Metabolic contribution to salt stress in two maize hybrids with contrasting resistance

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Metabolic contribution to salt stress in two maize hybrids with contrasting resistance

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Julia A. Richter a,∗ , Alexander Erban b , Joachim Kopka b , Christian Zörb a a b

University of Hohenheim, Institute of Crop Science, Quality of Plant Products, 70599 Stuttgart, Germany Max Planck Institute of Molecular Plant Physiology, 14476 Potsdam-Golm, Germany

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Article history: Received 30 September 2014 Received in revised form 8 January 2015 Accepted 10 January 2015 Available online xxx

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Keywords: Metabolomics Metabolite profiling Salt stress Salt resistance Zea mays

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1. Introduction

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Salt stress reduces the growth of salt-sensitive plants such as maize. The cultivation of salt-resistant maize varieties might therefore help to reduce yield losses. For the elucidation of the underlying physiological and biochemical processes of a resistant hybrid, we used a gas chromatography mass spectrometry approach and analyzed five different salt stress levels. By comparing a salt-sensitive and a salt-resistant maize hybrid, we were able to identify an accumulation of sugars such as glucose, fructose, and sucrose in leaves as a salt-resistance adaption of the salt-sensitive hybrid. Although, both hybrids showed a strong decrease of the metabolite concentration in the tricarboxylic acid cycle. These decreases resulted in the same reduced catabolism for the salt-sensitive and even the salt-resistant maize hybrid. Surprisingly, the change of root metabolism was negligible under salt stress. Moreover, the salt-resistance mechanisms were the most effective at low salt-stress levels in the leaves of the salt-sensitive maize. © 2015 Published by Elsevier Ireland Ltd.

Salt stress is a problem in many crops because it reduces growth [1] and therefore yield. With regard to increasing world food demand and the simultaneous prognosis of increasing soil salinization [2], attention should focused on improving salt resistance in important crop plants. Maize (Zea mays L.) is one of the main carbohydrate sources not only for human nutrition, but also for animal feed and bio-fuel production. Maize is furthermore considered a salt-sensitive crop plant [3]. It shows, on the one hand, a distinct reduction in leaf extensibility and reduced biomass production at salinity [4,5], resulting in enormous yield losses. On the other hand, the growth of the root system in which salt stress initially affects the plant appears to be less reduced than the growth of the maize shoot [6]. Mechanisms such as cell wall extensibility are partly responsible for such growth reduction and may be activated by the signaling from the roots to the shoot [7]. These findings underline the need to improve salt resistance in crop plants, especially in sensitive crops such as maize. In order to understand the integral mechanisms of the way that salt stress affects growth, we need to elucidate the biochemical and physiological reactions of maize under salt stress. In recent years, metabolite profiling has proved to be a powerful method, because it enables

∗ Corresponding author. Tel.: +49 711 459 22358; fax: +49 711 459 23946. E-mail address: juliaannika [email protected] (J.A. Richter).

the analysis of a large set of compounds with high throughput [8,9]. Metabolomics extensively reflect phenotypic changes in certain tissues. These metabolite changes are induced by stress-related reactions based on altered gene expression [8], post-transcriptional protein modification [10], and changes in protein function. Some valuable metabolic datasets exist for several model and crop plants grown under saline conditions and show that various pathways are affected. In the model plant Arabidopsis, Kim et al. [11] have studied the metabolite changes in cell cultures after salt-stress treatment by means of a gas chromatographic–mass spectrometry/liquid chromatographic–mass spectrometry (GC–MS/LC–MS) approach and determined that the methylation cycle, the phenylpropanoid pathway for lignin production, and the glycinebetaine biosynthesis are the mainly affected metabolic pathways. In crop plants, biochemical targets during salt stress are amino acid synthesis, the tricarboxylic acid (TCA) cycle, sugar biosynthesis, and polyol synthesis [12–14]. These metabolome studies demonstrate that plant metabolites react strongly to salt stress. However, a salt-sensitive plant species such as maize needs to get acclimated to salt stress during cultivation; otherwise, only shock reactions are detectable in metabolome studies. Therefore, a stepwise acclimation to hydroponic solution and to the salt stress is inevitable in our experimental design. Further investigations have shown that resistance adaptions are noticeable at the metabolic level in resistant varieties [13,14], but to date, no such data exist for salt-resistant maize. Amino acids and sugars serve as osmolytes, being widely accepted as resistance parameters indicating salt

http://dx.doi.org/10.1016/j.plantsci.2015.01.006 0168-9452/© 2015 Published by Elsevier Ireland Ltd.

