Research article
Comparative metabolic and ionomic profiling of two cultivars of Stevia rebaudiana Bert. (Bertoni) grown under salinity stress

https://doi.org/10.1016/j.plaphy.2018.05.001Get rights and content

Highlights

  • Fengtian showed increased levels of steviol glycosides, particularly rebaudioside A.

  • Salinity stress reduced stevia plant height and biomass, particularly in cultivar Shoutian-2.

  • Fengtian maintained higher K+/Na+ ratios as compared to Shoutian-2.

  • Amino acids and amines were the major osmotica in stevia under salinity stress.

  • Fengtian accumulated higher levels of proline and gluconate.

Abstract

This study provides a comprehensive investigation on the impact of increasing NaCl concentrations on hydroponically grown Stevia rebaudiana cultivars (Shoutian-2 and Fengtian). Growth parameters including plant height, biomass and physiological responses including osmotic potential were measured. In addition, the levels of steviol glycosides, elements and primary metabolites were measured and statistically evaluated. The cultivar Fengtian grew faster, accumulated less Na+ and compatible organic solutes, and more K+ in the leaves, as compared to the cv. Shoutian-2. Metabolite analysis identified 81 differentially accumulated metabolites, indicating an alteration in the metabolite phenotype of both cultivars upon exposure to salinity A general increase in many amino acids, amines, sugars and sugar phosphates with a concurrent decrease in most organic acids; including tricarboxylic acid (TCA) cycle intermediates, was observed. In the more salt tolerant cv. Fengtian, the levels of hexose phosphates and metabolites involved in cellular protection increased in response to salinity. These metabolites remained unchanged in the sensitive cv. Shoutian-2. Interestingly, salt treatment notably increased the rebaudioside A concentration by 53% while at the same time stevioside decreased by 38% in Fengtian which has important implications for controlling the relative amounts of reboudioside A and stevioside. The findings of this study leads to the conclusion that mild salinity stress can increase the yield of sweetener compounds, which is dependent on the cultivar and the level of salinity stress.

Introduction

Stevia rebaudiana Bert. (Bertoni) is a perennial shrub indigenous to Paraguay, South America. It is economically important in Asia and South America and grown for its non-carcinogenic and low-calorie sweeteners present in the leaves (Lemus-Mondaca et al., 2012). Stevia extracts have been used by traditional South American cultures for a range of medicinal applications, and studies have indicated that steviosides are beneficial to human health (Gardana et al., 2010 and references therein). These extracts were associated with antiproliferative effects in different cancer cells (López et al., 2016), and its antidiabetic (Zeng et al., 2013; Ritu and Nandini, 2016), antimicrobial (Atteh et al., 2008), anti-hyperglycemic, and antifungal activities (Lemus-Mondaca et al., 2012) have been investigated. Due to these various beneficial attributes, sweeteners produced from Stevia plants are gaining popularity. The US Food and Drug administration (US FDA), European Food Safety Authority (EFSA), Food Standards Australia New Zealand (FSANZ), the Joint FAO/WHO Expert Committee on Food Additives, and recently, the European Union (EU) have also considered the addition of steviol glycosides as a naturally occurring authorised sweetener for the food industry (Kubica et al., 2015).

Steviol glycosides (SGs) are the secondary metabolites responsible for the sweetness of Stevia (Gupta et al., 2014; Urban et al., 2015). Stevioside (ST) and rebaudioside A (RA) are the most abundant diterpenoid glycosides, but more than 30 additional SGs are currently known (Woelwer-Rieck et al., 2010). Recently, two minor diterpene glycosides, rebaudioside R and S were detected in the leaves of Stevia rebaudiana (Ibrahim et al., 2016). In dried leaves, ST represents 4–13% of SGs and is 110–270 times sweeter than conventional sugar (sucrose) (Tavarini and Angelini, 2012). In contrast, RA represents 2–4% of SGs but is 180–400 times sweeter than sucrose. As compared to ST, RA has an additional glucose monomer that gives it a higher sweetening potency and therefore is the most preferred component of the stevia leaf extracts (Lemus-Mondaca et al., 2012). The RA also lacks the bitter aftertaste usually found associated with steviosides (De Oliveira et al., 2007). Most importantly, purified RA has no effect on either blood pressure or glucose homeostasis (Carakostas et al., 2008). It is for this reason, the new cultivars of S. rebaudiana with a higher content of RA and a reduced content of ST are being developed by the plant breeders to improve the utilization of this source of natural sweeteners (Yadav et al., 2011).

Many countries have shown interest in the commercial cultivation of Stevia rebaudiana (Ramesh et al., 2006, 2007), but further research is required to better understand the physiological and biochemical responses to a range of abiotic stresses affecting SG production (Ren and Shi, 2012). The accumulation of such commercially important secondary metabolites is affected by abiotic stresses (Arbona et al., 2013), as SGs play important role in the adaptation of plants to stress environments via alleviating stress associated effects (Hill and Roessner, 2015; Ramakrishna and Ravishankar, 2011). Salinity is one of the major environmental stress factors that cause disturbances in plant growth and nutrient balance, reducing crop yields. It leads to alteration in metabolic processes, membrane disorganisation, and oxidative stress, reduction in cell division in addition to inducing water stress and ion toxicity (Blumwald, 2000; Aswathappa and Bachelard, 1986; Popp et al., 1990). The ultimate aim of salinity tolerance research is to increase the ability of plants to maintain growth and productivity in saline soils, thereby reducing the effects on growth and yield by introducing new traits like ion exclusion and tissue tolerance to osmotic stress (Roy et al., 2014). The accumulation of low-molecular compounds, termed as compatible solutes, is one important adaptation mechanism that plants exhibit in response to osmotic stress (Cao et al., 2017). Metabolite profiling has proven to be a powerful tool to gain an overview of biochemical changes occurring in important crops upon exposure to salt stress, and to identify pathways potentially involved in salinity tolerance (Dias et al., 2015; Natera et al., 2016; Shabala et al., 2016; Shelden and Roessner, 2013).

