Metabolic shifts associated with drought-induced senescence in Brachypodium

Plant Science 289 (2019) 110278

Contents lists available at ScienceDirect

Plant Science journal homepage: www.elsevier.com/locate/plantsci

Metabolic shifts associated with drought-induced senescence in Brachypodium

T

⁎

Amir H. Ahkamia, , Wenzhi Wangb, Thomas W. Wietsmaa, Tanya Winklera, Iris Langec, ⁎ Christer Janssona, B. Markus Langec, Nate G. McDowellb, a

The Environmental Molecular Sciences Laboratory (EMSL), Pacific Northwest National Laboratory (PNNL), Richland, WA, USA Earth Systems Science Division, Pacific Northwest National Laboratory (PNNL), Richland, WA, USA c Institute of Biological Chemistry and M.J. Murdock Metabolomics Laboratory, Washington State University, Pullman, WA, USA b

A R T I C LE I N FO

A B S T R A C T

Keywords: Drought Senescence Brachypodium Mortality Metabolites Model grasses Abiotic stress

The metabolic underpinnings of plant survival under severe drought-induced senescence conditions are poorly understood. In this study, we assessed the morphological, physiological and metabolic responses to sustained water deficit in Brachypodium distachyon, a model organism for research on temperate grasses. Relative to control plants, fresh biomass, leaf water potential, and chlorophyll levels decreased rapidly in plants grown under drought conditions, demonstrating an early onset of senescence. The leaf C/N ratio and protein content showed an increase in plants subjected to drought stress. The concentrations of several small molecule carbohydrates and amino acid-derived metabolites previously implicated in osmotic protection increased rapidly in plants experiencing water deficit. Malic acid, a low molecular weight organic acid with demonstrated roles in stomatal closure, also increased rapidly as a response to drought treatment. The concentrations of prenyl lipids, such as phytol and α-tocopherol, increased early during the drought treatment but then dropped dramatically. Surprisingly, continued changes in the quantities of metabolites were observed, even in samples harvested from visibly senesced plants. The data presented here provide insights into the processes underlying persistent metabolic activity during sustained water deficit and can aid in identifying mechanisms of drought tolerance in plants.

1. Introduction The effects of droughts on vegetation have increased in recent decades [1], with widespread impacts on crop yields observed [2] and further repercussions predicted [2–5]. While rising atmospheric CO2 levels enhance photosynthesis in most crops, the negative outcomes of droughts appear to outweigh these benefits [3]. The reduced water access for plants grown under drought conditions also inhibits nutrient absorption, which in turn results in compromised photoassimilate distribution and, ultimately, diminished growth [6,7]. Research on climate change and ensuing environmental perturbations indicates that drought effects and associated plant mortality may increase in the future due to warmer temperatures and potential reductions in seasonal rainfall in many regions of the world [8,9]. Thus, understanding the processes and signatures of crop mortality and survival under sustained drought is a growing scientific need. Brachypodium distchyon (L.) P. Beav. (Poaceae; henceforth Brachypodium) is a commonly used C3 model grass for energy and food

⁎

crops with a large number of important attributes, including small stature (≤15 cm at maturity) and a relative short life cycle (˜9-10 weeks); small diploid genome (˜270 Mb); large collections of natural accessions; recombinant inbred lines and mutants; complete genome sequence information; and high phenotypic diversity for agronomic traits such as biomass accumulation [10–14]. Importantly, a Brachypodium pan-genome based on de-novo genome assemblies from 54 diverse genotypes was constructed by the US Department of Energy Joint Genome Institute [15]. Brachypodium has been generally considered as a drought-tolerant grass [16]. It is also a wild, undomesticated grass species, which has not been subjected to significant human selection. Therefore, it represents a very promising system for identifying drought-tolerance mechanisms in grasses that may have been lost during the domestication of important cultured cereal crops like wheat and barley [17]. With the advent of high-throughput omics technologies, several studies of drought stress were conducted in Arabidopsis [18]), rice [19], and maize [20]. Moreover, an integrative analysis of transcriptional, metabolomic, and physiological responses to

Corresponding authors. E-mail addresses: [email protected] (A.H. Ahkami), [email protected] (N.G. McDowell).

https://doi.org/10.1016/j.plantsci.2019.110278 Received 2 June 2019; Received in revised form 5 September 2019; Accepted 15 September 2019 Available online 17 September 2019 0168-9452/ © 2019 Published by Elsevier B.V.

Plant Science 289 (2019) 110278

A.H. Ahkami, et al.

