J Plant Biotechnol (2023) 50:070-075
Published online May 2, 2023
https://doi.org/10.5010/JPB.2023.50.009.070
© The Korean Society of Plant Biotechnology
Correspondence to : e-mail: yck01000@jbnu.ac.kr, jhwanlee90@jbnu.ac.kr
swd@rda.go.kr
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
In legumes, soyasaponins, one of triterpenoid saponins, are major components of secondary metabolites with a more diverse array of bioactive chemicals. Although the biosynthetic pathway of soyasaponins has been largely studied in soybean, the study on the soyasaponin contents and biosynthesis-related gene expression in pea (Pisum sativum L.) is poorly understood. Here, we found the accumulation of only soyasaponin Bb component in the sprouts of two Korean domestic pea cultivars (Dachung and Sachul). This pattern was consistent with our observation that increased expression of PsUGT73P2 and PsUGT91H4 genes, but not PsCYP72A69, could be responsible for biosynthesis of only soyasaponin Bb in pea by examining their gene expression. However, gradual accumulation of soyasaponin Bb at developmental stages was not consistent with the expression of PsUGT73P2 and PsUGT91H4, suggesting that the changes of their protein activities may affect the accumulation patterns of soyasaponin Bb. We also revealed that the increased expression levels of PsUGT73P2 and PsUGT91H4 during light to dark transition led to increase of soyasaponin Bb contents. Collectively, our results provided a molecular basis of metabolic engineering for enhancing useful soyasaponin Bb metabolites in Korean domestic pea cultivars.
Keywords Pea, PsCYP72A69, PsUGT73P2, PsUGT91H4, Soyasaponin Bb
Legumes in the
In legumes, young germinated soybean sprouts contain a more diverse range of bioactive chemicals such as isoflavones, phenolic compounds and triterpenoids than their non-germinated counterparts (Choi et al. 2018; Ma et al. 2018; Mora-Escobedo et al. 2009; Wang et al. 2015). Of them, triterpenoid saponins are a main component of secondary metabolites, which are plentifully accumulated in plant specific tissues such as roots. The biosynthetic pathway of soyasaponins, one of triterpenoid saponins, has been extensively studied in soybean (Abe et al. 1993; Moses et al. 2014; Thimmappa et al. 2014). Previous reports have revealed that 2,3-oxidosqualane precursor is synthesized through the mevalonate pathway, and converted to soyasaponins through a biosynthetic pathway with at least 14 different enzymes, including one β-amyrin synthase (BAS), three cytochrome P450s (CYP450s), ten uridine 5’-diphosphate (UDP)-glycosyltransferases (UGTs), and one acetyltransferase. Structural diversity of the sugar numbers and types in triterpenoid saponins contributes to a variety of the biological and chemical activities (Bowles et al. 2005).
Because the common pea (
The sequence data of pea soyasaponin biosynthesis-related genes (
Table 1 Genes and oligonucleotides used for RT–qPCR analysis in this study
Gene name | Gene ID | Primers (5’ to 3’) | Direction |
---|---|---|---|
Psat7g228360 | TCCCTACTGGAAGCAAAGATGG | F | |
TTATGTTTCCACTTTACGTATAATAATTTGAGC | R | ||
Psatoss5882g0200 | CCTTTGTTTGCAGAACATTTTTTCAATG | F | |
TCAACCTAATAGCATTCCCAATCTCT | R | ||
Psat1g218280 | ATAGTGGATGAAGAAGGAAGTAGTTTTAG | F | |
TCATTCTTTGTTGGAATTGGAAGC | R | ||
Xm_051045246.1 | GCTCCCAGCAGTACAGGACTCT | F | |
CAACCTCCTTGGTACTCATCTTGCC | R |
Two pea cultivars, Dachung and Sachul, were kindly provided by the Division of Crop Foundation (National Institute of Crop Science, Rural Development Administration, Jeonju, Korea). The seeds were sterilized in 70% ethanol (EtOH) containing 0.5% Triton X-100 for 15 min, thoroughly washed once in water, soaked in water at 23°C for 24 h, and then transferred to Sunshine Mix 5 (Sungro, Agawam, MA, USA). Germinated peas were prepared by incubation at 23°C for 6, 9, 12, 15, and 18 days in a dark growth chamber. For light to dark treatment, pea seedlings grown for 9 days at 23°C under long-day (LD) conditions (16 h light/8 h dark at a light intensity of 120 μmol m-2 s-1) were transferred to continuous dark or light chambers for 1 and 2 days at 23°C. Seedlings harvested after each treatment were ground to a powder with liquid nitrogen in a mortar and stored at -80°C until use. All experiments were performed in three biological replicates (independently harvested samples).