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stress [15], but these findings needs to get proved for salt-resistant maize. Roots show a different metabolic reaction from the leaves of plants under salt stress in detail [12–14]. The reason that such a large metabolic reaction takes place in leaves with no growth reduction in roots [16] remains unclear. To date, no specific approach for maize including an investigation of its leaves and roots and further including a salt-sensitive and a salt-resistant genotype exits. Dose-dependent metabolite changes, possibly with threshold and plateau behaviors as reported for Lotus japonicus under salt stress [15], are moreover often disregarded. With the aim of identifying salt resistance mechanisms, such behavior should be addressed; otherwise, an interesting resistance mechanism might remain undetected, because the wrong salt-stress level might have inadvertently been chosen. Therefore, we investigate to exposure maize hybrids to control conditions, minor moderate and high salt concentrations in five low-increasing salt increments. For maize, the physiology and the biochemistry of salt resistance are only rudimentarily understood. Our aim has been to close this gap in our knowledge by providing physiological and biochemical data concerning the resistance mechanisms in maize root and leaves based on a metabolome study. We have grown two hybrids differing strongly in salt resistance, i.e., a salt-sensitive and a salt-resistant maize inbred line [16] under salt stress. The salt-sensitive hybrid Logo is a commercially cultivated hybrid (Limagrain, Chappes, France), which is used as double silage and corn type with no acknowledged resistance mechanisms to abiotic stress such as salt stress. As outcome of a screening for appropriate sensitive counterparts in former studies the used hybrid Logo showed a severe reaction to short term salt stress and therefore serves as a source for an appropriate salt-sensitive hybrid. The hybrid SR08 belongs to a closely related family of salt-resistant hybrids (SR), which was developed by crossing a distinct sodium-excluding inbred line (female) with different osmotic-resistant inbred lines (male) [16]. As a consequence the SR hybrids combine the two salt-resistance qualities Na+ exclusion and osmotic robustness [16]. Na+ exclusion which is inherited by one crossing parent, is especially a suitable quality for improving salt-resistance [17]. Moreover, the SR family is well characterized as showing marked differences in terms of growth reduction and hormonal responses under salinity as well as in growth promoting agents such as ␤-expansins in comparison to sensitive hybrids [5,7,18]. This metabolite study of two differently resistant maize hybrids might help to identify contrasting features with respect to metabolic alterations, thereby developing a clearer picture of the physiological and biochemical adjustments to salinity. New insights might help to provide useful tools for future breeding schemes or for the improvement of salt resistance by the screening of adequate physiological or biochemical characteristics. The GC/time-of-flight (TOF)-MS approach used here should enable us to answer following questions. (i) Which of the main pathways in maize leaves is affected during salt stress? (ii) Is root metabolism affected by long-term salt stress? (iii) Does the application of small salt-stress increments offer new insights into resistance mechanisms?

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2. Materials and methods

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2.1. Plant cultivation and processing

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Two hybrids of Z. mays L. cv. Logo and cv. SR08 [16] were cultivated in a hydroponic solution (0.2 mM KH2 PO4 , 1 mM K2 SO4 , 2 mM Ca(NO3 )2 , 2 mM CaCl2 , 0.5 mM MgSO4 , 150 ␮M Sequestrene® (Syngenta, Basel, Switzerland), 1 ␮M H3 BO3 , 2 ␮M MnSO4 , 0.5 ␮M ZnSO4 , 0.2 ␮M CuSO4 , 5 nM (NH4 )MoO), and 1 g quarz sand (SiO2 ) in a greenhouse. Five different salt-stress levels (0 mM (control),

Fig. 1. Acclimation procedure to hydroponic solution and to salt stress. Hydroponic solution was increased in 25% increments every 2nd day and NaCl was added every 2nd day in 25 mM NaCl increments until the final concentration was reached. Plants were treated with 0 (control), 25, 50, 75, 100 mM NaCl, for 7d.

