Drought-Induced Responses of Physiology ... - ACS Publications

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Drought-Induced Responses of Physiology, Metabolites, and PR Proteins in Triticum aestivum Zuzana Gregorová,† Jozef Kovácǐ k,*,‡,§ Bořivoj Klejdus,‡,§ Marína Maglovski,# Roman Kuna,† Pavol Hauptvogel,⊥ and Ildikó Matušíková# †

Faculty of Natural Sciences, Department of Botany and Genetics, Constantine the Philosopher University, Nábrežie mládeže 91, 949 74 Nitra, Slovak Republic ‡ Institute of Chemistry and Biochemistry, Faculty of Agronomy and §CEITEC − Central European Institute of Technology, Mendel University in Brno, Zemědělská 1, 613 00 Brno, Czech Republic # Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademická 2, P.O. Box 39A, 950 07 Nitra, Slovak Republic ⊥ National Agricultural and Food Centre − Research Institute of Plant Production, Bratislavská cesta 122, 921 68 Piešt’any, Slovak Republic S Supporting Information *

ABSTRACT: The impact of severe drought stress (13% soil moisture) on the physiological responses, metabolic profile, and pathogenesis-related (PR) proteins in wheat above- and below-ground biomass after 20 days of treatment was studied. Drought depleted growth, assimilation pigments, and majority of free amino acids in the shoots (but proline increased considerably, +160%). On the contrary, root growth parameters were elevated, and free amino acids did not decrease, indicating investment of metabolites into the growth of roots under water deficiency. Mineral nutrients were only slightly influenced. Profiling of pathogenesis-related (PR) proteins revealed that chitinases (EC 3.2.1.14) and glucanases (EC 3.2.1.39) were activated in wheat by drought. Individual isoforms and their activity were rather stimulated under drought, especially in shoots. The expression of selected genes is in agreement with enzymatic data and suggests an organ (tissue) specific- and opposing behavior of these two types of defense components in drought-stressed wheat. Metabolic analyses at the level of phenolics showed an increase in the free and bound fraction of phenolic acids almost exclusively in the shoots and flavonoid isoorientin increased considerably: protective action against oxidative stress and dehydration of the leaves seems to be the main reason for this finding. The role of PR proteins and phenolics in drought-stressed tissue is discussed. KEYWORDS: abiotic stress, antioxidants, enzymes, metabolome, soil limitation

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INTRODUCTION Drought is the major abiotic stress factor limiting crop productivity worldwide. Triticum species are important human food sources, accounting for more than half of total consumption.1 The capability to withstand limited water availability requires a complex combination of different genetic-, physiological-, and genome-based defense strategies to maintain functional integrity of cells and the whole organism.1 Disturbance of water balance evokes metabolic changes and accumulation of metabolites such as proline,2 simple low molecular weight antioxidants (ascorbic acid, glutathione, phenolics), and activation of detoxifying enzymes.3,4 However, a more detailed metabolic profile in drought-stressed plants including aboveand below-ground organs is not available. Glucanases (EC 3.2.1.39) and chitinases (EC 3.2.1.14) are hydrolases belonging to so-called pathogenesis-related (PR) proteins (group 2 and 3 of PR family, respectively). Research in model plants as well as in transgenic crops has confirmed that excessive accumulation of these proteins can strengthen the resistance to viruses and bacteria as well as fungi.5 Moreover, chitinase and glucanase induction can be triggered upon different abiotic environmental cues.6−8 Under water deficit, © XXXX American Chemical Society

chitinases have been observed to act in several plant species including Arabidopsis,9 but no deeper data are available in both above- and below-ground tissue. Similarly, glucanases can not only play a pivotal role (either alone or cooperating with chitinases) in defense against pathogens7,10 but also due to capability to decompose callose also in many other types of abiotic stresses including drought.6 The gene family of chitinases comprises up to 44 chitinases and 57 β-(1,3)glucanases in diploid rice11 (Oryza sativa; 2n = 24; haploid genome size of ∼0.4 Gb), though with increasing power of available bioinformatic methods these numbers are likely not definite. In bread wheat with a hexaploid genome (2n = 42; haploid genome size of ∼17 Gb), the profile of chitinases and glucanases is poorly known. Among the chromosome-based draft sequence data of the wheat genome (IWGSC)12 currently there are up to 184 sequences annotated as chitinases and 271 sequences as glucan endo-1,3-β-glucosidase(-like) proteins. On the other hand, the wheat proteome in the UniProt database Received: June 15, 2015 Revised: August 27, 2015 Accepted: September 2, 2015