Total RNA was extracted from the harvested samples using Trizol reagent (Invitrogen, Carlsbad, CA, USA) as previously described (Yun et al. 2021), and the quality and quantity of the resulting RNA was checked using a Nanodrop ND-2000 spectrophotometer (Nanodrop Technologies, Waltham, MA, USA). To analyze RNA expression, real time-quantitative PCR (RT-qPCR) analysis was performed.
Complementary DNA (cDNA) was synthesized from 5 µg RNA in accordance with the protocol of the ReverTra Ace qPCR RT Master Mix kit (Toyobo, Osaka, Japan), and RT-qPCR analysis was conducted in 96-well plates using a CFX real-time system (Bio-Rad, Hercules, CA, USA), THUNDERBIRD SYBR qPCR mix (Toyobo), and gene-specific RT-qPCR primers listed in Table 1 were designed using QuantPrime (http://quantprime.mpimp-golm.mpg.de/).
To prepare pea extracts, the pea seedlings harvested at different developmental stages or under light/dark transition conditions were dried in an oven at 57°C for 48 h, and then ground into powder using a blender (Electric Mixer SMX-W350, Seoul, Korea). As previously described (Yun et al. 2021), 20 ml of 80% methanol was added to 1 g of the ground samples and the mixtures were agitated for 24 h at 23°C. The supernatants were prepared by centrifugation (7,000 × g) for 5 min and then passed through Donex™ OnGuard™ II RP cartridges (Thermo Fisher Sci., MA, USA) to remove the hydrophobic substance. They were then filtered through a 0.22-μm filter unit. Soyasaponin contents were analyzed with a reverse-phase UHPLC (Dionex Ultimate 3000, Thermo Fisher Sci.) equipped with an Acclaim™ RSLC Polar Advangate II (2.2 μm 120 Å 2.1 × 150 mm) column. Following injection of 2 μL of sample, separated soyasaponin Aa, Ab, Ba, Bb, and Bb´ was detected using a CAD (Corona Veo, Thermo Fisher Sci.). The used reagents were UHPLC grade, and soyasaponin Aa, Ab, Ba, Bb, and Bb’ (ChemFaces, Wuhan, China) were used as reference substances. The student’s
We previously found that the accumulation patterns of soyasaponin Aa and Ab were different in the Socheongja and Haepum cultivars (Korean domestic soybean cultivars) (Yun et al. 2021). To investigate the accumulation patterns of soyasaponin contents in pea plants within the legumes, we prepared the sprouts of two Korean domestic pea cultivars (Dachung and Sachul) grown for 15 days at 23°C under darkness conditions, and analyzed their soyasaponin contents using UHPLC-CAD analysis. We found that only soyasaponin Bb was accumulated in two Korean domestic pea cultivars (Fig. 1A). Taken together with a previous data (Yun et al. 2021), this result indicated that different soyasaponin types were exclusively accumulated between soybean and pea sprouts, although they belong to legumes. Our data also suggested that the expression levels of soyasaponin biosynthetic genes or their protein activities may be different between soybean and pea plants.