25 mM, 50 mM, 75 mM, and 100 mM NaCl) were imposed on each of four biological replicates (n = 4). Plants were adapted to nutrient solution in 25% increments every second day (Fig. 1). This course of action enabled the osmotic adaption of the plants to the full-strength nutrient solution. Afterwards, salt-stress concentrations were also increased in 25 mM increments every second day for osmotic adaption. Full stress treatment of 100 mM NaCl was achieved after 7 days and for lower salt stress levels even after a shorter akklimation period at the same time point (Fig. 1). Young shoots (i.e., leaves that emerged after the achievement of 100% salt stress) and young root tips (i.e., the first 3 cm) were harvested and immediately frozen in liquid nitrogen. All samples were homogenized by being ground with liquid nitrogen and were immediately freeze-dried. Additionally, the remaining root material was dried at 80 ◦ C for the following Na+ analysis. 2.2. Atomic absorption spectrometry and calculation of Na+ uptake For the cation analysis, ca. 50 mg freeze-dried leaf material and ca. 50 mg dried roots were weighed out into 10 mL 69% nitric acid. Subsequently, samples were solubilized by microwave digestion (leaves: 15 min, 190 ◦ C, roots: 30 min, 190 ◦ C). The solutions were diluted and filtered, and Na+ analysis was carried out on an atomic absorption spectrometer (Thermo Fisher Scientific, 3300 series, Dreieich, Germany). Na+ uptake was calculated as the ratio of total plant Na+ -content (root plus shoot) to total fresh weight (adapted from Schubert et al. [16]). Results were visualized by the use of Excel (Microsoft, Redmont, USA). 2.3. Metabolite extraction and metabolite profiling Freeze-dried and homogenized sample material of roots and leaves, approximately 10 mg dry weight, were mixed with 360 mL pre-cooled methanol containing 0.02 mg/mL 13 C6 -sorbitol (Sigma–Aldrich, Steinheim, Germany) as an internal standard for the correction of volume errors. Samples were extracted at 70 ◦ C for 15 min. After a cooling step to room temperature, 200 ␮L CHCl3 was added, and afterwards, the solution was agitated at 37 ◦ C for 5 min. Finally, 400 ␮L H2 O was added in order to induce liquid phase separation. Samples were vortexed prior to centrifugation at 13,000 rpm for 5 min. A volume of 80 mL of the upper polar phase containing the primary metabolite fraction was dried in a vacuum concentrator (SpeedVac) overnight without heating and stored dry at −80 ◦ C. Chemical derivatization, i.e., methoxyamination and trimethylsilylation, and subsequent gas chromatography/time-of-flight-mass

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spectrometry-based metabolite profiling (GC/TOF-MS) was applied as described [15,19].

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2.4. Metabolite data processing

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GC-EI/TOF-MS chromatograms were acquired, visually controlled, baseline corrected and exported in NetCDF file format using ChromaTOF software (Version 4.22; LECO, St. Joseph, USA). GC-MS data processing into a standardized numerical data matrix and compound identification were performed using the TagFinder software [19]. Compounds were identified according to standardized guidelines [15] by mass spectral and retention time index matching to the reference collection of authenticated standard substances and of frequently observed not yet identified mass spectral tags from the Golm metabolome database, GMD (http://gmd.mpimpgolm.mpg.de) [20,21] and to the mass spectral collection of the NIST08 database (http://www.nist.gov/srd/mslist.htm). Laboratory and reagent contaminations were identified by non-sample control experiments and removed from further analysis. Numerical analyses were based on the peak height values of the recoded mass feature, i.e., the response values. These values were corrected for the dry weight of each sample and by the response of the internal standard, 13 C6 sorbitol, from each respective GC/TOF-MS chromatogram to obtain normalized responses. In this study the normalized responses were used to calculate ratios that were expresses as percentages relative to the average normalized response of each metabolite measured in the sensitive hybrid Logo under non-stressed control conditions, i.e., 100% concentration change. Prior to statistical assessment, hierarchical clustering and independent component analysis the response ratios of each of the 88 identified metabolites and nonidentified mass spectral tags from the leaf and root metabolite profiles were log10 -transformed of ratio normalized response to median per cluster, i.e., log10 (n) values.

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2.5. Statistical analyses and data visualization

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Root and shoot data were analyzed independently. Hierarchical clustering and visualization by heatmap-display were performed 210 with the MultiExperiment Viewer 4.9 [20]. Hierarchical clustering 211 was performed with the distance measure, Pearson’s square coeffi212 cient, and complete linkage. Independent component analysis was 213 Q3 carried out according to Schulz et al. [21] on the basis of log10 (n) 214 values. Analyses of variances (ANOVAs) and bar charts were gen215 erated with Origin 8G (OriginLab Corporation, Northampton, USA) 216 and positioned by Visio (Microsoft, Redmont, USA). For this pur217 pose metabolites and mass spectral tags were selected by 1-way 218 ANOVA with a significance threshold set to p < 0.05. Schemes of 219 reaction classes and the experimental design were generated by 220 Excel (Microsoft, Redmont, USA). The complete metabolite profiling 221 results including detected metabolites that were not significantly 222 changed according to ANOVA can be found in supplemental Table 223 1. 224 209

