Research article
Simultaneous determination of shikimic acid, salicylic acid and jasmonic acid in wild and transgenic Nicotiana langsdorffii plants exposed to abiotic stresses

https://doi.org/10.1016/j.plaphy.2016.02.040Get rights and content

Highlights

  • Salicylic, jasmonic and shikimic acids were simultaneously determined.

  • Heat stress exposition enhanced endogenous jasmonic acid levels.

  • gr-modified N. Langsdorffii plants were less affected by chemical and heat stresses.

  • rolC insertion determines the reduction of heat stress-induced oxidative stress.

Abstract

The presence and relative concentration of phytohormones may be regarded as a good indicator of an organism's physiological state. The integration of the rolC gene from Agrobacterium rhizogenes and of the rat glucocorticoid receptor (gr) in Nicotiana langsdorffii Weinmann plants has shown to determine various physiological and metabolic effects. The analysis of wild and transgenic N. langsdorffii plants, exposed to different abiotic stresses (high temperature, water deficit, and high chromium concentrations) was conducted, in order to investigate the metabolic effects of the inserted genes in response to the applied stresses. The development of a new analytical procedure was necessary, in order to assure the simultaneous determination of analytes and to obtain an adequately low limit of quantification. For the first time, a sensitive HPLC-HRMS quantitative method for the simultaneous determination of salicylic acid, jasmonic acid and shikimic acid was developed and validated. The method was applied to 80 plant samples, permitting the evaluation of plant stress responses and highlighting some metabolic mechanisms. Salicylic, jasmonic and shikimic acids proved to be suitable for the comprehension of plant stress responses. Chemical and heat stresses showed to induce the highest changes in plant hormonal status, differently affecting plant response. The potential of each genetic modification toward the applied stresses was marked and particularly the resistance of the gr modified plants was evidenced. This work provides new information in the study of N. langsdorffii and transgenic organisms, which could be useful for the further application of these transgenes.

Introduction

When in adverse or limiting conditions, plants activate a complex system of responses in order to alleviate cellular damage and to survive (Fuoco et al., 2013). Water deficiency, high temperatures and pollution represent the main stress factors for plants in relation to the expected climate changes. Heat stress conditions affect the cell membranes and the enzyme functionality while modifying the transpiration rate (Lipiec et al., 2013); water deficiency determines the inhibition of photosynthesis, the enhancement of respiration and the lack of mineral nutrients (Yordanov et al., 2000). Heavy metals, due to their widespread distribution and their persistency, represent one of the main issues for agriculture and land use. Heavy metals such as cadmium and chromium (Cr) induce enzyme inhibition, cellular oxidation and the alteration of metabolism (Obata and Fernie, 2012). Cr(VI) is the most toxic oxidation state of chromium, whose uptake was shown to influence the plant's growth, the production of many essential metabolites and enzymatic activity (Singh et al., 2015).

The use of genetic engineering to produce transformed stress-resistant organisms is increasingly gaining interest. Among the genetic modifications studied, the integration of the gene codifying for the rat glucocorticoid receptor (gr), which regulates genes controlling the development, metabolism and immune response, appears to be promising, inducing higher resistance against nematode infections and chemical stress in Nicotiana plants (Del Bubba et al., 2013). The rolC gene is a plant oncogene carried on plasmids of the plant pathogen Agrobacterium rhizogenes; after infection, the gene can be transferred to the plant genome causing hairy root disease and tumor formation. Multiple biochemical and physiological alterations have been observed in rolC transformed plants, including stimulation of alkaloid, anthraquinone and cytokinin production (Bulgakov et al., 2008, Kiselev et al., 2006). Enhancement of plant response to abiotic and biotic stresses has even been related with rolC gene insertion (Del Bubba et al., 2013, Intrieri and Buiatti, 2001). The Nicotiana genus (family of Solanaceae) includes small, well-characterized plants, traditionally used as biological models for genetic and physiological studies; the genetic rolC and gr modifications of Nicotiana langsdorffii plants were previously investigated, yielding interesting results for the production of plants resistant to different stresses (Del Bubba et al., 2013, Fuoco et al., 2013, Ranaldo et al., 2015). The biological state of plants can be monitored through different parameters such as their morphology, anatomy, physiology and biochemistry. Since the response to environmental stresses is controlled by the hormonal network, the presence and relative concentration of hormones may be regarded as a good indicator of an organism's physiological condition. Among the complex hormonal signaling system of plants, the following molecules have been recognized as central components of the adaptation response.