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DOI: 10.1021/acs.jafc.5b02951 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry contains only a single chitinase and up to 17 endoglucanase entries. Despite the intensive research, the study focused on the drought still needs attention.13,14 We therefore analyzed growth and physiological responses (biomass formation, photosynthetic pigments, mineral nutrients), metabolite profile (amino acids, phenolics), and the isoforms/activity/gene expression of glucanases and chitinases in a new Slovak wheat line after prolonged drought stress.

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MATERIAL AND METHODS

Plant Material, Cultivation, and Experimental Design. Wheat (Triticum aestivum) plants, line SK-196 that is currently under varietal testing in Slovakia, were sown in plastic pots (32.8 cm diameter and 25.8 cm depth) with commercial substrate BORA (BORA s.r.o., Slovakia) (measured pH of 7.4, 0.17% total N, 25 ppm P and 2.1 ppm Zn). Experiment in randomized complete block design with three replications, 6 pots per condition, was conducted at Faculty of Natural Sciences, University of Constantine the Philosopher in Nitra, Slovakia. A total of 20 seeds were planted per pot. One week after emergence, seedlings were thinned to ten per pot. The plants were grown in a temperature-controlled chamber at 18 °C between 6 p.m. and 6 a.m. and 22 °C between 6 a.m. and 6 p.m., relative humidity of 60% with a 16/8 h photoperiod with light intensity ranging from 300 to 400 μE/ m2/s. When tillering began, the plants were vernalized at 5 °C for 3 weeks in dim light. The pots were regularly irrigated twice a week with 750 mL of distilled water and once in 2 weeks fertilized using standard liquid fertilizer with micronutrients (Akrokompakt NPK 15-10-10, Agrona, Slovakia). The plants were treated preventively with insecticide Karate Zeon 5 CS (in May 2013). Water stress was applied at stage 5 on Feekes growth scale (leaf sheaths strongly erect15) by limiting the water supply to 250 mL of water each 48 h, while the control plants were kept well watered. Volumetric soil moisture content was measured for each pot at three different positions using a Theta Probe ML2 (Delta-T Device Ltd.) connected with an Infield 7 Data Logger (UMS GmbH München). The conductivity values measured for stressed plants dropped up to 13% (Figure 1A). Samples were taken 20 days after the start of the experiment. Roots were washed, dried using paper towels, and frozen immediately in liquid nitrogen. Samples were stored at −80 °C until analysis. Assay of Basic Physiology, Lipid Peroxidation, and Mineral Nutrients. Content of assimilation pigments (chlorophylls and total carotenoids) was determined according to Lichtenthaler and Wellburn16 on 8−10 leaves that were taken from 5 plants per replicate per treatment. The amount of reducing sugars was determined using the Anthron method.17 The level of malondialdehyde (MDA) was estimated using the thiobarbituric acid assay.18 For the quantification of mineral nutrients, dry samples were mineralized in the mixture of concentrated HNO3 and water (3 + 3 mL) using microwave decomposition (Ethos Sel Microwave Extraction Labstation, Milestone Inc.) at 200 °C over 1 h. Resulting clear solution was quantitatively placed in glass flasks and diluted to a final volume of 20 mL. All measurements were carried out using an atomic absorption spectrometer AA30 (Varian Ltd.; Mulgrave, Australia) and the airacetylene flame.19 Assay of Chitinase and Glucanase Proteins. Total proteins were extracted from roots and leaves.20 Aliquots (20 μg)21 were separated in 12.5% polyacrylamide gels with 0.01% (w/v) glycol chitin as an enzyme substrate. Profiles of total chitinases as well as profiles of acidic/neutral and basic/neutral chitinase isoforms after separation were stained for enzyme activity with 0.01% (w/v) Fluorescent Brightener 28 (Sigma) and subsequent illumination under UV light as described previously.8 The gels were photographed (BioDoc-It 210 Imaging System, UVP, California, USA) and stained with Coomassie Brilliant Blue R250. The sizes of individual chitinase isoforms were determined based on a co-separated molecular standard (Page Ruler Plus Prestained Protein Ladder, Thermo Scientific) by measuring migration distances of protein bands. Gel images were processed using