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3. Results

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3.1. Plant growth reduction under salinity

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Maize hybrids react differently under salt stress. Fig. 2 is representative for the reactions of the sensitive and the resistant maize under increasing salinity levels. The sensitive hybrid Logo reacted with greater growth reduction after 7 days of salt treatment compared with the resistant hybrid SR08. This is consistent with former findings [16]. The resistant hybrid was formerly characterized as a sodium excluder at the level of roots and as an includer of sodium into vacuoles in the leaf cells [16]. Sodium uptake of the two hybrids

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Fig. 2. Representative photographs of the two hybrids showing a growth reduction (A) Logo (salt-sensitive) and (B) SR08 (salt-resistant) with increasing salt stress levels. Plants were grown in hydroponic solution and treated with 0 (control), 25, 50, 75, 100 mM NaCl respectively for 7d; see Section 2.

Logo and SR08 is shown in Fig. 3. The lower dotted line shows a higher capacity of the resistant hybrid to exclude sodium at the level of the root surface. The higher capacity of vacuoles, especially in the xylem parenchyma cells of the resistant hybrid, has been shown elsewhere [16]. 3.2. The metabolites of Logo and SR08 react differently on the salt-stress gradient Young leaves and young roots, completely developed under salt stress, were analyzed by GC/TOF-MS-based metabolite profiling. In total, 61 metabolic components were detected in the leaves and 60 metabolites were detected in roots. In leaves, 17 metabolites belonged to frequently observed but not yet identified mass spectral tags and 14 in roots. In addition, 27 metabolites were found exclusively in leaves and 27 different were found exclusively in roots. The normalized average responses of metabolites in young leaves are shown in a heatmap (Fig. 4). The means of the root data are not shown, because independent component analysis (ICA; Fig. 6) did not indicate different clusters within the independent components. As no clear fringed clusters were visible in the ICA, no dominating experimental factor was indicated meaning that the differences between the two variables, genotype and salt stress

Fig. 3. Uptake of Na+ ions by the maize hybrids Logo and SR08. A lower uptake indicates a greater Na+ -exclusion. Values were calculated as the ratio of total plant Na+ -content to fresh weight (adapted of Schubert et al. [16]). Plants were treated with 0 (control), 25, 50, 75, 100 mM NaCl, for 7d, respectively; see Fig. 1 Circles, Logo (salt-sensitive); triangles, SR08 (salt-resistant). n = 4.

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metabolite increases or decreases (class II) are likely associated with a metabolic resistance mechanism. The third reaction type (class III, Fig. 7) is characterized by no reaction to increasing salt stress of both hybrids, but the metabolite concentration of the two hybrids lies at differently constant levels. These differences of class III are genotypic based and might contribute to salt resistance on the one hand. On the other hand, they might be constant off-sets and are not correlated to the salt concentration but are always higher or lower, respectively, in the sensitive compared to the resistant hybrid. Nevertheless, the metabolites of class III might be useful as possible metabolic markers for the maize hybrids, because they are constant with or without salt stress.

3.3. Genotypic and salt-stress-related metabolic changes in leaves

Q8 Fig. 4. Hierarchical clustering of metabolites in young leaves of maize. Logo, saltsensitive hybrid; and SR08, salt-resistant hybrid. The log ratios of 61 metabolites and non-identified mass spectral tags are shown. Blue: low content of the metabolites; red: high content of the metabolites. Hierarchical clustering was formed by Pearson’s square coefficient. Hierarchical trees were calculated by complete linkage. Plants were treated with 0 (control), 25, 50, 75, 100 mM NaCl respectively for 7d; see Fig. 1. n = 4. (For interpretation of the references to color in figure legend, the reader is referred to the web version of the article.)

