Nutrient homeostasis under salt acclimatization
Salinity-induced nutritional disorders are typically discussed as deficiencies or changes in the requirement for nutrients. However, the influence of salt stress on plant nutrition is highly variable, and depends on the genotype, tissue, growth conditions and chemical characteristics of the soil (Grattan and Grieve, 1999). Exposure of L. japonicus plants to increasing concentrations of salts led, as expected, to increases in shoot Na+ with concomitant decreases in shoot K+ (Figure 2). Reduction in plant K+ is the most commonly recognized nutritional change under salt stress, and has been implicated in growth and yield reduction in crops (Grattan and Grieve, 1999). However, many glycophytes are able to substitute Na+ for K+ without negative effects on growth (Marschner, 1995). Increases in calcium, magnesium and manganese rule out an NaCl-induced deficiency of these elements in L. japonicus shoots under our experimental conditions (Figure 2). The same applies to iron and boron, which appeared to be under homeostatic control. However, we found decreased concentrations of zinc, phosphorus and sulphur under severe salt stress (Figure 2). Salt-induced deficiency of these elements has been reported previously in other species, and could arise from changes in nutrient availability, uptake, transport or partition within the plant (Grattan and Grieve, 1999). As it is generally accepted that an increased supply of nutrients does not necessarily improve the growth of salt-stressed plants under nutrient-sufficient conditions, it is difficult to assess whether a real deficiency exists in our experiments (Grattan and Grieve, 1999). In addition, molybdenum was the most salt-sensitive of all micronutrients in our ionomic profile, decreasing even under mild treatments (Figure 2). To our knowledge, a connection between salinity and Mb content has not been recognized previously. Currently, we have no evidence that salt-acclimatized L. japonicus plants reach critical molybdenum deficiency levels, and further analyses will be required to assess this possibility. Interestingly, shoot nitrate reductase activity vital for nitrate assimilation has been shown to decrease under salt stress in tomato (Debouba et al., 2007). As molybdenum is a co-factor of nitrate reductase, our data may provide an explanation for these results.
Salinity-induced changes of gene expression
Major changes in the expression of genes involved in amino acid metabolism as well as nitrogen and organic compound transport, including putative sucrose, amino acid and organic acid transporters, indicated profound changes in central metabolism under long-term salt stress. General metabolic changes were also reflected within the miscellaneous gene group, which included large enzyme families including peroxidases, lipases, glucosidases, glycosyl- and glutathione-S-transferases, cytochrome P450s and oxidases, linked to a myriad of cellular processes (Figure S1). Further global control of the acclimatization response is reflected by changes in the transcription and RNA processing, signalling and hormone metabolism categories (Figure 4c).
We identified new molecular candidates that may represent important factors in salt acclimatization in legumes, and further analysed the expression of selected genes within the transcription, transport and signalling functional groups. We also identified a multitude of genes from L. japonicus that are homologous to genes that are stress-regulated in other species, not only in the stress and defence-related group but also in other functional categories. For example, many probesets representing genes from secondary metabolism were transcriptionally regulated by salt stress, including flavonoid, phenylpropanoid and phenol metabolism (Figure S1), which have been previously correlated with biotic and abiotic stress responses (Kliebenstein, 2004; Walia et al., 2005). In addition, we found many transcriptionally regulated genes relating to the cell wall, including expansins, cellulose synthases and glycosyl transferases. In contrast to results reported for rice (Walia et al., 2005), most L. japonicus genes in this functional group were down-regulated by salt stress (Table S2 and Figure S1). This observation may be a consequence of the decreased growth and reduced requirement for cell-wall synthesis under long-term salt acclimatization.
Some transcriptional changes were reflected in the metabolomic data. For instance, proline accumulation in plants is common under salt stress (Figure 6c), and seven up-regulated probesets encode proteins that are putatively involved in proline metabolism: Δ-1-pyrroline 5-carboxylase synthetases (P5CS, probesets Ljwgs_006172.2, Ljwgs_032463.1, Ljwgs_053689.1 and chr1.CM0147.99) and Δ-1-pyrroline-5-carboxylate dehydrogenases (P5CDH, probesets chr4.CM0170.37, Ljwgs_019593.1_s and Ljwgs_052588.1_s). We also found induction of two probesets encoding putative myo-inositol-1-phosphate synthases (Ljwgs_091497.1_s and chr4.CM0307.12), concomitant with changes in osmoprotectants from the inositol family (see below). The activity of this enzyme was found to be a rate-limiting step in the biosynthesis of inositol-containing compounds, and is known to be involved in the salt-stress responses of halophytes (Nelson et al., 1998). The depletion of asparagine may be under transcriptional control, as indicated by down-regulation of asparagine synthase 1 (LjAS1, probesets gi897770, gi897770_s and chr5.CM0071.60_s) and up-regulation of two putative asparaginases (Ljwgs_021574.1 and chr5.CM0096.107). In addition, the slight decrease in galactinol was paralleled by down-regulation of a putative galactinol synthase (chr1.CM0122.56).