Figure 1. (A) Soil moisture content of the substrate in the pots of control plants (empty symbols) and drought-stressed plants (filled symbols) measured in the period of 20 days (axis x). Three replicates are represented by different symbols, while each symbol indicates average value taken at 3 distinct positions in a pot (respectively). (B) Phenotype of experimental wheat plants watered regularly (controls) and after drought stress (drought) applied by withholding water for the period of 20 days. Lengths of roots were significantly affected; arrows indicate 20 cm on the ruler. Scion Image software (http://www.scioncorp.com). Backgroundcorrected integrated density (ID) of chitinase bands was calculated in areas of constant size: ID = N*(mean-background) where N is the number of pixels in the selected area and background is the modal gray value (pixels). Total chitinase activity in samples was measured fluorimetrically using 4-methylumbelliferyl-β-D-N,N′,N″-triacetylchitotrioside [4-MU(GlcNAc)3] as substrate, as described previously.22 For the analyses of glucanase enzymes, protein samples were separated in 12.5% polyacrylamide gels containing 0.01% (w/v) laminarin (Sigma), as described above. Activities of individual isoforms in profiles of all active glucanases in samples were detected by boiling of the gels for 10 min in 1 M NaOH containing 0.1% (w/v) 2,3,5triphenyltetrazolium chloride.8 The sizes of individual isoforms were determined directly based on a coseparated molecular standard (Page Ruler Plus Prestained Protein Ladder, Thermo Scientific). The gels were scanned; intensity of the bands was taken as a measure of the glucanase activity and determined as described above. The activity of total β-(1,3)-glucanases in protein samples was measured with DNS method by measuring total reducing sugar content.23 Analyses of Gene Expression by qRT-PCR. Total RNA was isolated using the RNease Plant Mini Kit (Quiagen), quantified spectrophotometrically (BioSpec-nano, Shimadzu Biotech), and stored at −80 °C until use. cDNA was made from 1 μg RNA using the Maxima H Minus First Strand cDNA Synthesis Kit and OligodT primers (Thermo Scientific) as per manufacturer’s instructions and stored at −20 °C until use. Among the wheat chitinase (6) and glucanase (7) genes available in the NCBI gene bank (to the July 2013), PCR conditions were optimized for those listed in Table S1. B


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Journal of Agricultural and Food Chemistry

Figure 2. Growth and physiological parameters in wheat control (watered) plants (white columns) or exposed to drought (black columns) over 20 days. Data are means ± SEM (n = 10 for dry mass and length, n = 5 for the content of total reducing sugars, n = 4 for the content of malondialdehyde). Assimilation pigments were quantified in the apical parts of leaves (chlorophyll a (Chl a), chlorophyll b (Chl b), total chlorophyll (Chl a + Chl b = ChlTot), content of total carotenoids (Car), ratio of chlorophylls Chl a/b, and ratio of total Chl to Car content, n = 7). The significance of difference between control and drought-stressed plants is indicated with * at p < 0.05, ** at p < 0.01 and *** at p < 0.001.