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do not represent the major variances in the data set. The detailed charts Figs. 8 and 9 show only those metabolites with a significant ANOVA. Non-significant metabolites are presented at supplemental Table 1. Because of the close-meshed NaCl additions in small increments of 25 mM NaCl (Fig. 1), an analysis of the different reaction patterns of the two hybrids Logo and SR08 was possible. Thus, we were able to define three different reaction classes according to the simplified trends of metabolite changes in the two hybrids treated with the given salt-stress gradient (Fig. 7). One possible reaction was an exclusive salt-stress reaction without any resistant-based reactions (class I). This reaction type stands out because of a parallel development of the metabolite concentrations of both hybrids with increasing salt-stress level. As a physiological consequence, possible differences of the unstressed control are generally based on genotypic differences but not on salt stress. A different effect (class II) is characterized by an opposing reaction of the metabolite concentration between the two hybrids with increasing salt stress. A so-called resistant-based reaction to salt stress (class II) is of great interest, because it helps to explain the reasons for the salt resistance of the hybrid SR08. Different

The identified and significant metabolites in the shoot particularly belong to the TCA cycle and the raffinose pathway. Additionally, several other metabolites such as threonic acid, quinic acid, galactaric acid and 3-phosphate glyceric acid are affected. All metabolites decrease in the TCA cycle with increasing salt stress. This biochemical cycle provides precursors for several metabolic pathways. The decreasing TCA-cycle metabolites are: aconitic acid, 2-oxo-glutaric acid, succinic acid and malic acid (Fig. 8). Hence, all detected TCA-cycle metabolites represent a class I reaction. Furthermore, malic acid and citric acid exhibit a transient peak with 50% and 10% at the salt-stress level of 50 mM NaCl in the hybrid Logo (class II), respectively. The second main effected pathway is the raffinose pathway. The mono- and disaccharides sucrose, fructose, glucose, and galactose all show a class II reaction (Fig. 9). The concentration of sucrose and fructose in the hybrid Logo increases strongly with increasing salt stress up to 250% and 300%, respectively, compared with 100% in the Logo control. This large increase is also present for glucose and galactose even at 75 mM NaCl. However, the concentration changes of the mono- and disaccharides of the resistant hybrid SR08 are completely different. All identified mono- and disaccharides greatly increase at the saltstress level of 50 mM NaCl (Fig. 9). A decrease up to 400% at 50 mM NaCl is detected for fructose, and afterwards, the concentration of fructose decreases to the level of the hybrid Logo at 100 mM NaCl. This transient peak is an unambiguously different genotypic specific reaction to salt stress (class II). On leaving the major carbohydrate cycle and going one step further along the raffinose pathway, the concentrations of polyol myo-inositol and galactinol can be seen to react differently to the salt stress, respectively. These two compounds increase with increasing salt stress both in the hybrid Logo and the hybrid SR08. The sugar alcohol galactinol increases for both hybrids without the peak at 50 mM NaCl, unlike its substrate, namely the monosaccharides (Fig. 9). Galactinol increases in the sensitive hybrid Logo up to 400% compared with the control, whereas the resistant hybrid shows only an increase of 190% (class II). The final product, the trisaccharide raffinose (Fig. 9), increases up to 490% with increasing salt stress in both hybrids. Therefore, this parallel increase in both genotypes is a typical class I reaction with no influence on the factor genotype (Fig. 9). Other than the metabolites of the TCA cycle and the raffinose pathway, only four further identified and significantly different metabolites occur in the leaves, namely quinic acid, galactaric acid, threonic acid, and glyceric acid-3-phosphate (see supplemental Table 1, Fig. 4). The polyol quinic acid shows the highest concentration peak in SR08 with 100% at 50 mM NaCl (class II). The polyhydroxy acid galactaric acid remains at the same concentration level, independently of the salt-stress level but 60% higher than that in SR08 (class III). Threonic acid, as part of ascorbic acid metabolism, and 3-phosphate glyceric acid (carbon fixation) exhibit only small

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Fig. 5. Independent Component Analysis (ICA) of 56 identified metabolites and nonidentified mass spectral tags of young leaves. Independent Component values were calculated from log ratios. Plants were treated with 0 (control), 25, 50, 75, 100 mM NaCl respectively for 7d; see Fig. 1. (a) Cluster of metabolites from control treatment and low salt treatment (0, and 25 mM NaCl) of both hybrids (Logo; triangles and SR08; circles). (b) Cluster of metabolites from high salt treatment (50, 75, and 100 mM NaCl) of salt-sensitive hybrid (Logo). (c) Cluster of metabolites from high salt treatment (50, 75, and 100 mM NaCl) of salt-resistant hybrid (SR08).

