contains significant amounts of biotin; however, the influence of environmental conditions on the biotin content in plants remains largely unclear. In the present study, we investigated the effects of different cultivation conditions on the biotin content and biotin synthesis in pea sprouts (Pisum sati-vum). In the experiment, the pea sprouts were removed from their cotyledons and cultivated by hydroponics under five different lighting and temperature conditions (control [25˚C, 12-h light/12-h dark cycle], low light [25˚C, 4-h light/20-h dark cycle], dark [25˚C, 24 h dark], low temperature [12˚C, 12-h light/12-h dark cycle], and cold [6˚C, 12-h light/12-h dark cycle]) for 10 d. Compared to the biotin content of pea sprouts under the control condi-tions, the biotin contents of pea sprouts under the low-light, dark, and cold conditions had significantly decreased. The dark group showed the lowest biotin content among the groups. Expression of the biotin synthase gene (bio2) was also significantly decreased under the dark and cold conditions compared to the control condition, in a manner similar to that observed for the biotin content. No significant differences in the adenosine triphosphate content were observed among the groups. These results indicate that environmental conditions such as light and temperature modulate the biotin content of pea plant tissues by regulating the expression of biotin synthase.Key Words biotin, biotin synthase, Pisum sativum, pea sprout, environment

Biotin is an essential component for organisms, and functions as a cofactor for enzymes that are involved in carboxylation, decarboxylation, and transcarboxylation reactions (1). In humans, biotin is a member of the B-complex vitamin and has critical roles in many meta-bolic processes, including gluconeogenesis and energy production, as well as in the synthesis and metabo-lism of fatty acids and amino acids. Furthermore, the vitamin has functions in glucose homeostasis, such as enhancing insulin secretion (2, 3) and reducing insulin resistance (4). Biotin also regulates the expression of various genes, including the genes for glucokinase and phosphoenolpyruvate carboxykinase (5–7). Biotin is ubiquitous in food, and the body requires relatively little biotin to function normally.

Animals obtain biotin from their diets or from germs in their intestines, while microorganisms and plants can synthesize it from pimeloyl-coenzyme A (CoA) and alanine through a four-step series of reactions. The details of the biotin biosynthetic pathway have been characterized in Escherichia coli. In microbes, the steps

are generally catalyzed by four enzymes, namely 7-keto-8-aminopelargonic acid (KAPA) synthase, 7,8-diami-nopelargonic acid (DAPA) synthase, dithiobiotin (DTB) synthase, and biotin synthase, which are encoded by the bioF, bioA, bioD, and bioB genes in E. coli, respectively. In E. coli, the five genes that are involved in biotin synthesis (bioA, bioB, bioC, bioD, bioF) are organized in a bidirec-tional operon and are negatively regulated by biotinyl-5′-adenylate (8–11). Transcription of the biotin operon is directly regulated by a biotin-protein ligase, BirA, which is an enzyme protein that catalyzes the covalent attachment of biotin to its acceptor proteins (12). The operon transcription is sensitive not only to the intracel-lular biotin concentration, but also to the levels of cog-nate proteins that require biotin as a cofactor (13).

Plants synthesize biotin through a pathway that is similar to the pathway in E. coli, and the enzymes involved in the pathway have been identified in Arabi-dopsis thaliana, a model organism for the study of plant biology. The process is carried out by enzymes that are encoded by the bio4, bio1, bio3, and bio2 genes, which are orthologs of the bacterial bioF, bioA, bioD, and bioB genes, respectively. The pathway for biotin synthesis in plants is summarized in Fig. 1. The first step is catalyzed