Table 1. Accumulation of Free Amino Acids (μmol g−1 DW) in Wheat Control (Watered) Plants or Exposed to Drought Over 20 Daysa shoots control aspartic acid glutamic acid serine histidine glycine threonine arginine alanine tyrosine cysteine valine methionine phenylalanine isoleucine leucine lysine proline sum

7.30 5.79 33.8 24.6 1.51 14.7 4.92 26.3 7.38 13.4 28.5 0.24 13.3 16.6 12.3 0.78 20.1 232.7

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.79 0.62 1.02 1.46 0.076 0.80 0.37 1.02 1.33 0.95 1.55 0.019 0.64 1.37 0.97 0.081 3.14 8.65

roots drought 8.96 4.40 12.3 14.8 1.66 6.30 3.51 14.9 2.80 5.48 11.9 0.19 6.11 6.23 3.36 0.61 52.3 156.0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

control

1.07 0.13* 1.47*** 1.77** 0.12 1.24*** 0.33** 0.76*** 0.30** 0.57*** 0.65*** 0.023 1.18*** 1.22*** 0.30*** 0.12 6.49** 7.94***

7.01 0.74 3.06 2.68 0.51 1.30 1.07 2.78 0.22 0.66 1.50 0.065 0.29 0.89 0.75 0.18 1.30 25.0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.05 0.063 0.17 0.20 0.034 0.15 0.12 0.077 0.013 0.14 0.087 0.013 0.013 0.050 0.19 0.015 0.10 1.93

drought 6.53 0.85 2.80 4.06 0.56 1.23 1.37 2.52 0.34 0.53 1.55 0.073 0.48 0.78 0.69 0.22 1.24 25.8

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.79 0.13 0.21 0.30** 0.040 0.17 0.11 0.20 0.050* 0.087 0.19 0.008 0.060** 0.086 0.17 0.031 0.19 1.05

Data are means ± SEMs (n = 4). The significance of difference between control and drought-stressed plants is indicated with * at p < 0.05, ** at p < 0.01, and *** at p < 0.001. a

started with initial incubation at 50 °C for 2 min, followed by denaturation at 95 °C for 10 min. Forty-five cycles of amplification were performed using a thermal cycling profile of 95 °C for 10 s and 60 °C for 30 s. Subsequently, a melting curve was recorded by holding at 95 °C for 10 s, cooling to 60 °C, and then heating at 0.1 °C/s up to 95 °C. The amplification and melting curve data were collected and analyzed using the LightCycler Nano software 1.0. Quantification of Metabolites. Soluble phenols were extracted with 80% methanol and quantified using Folin-Ciocalteu method with

Primers were from the literature or were designed using IDTPrimerQuest Input (http://eu.idtdna.com/PrimerQuest/Home/ Index). Actin (GenBank accession AB181991) and β-tubulin (U7689524) were used as internal standards. Reaction mixtures contained 5 μL of 2X SYBR Green PCR Master Mix (Life Technologies), 0.3 μL of 10 μM of both forward and reverse gene specific primers, nuclease-free water, and 1 μL of 1:3 diluted cDNA. The qPCR was carried out in LightCycler Nano (Roche) real-time PCR system in duplicate in 8 well PCR strips. The PCR program C


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Journal of Agricultural and Food Chemistry gallic acid as standard with detection at 750 nm.18 In these extracts, selected cinnamic and benzoic acid derivatives were also measured by liquid chromatography with mass spectrometry detection (LC-MS/ MS) using device Agilent 1200 Series Rapid Resolution LC system coupled online to Agilent 6460 Triple quadrupole detector with Agilent Jet Stream Technologies: compounds were identified based on the specific m/z values and retention time and quantified using commercially available standards.25 Flavonoid glycoside isoorientin was also quantified.26 Thereafter, pellet was 5 times resuspended in 80% methanol followed by centrifugation to remove soluble phenols, dried, and dissolved in 2 M NaOH to release cell wall-bound phenols as reported earlier.19 Quantifications of the sum of phenols with the Folin-Ciocalteu method and individual metabolites with LC-MS/MS were the same as above. Free amino acids were extracted with 80% aqueous ethanol and analyses were performed on an HP 1100 liquid chromatograph (Hewlett-Packard, Waldbronn, Germany) with fluorometric detector FLD HP 1100 and using precolumn derivatization with ophtalaldehyde and 9-fluorenylmethyl chloroformate.25 Statistical Analyses. Differences between control and droughtstressed plants were evaluated by Student’s t-test (Microsoft Excel 2003); homogeneity of variance was checked by Levene’s test. Number of repetitions in table/figure legends indicates individual plants analyzed for the given parameter.