Based on the soyasaponin biosynthesis pathway identified in soybean plants (Abe et al. 1993; Moses et al. 2014; Thimmappa et al. 2014), we focused on four genes (
To investigate whether soyasaponin Bb accumulation is affected by the expression changes of soyasaponin biosynthetic genes, we examined the soyasaponin Bb contents and the expression levels of three soyasaponin biosynthetic genes (
Because light is important for inducing or regulating plant metabolism, two pea cultivar seedlings (Dachung and Sachul) grown for 9 days at 23°C under LD conditions were transferred to continuous dark or light chambers for 1 and 2 days, and then soyasaponin Bb contents and three soyasaponin biosynthetic genes were measured. We found that the accumulation patterns of soyasaponin Bb increased for 1 and 2 days in Dachung and Sachul cultivars, respectively (Fig. 3A). Furthermore, the light to dark transition differentially affected the expression levels of three soyasaponin biosynthetic genes. The expression of
Soyasaponins, one of the triterpenoid saponins, are used as medicinal and food ingredients, and also served as plant secondary metabolites made against biotic and abiotic stress conditions. Accumulations of soyasaponins, and the polymorphisms and the expression levels of their biosynthetic genes have been extensively studied in soybean plants. However, the study on the soyasaponin contents and their biosynthetic gene expression in pea have not yet been known. Here, we found that only soyasaponin Bb was exclusively accumulated in the sprouts of two Korean pea cultivars (Dachung and Sachul), compared with our previous study in soybean sprouts (Fig. 1A) (Yun et al. 2021). Furthermore, these accumulation patterns of soyasaponin Bb were correlated with differential expression patterns between
This work was carried out with the support of “Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01596502 to J. H. Lee and Project No. PJ01706902 to W. D. Seo), Rural Development Administration, Republic of Korea. Additionally, this study was supported by the 2023 RDA Fellowship Program of the National Institute of Crop Science, Rural Development Administration, Republic of Korea.
The authors declare that they have no conflict of interest.
J Plant Biotechnol 2023; 50(1): 70-75
Published online May 2, 2023 https://doi.org/10.5010/JPB.2023.50.009.070
Copyright © The Korean Society of Plant Biotechnology.
Gang Deok Han ・HanGyeol Lee ・Jae-Hyeok Park ・Young Jae Yun ・Gee Woo Kim・Sangyun Jeong ・ So-Yeon Moon ・Hye-Young Seo ・Young-Cheon Kim・Woo Duck Seo ・Jeong Hwan Lee
Division of Life Sciences, Jeonbuk National University, 567 Baekje-daero, Deokjin-gu, Jeonju, Jeollabuk-do 54896, Republic of Korea
Division of Crop Foundation, National Institute of Crop Science, Rural Development Administration, Jeollabuk-do 55365, Republic of Korea
Correspondence to:e-mail: yck01000@jbnu.ac.kr, jhwanlee90@jbnu.ac.kr
swd@rda.go.kr
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
In legumes, soyasaponins, one of triterpenoid saponins, are major components of secondary metabolites with a more diverse array of bioactive chemicals. Although the biosynthetic pathway of soyasaponins has been largely studied in soybean, the study on the soyasaponin contents and biosynthesis-related gene expression in pea (Pisum sativum L.) is poorly understood. Here, we found the accumulation of only soyasaponin Bb component in the sprouts of two Korean domestic pea cultivars (Dachung and Sachul). This pattern was consistent with our observation that increased expression of PsUGT73P2 and PsUGT91H4 genes, but not PsCYP72A69, could be responsible for biosynthesis of only soyasaponin Bb in pea by examining their gene expression. However, gradual accumulation of soyasaponin Bb at developmental stages was not consistent with the expression of PsUGT73P2 and PsUGT91H4, suggesting that the changes of their protein activities may affect the accumulation patterns of soyasaponin Bb. We also revealed that the increased expression levels of PsUGT73P2 and PsUGT91H4 during light to dark transition led to increase of soyasaponin Bb contents. Collectively, our results provided a molecular basis of metabolic engineering for enhancing useful soyasaponin Bb metabolites in Korean domestic pea cultivars.
Keywords: Pea, PsCYP72A69, PsUGT73P2, PsUGT91H4, Soyasaponin Bb
Legumes in the
In legumes, young germinated soybean sprouts contain a more diverse range of bioactive chemicals such as isoflavones, phenolic compounds and triterpenoids than their non-germinated counterparts (Choi et al. 2018; Ma et al. 2018; Mora-Escobedo et al. 2009; Wang et al. 2015). Of them, triterpenoid saponins are a main component of secondary metabolites, which are plentifully accumulated in plant specific tissues such as roots. The biosynthetic pathway of soyasaponins, one of triterpenoid saponins, has been extensively studied in soybean (Abe et al. 1993; Moses et al. 2014; Thimmappa et al. 2014). Previous reports have revealed that 2,3-oxidosqualane precursor is synthesized through the mevalonate pathway, and converted to soyasaponins through a biosynthetic pathway with at least 14 different enzymes, including one β-amyrin synthase (BAS), three cytochrome P450s (CYP450s), ten uridine 5’-diphosphate (UDP)-glycosyltransferases (UGTs), and one acetyltransferase. Structural diversity of the sugar numbers and types in triterpenoid saponins contributes to a variety of the biological and chemical activities (Bowles et al. 2005).