Salicylic acid (SA) and jasmonic acid (JA) are hormones involved in plant growth and development; recent studies demonstrated that they are implicated as signaling compounds in response to both biotic and abiotic stresses (Clarke et al., 2009, Maksymiec, 2007, Maksymiec et al., 2005, Metwally et al., 2003, Pál et al., 2005); many studies showed that the relative concentrations of JA and SA are affected during drought, chemical and temperature stresses (Clarke et al., 2009, De Ollas et al., 2013, He et al., 2014, Maksymiec et al., 2005, Pál et al., 2005, Wang et al., 2010). Shikimic acid (SHA) is an important intermediate in plant metabolism and a key molecule in the biosynthesis of numerous secondary metabolites. The SHA pathway represents the central point for the production of many compounds involved in the principal functions of plant life, including defense, such as flavonoids, lignins, indole derivatives and many aromatic alkaloids. The SHA pathway leads also to the production of SA, through the first step of the phenylpropanoid pathway or directly from isochorismate. The targeted determination of these three compounds could be very useful in the investigation of the effects of rolC and gr insertion in N. langsdorffii and in the better comprehension of plant response towards abiotic stresses. The aim of this paper is to investigate the effects of Cr(VI) exposure, water deficiency and high temperature on wild and transgenic N. langsdorffii plants, through the analysis of selected metabolites, in order to highlight the influence of the inserted transgenes (rolC and gr genes) on plant stress responses. The morphological and physiological effects of rolC and gr insertion in N. langsdorffii plants, exposed to heat, water and chemical stresses, have been the subject of other studies (Bogani et al., 2015, Ancillotti et al., 2015).

Taking advantage from the use of HPLC-HRMS technology, the quantitative determination of SA, SHA and JA was performed. The development of a new analytical procedure was necessary, in order to assure the simultaneous determination of analytes, due to the limited available plant material, and to obtain a limit of quantification adequately low to fulfill the analyte concentrations. The use of a high-resolution detector permitted the accurate measurement of metabolite masses and the discrimination between the analytes and potential interfering compounds; therefore the sample treatment procedure was fast, not requiring the purification step, which is generally essential for biological matrices analyses.

To our knowledge, no method for the simultaneous determination of these three compounds has been reported yet. The comparative evaluation of phytohormonal changes, induced by different abiotic stress factors, in wild type and in gr and rolC plants, allowed us to study the different metabolic mechanisms involved in stress response, in order to identify the organisms more promisingly resistant to the applied stresses.

Section snippets

Chemicals

SHA, JA, SA, salicylic acid phenyl13C6 (SA13C6) and acetic acid HPLC grade were purchased from Sigma Aldrich® (Buchs, Switzerland). HPLC/MS-grade methanol (MeOH) and acetonitrile (ACN) were obtained from Romil LDT (Cambridge, U.K.). Hydrochloric acid (HCl) 37% ACS was purchased from Carlo Erba Reagents (Milano, Italy). Ultrapure water (18.2 MΩ cm, 0.01 TOC) was produced using a Purelab Ultra System (Elga, High Wycombe, U.K.).

Stock and working solutions

Stock standard solutions (10 μg/μL) of SHA, JA, SA and SA13C6 were

Effect of rolC and gr insertion

The gr-modified plants were obtained by inserting a rat gene encoding the glucocorticoid receptor, containing the constitutive CaMV promoter; it is possible that plant phyto-steroids, and particularly brassinosteroids, which showed to induce tolerance towards oxidative, drought and heavy metal stresses (Bartwal et al., 2013), have sufficient affinity with the rat glucocorticoid receptor protein and, therefore, can be activated by this signaling chain in gr transgenic plants. The gr insertion in

Conclusions

The method presented in this work enabled the simultaneous and sensitive determination of three relevant plant metabolites, which proved to be useful in the assessment of the plant metabolic and biochemical status. The investigation of wild and genetically modified plants, exposed to three abiotic stresses, allowed the evaluation of the combined effect of transgenes and stresses on plant response mechanisms. The three genotypes showed to induce different responses during stress exposure;

Contributions

Elisa Scalabrin conducted the experiments, analyzed the data and wrote most of the manuscript. Marta Radaelli helped in plant sample preparation and revised the manuscript. Gabriele Capodaglio conceived and designed the experiments.

Acknowledgments

This work was supported by the PRIN grant number 20098TN4CY from the Italian Ministry of Education, University and Research (MIUR). The authors thank Marcello Buiatti and Patrizia Bogani for sample supply and Daniela Almansi for the English editing.

References (41)

  • E. Sánchez-Rodríguez et al.

    Differential responses of five cherry tomato varieties to water stress: changes on phenolic metabolites and related enzymes

    Phytochemistry

    (2011)
  • C. Shan et al.

    Jasmonic acid regulates ascorbate and glutathione metabolism in Agropyron cristatum leaves under water stress

    Plant Sci.