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differences between the variables and are therefore thought to have a lower relevance for salt resistance. 3.4. Few metabolites in the roots are influenced by salt stress

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Only 13 metabolites of the roots changed significantly (p < 0.05) in concentration with increasing salt stress in both hybrids (supplemental Table 1). We applied a full statistic evaluation of all metabolites by the use of ANOVA. The outcome of the ANOVA was critically evaluated subsequently for each of these 13 metabolites. We consider that a significant but small change of a metabolite should not be discussed as a severe physiological relevance. Therefore, we tend to apply a conservative approach and will not discuss 10 out of the 13 metabolites, as these are not convincing indicators of a biochemical or physiological adaption. The remaining three metabolites consists of one not yet identified metabolite A174005, cellobiose, and 2,4-dyhdroxy-butanoic acid (supplemental Fig. 1). The metabolite A174005 similarly increases with increasing salt stress in both hybrids up to 180% (class I). The concentration of the disaccharide cellobiose differs between the two hybrids but remains unchanged with increasing salt stress (class III). The concentration of the metabolite 2,4-dyhdroxy-butanoic acid varies by 250% between Logo and SR08 (class III) and further falls in SR08 at 75 mM NaCl down to 100% (class II). This metabolite switches from class III to class II with increasing salt stress.

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4. Discussion

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Fig. 6. Independent Component Analysis (ICA) of 56 identified metabolites and nonidentified mass spectral tags of young roots. Independent Component values were calculated from log ratios. Plants were treated with 0 (control), 25, 50, 75, 100 mM NaCl respectively for 7d; see Fig. 1. (a) No cluster differentiation between metabolites from control treatment, salt treatment or different hybrids (Logo and SR08). n = 4.

affects the metabolome of the two hybrids differentially at a saltstress level of 50 mM NaCl. The heatmap (Fig. 6) clearly shows this phenomenon (coloring the difference of SR08 at a concentration of 50 mM NaCl) with the interpretation being that the concentration of 50 mM NaCl induces a maximum of metabolite changes, as is also supported by Fig. 9. The differences of Logo and SR08 are of special

4.1. Impact of salt stress on the two hybrids with contrasting salt resistance The lower growth reduction (Fig. 2) and the lower Na+ uptake of the hybrid SR08 in comparison with the hybrid Logo (Fig. 3) confirm the better resistance of the hybrid SR08, as shown previously [16]. Basically, the independent component analysis (ICA) of the metabolites in leaves shows that treatment with NaCl up to 25 mM induces no major metabolite differentiation. All these metabolites can be centered in one cluster. For higher salt-stress levels (50–100 mM) both hybrids react differently in comparison with each other. Therefore, the ICA shows different clusters for SR08 and Logo (Fig. 5). We can deduce that salt stress particularly

Fig. 7. General reaction classes of single metabolites for both hybrids with increasing salt stress. Logo, salt-sensitive; SR08, salt-resistant maize hybrid. (a) Class I: set of metabolites which reacts to salt stress but not with genotypic difference. (for metabolite examples see Section 3). (b) This set of metabolites reacts no genotypic differences; class II: reaction on salt stress with differences in the salt sensitive and the salt resistance genotype; class III: no reaction on salt stress but different metabolites concentrations in the genotypes.

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Fig. 8. Physiological pathway of the TCA cycle with changes of relevant metabolites in shoots of sensitive and resistant maize hybrids Logo and SR08. Plants were treated with 0 (control), 25, 50, 75, 100 mM NaCl respectively for 7d; see Fig. 1. Filled dots; hybrid Logo (salt sensitive), empty dots; hybrid SR08 (salt resistant). The error bars indicate the standard errors of the mean between biological replicates (n = 4). 100% represents the control (0 mM NaCl) of Logo.

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interest, because the above-mentioned peak of the metabolites at 50 mM only appears in SR08. This suggests an adaptive biochemical reaction to osmotic changes or as a result of physiological reactions to an increasing sodium uptake into the vacuole or even into the cytoplasm. The physiological reaction to an increased sodium uptake by, for example, the Na+ /H+ antiporter in the tonoplast, which is able to include sodium in vacuoles, has previously been reported [22,23]. A sodium uptake with a subsequent inclusion into vacuoles is present at 50 mM NaCl concentrations in the nutrient solution (Figs. 4 and 9). If the salt stress increases to a greater level, the vacuoles of these cells are no longer able to harbor the uptaken sodium, and therefore, sodium breaks through into the cytoplasm. This causes damage to the cell, with the known stress symptoms such as an upregulation of osmolytica. The metabolite profile of some monosaccharides shows this phenomenon (Fig. 9). At higher salt concentrations, the plants are not able to maintain the provision of high sugar concentrations, and therefore, the concentration drops to a lower level. 4.2. Metabolites of roots and shoots are differentially affected by salt stress Some metabolite reactions to salt stress are reported to occur in the roots [12–14], even if these changes in roots are regularly lower than in shoots. Salt stress has been formerly reported to affect mainly shoot growth [16,24]. Our metabolite profiling approach