Mineral nutrients in drought-stressed plants have not been extensively studied yet. Analyses of changes at the level of various macro- and micronutrients showed significant depletion of Ca in the shoots and Na in the roots only (Figure 3). It is evident that despite severe soil water deficiency, tissues retain mineral nutrients effectively.

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RESULTS AND DISCUSSION Drought Affected Wheat Growth and Physiology More Negatively in the Shoots than Roots. The drought evoked in the experimental soil (Figure 1A) suppressed shoot biomass at the level of length and dry weight (Figure 1B and Figure 2). In contrast, a more developed root system was retained in the stressed wheat plants (Figure 1B) due to wellcontrolled cell expansion for reaching deeper water resources.27 Leaf necroses were observed and the amount of chlorophylls decreased (Figure 2) as observed by others.28 The relatively low levels of MDA appeared unaffected by water shortage (Figure 2), contrasting to earlier study.29 Previously, wheat genotypes with lower MDA content (such as our Slovak wheat line) have been indicated as more tolerant to drought,30 and it seems that our present wheat line is also tolerant considering duration and intensity of the water deficiency. The profile of free amino acids revealed a decrease in the majority of detected compounds in the shoot biomass, while the amount of proline increased considerably in response to drought (+160%, Table 1). This is in line with the abovementioned decrease in assimilation pigments and overall growth. On the other hand, elevated proline accumulation may serve as a protective mechanism aimed at maintaining protein structures and ROS balance. A similar increase in proline has been found in four wheat cultivars under soil drought stress,29 while application of PEG did not affect it significantly.31 At the root level, only negligible changes of individual amino acids were observed (significant increase of three compounds, Table 1). The amount of proline was not affected which contradicts data from PEG-treated wheat.31 Based on the significant increase in root dry biomass (Figure 2) we assume shoot-to-root amino acids translocation aimed at maintaining root growth allowing “search” for water in the soil. On the contrary to previous study with barley exposed to nitrogen deficiency,26 not only root length but also root dry biomass increased in the present study (Figure 2), indicating investment of metabolites into the growth. Further research focused on phloem exudates is needed to verify this aspect and its regulation by drought.

Figure 3. Quantitative changes of selected mineral nutrients in wheat control (watered) plants (white columns) or exposed to drought (black columns) over 20 days. Data are means ± SEM (n = 4). Explanation of statistics is the same as in Figure 2.

Drought Variably Influenced PR Protein Profile. Only a few studies reported the activity of chitinases under abiotic stress.7,8 Our experiment revealed the presence of at least 7 active chitinase isoforms in wheat (5 acidic and 2 basic ones) in shoots and 10 (6 acidic and 4 basic) isoforms in roots (Figure 4). Previously, up to six different chitinase activity peaks were separated by polyacrylamide gel electrophoresis in wheat plants.32 In our Slovak wheat line, drought conditions induced chitinases mainly in roots, mainly at the level of acidic isoforms of ∼40 kDa (isoforms D and F, Figure 4A). In shoots, small (but significant) induction of a single basic isoform “b” was not reflected into a change of total enzyme activity (Figure 5). No chitinase was newly induced by water shortage in either shoots or roots. These data are in agreement with transcript levels that were elevated for 3 of 4 tested chitinase genes in roots but mostly repressed in shoots (except for a single gene CD490414) (Figure 7). In the literature, chitinases are rarely explicitly mentioned in high-throughput drought research. An acidic chitinase has been identified as drought responsive in a proteomic study.33 Our observations fit well with transcriptomic data of Ergen et al.,14 who identified 18 chitinase gene representatives differentially expressed in wild emmer D