    (2010)
  • L. Torras-Claveria et al.

    Analysis of phenolic compounds by high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry in senescent and water-stressed tobacco

    Plant Sci.

    (2012)
  • M.S. Zawoznik et al.

    Endogenous salicylic acid potentiates cadmium-induced oxidative stress in Arabidopsis thaliana

    Plant Sci.

    (2007)
  • A. Bartwal et al.

    Role of secondary metabolites and brassinosteroids in plant defense against environmental stresses

    J. Plant Growth Regul.

    (2013)
  • P. Bogani et al.

    The rat glucocorticoid receptor integration in Nicotiana langsdorffii genome affects plant responses to abiotic stresses and to arbuscular mycorrhizal symbiosis

    Plant Biotechnol. Rep.

    (2015)
  • V.P. Bulgakov et al.

    Suppression of reactive oxygen species and enhanced stress tolerance in Rubia cordifolia cells expressing the rolc oncogene

    Mol. Plant. Microbe. Interact.

    (2008)
  • S.M. Clarke et al.

    Jasmonates act with salicylic acid to confer basal thermotolerance in Arabidopsis thaliana

    New Phytol.

    (2009)
  • C. De Ollas et al.

    Jasmonic acid transient accumulation is needed for abscisic acid increase in citrus roots under drought stress conditions

    Physiol. Plant

    (2013)
  • Q. He et al.

    Endogenous salicylic acid levels and signaling positively regulate Arabidopsis response to polyethylene glycol-simulated drought stress

    J. Plant Growth Regul.

    (2014)
  • Cited by (34)

    • Influence of delta-hexachlorocyclohexane (δ-HCH) to Phytophthora ×alni resistant Alnus glutinosa genotypes − Evaluation of physiological parameters and remediation potential

      2022, Ecotoxicology and Environmental Safety
      Citation Excerpt :

      Their primary task is the regulation of physiological processes such as germination, growth, development, photosynthesis or reproduction (Davies, 2010; Dilworth et al., 2017). Simultaneously they are involved in a plant's response to abiotic or biotic stress stimuli (Cortleven et al., 2019; de la Torre-González et al., 2017; Fujita et al., 2006; Saini et al., 2021; Scalabrin et al., 2016; Segarra et al., 2006; Wang et al., 2020; Zhang et al., 2019; Zhao et al., 2019). The significant representatives are auxins, gibberellins, cytokinins, brassinosteroids, strigolactone, ethylene and abscisic, jasmonic and salicyl acid.

    • Metabolic regulation of α-Ketoglutarate associated with heat tolerance in perennial ryegrass

      2022, Plant Physiology and Biochemistry
      Citation Excerpt :

      GSH, a key product of glutathione metabolism, is a typical antioxidant that improves the antioxidant capacity of plants (Noctor et al., 2012). Shikimate, the precursor of phenylalanine synthesis, is related to the synthesis of secondary metabolites and the antioxidation and ROS clearance in plants (Scalabrin et al., 2016) AKG can react with H2O2 during the TCA cycle to form succinate, CO2 and H2O, scavenging ROS and improving the antioxidant capacity of the TCA cycle (Liu et al., 2018). AKG-enhanced AsA, GSH, succinate, and shikimate enhance antioxidant capacity in the AsA-GSH and decrease H2O2 content, preventing heat-induced ROS damages in perennial ryegrass (Fig. 7).

    • A phenylalanine ammonia lyase from Fritillaria unibracteata promotes drought tolerance by regulating lignin biosynthesis and SA signaling pathway

      2022, International Journal of Biological Macromolecules
      Citation Excerpt :

      Finally, the flavonoid segment was resolved in ethyl acetate and was determined. The salicylic acid contents were determined using HPLC method at absorption wavelength of 306 nm [41]. Firstly, 0.1 g leaf was grinded with liquid N2, and the salicylic acid content was ultrasonic-extracted with 2 mL of 90% methanol.

    • CIP elicitors on the defense response of A. macrocephala and its related gene expression analysis

      2020, Journal of Plant Physiology
      Citation Excerpt :

      The volume was adjusted to 4 mL with ether, and the mixture was allowed to stand in a refrigerator for 12 h, centrifuged at 8000 rpm for 15 min, and the upper layer of diethyl ether was removed. The lower layer was extracted with diethyl ether two to three times, and finally the ether phase was combined and dried in a water bath at 40℃ (Scalabrin et al., 2016). The residue was dissolved in 1 mL of mobile phase (50 % methanol: 50 % PH 3.2 acetic acid) and passed through a 0.45-μm microporous membrane for HPLC.

    View all citing articles on Scopus
    View full text