Approximately one third of the probesets identified in L. japonicus showed a significant hit (E value ≤1e-5) when matched to those reported under long-term salt stress in A. thaliana, suggesting a certain degree of inter-species similarity in the molecular responses (Table S5) (Sottosanto et al., 2004). Future comparative systems analysis under well-controlled environmental and nutritional conditions will be required to unravel inter-species conservation of salt-stress acclimatization mechanisms.
Salinity-induced changes of the metabolic phenotype
Results from the non-targeted metabolite profiling demonstrated a major and reproducible change of the metabolic phenotype in the course of salt acclimatization, which was most evident for amino acid, sugars and polyols and organic acid metabolism (Figure 6c).
Accumulation of amino acids and other nitrogen-containing compounds is a remarkable biochemical feature of almost all plant stress responses reported so far. This change in nitrogen metabolism has been interpreted as an accumulation of compatible solutes, generation of carbon and nitrogen reserves for future needs, a sink for detoxification of excess nitrogen or for redox potential cycling (Gilbert et al., 1998; Rabie, 1999). This broad response reflects a tightly controlled metabolic shift, and is inconsistent with nitrogen deficiency as a mechanism of salt injury (Grattan and Grieve, 1999). Although it has been extensively shown that nitrogen content may be affected under salinity due to alterations in NO3− uptake, non-nodulated salt-stressed legumes exhibited accumulation of NH4+ and even increased total nitrogen content (Huq and Larher, 1983; Speer et al., 1994). Therefore, the accumulation of nitrogen-containing compounds may represent a response to a decrease in nitrogen demand caused by reduced growth rates under stress. This contention is in line with the hypothesis of reduced assimilation of inorganic nitrogen in salt-acclimatized L. japonicus plants (see below). In addition, the enhanced expression of most of the genes involved in amino acid metabolism (Figure S1) indicates the requirement for de novo synthesis already observed in other plant species (Gilbert et al., 1998).
Notable exceptions to the general amino acid behaviour were the two amides glutamine and asparagine. The observed decrease in both glutamine and glutamate may suggest a reduced capacity of NH4+ assimilation through the glutamine synthetase/glutamate synthase pathway, as they play a pivotal role in this process (Forde and Lea, 2007). Indeed, these enzymatic activities have been reported to decrease under salinity (Debouba et al., 2007). Although we did not find any regulation of the genes involved in this pathway, we demonstrated consistently reduced expression of two probesets encoding putative nitrite reductases (gi9968472 and chr4.CM0227.40_s, Table S2), which are also involved in nitrogen assimilation, and also down-regulation of genes encoding putative NO3− and NH4+ transporters. In contrast, the depletion of asparagine was paralleled by transcriptional changes of genes putatively involved in its metabolism (see above). As a major nitrogen transport compound of L. japonicus (Waterhouse et al., 1996), the transcriptional control of asparagine levels in the shoot may also reflect decreased inorganic nitrogen assimilation.
Along with nitrogen-containing compounds, sugars and polyols also increase under stress and are known to have protective roles as osmoprotectants (Munns, 2005; Orcutt and Nilsen, 2000). Several compounds related to these chemical groups accumulated in salt-acclimatized L. japonicus plants, including the disaccharides maltose and sucrose and the polyols arabitol and erythritol (Figure 6c and Table S4). We also identified the salt-induced methylated inositols pinitol and ononitol, in line with transcriptional changes of the myo-inositol pathway as described above.
The general depletion in organic acids observed in L. japonicus shoots has been demonstrated previously in salt-stressed roots and nodules of the legume alfalfa (Fougere et al., 1991). This phenomenon may reflect an increased energy demand that is met by intensified respiration, carbon allocation to amino acid or sugar pools that are required as compatible solutes, or transport from shoots to roots as carbon source. It is interesting to consider a possible role in compensation for uneven charges entering the plant; organic acids are known to counterbalance unequal uptake of ions as they occur as carboxylic anions under physiological pH (Hinsinger et al., 2003; Marschner, 1995). Moreover, many organic acids accumulate in other species under a variety of stress regimes, such as drought, cold and heat, suggesting that this metabolic change is due to ionic misbalance rather than a decrease in the amount of fixed carbon under stress conditions (Kaplan et al., 2004; Timpa et al., 1986). Notable exceptions to the general depletion of organic acids were glucuronic and gulonic acids (Table S4). As glucuronate is involved in the myo-inositol oxidation pathway that synthesizes nucleotide sugars for cell-wall polysaccharides, the accumulation of glucuronic acid may reflect an inhibition of cell-wall biosynthesis due to decreased growth (Kanter et al., 2005). Alternatively, it might represent an important feature of ascorbic acid metabolism, in line with the parallel increase in gulonic acid. Glucuronic and gulonic acids are intermediates of the uronic pathway that synthesizes ascorbate from myo-inositol (Ishikawa et al., 2006).