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Comparing the Defense Strategy of Tissues/Organs. The activity of glucanases and chitinases differed in shoot and root tissue (Figures 5 and 6). Furthermore, their often synergistic action described during biotic stress10 does not seem to be applicable under drought. Roots responded by an activation of chitinases at both enzyme- and gene activity levels and by repression of two glucanase isoforms (Figures 4A and 5). In shoots the glucanase enzyme isoforms were significantly induced, while the examined genes behaved adversely (Figure 4B). Here we note that data can depend not only on organ type or experimental conditions but also might be influenced by the presence of highly similar homologues in the allohexaploid wheat. Genome-specific primers would be very helpful for exploring the gene families of PRs in wheat. At the same time, PR proteins can be constitutively expressed in tissue at very low levels;10 therefore, their research might be hampered by detection limit of the approach applied, too. The opposing expression of studied drought-responsive genes in the two organ types might imply specific role of chitinases and glucanases in drought adaptation. It is proposed that chitinases alter the flexibility-elasticity of the cell wall38 enabling it to withstand the shrinking of cells during dehydration. They also may reduce the risk of plant infection by pathogenic microbes naturally persisting in the soil. On the contrary, the action of glycoside-hydrolyzing, cell wall polysaccharide modifying enzymes like glucanases is suggested to affect accumulation of soluble sugars under water stress.38 Wheat genotypes with higher concentrations of small organic osmolytes (including sugars released by glucanases) revealed better tolerance to drought.30 Besides, accumulated PRs can serve as storage proteins under conditions of growth limitations such as reduction of photoperiod, low temperatures, or nutritional/water deficiency.6,39 Intriguingly, altered activities of defense proteins in droughtstressed plants are supposed to be associated with overlapping defense responses to water limitation and pathogen attack.40 Correlation between the isoform banding pattern and activity of chitinases and the genetic resistance of plants to biotic32 or abiotic8 stress has been suggested but is not clearly established. Furthermore, kinetics and amplitude of their action has been suggested to contribute to overall susceptibility/resistance of plants.8 However, though PRs can help plants to endure and recover from mild- or short-term stress, at terminal drought their accumulation can have negative influence on growth,6 possibly due to additional costs for their synthesis in damaged plants. Responses of Phenolics to Drought Differed in Shoots and Roots. Phenolic metabolites are abundant plant compounds induced by various stress stimuli.19,26 In our study, total phenols (Folin-Ciocalteu assay) were affected only in the shoots in the free fraction (Table 2) that is in accordance e.g. with no or slight changes reported in various barley cultivars:35 we note a strong quantitative discrepancy with our values (10 mg/g DW = ∼ 1 mg/g FW) which is physically impossible (ca. 500 mg/g FW, cf. ref 35). In rice grains, prolonged drought even depleted the amount of total phenols,41 indicating tissue and/or species-specific differences. Surprisingly, detailed quantification of individual metabolites under drought has only rarely been reported.42−44 Infusion prepared from peppermint grown at various levels of soil moisture revealed that strong drought (12%) depleted phenolics more than moderate stress.43 This could be a reason why we observed a relatively slight increase in phenolic acids

Figure 4. Representative photographs of in-gel detection of the activities of the studied PR proteins in wheat plants grown under normal (control) or drought conditions. Protein extracts from roots and shoots were separated in SDS-PAGE and subsequently renatured for detection of total chitinases (A) and glucanases (B) enzymes profiles (size of isoforms in kDa). Chitinases were also separated for more detailed analysis of acidic- (upper case letters) as well as basic (lower case letters) isoforms, respectively (A). Detected isoforms are indicated with arrows. Size of individual glucanase isoforms was determined by coseparation of a molecular weight standard (Page Ruler Plus Prestained Protein Ladder, Thermo Scientific). Molecular sizes of chitinase isoforms were determined based on Coomassiestained protein profiles.

