Improved phosphorus nutrition by arbuscular

0 downloads 0 Views 1MB Size Report
increased stevioside concentration in Stevia rebaudiana. (Mandal et al. 2013), increased ... 2013; Schweiger and Müller 2015; Welling et al. 2016; Kapoor et al.
Plant Soil https://doi.org/10.1007/s11104-018-3861-9

REGULAR ARTICLE

Improved phosphorus nutrition by arbuscular mycorrhizal symbiosis as a key factor facilitating glycyrrhizin and liquiritin accumulation in Glycyrrhiza uralensis Wei Xie & Zhipeng Hao & Meng Yu & Zhaoxiang Wu & Aihua Zhao & Jinglong Li & Xin Zhang & Baodong Chen

Received: 21 May 2018 / Accepted: 18 October 2018 # Springer Nature Switzerland AG 2018

Abstract Background and aims Liquorice (Glycyrrhiza uralensis Fisch.) is an important medicinal plant as it accumulates active ingredients, glycyrrhizin and liquiritin, in its roots. Arbuscular mycorrhizal (AM) symbiosis and phosphorus (P) nutrition both affect the accumulation of glycyrrhizin and liquiritin in liquorice roots and it is well known that AM symbiosis mediates P nutrition in many plant species. However, whether AM symbiosis affects the accumulation of glycyrrhizin and liquiritin in G. uralensis through P nutrition is largely unknown. Responsible Editor: Tatsuhiro Ezawa. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11104-018-3861-9) contains supplementary material, which is available to authorized users. W. Xie : Z. Hao (*) : M. Yu : Z. Wu : A. Zhao : J. Li : X. Zhang : B. Chen State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, 18 Shuangqinglu, Haidian District, Beijing 100085, China e-mail: [email protected]

Methods In order to compare the P addition and AM effects on plant performance, we carried out a controlled-environment experiment in which non-AM plants were subjected to different P addition levels compared with an AM inoculated treatment with no P addition. Plant dry weight, stomatal conductance and photosynthetic rate, root P, carbon (C) and nitrogen (N) concentrations, glycyrrhizin and liquiritin concentrations, as well as the expression of glycyrrhizin and liquiritin biosynthesis genes were measured. Results Both P addition and AM inoculation improved plant growth and photosynthesis traits. When root P concentration of non-AM plants matched that of AM plants, both plants showed similar glycyrrhizin and liquiritin concentrations, C:N ratios and biosynthesis gene expressions. Conclusions The results suggested that improved P nutrition by AM symbiosis was of primary importance for facilitating glycyrrhizin and liquiritin accumulation in G. uralensis plants. This confirmed the role of AM symbiosis improving plant P uptake in the regulation of secondary metabolite biosynthesis.

W. Xie : M. Yu : A. Zhao : J. Li : B. Chen (*) University of Chinese Academy of Sciences, Beijing 100049, China e-mail: [email protected]

Keywords Liquorice . Arbuscular mycorrhiza . Nutrient uptake . C:N:P ratio . Secondary metabolite . Gene expression

Z. Wu Jiangxi Engineering and Technology Research Center for Ecological Remediation of Heavy Metal Pollution, Institute of Biology and Resources, Jiangxi Academy of Sciences, Nanchang 330096, China

Abbreviations AM Arbuscular mycorrhiza HMGR 3-Hydroxy-3-methylglutary CoA reductase gene

Plant Soil

FPS SQS β-AS CYP88D6 CYP72A154 UGAT CHS P C N MVA

Farnesyl diphosphate synthase gene Squalene synthase gene β-amyrin synthase gene Cytochrome P450 monooxygenase 88D6 gene Cytochrome P450 monooxygenase 72A154 gene UDP-dependent glucuronosyltransferases gene Chalcone synthase gene Phosphorus Carbon Nitrogen Mevalonic acid