with two hybrids differing in salt resistance and at five different salt-stress levels has enabled us to distinguish between control plants and salt-stressed plants with regard to their leaves and roots (Fig. 5). The two analyzed hybrids showed a growth reduction primarily in their shoots but not in their roots [16]. Therefore, the main metabolite reaction is also expected in the leaves. These results provide strong evidence that the physiological and biochemical metabolic reactions take place mostly in the shoot. This is a surprise, inasmuch as the NaCl is supplied directly to the roots, which are directly in contact with the toxic sodium ions. This finding is consistent with the analysis of the root metabolite profile (Fig. 6), even down to the level of single metabolites (data not shown). Different authors have detected some metabolites that change in roots after application of salt stress in poaceae. Wu et al. [13] found by an application of severe salt stress (300 mM NaCl) over a long period of three weeks a decrease of the metabolites of different metabolic pathways including glycolysis, TCA cycle, amino acids synthesis, and sugar- and polyols pathways in barley. But Widodo et al. [14] demonstrated in a comparative study of a salt-sensitive and a salt-resistant barley genotype by comparing severe salt stress (100 mM NaCl) to control conditions, that metabolite changes are more distinct in leaves than in roots and become visible at a longer stress period of three weeks. It can be assumed that roots are first affected at high and maybe toxic salt stress conditions or of a prolonged salt stress period. Besides the later toxic effects, the occurrence of root metabolites might be

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Fig. 9. Physiological scheme of the raffinose pathway including the concentration changes of the relevant metabolites in the shoot of the two maize hybrids Logo and SR08 with increasing salt stress levels. Filled dots; hybrid Logo (salt-sensitive), empty dots; hybrid SR08 (salt-resistant). Plants were treated with 0 (control), 25, 50, 75, 100 mM NaCl respectively for 7d; see Fig. 1. The error bars indicate the standard errors of the mean between biological replicates (n = 4). 100% represents the control value (0 mM NaCl) of Logo. Blue arrows show raffinose pathway with changes of relevant metabolites in shoots of two maize hybrids Logo and SR08. A “+” combines substrates forming a possible metabolic product. (For interpretation of the references to color in figure legend, the reader is referred to the web version of the article.)

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a result from a translocation of these metabolites from leaves to roots. This assumption is supported through the fact that there are only a few metabolites changed which can be found exclusively in roots and not in leaves ([12–14], Fig. 4). The salt-sensitive species maize may show these effects earlier or at low salt concentrations. Gavaghan et al. [12] showed under comparable conditions to this experiment an increase of alanine, ␥-amino-N-butyric acid, malic acid, succinate and sucrose and a decrease of acetoacetate and glucose. If there is a metabolite such as is ␥-amino-N-butyric acid only present in root it could be concluded that no translocation effect occurs to the leaves. Another explanation for the change of metabolites in roots in different investigations could be due to harsh stress conditions which caused physiological toxicity effects which may not directly correspond to salt stress resistance mechanisms. Only three exceptions of metabolites that react to the salt-stress gradient in roots have been found in this investigation (supplemental Table 1, supplemental Fig. 1). Nevertheless, the three compounds 2,4-dihydroxy-butanoic acid, cellulose, and a not yet identified metabolite show distinct genotypic differences between the contrasting hybrids; these could be taken into account. The metabolite 2,4-dihydroxy-butanoic acid has a much higher concentration in SR08 than in Logo; this might be attributable to the salt resistance of SR08. In contrast, cellobiose has a higher concentration in the salt-sensitive hybrid Logo. Taken together with ambiguous findings in the literature, we conclude that the metabolite changes in roots of maize do not contribute greatly to salt-resistance mechanisms because the maize hybrids try to establish a metabolic homeostasis within the root under increasing salt concentrations.