wheat upon quick dehydration, mostly in roots. However, the published transcriptomic data on chitinase activity under drought are inconsistent: induction34,35 or unaffected expression36 are known. Regulation of individual chitinase genes may depend on genotype, tissue type, and time point.13,14 Enhanced chitinase activity has been suggested to coincide with drought tolerance of durum wheat13 and Tibetan wild barley genotypes.35 Similarly to chitinases, the profile and activity of glucanases in wheat, especially under abiotic stress, is poorly known.7,35 Our analyses identified up to 4 different enzyme fractions in wheat tissue. In shoots, each isoform fraction (∼30, 45, 68, and 150 kDa) showed an enhanced accumulation by drought (Figure 4B) that resulted in increased overall glucanase activity (Figure 6). Such induction by drought has been reported previously.14 Only one of the 3 glucanase genes we studied was activated in shoots by drought (AF112967), the other was repressed (DQ090946), while the third was unaffected (Figure 7). In contrast, in root samples the overall activities of glucanases did not alter markedly (Figure 6), possibly due to differential effect of drought on individual isoforms (Figures 4B and 6) as reported previously.14,37 Suppression of the activity of some of the glucanase isoforms has been associated with abscisic acid-mediated callose deposition leading to increased water holding capacity.35 E


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Figure 5. Quantification of the activities of individual chitinase isoforms (total, acidic and basic) in protein extracts from roots and shoots of wheat plants grown under normal and drought conditions. Values are expressed as average raw integrated densities of pixels (ID) ± SE for n ≥ 3. Total chitinase enzyme activity of samples measured fluorimetrically is also given. Explanation of statistics is the same as in Figure 2 (n.q. − isoform was nonquantifiable due to high background).

Figure 6. Quantification of activities of individual glucanase isoforms in extracts from roots and shoots of wheat plants grown under normal and drought conditions. Values are expressed as average raw integrated densities of pixels (ID) ± SE for n ≥ 3. Enzyme activity values in samples measured with DNS method are also shown. Explanation of statistics is the same as in Figure 2 (n.q. − isoform that was nonquantifiable due to high background).

phosphate pathway forming not only NADPH but also precursors for phenolics such as erythrose 4-phosphate.45 This fact was even more visible at the level of isoorientin, a flavonoid glycoside from the luteolin family, which increased 3 times in the shoots but not in the roots (Table 2). Unlike free phenolic acids serving as a storage pool (for more complex compounds) or as antioxidants, cell wall-bound

(Table 2) at an ca. 15% soil moisture (Figure 1A). However, stimulation of acids was clearly significant at the level of sum (+44%) and individual metabolites in the shoots but only occasional in the roots. These data fit well with the increase in proline and support, at least partially, a metabolic connection between proline and phenolics: biosynthesis of proline depletes the amount of NADPH, leading to stimulation of pentose F


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growth and biomass that was also visible at the level of amino acids changes and shoot-to-root translocation is expected. At the same time, mineral nutrients were not affected extensively. Chitinases and glucanases were activated in wheat under drought. Directly as components of defense mechanisms and/ or in morphophysiological adjustments of cells during water shortage they contribute to plant capability to survive. Though individual glucanases and chitinases were identified here as drought-responsive, their numbers even under slightly different conditions, another growth stage, and/or using other approaches is likely much larger and requires further research at both genome and proteome levels. Detailed quantification of phenolic metabolites revealed their increase in the free and bound fraction almost exclusively in the shoots, indicating protective action under water deficiency.

Figure 7. Relative gene expression data by means of the fold change values on a vertical scale for the set of studied PR genes under drought conditions in roots (black columns) and shoots (gray columns). Significant changes (p ≤ 0.05) are indicated with asterisks. Sequences of primers are shown in Supplementary Table S1.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b02951. Supplementary Table S1 (PDF)

compounds contribute to fortification by forming bridges with carbohydrates.4 In agreement with data from drought-stressed triticale,4 cell wall-bound acids (revealed by alkaline hydrolysis) were quantitatively typically more abundant than respective free counterparts (Table 2). This was mainly visible at the level of lignin precursors including ferulic, p-coumaric, and sinapic acids. Overall, our data clearly confirm that synthesis of free and wall-bound phenols in the above-ground biomass of wheat may contribute to protection of photosynthetic organs against oxidative stress and dehydration of the leaves. In conclusions, prolonged soil drought stress depleted shoot growth and assimilation pigments in wheat, but stimulated root

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AUTHOR INFORMATION

Corresponding Author

*Phone: 420 545 133281. Fax: 420 545 212044. E-mail: [email protected]. Corresponding author address: Institute of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemědělská 1, 613 00 Brno, Czech Republic.