Fig. 1. Experimental design and phenotypic responses of plants grown under control and drought conditions. Abbreviations: DAS, Days After Sowing; Pre-C, PreControl; ED, Early Drought; LD, Late Drought; PD, Post Drought.

leucine, isoleucine, and valine under drought stress [42]. Understanding of the metabolic shifts associated with plant exposure to severe drought is poorly understood, but evidence suggests widespread shifts in metabolites may enable maintenance of metabolism and turgor during drought [43]. Drought induces several responses in plants, including leaf senescence, which helps to alleviate water loss and contributes to nutrient remobilization to younger leaves or sink tissues [44]. These processes involve the induced expression of senescence-asscoated genes (SAGs) [45]. Although accelerated senescence in response to drought is naturally ideal for plant survival, it can have devastating effects on crop productivity including reductions in canopy size and photosynthesis rate as well as critical decreases in crop yield and biomass [46]. The suppression of drought-induced leaf senescence (by expression of a gene mediating cytokinin biosynthesis) was shown to result in outstanding drought tolerance [47]. While a key consideration with respect to abiotic stress tolerance in crops is their genetic programming to undergo accelerated senescence in response to drought [48], our knowledge of the metabolite shifts associated with drought-induced senescence per se has remained fragmentary. The main objective of this study was to better understand the shifts in metabolites when plants undergo prolonged drought stress to the point of mortality. We aimed to document dynamic changes in metabolites as the final product or ultimate response of Brachypodium to severe drought-induced senescence. The derived information can be used to obtain predictive understanding of drought acclimation and to improve drought tolerance in food and bioenergy crops.

drought in switchgrass revealed non-linear relationships, suggesting critical thresholds in drought stress responses in monocots [21]. However, much less information is available for the important genomics model grass Brachypodium [16,17,22,23]. In one study, comparative analyses of drought stress responses among four Brachypodium varieties contrasting in drought resistance were conducted and multiple physiological parameters, including antioxidants and numerous metabolites, were determined [22]. The value of metabolomics to investigate Brachypodium responses to drought stress [24], and its application to separate Brachypodium ecotypes into distinct drought tolerance groupings has also been reported [25]. Plant responses to drought stress involve changes in various metabolic pathways [26–29]. Raffinose and L-proline accumulate to high levels over the course of several days of abiotic stress exposure, whereas alterations in central carbohydrate metabolism occur more quickly [30]. Metabolome analyses indicated amino acids, organic acids, sugars and fatty acids as the main metabolites that change upon water deficiency in wheat [31,32], rice seedlings [26], barley [33] and various Triticeae species [34]. To tolerate drought stress, plants have evolved adaptive mechanisms such as the accumulation of compatible solutes (including sucrose, fructose and glucose) in the cytoplasm [35–37]. Stress-inducible galactinol synthase was shown to play a role in the accumulation of galactinol and raffinose under abiotic stress conditions, and both metabolites may function as osmoprotectants in droughtstress responses [38,39]. Other osmolytically active metabolites, such as proline, also contribute to drought stress tolerance [40,41]. Using a targeted GC–MS approach, several cultivars of bread wheat differing in water stress tolerance showed significantly higher levels of amino acids, most notably proline, tryptophan, and the branched chain amino acids 2

Plant Science 289 (2019) 110278

A.H. Ahkami, et al.

speed setting of 4) at 23 °C for 20 min. The reaction vial was centrifuged for 2 min at 3500 x g and supernatants (lower chloroform/methanol phase and upper water/methanol phase) were transferred separately to 2 ml glass vials. To the residue 200 μl of chloroform were added and the mixture was vigorously shaken as above at 23 °C for 2 min. Extracts were centrifuged again for 15 min at 3500 x g and the supernatant transferred to the vial containing the first chloroform/methanol extract. The chloroform/methanol extract was evaporated using an EasyBio concentrator (Genevac, Ipswich, UK) for 30 min (program: BP, 75 °C), while the water/methanol extract was evaporated with a Vacufuge Plus 5305 concentrator (Eppendorf, Hamburg, Germany) for 2.5 h at 45 °C. Samples were derivatized by adding 20 μl of a 20 mg/ml solution of methoxyamine hydrochloride in pyridine (Sigma-Aldrich, St. Louis, MO, USA) and shaking gently at 30 °C for 90 min. Then, 50 μl of Nmethyl-N-(trimethylsilyl) trifluoroacetamide (Sigma-Aldrich, St. Louis, MO, USA) were added and samples incubated at 37 °C for another 30 min. Reaction mixtures were transferred to GC vials with glass inserts. We used a just-in-time approach, where derivatization was timed to occur directly before sample injection (with “waiting times” of < 1 h). Samples were processed and analyzed in random order. The GC–MS instrumentation consisted of a model 6890 GC coupled to a model 5975 mass selective detector MSD (Agilent Technologies, Santa Clara, CA, USA). Analytes were separated over a DB-5MS + DG column (30 m x025 mm; 0.25 μm film thickness; Agilent Technologies, Santa Clara, CA, USA). The initial oven temperature was set to 60 °C. During a sample run, the oven temperature was increased by 3 °C per min from 60 to 320 °C, which was then held for 10 min for a total run time of 97.67 min. The other settings were as follows: inlet temperature 250 °C; split ratio 10:1; He as carrier gas; initial flow 1.2 ml/min; MSD transfer line temperature at 290 °C, quadrupole at 150 °C and source at 250 °C; and the solvent delay for data acquisition was 7.3 min. The method was retention time locked with myristic acid-d27 (42.020 min). For absolute quantitation a unique target ion was selected for each peak and a calibration curve obtained with varying concentrations of each authentic standard. A custom report macro was generated to export raw data into a Microsoft Excel-compatible format.