* To whom correspondence should be addressed.E-mail: sone@unii.ac.jp

Kamiyama S et al.20

by KAPA synthase (Bio4) in the cytosol and produces 7-keto-8-aminopelargonic acid from pimeloyl-CoA and alanine (14). The second and third steps are catalyzed by a bifunctional fusion protein comprised of DAPA syn-thase (Bio1) and DTB synthase (Bio3), which produce DTB in the mitochondria (15). The final step, conver-sion of DTB to biotin, occurs in the mitochondria with a stepwise reaction catalyzed by biotin synthase (Bio2) (14, 16), which then forms the thiophane ring of biotin through the oxidative addition of a sulfur atom between the C6 methylene and C9 methyl groups of DTB (17). Bio2 is a member of the radical S-adenosyl-l-methio-nine (SAM) superfamily of enzymes that interact with iron-sulfur clusters and convert SAM to methionine and a highly reactive 5′-deoxyadenosyl radical, which is used to form the C-S bonds in biotin. The plant Bio2 enzyme requires mitochondrial targeting for the reaction (18), and some mitochondrial proteins such as adrenodoxin, adrenodoxin reductase, and cysteine desulfurase are essential for the reaction in plants (19). Cysteine desul-furase (Nfs1-Isd11 protein complex) provides sulfur for the formation of [2Fe-2S] clusters by converting cys-

teine to alanine, and one of the sulfur molecules in these clusters is thought to serve as the sulfur donor for bio-tin (20). The step catalyzed by Bio2 is considered to be a rate-limiting step in the biotin synthesis pathway (21).

Patton et al. reported that the expression of the plant biotin synthase gene (bio2) is regulated by the cellular concentration of biotin (16). In Arabidopsis, a biotin limiting condition was able to induce 5-fold increase in bio2 expression. They also showed that the expression of bio2 changes during the light/dark cycle and that the trend is reproducible and consistent with the regula-tion by light or circadian rhythms (16). Previously, we observed that the biotin content in vegetables in Japan is dependent on the climate conditions of the cultiva-tion area (22). In the present study, we investigated the effects of different cultivation conditions on the biotin content of green vegetables, specifically the biotin con-tent of pea sprouts (Pisum sativum). We found that the biotin content decreases under either low temperature or decreased light duration conditions. The expression of the biotin synthase (bio2) gene was also altered in a manner similar to that for the biotin content. Here, we discuss the role of biotin synthase in biotin synthesis in plants.

MATERIALS AND METHODS

Plant materials and growth conditions. Pea (P. sati-vum L.) seeds were obtained from a local market in Niigata City, Japan. The seeds were fully rinsed with distilled water and soaked in distilled water for 24 h in a bowl. Then, the seeds were placed in a container and distilled water was added until it reached one-third of the height of the seeds. After germination, the sprouts were removed from their cotyledons and divided into the following five groups: control (25˚C, 12-h light/12-h dark cycle), low light (25˚C, 4-h light/20-h dark cycle), dark (25˚C, 24 h dark), low temperature (12˚C, 12-h light/12-h dark cycle), and cold (6˚C, 12-h light/12-h dark cycle). Each group was cultivated by hydroponics with cultivation water (hydroculture cultivation solu-

Table 1. Composition of the cultivation water.

Ingredient Concentration (mg/L)

Nitrogen 50Phosphate 25Potassium 25Magnesium 0.25Manganese 0.1Boron 0.25Iron 0.05Zinc 0.11Copper 0.11Molybdenum 0.01

The composition of cultivation water (Hydroculture cul-tivation solution) is shown according to the manufac-turer’s specifications (Iris-Oyama, Co. Ltd.). The concen-tration of biotin in the cultivation water was below the detection limit of the bioassay.

Fig. 1. The biotin synthesis pathway in plants. Plants synthesize biotin from pimeloyl-CoA and l-alanine through four sequential reactions catalyzed by Bio4, Bio1, Bio3, and Bio2. The biotin synthase Bio2 converts dethiobiotin to biotin in mitochondria with a stepwise reaction that includes 9-mercaptodethiobiotin as an intermediate. Bio2 cleaves S-adenosyl-l-methionine and generates a 5′-deoxyadenosyl radical and methio-nine to form the C-S bonds of biotin. CoA, coenzyme A; Pi, inorganic phosphate; SAM, S-adenosyl-l-methi-onine; 5′-Ado•, 5′-deoxyadenosyl radical.

Effects of Light and Temperature on Biotin Synthesis 21

tion, Iris-Oyama Co. Ltd., Sendai, Japan) for 10 d in temperature-controlled chambers (BR-63SB, Hoshizaki Co. Ltd., Toyoake, Japan) under fluorescent lights (RB37 Photosynthesis Stimulant, Toshiba Lighting & Technol-ogy Corporation, Yokosuka, Japan). The composition of the cultivation water is shown in Table 1. After the experimental period, plants were harvested and stored at 280˚C until use.