4.3. Effects on metabolites in shoots mainly affects the TCA cycle and sugar metabolism A central result of this investigation is a consistent reduction in the concentrations of the metabolites of the TCA cycle with increasing salt stress in both hybrids. A reduction of some of these metabolites has formerly been reported to occur during salt stress [12–14]. The reduced carbon catabolism might be one reason for the accumulation of compatible solutes acting as osmoprotectants [25]. As a result of the reduced metabolites of the TCA cycle, less energy in the form of reduction equivalents such as NADH, FADH2 , and ATP is available for plant growth. A limiting situation is unfavorable for a crop plant under saline conditions, apart from its being sensitive or resistant. Obviously, the salt-resistant hybrid is much better able to grow with limiting energy conditions in comparison with the salt-sensitive hybrid Logo. An eye-catching difference between the two hybrids appears in the concentration change of the soluble sugars. An increase of soluble sugars is reported for several salt-resistant genotypes of original glycophytes such as rice [26–28] wheat [29], and poplar [30]. Several reasons are assumed for the increase of soluble sugars during salt stress. The mono- and disaccharides could have an osmolytic effect together with other compatible solutes [31], contribute to the stabilization of membrane structures [32], and are involved in stress signaling while regulating genes concerned with salt-resistance mechanisms [33]. These soluble sugars increase with salinity in the salt-sensitive hybrid Logo (Fig. 9). Moreover, a transient peak is significant at the 50 mM NaCl level for SR08 as

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discussed above. On the one hand, a higher concentration of the sugars clearly enhances the resistance to salt stress in the hybrid SR08. On the other hand, this system might be efficient, especially at a certain NaCl-level of up to 50 mM NaCl when sodium is taken up into the cytoplasm. Therefore, we conclude that a breeding program is the more effective method in salt-sensitive plants such as maize, if the testing concentration does not exceed a level of 50–75 mM NaCl. This salinity level of 50 mM NaCl corresponds to a soil salinity conductance of 4 dS m−1 [6]. The peak behavior of osmolytical sugars might have been overlooked in investigations with high salt-stress levels. The resistance mechanisms would have been overlooked in these cases. For the salt-resistant hybrid used in this study, sodium exclusion and inclusion mechanisms (with regard to cytoplasm) are effective in SR08 [16]. In addition to its ion uptake, SR08 shows a lower growth reduction, even on higher salt-stress levels, in comparison with recent maize varieties. We add herewith a valuable piece to overall picture of the metabolic adjustment of salt resistant maize. Myo-inositol is connected to sugar metabolism by galactinol synthase (GolS), which generates galactinol from the substrates UDP-galactose and myo-inositol. Both myo-inositol and galactinol belong to the polyols that act as compatible solutes. Inositol takes a central position in cellular metabolism, but its meaning in response to abiotic stress begins with its derivates [34]. The enzyme ␣-galactosidase (␣-GAL) catalyzes the reaction between galactinol and sucrose and forms the trisaccharide raffinose. Raffinose belongs to the raffinose family with further derivates such as stachyose and verbascose. The oligosaccharides of the raffinose family are reported to be enriched during salt stress and act therein as osmoprotectants [35,36] and antioxidants [37] and might even function as signals in response to stress [34]. Raffinose, myo-inositol and galactinol, which are all connected to the raffinose pathway, increase linearly with increasing salt stress (Fig. 9). We assume that the plants increase these compatible solutes for coping with salt stress. In plants, myo-inositol, galactinol, and raffinose have been shown to be important for the improvement of stress resistance in plants [38–41]. Galactinol might improve the salt resistance of SR08, even at higher salt-stress levels, because this metabolite accumulates more strongly in SR08 than in Logo with increasing salt stress. A resistance effect of myo-inositol and raffinose can be ruled out, because no differences between SR08 and Logo occurred. Moreover, the raffinose precursors myo-inositol and sucrose are supposed to regulate raffinose accumulation [42]. Although the transient peak behavior of the mono- and disaccharides is not visible in the linear increase of galactinol and raffinose, this demonstrates the prime importance of myo-inositol as a key regulator in comparison with sucrose for the raffinose pathway.

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In this salt-stress experiment, maize was stepwise adapted to ion stress and treated with slight different NaCl levels. The subsequent GC/TOF-MS approach enabled us to draw certain conclusions: (i) The TCA cycle and sugar metabolism are the main affected biochemical pathways in maize leaves during salt stress; (ii) root metabolism is not affected by the long-term salt stress and is therefore negligible for similarly motivated analyses; (iii) the application of small salt-stress increments of 25 mM NaCl offer new insights into resistance mechanisms, which would otherwise remain hidden. Uncited references [43–45].

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We are grateful to Prof. Sven Schubert for providing the SR08 hybrids. Limagrain, Edemissen, Germany, is acknowledged for providing seeds of maize cv. Logo. The work was supported by a research grant (ZO118/6) of the Deutsche Forschungsgemeinschaft Q5 (DFG) which is gratefully acknowledged. Appendix A. Supplementary data

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