Gradual step increase in salts compared to initial acclimatization
The salt-stress physiology of glycophytes is currently interpreted in terms of the biphasic growth model (Munns, 2002, 2005). In essence, this model proposes two growth inhibition phases in response to a gradual increase of salinity. In the first phase, growth inhibition is caused by decreased osmotic potential and thus reduced water availability. After prolonged exposure to salinity, accumulation of ions within plant tissues triggers a second mode of growth inhibition caused by ion toxicity. The biphasic growth model formalizes three essential aspects of salinity: (i) that plant salt-stress physiology ultimately depends on toxicity of ions per se, (ii) that the time of exposure is an important variable, and (iii) that appropriate experimentation requires long-term progressive acclimatization to differentiate between (a) hyperosmotic and hyperionic stresses, and (b) physiological acclimatization responses and cellular damage or senescence. In this work, we compared the experimental approach of the biphasic growth model, i.e. the conventional gradual step increase design, to an alternative experimental design based on initial acclimatization. The rationale behind the initial acclimatization is that, given a particular genotype and environment, a range of non-lethal soil salt concentrations should exist that allow the genotype to geminate and develop. Such an experimental design mimics natural and agricultural topsoil-associated salinity, where not only successful plant growth but also establishment on salt-rich soils is required.
Nutrient, transcriptome and metabolome profiling data were used to test whether both experimental designs equally address salt-stress physiology. Firstly, non-supervised analysis suggested that changes do not cluster according to the experimental approach but to the salt-stress dose (Figures 3a and 6a). Secondly, we found only rare statistical evidence for a qualitative differentiation between the acclimatization regimes. For example, under ia50 and ia75 treatments, zinc content decreased while glucose and fructose were increased, a behaviour that is not observed in gradual acclimatization (Figures 2 and 6c). However, it cannot be ruled out that such a difference is due to the differential stress doses perceived by plants between the two designs, as described previously (see Results). Other changes showed only quantitative trends differing between the experimental approaches, with a few changes arising specifically in ia75 plants, probably due to the highly stressful nature of this treatment. Considering the broad scope of the profiling techniques used herein, it could be argued that no major differential effects were observed between the gradual acclimatization and the initial acclimatization experiments. Our results reveal a general equivalence of both salt regimes, supporting the use of the gradual acclimatization approach and the biphasic growth model interpretations, despite previous concerns raised against them (Neumann, 1997).
The stress acclimatization process requires fine tuning of responses
Given that plants acclimatized to a non-lethal salt-stress dose may have reached a stable physiological state essential for survival, it could be argued that most, if not all, of the molecular and metabolic responses reflect the set of plastic physiological changes that, as a whole, allow the plant to cope with the environmental constraint (Lichtenthaler, 1996). If this is the case, how are the various traits regulated and coordinated within the plant in response to changes in the stress dose? From a molecular perspective, a temperature-dependent adjustment has been described for expression of CBF/DREB transcription factors controlling cold-stress responses in A. thaliana, demonstrating that stress sensing and response mechanisms are not binary on/off systems (Zarka et al., 2003). Such ‘rheostat’ control of responses may be interpreted in a simple deterministic model, where all available traits needed to cope with the stress are elicited in a strictly linear dose-dependent manner (Figure 7a). Based on the results of our work, two refinements of this model are necessary (Figure 7b). First, some responses may reach a systems constraint, such as a concentration plateau (trait A). These constraints may be of a genetic or metabolic nature and reflect limitations in the minimal or maximal elicitation of transcript or metabolite pools. Second, some responses are not required at low salt concentrations and will be activated only when a threshold is reached (trait C), suggesting that sensitive responses at the molecular and metabolic systems level are sufficient to compensate for the induced change under low stress intensities. We propose that most major plastic changes under salt acclimatization may be qualitatively explained by this fine tuning model, which includes linear, plateau and threshold dose-dependent responses (Figure 7b). Whether this model may fundamentally be applied to other abiotic stresses remains to be determined.
Figure 7. Models of dose-dependent salt-stress acclimatization responses. (a) Strictly linear dose-dependent responses. (b) Fine-tuning model, including linear, plateau and threshold dose-dependent responses. A, B and C represent measurable molecular and metabolic plant traits.
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In conclusion, we performed a systems investigation of salt acclimatization in L. japonicus, designed to be as non-biased and comprehensive as possible given the current technological limitations. We found a complex pattern comprising ionomic, transcriptomic and metabolomic responses to salt stress, some of which have not been described in plants before. In addition, we showed that the general transcriptional regulation under long-term salinity is mainly dominated by ion accumulation and toxicity rather than osmotic effects, in line with the proposed physiological biphasic growth model (Munns, 2002, 2005). Finally, we demonstrated the need to refine simple dose-dependence models of salt acclimatization. We now venture to predict that molecular and metabolic differences in acclimatization responses between tolerant and sensitive cultivars may be characterized by three possible features within the framework of the fine-tuning model. Increased tolerance may arise from: (i) changes in the slope of dose-dependent responses, (ii) changes in the upper or lower concentration constraints of a response, or (iii) a shift in the stress dose threshold of factors that do not respond at low stress levels.