Determination of the biotin concentration in the plants. The biotin concentration was determined by a bioassay using Lactobacillus plantarum (ATCC8014), as described previously (2). Briefly, 0.2 g of the sample tissues (leaf and stem 1 : 1) were homogenized with 5 mL of 100 mm phosphate buffer (pH 7.2) using a Glass Teflon Homogenizer. The homogenates were hydrolyzed with an equivalent volume of 2.8 m H2SO4 at 120˚C for 1 h and subsequently neutralized with 4.5 m NaOH. Sample solutions were diluted with 10 volumes of distilled water and sterilized at 121˚C for 10 min. An aliquot (180 mL) of bacterial solution in an assay medium for the deter-mination of d-biotin (Nissui Co., Ltd, Japan) was dis-pensed in a 96-well plate and 20 mL of sample solution was added to each well. The plate was incubated at 37˚C for 24 h and the absorbance was measured at 655 nm using a microplate reader (model 550, Bio-Rad Labo-ratories, Philadelphia, PA); finally, the absorbance was compared against the biotin standard curve.

Determination of the adenosine triphosphate (ATP) con-centration in the plants. We homogenized 0.2 g of the tissue samples (leaf and stem 1 : 1) with 2 mL of 10 mm HEPES buffer (pH 7.2) using a Glass Teflon Homog-enizer. The ATP concentration in the homogenate was quantified immediately by a luciferin-luciferase assay using a Luciferol 250 kit and Lumitestor C-100 lumi-nometer (Kikkoman, Chiba, Japan). The ATP concentra-tion was determined by a standard curve using standard ATP solutions.

Primers for polymerase chain reaction (PCR). The partial bio2 gene sequence of P. sativum was obtained by TBLASTN searches of the GenBank database from the National Center for Biotechnology Infor-mation (www.ncbi.nlm.nih.gov) as a query for the coding region sequence of Arabidopsis thaliana Bio2 gene (NM_129897). Consequently, we obtained two expressed sequenced tag (EST) sequences for the P. sati-vum bio2 gene (GeneBank accession No: FG533666 and GH720802). A primer set (forward sequence, 5′-TGTTC-GTGATGCAGGGATTA-3′; reverse sequence, 5′-AACCT-CACCATTGCTTTTGG-3′; amplification length, 246 base pairs) was designed from the identical part of the EST sequences by using the primer3 program (http://primer3.sourceforge.net). Similarly, the P. sativum actin gene sequence (GeneBank accession No: X67666) was obtained from the GenBank database, and the primer set sequence was designed by using the primer3 program (forward sequence, 5′-GTTTGGATCTTGCTGGTCGT-3′; reverse sequence, 5′-GAACCTCTCAGCTCCGATTG-3′; amplification length, 236 base pairs).

Preparation of cDNA samples and standards for real-time PCR. Total RNA was extracted from the tissue samples

by using Trizol reagent (Life Technologies Corporation, Carlsbad, CA). Each of the 0.05 g tissue samples was homogenized in 1 mL Trizol with a polytoron PT 10-35 homogenizer (Kinematica, Lucerne, Switzerland), and the total RNA was prepared in accordance with the manufacturer’s protocol. First-strand cDNA was synthe-sized using Superscript III first-strand synthesis Super-Mix (Life Technologies Corporation) with an oligo (dT)20 primer.

Standards for real-time PCR were prepared by PCR amplification with a cDNA template and the primers described above. PCR was performed using Takara Ex Taq HotStart version (Takara Bio Inc., Otsu, Japan) and GeneAmp 9700 PCR system (Life Technologies Corpora-tion). The PCR conditions were as follows: first denatur-ation at 94˚C for 1 min; subsequent 35-cycle reactions at 94˚C for 15 s, 60˚C for 30 s, and 72˚C for 30 s; and a final elongation step at 72˚C for 7 min. The ampli-fied products were separated in 2% agarose gel and the DNA was purified from each band on the gel using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). The DNA concentrations in the standard solutions were determined by their absorbance at 260 nm, and we diluted them to the appropriate concentrations with EASY Dilution solution (Takara Bio).

