Nodule carbohydrate metabolism and polyols involvement in the response of Medicago sativa to salt stress

https://doi.org/10.1016/j.envexpbot.2012.08.009Get rights and content

Abstract

Alterations of plant growth, chlorophyll fluorescence parameters, nodule carbon metabolism and polyols concentration as result of salt stress were examined in alfalfa (Medicago sativa). Plants, in symbiosis with Sinorhizobium meliloti GR4 strain, were grown under controlled conditions for 35 days (DAS) and subjected to 150 mM of NaCl stress. Plant biomass (PDW) and nitrogen fixation rate (NFR) were markedly affected by salt stress conditions; the highest reductions of PDW (50%) and NFR (40%) were registered at 84 DAS and 56 DAS, respectively. In addition, salinity affected the chlorophyll fluorescence parameters, decreased initial chlorophyll fluorescence (F0) and increased the optimum quantum yield of PSII (Fv/Fm ratio). The enzyme activities sucrose synthase activity and phosphoenolpyruvate carboxylase, responsible for the carbon supply to the bacteroids by the formation of dicarboxylates, were drastically inhibited by salinity, mainly at 56 DAS with the beginning of flowering. The content of total soluble sugars and proline increased under salt stress, and these concentrations were higher in nodule than in leaf. This last result suggests that the nodule is an organ specially protected in order to maintain its functioning, even under stress conditions. Besides, the content of myoinositol and pinitol in leaves and nodules changed with the plant growth stage and the saline treatment. Under salinity stress, the concentrations of pinitol in nodule were higher than in leaf, which supports the central function of this molecule in the adaptive response of nodules to salt stress. The increase of pinitol synthesis in nodule of M. sativa under salt stress could be one of the adaptive features used by the plant.

Highlights

► NaCl altered the content of compatible osmolytes and enzyme activities in M. sativa. ► Salt stress decreased Fo and increased the quantum efficiency of PSII (Fv/Fm ratio). ► The content of polyols was related with the response of the symbiosis to salt stress. ► Nodules increased the synthesis of pinitol in response to salt stress.

Introduction

Salinity is one of the major abiotic factors limiting global agricultural productivity, and it is estimated that one-third of the world's irrigated land are unsuitable for crops (Frommer et al., 1999). Salt stress drastically affects the photosynthesis (Soussi et al., 1998), the nitrogen metabolism (Cordovilla et al., 1994), the carbon metabolism (Delgado et al., 1993, Balibrea et al., 2003) and the plant nutrition (Mengel and Kirkby, 2001). Legumes are classified as salt-sensitive crop species (Läuchli, 1984) and their production is particularly affected by salt stress because these plants depend on symbiotic N2 fixation for their nitrogen requirement (Elsheikh and Wood, 1995). The limitation of productivity is associated with a lower growth of the host plant, poor development of the root nodules (Georgiev and Atkins, 1993) and consequently with a reduction of the nitrogen-fixation capacity (Ben Salah et al., 2011).

Under the variation of saline environments, plants develop different adaptive mechanisms (Rhodes et al., 2002, Sairam et al., 2006), some of which include the synthesis and accumulation of low-molecular weight organic compounds in the cytosol and organelles (Ashraf and Harris, 2004, Sairam et al., 2006). These compounds, collectively called compatible osmolytes, are simple sugars, disaccharides, sugar alcohols or polyols, amino acids and sulfonium compounds (Ashraf and Harris, 2004, Bartels and Sunkar, 2005, Ashraf and Foolad, 2007). A major function of the accumulation of the compatible osmolytes is the osmotic adjustment to counteract the high concentrations of inorganic salts in the vacuole and in the root medium (Zhu, 2001, Rhodes et al., 2002). Another function of the compatible osmolytes is the osmoprotection which may occur at lower salt concentrations. This role involves the protection of thylakoids and the plasma membrane, as well as the stabilization of proteins. Under salinity stress these compounds also act as a sink of energy or reducing power, as a source of carbon and nitrogen, or scavenging reactive oxygen species (Bartels and Sunkar, 2005, Sairam et al., 2006).

Soluble carbohydrates and their polyol derivatives are the most common osmolytes accumulating in plants in response to low water potentials. Cyclic polyols involve myoinositol, ononitol and pinitol. Myoinositol is derived from glucose-6-phosphate and can be further methylated to sequoyitol or ononitol, which are epimerized to d-pinitol (Loewus and Loewus, 1980). An increase in pinitol content has been shown to occur in plants subjected to a water deficit (Streeter et al., 2001, Matos et al., 2010) as well as to a high salinity (Sengupta et al., 2008). Salinity induces the expression of genes, one of which has been shown to be involved in pinitol synthesis (Vernon and Bohnert, 1992). Thus, the introduction of genes involved in the synthesis of proline (P5CF127A), betaines (betA) and polyols (mt1D, imt1) into plants contributes to abiotic stress tolerance (Rathinasabapathi, 2000, Chen and Murata, 2002). It has been also suggested that exogenous application of compatible solutes is an alternative approach to improve crop productivity under saline conditions (Makela et al., 1999, Chen and Murata, 2002).

The objective of the present work was to investigate changes induced by salinity on the content of some compatible osmolytes and enzyme activities of carbon metabolism in Medicago sativa grown under symbiotic conditions. In addition, the content of the polyols myoinositol and pinitol, as well as their relation with the adaptation of the symbiosis to salt stress, was also evaluated.

Section snippets

Biological material and growth conditions

Seeds of M. sativa (var. Aragon) were surface sterilized by immersion in 5% NaClO for 3 min and germinated in 0.8% water-agar plates at 28 °C in darkness. Two days after, seedlings were transferred to individual pots of about 200 ml containing a vermiculite–perlite mixture (3:1) and watered with N-free nutrient solution (Rigaud and Puppo, 1975). Each seedling, inoculated with 1 ml of a stationary culture of Sinorhizobium meliloti GR4 strain (ca. 109 cell ml−1), was grown in a controlled environmental

Results

Plant biomass and nitrogen fixation rate were markedly affected by salt stress conditions (Fig. 1). In control (0 mM NaCl) and treated plants (150 mM NaCl), the plant dry weight (PDW) increased with plant age, although plants grown without salt showed values of PDW higher than salinized plants in all harvests. At fructification stage (84 days after sowing, DAS) PDW decreased about 50% with the saline treatment. The NFR was maximal at the beginning of flowering stage (56 days after sowing, DAS) in

Discussion

Several papers have been written regarding the effect of salt stress on nitrogen fixation and on the enzymes of the carbon metabolism in nodules (Lopez et al., 2008, Lopez and Lluch, 2008) as well as on the accumulation of sugars and other compatible solutes (Bartels and Sunkar, 2005, Khadri et al., 2007), but the mechanisms implicated in these processes remain unclear. In the present work, we examined the changes in the content of polyols (myoinositol and pinitol), the presence of soluble

Acknowledgments

Financial support was obtained through the Andalusian Research Program (AGR-139) and the Spanish Ministry of Education and Science AGL2006-01279. The authors are grateful to anonymous reviewers for making valuable suggestions to earlier drafts of this study.

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