Because the common pea (
The sequence data of pea soyasaponin biosynthesis-related genes (
Table 1 . Genes and oligonucleotides used for RT–qPCR analysis in this study.
Gene name | Gene ID | Primers (5’ to 3’) | Direction |
---|---|---|---|
Psat7g228360 | TCCCTACTGGAAGCAAAGATGG | F | |
TTATGTTTCCACTTTACGTATAATAATTTGAGC | R | ||
Psatoss5882g0200 | CCTTTGTTTGCAGAACATTTTTTCAATG | F | |
TCAACCTAATAGCATTCCCAATCTCT | R | ||
Psat1g218280 | ATAGTGGATGAAGAAGGAAGTAGTTTTAG | F | |
TCATTCTTTGTTGGAATTGGAAGC | R | ||
Xm_051045246.1 | GCTCCCAGCAGTACAGGACTCT | F | |
CAACCTCCTTGGTACTCATCTTGCC | R |
Two pea cultivars, Dachung and Sachul, were kindly provided by the Division of Crop Foundation (National Institute of Crop Science, Rural Development Administration, Jeonju, Korea). The seeds were sterilized in 70% ethanol (EtOH) containing 0.5% Triton X-100 for 15 min, thoroughly washed once in water, soaked in water at 23°C for 24 h, and then transferred to Sunshine Mix 5 (Sungro, Agawam, MA, USA). Germinated peas were prepared by incubation at 23°C for 6, 9, 12, 15, and 18 days in a dark growth chamber. For light to dark treatment, pea seedlings grown for 9 days at 23°C under long-day (LD) conditions (16 h light/8 h dark at a light intensity of 120 μmol m-2 s-1) were transferred to continuous dark or light chambers for 1 and 2 days at 23°C. Seedlings harvested after each treatment were ground to a powder with liquid nitrogen in a mortar and stored at -80°C until use. All experiments were performed in three biological replicates (independently harvested samples).
Total RNA was extracted from the harvested samples using Trizol reagent (Invitrogen, Carlsbad, CA, USA) as previously described (Yun et al. 2021), and the quality and quantity of the resulting RNA was checked using a Nanodrop ND-2000 spectrophotometer (Nanodrop Technologies, Waltham, MA, USA). To analyze RNA expression, real time-quantitative PCR (RT-qPCR) analysis was performed.
Complementary DNA (cDNA) was synthesized from 5 µg RNA in accordance with the protocol of the ReverTra Ace qPCR RT Master Mix kit (Toyobo, Osaka, Japan), and RT-qPCR analysis was conducted in 96-well plates using a CFX real-time system (Bio-Rad, Hercules, CA, USA), THUNDERBIRD SYBR qPCR mix (Toyobo), and gene-specific RT-qPCR primers listed in Table 1 were designed using QuantPrime (http://quantprime.mpimp-golm.mpg.de/).