Quantitative analysis of biotin synthase gene expression by real-time PCR. The amount of bio2 transcripts in the cDNA samples was determined by real-time PCR. Real-time PCR was performed using a SYBR Premix Ex TaqTM

(Perfect Real Time; Takara Bio) and PicoReal 96 real-time PCR system (Thermo Fisher Scientific, Waltham, MA) through 40 cycles of denaturation at 95˚C for 5 s and extension at 60˚C for 30 s. The specificity of the amplification was confirmed by melting curve analyses. The relative amount of bio2 transcripts was determined from the standard curve and normalized with respect to the actin transcripts present in the same cDNA.

Statistical analysis. All values are expressed as the mean6the standard error of the mean (SE). The data were analyzed using one-way analyses of variance cou-pled with Bonferroni’s post hoc tests using the StatView 5 program (SAS Institute Inc., Cary, NC). A p-value of less than 0.05 was considered statistically significant.

RESULTS

The appearance of pea sprouts cultivated under different growth conditions

To investigate the effects of the growth conditions on biotin synthesis in the pea plant, we cultivated the plants using hydroponics under the following five growth conditions: control (25˚C, 12-h light), low light (4-h light), dark (no light), low temperature (12˚C), and cold (6˚C). The light duration of the low light group was one-third that of the control group, which reflected the climate conditions of an area receiving snowfall in winter in Japan (Niigata). The temperatures of the low-temperature and cold groups assume the temperature conditions inside the house and outside during winter, respectively.

Because pea seeds contain considerable amounts of

Kamiyama S et al.22

biotin (16 mg/100 g) and are a major nutrient source for the sprouts, we removed the cotyledon from each sprout before the experimental period (for 10 d). The appearance of the sprouts cultivated under the differ-ent growth conditions is shown in Fig. 2. Compared to the control group, the sprouts in the dark group had smaller, yellowish leaves and longer stems. In contrast, the sprouts in the cold group had smaller, yellowish leaves and shorter stems compared to those in the con-trol group. The average heights of sprouts were as fol-lows: control, 20.660.7 cm; low light, 23.061.9 cm; dark, 26.263.3 cm; low temperature, 19.962.9 cm; cold, 17.364.5 cm (n55). The weight percentages of leaves in each sprout sample were approximately 29, 25, 5, 27, and 9%, respectively.Growth conditions affect the biotin content in pea sprouts

The biotin content in the pea sprouts of each group was determined by using bioassays. The results showed that the biotin content in the sprouts was significantly reduced by the decreased light durations (Fig. 3). Fur-thermore, the sprouts in the cold group had significantly lower biotin content than did the sprouts in the control group. The effect was greater for sprouts exposed to decreased light durations than it was for those exposed

to lower temperatures, regarding the biotin content in the sprouts. The dark group showed the lowest biotin content among the groups, with the value being less than half of that for the control group. These results indicate that the biotin content in the pea sprout is affected by the growth conditions, especially tempera-ture and the duration of light.

In this experiment, we used both leaf and stem (1 : 1) tissues as the sample. It is worth noting that the concentration of biotin in pea leaf was four times higher than in the stem (8.2461.73 mg/100 g and 1.8660.64 mg/100 g, respectively). Since the sprouts in the dark and cold groups had smaller leaves than those in the control group, the biotin contents of whole sprouts might be more severely decreased in these groups than in the control group.The ATP concentration does not affect the biotin content in pea sprouts

ATP is a high-energy molecule found in all organ-isms and is used in various biochemical reactions in the cells. In plant cells, ATP is produced in the cristae of mitochondria and chloroplasts where the Bio1, Bio3, and Bio2 enzymes are localized. Because ATP is required in the reaction of the third step of biotin synthesis and

Fig. 2. The appearance of pea sprouts cultivated under different growth conditions. The pea sprouts, which had been removed from their cotyledons, were cultivated by hydroponics for 10 d under different growth conditions in temperature-controlled chambers with fluorescent lights. Control: 25˚C, 12-h light/12-h dark cycle; low light: 25˚C, 4-h light/20-h dark cycle; dark: 25˚C, 24 h dark; low temp.: 12˚C, 12-h light/12-h dark cycle; and cold: 6˚C, 12-h light/12-h dark cycle. Upper panels, overall views of sprouts; lower panels, magnified views of their leaves.