Recently, Zeng et al. (2013) reported that salt stress for four weeks changed growth and physiological responses as well as glycoside contents of Stevia rebaudiana. Their study showed a decrease in total dry weight and chlorophyll and an increase in proline concentration in response to with increasing salt concentrations (60, 90, and 120 mM). Both RA and ST concentration also decreased with increasing salt concentrations and the ratio of RA/ST of salt-treated plants changed. Their study indicated that this plant could tolerate salt stress, and there is a possibility of optimising the SG composition by using saline soil for growing S. rebaudiana. Another recent study found that stevia copes well with mild (34 and 90 mM), short-term (16 and 25 days) salinity stress, which did not change chlorophyll, RA or ST concentrations, but changed tissue ion concentrations (Cantabella et al., 2017).

In this study, we investigated the growth, physiological and biochemical changes induced by salinity stress in two cultivars of Stevia rebaudiana (cv. Shoutian-2 and Fengtian) which showed contrasting salt responses. As previous studies investigated the effects of short-term and mild salinity stress (Zeng et al., 2013; Cantabella et al., 2017), we focussed instead on investigating long-term exposure to salinity stress (8 weeks treatment) at three levels of salinity, ranging from mild to severe (50 mM, 100 mM, 200 mM). We determined differential changes in plant height, biomass accumulation, osmolarity, chlorophyll, RA, ST, ion and primary metabolite concentrations in both salt stressed and control plants of two cultivars of Stevia rebaudiana. The aims of this study were to investigate the effects of salinity stress on the plant phenotype (growth and physiology), as well as the metabolome and ionome. We identified a cultivar that is tolerant to salinity stress whilst maintaining high yields of SG's, suitable for the food industry.

Section snippets

Plant growth conditions and treatments

Seeds of the two cultivars of Stevia rebaudiana, cv Shoutian-2 (C1) and Fengtian (C2), were supplied by Mr Andrew Rank (Central Queensland University, Rockhampton, Australia). The seeds were sown in a potting media containing a mixture of washed river sand, commercial potting mix and coconut peat (4:3:3 v/v). These cultivars were selected based their high SG content (Midmore et al., 2012). Fifteen days after germination, seedlings were transplanted into small plastic pots (5 cm × 10 cm)

Impact of salinity on plant growth and biomass accumulation

Both cultivars of Stevia rebaudiana showed similar height growth response when exposed to salinity for 4 weeks. The plants showed no signs of stress within the first 4 weeks of salinity treatment. After 5 weeks of treatment, leaf chlorosis and a loss of viability (35% and 30% survival) were observed in the 100 mM and 200 mM salt-treated plants respectively. With an increase in salt concentrations, both Fengtian and Shoutian-2 plants showed slow and stunted growth compared to control plants. By

Exposure to salinity stress leads to a reduction in plant height and biomass

Strategies of plants to cope with saline environments include salt exclusion or sequestration, tissue tolerance to accumulated ions and reduced loss of K+, osmotic adjustment and control of water homeostasis, biochemical and molecular responses, and changes in growth and development (Tester and Davenport, 2003; Shabala and Cuin, 2006; Munns and Tester, 2008; Sanchez et al., 2008). In this study, growth and biomass production were severely affected by salinity stress in both cultivars of Stevia

Conclusions

In conclusion, salinity stress of Stevia resulted in a general increase in the levels of many amino acids, amines, sugars and sugar phosphates, with a concurrent decrease in most organic acids including TCA intermediates. The metabolites involved in salinity response differed between the two cultivars, with results suggesting that the differences in salt response of Shoutian-2 and Fengtian were due to differences in the accumulation of ions and organic solutes. The more salt tolerant cultivar

Contributions

M.D., N.A. and U.R. conceived and designed the experiments. M.D. performed the Stevia growth, salinity and ionomic experiments. D.D. and N.J. performed the metabolite profiling analysis. D.C. performed the steviol glycoside measurements and N.A. and M.D. performed the statistical analysis. M.D., N.A., C.H., D.D., N.J., D.C., D.M. and U.R. analysed and interpreted the data. All authors read and approved the manuscript.

Conflicts of interest

The authors declare that they have no conflict of interest.

Acknowledgment

The research was financially supported by the Endeavour Research Fellowship (Department of Employment, Education and Workplace, Australian Government) [grant no. 3416_2012] to A/Prof Debnath. This study was also supported by the grants through the Australian Centre for Plant Functional Genomics (ACPFG), Australian Research Council [grant no. FT130100326] and The Grains Research and Development Corporation (GRDC). The authors gratefully acknowledge Metabolomics Australia, School of BioSciences,

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