To prepare pea extracts, the pea seedlings harvested at different developmental stages or under light/dark transition conditions were dried in an oven at 57°C for 48 h, and then ground into powder using a blender (Electric Mixer SMX-W350, Seoul, Korea). As previously described (Yun et al. 2021), 20 ml of 80% methanol was added to 1 g of the ground samples and the mixtures were agitated for 24 h at 23°C. The supernatants were prepared by centrifugation (7,000 × g) for 5 min and then passed through Donex™ OnGuard™ II RP cartridges (Thermo Fisher Sci., MA, USA) to remove the hydrophobic substance. They were then filtered through a 0.22-μm filter unit. Soyasaponin contents were analyzed with a reverse-phase UHPLC (Dionex Ultimate 3000, Thermo Fisher Sci.) equipped with an Acclaim™ RSLC Polar Advangate II (2.2 μm 120 Å 2.1 × 150 mm) column. Following injection of 2 μL of sample, separated soyasaponin Aa, Ab, Ba, Bb, and Bb´ was detected using a CAD (Corona Veo, Thermo Fisher Sci.). The used reagents were UHPLC grade, and soyasaponin Aa, Ab, Ba, Bb, and Bb’ (ChemFaces, Wuhan, China) were used as reference substances. The student’s
We previously found that the accumulation patterns of soyasaponin Aa and Ab were different in the Socheongja and Haepum cultivars (Korean domestic soybean cultivars) (Yun et al. 2021). To investigate the accumulation patterns of soyasaponin contents in pea plants within the legumes, we prepared the sprouts of two Korean domestic pea cultivars (Dachung and Sachul) grown for 15 days at 23°C under darkness conditions, and analyzed their soyasaponin contents using UHPLC-CAD analysis. We found that only soyasaponin Bb was accumulated in two Korean domestic pea cultivars (Fig. 1A). Taken together with a previous data (Yun et al. 2021), this result indicated that different soyasaponin types were exclusively accumulated between soybean and pea sprouts, although they belong to legumes. Our data also suggested that the expression levels of soyasaponin biosynthetic genes or their protein activities may be different between soybean and pea plants.
Based on the soyasaponin biosynthesis pathway identified in soybean plants (Abe et al. 1993; Moses et al. 2014; Thimmappa et al. 2014), we focused on four genes (
To investigate whether soyasaponin Bb accumulation is affected by the expression changes of soyasaponin biosynthetic genes, we examined the soyasaponin Bb contents and the expression levels of three soyasaponin biosynthetic genes (
Because light is important for inducing or regulating plant metabolism, two pea cultivar seedlings (Dachung and Sachul) grown for 9 days at 23°C under LD conditions were transferred to continuous dark or light chambers for 1 and 2 days, and then soyasaponin Bb contents and three soyasaponin biosynthetic genes were measured. We found that the accumulation patterns of soyasaponin Bb increased for 1 and 2 days in Dachung and Sachul cultivars, respectively (Fig. 3A). Furthermore, the light to dark transition differentially affected the expression levels of three soyasaponin biosynthetic genes. The expression of
Soyasaponins, one of the triterpenoid saponins, are used as medicinal and food ingredients, and also served as plant secondary metabolites made against biotic and abiotic stress conditions. Accumulations of soyasaponins, and the polymorphisms and the expression levels of their biosynthetic genes have been extensively studied in soybean plants. However, the study on the soyasaponin contents and their biosynthetic gene expression in pea have not yet been known. Here, we found that only soyasaponin Bb was exclusively accumulated in the sprouts of two Korean pea cultivars (Dachung and Sachul), compared with our previous study in soybean sprouts (Fig. 1A) (Yun et al. 2021). Furthermore, these accumulation patterns of soyasaponin Bb were correlated with differential expression patterns between
This work was carried out with the support of “Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01596502 to J. H. Lee and Project No. PJ01706902 to W. D. Seo), Rural Development Administration, Republic of Korea. Additionally, this study was supported by the 2023 RDA Fellowship Program of the National Institute of Crop Science, Rural Development Administration, Republic of Korea.
The authors declare that they have no conflict of interest.
Table 1 . Genes and oligonucleotides used for RT–qPCR analysis in this study.
Gene name | Gene ID | Primers (5’ to 3’) | Direction |
---|---|---|---|
Psat7g228360 | TCCCTACTGGAAGCAAAGATGG | F | |
TTATGTTTCCACTTTACGTATAATAATTTGAGC | R | ||
Psatoss5882g0200 | CCTTTGTTTGCAGAACATTTTTTCAATG | F | |
TCAACCTAATAGCATTCCCAATCTCT | R | ||
Psat1g218280 | ATAGTGGATGAAGAAGGAAGTAGTTTTAG | F | |
TCATTCTTTGTTGGAATTGGAAGC | R | ||
Xm_051045246.1 | GCTCCCAGCAGTACAGGACTCT | F | |
CAACCTCCTTGGTACTCATCTTGCC | R |
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