Effects of Light and Temperature on Biotin Synthesis 23

when biotin attaches to its acceptor proteins in the cells, the ATP concentration in the sprouts of each group was measured by using luciferin-luciferase assays. As shown in Fig. 4, no significant differences in the ATP concen-tration were observed among the groups. This suggests that the ATP concentration is unlikely to be a major regulator of biotin synthesis in pea sprouts.Expression of the bio2 gene in pea sprouts is suppressed under either low light or low temperature

Bio2 is the key enzyme involved in the final step of biotin synthesis, namely conversion of DTB to biotin. This step includes reactions that form a thiophene ring via the addition of a sulfur atom and is considered to be a rate-limiting step for biotin synthesis (21). In Ara-bidopsis, the expression of bio2 is reportedly regulated in response to development, light, the circadian period, and biotin starvation (16). Accordingly, the expression sta-tus of bio2 in the sprouts of each group was determined by quantitating the transcripts with real-time PCR. As

shown in Fig. 5, the gene expression of bio2 was depen-dent on both the temperature and light conditions. The bio2 expression decreased with either decreased light durations or lower temperatures, in a manner similar to that observed for the biotin content (Fig. 3). In the dark and cold groups, the amounts of bio2 transcripts were approximately half of those in the control group. Collec-tively, these results indicate that environmental stimuli, such as the duration of light and temperature, are able to modulate the biotin content of pea plants by regulat-ing the expression of biotin synthase.

DISCUSSION

Our findings demonstrate that the cultivation envi-ronment affects biotin synthesis in the pea plant. In par-ticular, expression of the bio2 gene is significantly mod-ulated by the temperature and duration of light (Fig. 5). The Bio2 enzyme is considered to be a rate-limiting enzyme in the biotin synthesis pathway (21). Because biotin-dependent carboxylases are involved in various processes such as energy metabolism, gluconeogen-esis, and fatty acid synthesis in plants, it is believed that the need for biotin increases in light conditions rather than in dark conditions. In Arabidopsis, Patton et al. also reported that bio2 expression is regulated in response to light or circadian rhythms (16). How the expression of bio2 is regulated remains unclear; however, a num-ber of genes have promoter elements that direct either developmental or light-regulated expression (23, 24). In bacteria, genes involved in biotin synthesis consti-tute a gene cluster, and the transcription of these genes is regulated by a biotin operon that is sensitive to both the intracellular biotin concentration and the levels of cognate proteins that require biotin (13). In Arabidopsis, the genomic loci of genes involved in biotin synthesis

Fig. 3. Biotin content of pea sprouts cultivated under different growth conditions. The biotin content in the pea sprouts of each group was determined by bioas-says using Lactobacillus plantarum. Values shown are the mean6SE of the biotin content per 100 g of tissue obtained from 5 samples. Mean values indicated by dis-similar letters are significantly different (p,0.05).

Fig. 4. ATP concentration in pea sprouts cultivated under different growth conditions. The ATP concentra-tion in the pea sprouts of each group was determined by using luciferin-luciferase assays. Values shown are the mean6SE of the ATP concentration per 1 g of tis-sue obtained from 5 samples.

Fig. 5. Expression of the bio2 gene in pea sprouts cul-tivated under different growth conditions. The amount of bio2 transcripts in the pea sprouts of each group was determined by real-time PCR. The relative amount of bio2 transcripts was determined from the standard curve and normalized with respect to the actin tran-scripts present in the same cDNA. Values shown are the mean6SE of the bio2/actin ratio obtained from triplicate measurements of three independent samples. Mean values indicated by dissimilar letters are signifi-cantly different (p,0.05).

Kamiyama S et al.24

are dispersed, although the bio1 and bio3 genes have a bifunctional locus and produce a fusion protein that catalyzes two sequential reactions in the biotin synthe-sis pathway (15). Further analyses are required to eluci-date the details of how genes involved in biotin synthesis are regulated in plants.

