Triterpenoid gene expression and phytochemical content in Iranian licorice under salinity stress

Abstract

Licorice is a well-known medicinal plant, containing various secondary metabolites of triterpenoid and phenolic families. The aim of this study is to evaluate the effect of salinity stress on the expression of key genes involved in the biosynthetic pathway of triterpenoids such as glycyrrhizin, betulinic acid, soyasaponins, and phytosterols in licorice root, as well as providing a phonemic platform to characterize antioxidant properties, glycyrrhizin, and total phenolic content. This study also includes measuring the gene expression level and glycyrrhizin content in leaves and roots of control plants. The studied genes included squalene synthase (SQS1 and SQS2), β-amyrin synthase (bAS), lupeol synthase (LUS), cycloartenol synthase (CAS), β-amyrin 11-oxidase (CYP88D6), and β-amyrin 24-hydroxylase (CYP93E6). Our results revealed that all of the mentioned genes were upregulated following the stress condition with different transcription rates. The highest increase (12-fold) was observed for the expression of the LUS gene, which is related to the betulinic acid pathway. Also, the highest content of glycyrrhizin was observed at 72 h posttreatment, which was consistent with the upregulated transcription levels of the glycyrrhizin pathway genes especially SQS1 and CYP88D6 at the same time. Correlation and stepwise regression analysis proved the key role of SQS1 gene in the biosynthetic pathway of glycyrrhizin. Antioxidant activity and phenolic content also were increased following stress condition. A comparison between the expression levels of SQS1 and other genes involved in the production of glycyrrhizin, phytosterols, and soyasaponins revealed a similar transcription trend, which shows the gene expression in the roots was significantly higher than the leaves. In contrast, SQS2 and LUS genes displayed a higher expression in leaf tissues. The genes related to betulinic acid biosynthetic pathway exhibited an expression rate different from other triterpenoid pathway genes, which could be observed in the leaves and roots of control plants and the roots of salt-treated plants. Furthermore, results showed that these two SQS genes have different expression rates due to different plant tissues (roots and leaves) and stress conditions. Importantly, in contrast to previous reports, we detected the glycyrrhizin in leaf tissues. This result may indicate the presence of a different genetic background in native Iranian licorice germplasm. Keywords Gene expression . Glycyrrhiza glabra . Glycyrrhizin . Quantitative real-time PCR . Secondary metabolites Abbreviations bAS β-Amyrin synthase CAS Cycloartenol synthase CYP88D6 β-Amyrin 11-oxidase CYP93E6 β-Amyrin 24-hydroxylase FDP Farnesyl diphosphate GA3 Gibberellic acid HPLC High-performance liquid chromatography LUS Lupeol synthase MeJA Methyl jasmonate OSCs Oxidosqualene cyclases QRT-PCR Quantitative reverse transcription PCR SQS Squalene synthase

Bacillus cereus Enhanced Medicinal Ingredient Biosynthesis in Glycyrrhiza uralensis Fisch. Under Different Conditions Based on the Transcriptome and Polymerase Chain Reaction Analysis

“… Behdad et al (2020) found that 100 mM NaCl significantly upregulated the expression level of key genes (β -AS , CYP88D6 , and CYP72A154 ) in Glycyrrhiza glabra L. involved in triterpenoid saponin biosynthesis and directly increased the content of glycyrrhizic acid. Shirazi et al (2019) also found that salt stress upregulated the expression level of key genes (including SQS , β -AS , LUS , CAS , CYP88D6 , and CYP93E6 ) in Iranian licorice. Moreover, Wang et al (2020) reported that 200 mM NaCl upregulated the expression level of key enzymes (β-AS, CYP88D6, and CYP72A154) involved in the glycyrrhizic acid biosynthesis pathway in G. uralensis .…”

“…The content of compounds and the expression level of key genes have significantly changed in glycyrrhizic acid and liquiritin biosynthesis pathway during abiotic stress, salt stress specifically caught our attention ( Wang et al, 2015 , 2017 ; Zhou et al, 2017 ; Yeo et al, 2018 ; Kaur and Ganjewala, 2019 ). Shirazi et al (2019) reported that salt stress significantly upregulated the expression level of SQS , β -AS , CAS , LUS , and some CYPs genes and increased the content of glycyrrhizin in Glycyrrhiza glabra , which illustrated that salt stress inhibited in the glycyrrhizin biosynthesis pathway. In conclusion, we provided a basis for further studies concerned with the relation between salt stress and glycyrrhizic acid pathway and liquiritin pathway in G. uralensis .…”

Transcriptome Meta-Analysis Associated Targeting Hub Genes and Pathways of Drought and Salt Stress Responses in Cotton (Gossypium hirsutum): A Network Biology Approach

“…SMRGs including MYB15 ( Marchev et al, 2020 ), NAC29 ( Huang et al, 2015 ; Wang T. T. et al, 2019 ), TLP3 ( Ibrahim and Jaafar, 2013 ; Bao et al, 2014 ), HFB2A ( Paupiere et al, 2020 ), WRKY27 , OLE16 ( Shao et al, 2019 ), BBE8 ( Daniel et al, 2017 ), LEA3 ( Zhao et al, 2011 ), CHSY ( Dao et al, 2011 ), DRE1D ( Agarwal et al, 2017 ), Malate synthase ( de Kraker and Gershenzon, 2011 ), cycloartenol synthase ( Jin et al, 2017 ) and CASL1 ( Taura et al, 2007 ) in drought enhances secondary metabolite responsive pathways and exert to provide tolerance in the adverse condition in drought stress condition. In salt, several identified genes such as TLP ( Zare-Hassani et al, 2019 ; de Jesus-Pires et al, 2020 ), WRKY6 ( Robatzek and Somssich, 2001 ; Afrin et al, 2015 ), MMP-2 ( Guo et al, 2019 ), peroxidase 53 ( Novo-Uzal et al, 2014 ), bHLH25 ( Goossens et al, 2019 ; Meraj et al, 2020 ; Yang et al, 2020 ), malate synthase ( Brito et al, 2020 ), carboxylesterase ( Wang et al, 2015 ; Cao et al, 2019 ), 1-deoxy-D-xylulose-5-phosphate synthase 2 ( Paetzold et al, 2010 ), NAC ( He et al, 2019 ), DCS2 ( George et al, 2017 ), XTH ( Choi et al, 2011 ; Guerriero et al, 2018 ), AtHB-7 ( Olsson et al, 2004 ), NCED5 ( Udomchalothorn et al, 2017 ), CAS ( Shirazi et al, 2019 ), ERF ( Meraj et al, 2020 ), galactinol synthase ( Patel et al, 2020 ), EXPA7 ( Jadamba et al, 2020 ) and glutathione S-transferase ( Nazar et al, 2011 ; Czerniawski and Bednarek, 2018 ) are SMRGs exhibited crucial role in providing resistance under salt stress condition. Overall, these putative hub genes might have putative functions in drought and salt stress responses in cotton.…”

Metabolome and Transcriptome Analyses of Cucurbitacin Biosynthesis in Luffa (Luffa acutangula)

“…Cycloartenol synthase (CAS) converts 2,3-oxidosqualene to cycloartenol through the protosteryl cation intermediate, and was the basal plant OSC from which others derived ( Phillips et al, 2006 ). Some studies have shown that abiotic stress can up-regulate the CAS genes ( Babiychuk et al, 2008 ; Shirazi et al, 2019 ). However, some studies reported no significant change in CAS expression under drought and salt stress ( Basyuni et al, 2009 ; Nasrollahi et al, 2014 ).…”