Table 2. Accumulation of Phenolic Acids (μg g−1 DW), Flavonoid Isoorientin (μg g−1 DW), and Total Phenols (Assay by FolinCiocalteu, mg g−1 DW) in Wheat Control (Watered) Plants or Exposed to Drought over 20 Daysa shoots f ree compounds gallic acid p-hydroxybenzoic acid p-hydroxybenzoic aldehyde protocatechuic acid salicylic acid syringic acid vanillic acid vanillin ferulic acid p-coumaric acid sinapic acid sum of phenolic acids isoorientin total phenols alkaline hydrolysis gallic acid syringic acid salicylic acid ferulic acid p-coumaric acid sinapic acid caffeic acid sum of phenolic acids total phenols

control

roots drought

0.16 5.84 3.49 1.25 4.99 11.2 6.19 4.73 5.26 2.91 1.54 47.5 158.4 10.3

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.025 0.14 0.26 0.10 0.31 0.91 0.50 0.49 0.48 0.37 0.33 1.21 12.6 0.59

0.15 8.48 2.93 1.46 5.14 16.8 13.1 3.85 8.98 6.82 1.37 68.8 477.3 12.0

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.028 0.72** 0.30 0.15 0.63 0.86** 1.81** 0.25* 0.86** 0.53*** 0.19 2.67*** 26.2*** 0.56*

0.13 22.9 2.28 390.0 556.1 5.10 5.91 982.6 2.57

± ± ± ± ± ± ± ± ±

0.011 2.25 0.22 24.3 34.9 0.22 0.37 18.8 0.12

0.14 33.5 2.16 481.4 676.2 9.44 8.96 1211.8 2.63

± ± ± ± ± ± ± ± ±

0.020 2.64** 0.19 31.8* 35.9* 1.03** 0.68** 61.3** 0.11

control

drought

0.15 4.47 10.7 0.55 0.62 0.35 9.64 14.3 2.96 31.3 0.48 75.7 15.1 4.72

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.023 0.22 0.46 0.04 0.027 0.040 0.65 1.90 0.15 2.77 0.034 4.02 1.04 0.28

0.27 4.61 10.0 0.69 0.68 0.31 10.1 13.2 3.66 32.4 0.56 76.2 16.4 5.67

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.019** 0.34 0.51 0.028* 0.021 0.024 0.24 1.55 0.18** 2.20 0.039 1.99 1.85 0.80

0.26 32.5 1.49 395.3 2882.7 26.1 18.3 3356.8 13.0

± ± ± ± ± ± ± ± ±

0.04 2.23 0.03 12.1 151.8 2.85 1.37 137.6 1.12

0.25 26.5 1.26 372.1 2704.1 21.5 20.4 3144.2 12.3

± ± ± ± ± ± ± ± ±

0.03 4.40 0.21 15.0 329.4 2.94 2.09 328.9 0.54

Data are means ± SEMs (n = 4). Free compounds means 80% methanolic extracts and alkaline hydrolysis means cell wall-bound compounds. The significance of difference between control and drought-stressed plants is indicated with * at p < 0.05, ** at p < 0.01, and *** at p < 0.001. a

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Gregorová and Kuna received funding for equipment from European Community under project no 26220220180: Building Research Centre “AgroBioTech”. The work of ́ Maglovski, Hauptvogel, and Matušiková was supported by the Slovak Research and Development Agency under the contract no. APVV-0197-10. Kovácǐ k received funding from OP Education for Competitiveness (European Social Fund and the state budget of the Czech Republic) CZ.1.07/2.3.00/ 30.0017 Postdocs in Biological Sciences at MENDELU. Klejdus and Kovácǐ k received funding for metabolic analyses with research infrastructure supported by the CEITEC − Central European Institute of Technology (project CZ.1.05/1.1.00/ 02.0068 financed from European Regional Development Fund). Sponsors had no involvement in the present study. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Silvia Mihaličová, Ph.D. for proofreading the manuscript.

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