The biotin content in the pea sprouts cultivated with-out cotyledons was sensitive to temperature and the duration of light (Fig. 3). Previously, we observed that these stresses have less of an effect on the biotin con-tent in pea sprouts cultivated with cotyledons (22). Unlike bacteria, plant cells in the leaves, roots, stems, and flowers contain high amounts of free biotin in the cytoplasm. In pea leaves, over 80% of the biotin is in an unbound form and the rest is bound to biotin-requiring carboxylases (25). In contrast, much of the biotin in mature pea seeds is protein-bound due to a seed-specific biotinylated protein called SBP65 that functions for bio-tin storage and does not have catalytic activity (26–28). SBP65 has properties similar to late embryogenesis abundant (LEA) proteins, which are synthesized in only late embryogenesis and serve to protect other proteins from cold and osmotic stress. SBP65 is presumed to pre-vent biotin depletion in seeds, and the spatial and tem-poral expression of SBP65 is dependent on the osmotic environment of the developing embryos (28). Under water or cold stress conditions, a growth factor, namely abscisic acid, regulates the expression of several genes, including those for LEA proteins, to obtain stress toler-ance in plants. Abscisic acid also regulates the expres-sion of the sbp65 gene in a restricted fashion, i.e., only during particular stages of embryo development, and consequently, SBP65 plays a role in late embryo devel-opment, rather than early germination, by controlling the free biotin pool (28). In Arabidopsis, bio2 mutant embryos typically arrest at the globular stage in embryo-genesis because there is insufficient biotin to support the rapid cell division and increased lipid biosynthesis that are associated with the later stages of development (29). The amount of free-form biotin in the leaves and stems likely changes in response to different cultivation condi-tions after the stores of maternal SBP65-bound biotin in the albumen or cotyledons are depleted.

While pea seed contains a high amount of biotin, it is hypothesized that the biosynthesis of biotin is associated with photosynthesis in plant. Environmental conditions such as light and temperature could affect the biosyn-thesis of biotin in pea sprouts; however, the factors that regulate biotin biosynthesis in this plant remain unknown. Biotin synthesis requires energy investments from ATP, SAM, and other reducing equivalents such as nicotinamide adenine dinucleotide phosphate. To date, the origin of pimeloyl-CoA, a precursor for biotin syn-thesis, remains largely unclear. In E. coli, it has been suggested that the pimelic acid itself is not a precursor for biotin (30), but that the pimeloyl-acyl carrier protein (ACP) that is synthesized by a modified fatty acid syn-thetic pathway is instead the substrate for biotin syn-thesis (31). In contrast, plants may use pimelic acid as a carbon source for biotin, although the corresponding

gene for pimeloyl-CoA synthetase has not been identi-fied in plants. In the subsequent steps of biotin synthesis, the conversion of DAPA to DTB, which is catalyzed by DTB synthase (Bio1), requires ATP to attach a carbon in the mitochondria. Furthermore, biotin synthase (Bio2) reductively cleaves SAM and generates a 5′-deoxyadeno-syl radical as a strong oxidant to form the thiophane ring of biotin. In plants, these reactions occur in the mito-chondria (18, 32), and some mitochondrial proteins such as adrenodoxin (adrenal ferredoxin), adrenodoxin reductase, and cysteine desulfurase are required (19). By using a reconstituted system with mitochondrial deter-gent extracts, Mühlenhoff et al. reported that efficient assembly of biotin synthase in Saccharomyces cerevisiae requires anaerobic conditions, dithiothreitol, cysteine, ATP, and nicotinamide adenine dinucleotide (33). In this study, we could not determine the detailed effects of these factors, but the levels of ATP in the sprout did not correlate with either the biotin content or the expres-sion of bio2 (Fig. 4). Because the expression of bio2 is increased in response to biotin starvation in Arabidopsis (16), the expression level of bio2 may be a crucial factor for the biotin content in plants. Thus, the biotin content in vegetables may be dependent on the climate condi-tions of the cultivation area. Our findings also provide information on the relationship between the expression of bio2 and the biotin content in vegetables.

AcknowledgmentsBoth S. K. and R. O. contributed equally to this study.

A. M., M. A., and M. I. assisted in the experiments. S. K. prepared and revised the manuscript. H. S. designed the study. This work was supported by Grant-in-Aid for Sci-entific Research (KAKENHI) Grant Number 21700776 from the Japan Society for the Promotion of Science (JSPS).

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