Introduction Plants accommodate microorganisms in and on their roots that helps plants with nutrient acquisition and affects root physiological activities (Hacquard et al. 2015). Most vascular plants form symbiotic associations with arbuscular mycorrhizal (AM) fungi (Walder and van der Heijden 2015). In the AM symbiosis, AM fungi provide mineral nutrients, particularly phosphorus (P), to host plants in exchange for carbohydrates (Smith and Read 2008; Walder and van der Heijden 2015). Plants can invest 1030% of their photosynthetic products to their fungal partners, and in return receive up to 90% of their mineral nutrients from AM fungi (Walder and van der Heijden 2015). Besides the nutritional benefits for host plants, AM symbiosis also affects plant secondary metabolism, which can be directly or indirectly associated with plant defense systems (Gianinazzi et al. 2010; Sharma et al. 2017; Avio et al. 2018; Tavarini et al. 2018). Therefore, AM symbiosis is potentially important for plant cultivation, especially for those plants growing in nutrient-poor soils where P is usually a limiting macronutrient, which can potentially reduce the fertilizer application (Wassen et al. 2005; Kiers and Denison 2008; Li et al. 2016a). Glycyrrhiza species are perennial Fabaceae herbs widely growing in arid and semi-arid regions of the world (Rizzato et al. 2017). As one of the Glycyrrhiza species most widely used in commercial products, liquorice (Glycyrrhiza uralensis Fisch., G. uralensis) becomes the main cultivar in glycyrrhiza cultivation (Pan et al. 2006; Mochida et al. 2017; Rizzato et al. 2017). Glycyrrhizin (C42H62O16) and liquiritin (C21H22O9),

which belong to triterpene saponins and flavonoids respectively, are two major secondary metabolites rich in the roots and stolons of liquorice plants (up to 8% of the total dry weight) and play important physiological roles in protecting plants from biotic and abiotic stresses (Afreen et al. 2005; Hayashi and Sudo 2009). Glycyrrhizin and liquiritin also have long been used as herbal medicines since they exhibit a wide range of pharmacological effects, such as anti-inflammatory, immunomodulatory and antiviral activities (Hayashi and Sudo 2009; Hosseinzadeh and Nassiri-Asl 2015). Moreover, glycyrrhizin is also a sweetener in the food industries because of its higher sweetness compared to sucrose (Seki et al. 2011). The market value of liquorice root extract in global trade had grown from US $149.5 million in 2001 to 403.6 million in 2014, and the market demand for high quality liquorice is also increasing steadily (Vasisht et al. 2016). Currently, cultivated liquorice plants are the main source of glycyrrhizin and liquiritin due to exhausted natural resources (Hayashi and Sudo 2009; Wang et al. 2013; Chen et al. 2017). According to previous studies, glycyrrhizin is synthesized mainly via the cytosolic mevalonic acid (MVA) pathway, and most of the key genes involved in this process have been successfully cloned and characterized, including genes encoding 3-Hydroxy-3methylglutary CoA reductase (HMGR) (Chappell et al. 1995; Mochida et al. 2017), farnesyl diphosphate synthase (FPS) (Li et al. 2013; Li et al. 2017a), squalene synthase (SQS) (Hayashi 2009; Li et al. 2017a), βamyrin synthase (β-AS) (Shen et al. 2009; Mochida et al. 2017), cytochrome P450 monooxygenase 88D6 (CYP88D6) (Seki et al. 2008) and 72A154 (CYP72A154) (Seki et al. 2011), as well as UDPdependent glucuronosyltransferases (UGAT) (Xu et al. 2016). The biosynthesis of liquiritin starts with the universal precursor acetyl-CoA, which also involved in glycyrrhizin biosynthesis, and followed by biosynthesis processes involving a series of key genes such as chalcone synthase gene CHS (Winkel-Shirley 2002; Zhang et al. 2009; Zhou et al. 2017). Previous studies, including our own results have shown that expressions of these functional genes were closely associated with glycyrrhizin and liquiritin accumulation in liquorice plants, while AM inoculation could up-regulate the gene expressions, suggesting important roles of transcriptional level regulation by AM symbiosis in secondary metabolite biosynthesis (Hayashi 2009; Nasrollahi et al. 2014; Li et al. 2017a; Xie et al. 2018).

Plant Soil

Actually, it has been well demonstrated that AM symbiosis substantially influence the accumulation of secondary metabolites in plants (Zeng et al. 2013; Welling et al. 2016; Kapoor et al. 2017; Avio et al. 2018). For example, AM symbiosis significantly promoted plant growth and increased stevioside concentration in Stevia rebaudiana (Mandal et al. 2013), increased glycyrrhizin and liquiritin concentrations in Glycyrrhiza uralensis (Liu et al. 2007; Chen et al. 2017), as well as the accumulation of phydroxybenzoic acid and rutin in Viola tricolor (Zubek et al. 2015). In general, the positive influence of AM symbiosis on plant growth and secondary metabolite accumulation could principally attribute to nutritional benefits and non-nutritional mechanisms, such as changes in phytohormone levels and regulation of biosynthesis genes (Mandal et al. 2013; Schweiger and Müller 2015; Welling et al. 2016; Kapoor et al. 2017). Our previous study showed that AM symbiosis induced a notable increase of root biomass and root glycyrrhizin and liquiritin concentrations. Moreover, glycyrrhizin and liquiritin concentrations were closely related with root P concentration, C:N ratio and glycyrrhizin and liquiritin biosynthesis gene expressions (Xie et al. 2018). However, the key mechanisms underlying the AM effects on glycyrrhizin and liquiritin accumulation are still unclear. Improved P nutrition is thought to be the primary benefit of AM symbiosis to host plants that fundamentally influences plant growth (Clark and Zeto 2000; Smith and Read 2008; Urcoviche et al. 2015). Phosphorus nutrition is also one of the important factors influencing plant secondary metabolisms as P directly involves in the biosynthesis of key precursors for secondary metabolites, including terpenoids and flavonoids (Welling et al. 2016; Kapoor et al. 2017). However, some reports indicated that AM induced changes in plant secondary metabolism were only partially P-mediated or even independent of P supply (Toussaint et al. 2007; Schweiger et al. 2014a), suggesting the uncertain role of P in plant secondary metabolism. On the other hand, secondary metabolite biosynthesis gene expressions also influence secondary metabolite production (Kapoor et al. 2017). It has been reported that the expression of plant secondary metabolite biosynthesis genes can be affected by abiotic factors including P availability (Lillo et al. 2008; Jia et al. 2015; Pant et al. 2015; Liu et al. 2016), while mycorrhizal plants often contain more P in their tissues than non-mycorrhizal plants (Smith and Read 2008; Zubek et al. 2015; Kapoor et al. 2017), which could essentially influence the secondary metabolite

biosynthesis gene expressions. Furthermore, improved P nutrition by AM symbiosis could influence other nutrient uptake and assimilation, such as C and N, and finally affect nutrient allocation and stoichiometric ratio (Chen et al. 2010; Gao et al. 2017; Liu et al. 2017). Therefore, it is highly possible that AM regulation of biosynthesis gene expressions and nutrient allocation through improved plant P nutrition could substantially influence plant secondary metabolite accumulation. This P-related AM effects are of interest for management of plant cultivation especially in P-deficient soils, where the use of P fertilizers could be at least partly replaced by AM fungi (Schweiger and Müller 2015; Urcoviche et al. 2015). It has been estimated that inoculation with AM fungi could supplement a reduction of 80% of the recommended P fertilizer in a given field condition (Jakobsen 1995; Gianinazzi et al. 2010). In this study, we hypothesize that AM symbiosis can increase glycyrrhizin and liquiritin concentrations in G. uralensis plant mainly through improving plant P nutrition. To test this hypothesis, we carried out an experiment with different P supply to non-AM plants in order to obtain a similar P status and plant biomass with AM plants. We predicted that AM and non-AM plants would exhibit similar biosynthesis gene expressions, nutrient concentrations and glycyrrhizin and liquiritin concentrations when they reach similar P concentrations. We quantified root nutrient concentrations and expression of glycyrrhizin and liquiritin biosynthesis genes, and calculated nutrient stoichiometric ratios. We also measured plant stomatal conductance and net photosynthetic rate to interpret the P addition and AM inoculation effects on plant performance.

Materials and methods Host plant, AM fungal inoculum and growth substrate Newly harvested seeds (9.8 g per thousand seeds) of Glycyrrhiza uralensis Fisch. were collected from the Minqin cultivation base of Gansu Province in China. Seeds of liquorice were scarified in sulfuric acid (50%) for approximately 30 min, surface-sterilized with 10% H2O2, and then washed several times with sterile water. Pre-treated seeds were then pre-germinated on moist filter paper in darkness for about 2 days. Germinated seeds with uniform radicle were used for following experiment.

Plant Soil

The AM fungus Rhizophagus irregularis (R. irregularis) Schenck & Smith BGC AH01 was provided by the Beijing Academy of Agriculture and Forestry. The fungus was propagated on Sorghum bicolor L. in a sandy soil for about 12 weeks, and a mixture of infected roots, hyphae, spores and sandy soil was used as the fungal inoculum (approximately 60 spores/g soil). A soil of low fertility collected from Erdos (39°53' N, 110°1' E), Inner Mongolia, China, was mixed with sand (2:1, w/w) as the growth substrate. The soil was passed through a 2 mm sieve and sterilized by γ-radiation (20 kGy) before use; the sand (