Table 1 Harvesting time-points and level of supplied water in each stage. Abbreviations: DAS, Days After Sowing. Harvesting Time and Condition Time

Watering Supply

Pre-Control (Pre-C) Early Drought (ED)

34 DAS 42 DAS

Late Drought (LD) Post Drought (PD)

50 DAS 63 DAS

Regular water amount (50 ml per pot) Reduced water to half (25 ml) for 6 days and then to one-fifth of original amount (10 ml) for 2 days Withheld water (0 ml) for 6 days Withheld water (0 ml) for 19 days

2. Materials and methods 2.1. Experimental design, growth conditions and harvest Brachypodium (Brachypodium distachyon accession Bd21) was grown in growth chambers (Percival Scientific, Inc) under 16 h/8 h light-dark regime (with light intensity of 250 μmol m−2 s-1), temperature of 24 °C day/18 °C night, and relative humidity of 60%. Plants were grown in 9 cm (3.5") size pots containing commercial soil (Sun Gro® Horticulture, Metro-Mix 360 growth mix). Control plants were supplied with 50 ml water per pot [24] during the entire experimental period. Starting at booting stage [49] (BBCH scale 45 [50];), we reduced water in the drought replicates from 50 ml to 25 ml, 10 ml, and zero ml on the subsequent watering days (36 days after sowing (DAS), 40 DAS and 44 DAS, respectively). Pots were randomized and moved to different locations within the growth chamber biweekly. Plant growth and developmental characteristics were monitored daily. Five to eight biological replicates were harvested at four time points: 34 DAS as control sample prior to drought treatment (Pre-C), 42 DAS as Early-Drought (ED), 50 DAS as Late-Drought (LD), and 63 DAS as Post-Drought (PD). (See Fig. 1 and Table 1 for details). Whole leaves were harvested from control and drought-treated samples during each collection and used directly for morphological and physiological analysis. Samples for metabolite analysis were snap-frozen in liquid nitrogen, freeze-dried, and stored at −80 °C until further processing.

2.4. GC–MS data processing and statistical analysis

2.2. Morphology, leaf water potential and C/N ratio analysis

Raw data were processed with ChemStation version E.02.00.493 (Agilent Technologies, Santa Clara, CA, USA). Authentic standards were combined in bins of 4–6 compounds and aliquots injected onto the GC. A spectral database covering a total of 83 compounds was developed based on retention time, quantifier ion and three qualifier ions (details in Supplementary Table S1). Unknowns were reported based on community standards as formulated under the Minimum Information About a Metabolomics Experiment (MIAMET) guidelines [53]. Raw data were adjusted for differences in sample amounts (variance introduced by weighing out samples) and internal standard signal intensity (variance introduced by injection). The normalized data were log10-transformed to facilitate downstream statistical analysis. Factorial analysis of variance (Student’s t-test) and Principal Component Analysis (PCA) were performed with the Microsoft Excel add-on software package StatistiXL (Nedlands, WA, Australia).

On all harvested plants we counted the number of leaves, panicles and tillers, followed by fresh weight measurement. Water potential was measured on a distal green tiller of each plant with a Scholander-type pressure chamber (PMS, Corvallis, OR, USA). Percentage of canopy senescence was recorded visually as the fraction of the plant canopy that was no longer green [51]. The relative content of total carbon and total nitrogen was measured according to Ahkami (2010) [52] using freeze-dried, powdered samples by a VarioEL Cube Elemental Analyzer (Elementar Analysensysteme GmbH, Langenselbold, Germany). 2.3. Extraction of plant material, derivatization of metabolites, and acquisition of GC–MS data Plant material was shock-frozen in liquid nitrogen, lyophilized to dryness, and ground to a fine powder using mortar and pestle in the presence of liquid nitrogen. Aliquots (˜ 10 mg) of the ground material were further homogenized by cryogenic milling (MM400, Retsch, Haan, Germany) and transferred to 8 ml screw-cap glass vials. To this homogenate 400 μl of chloroform (including myristic acid-d27 (SigmaAldrich, St. Louis, MO, USA) as internal standard; concentration 0.05 mg/ml) and 960 μl of methanol (including salicylic acid-d4 (Sigma-Aldrich, St. Louis, MO, USA) as internal standard; concentration 0.01 mg/ml) were added and the mixture kept at 60 °C for 15 min. Then, 640 μl of water were added, the mixture was vigorously shaken (multi-tube vortexer, ThermoFisher Scientific, Waltham, MA, USA;

2.5. qRT-PCR Frozen leaf tissue was homogenized using liquid nitrogen, mortar and pestle. Approximately 100 mg of the resulting leaf homogenate from each sample was used for RNA isolation using the GeneJET Plant RNA Purification Kit (ThermoFisher Scientific, Waltham, WA, USA) according to the manufacturer’s protocols. Total RNA was subjected to reverse transcription using the High Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific, Waltham, WA, USA) according to the manufacturer’s instructions. Quantitative PCR was conducted on a StepOnePlus Real-Time PCR system using Power SYBR 3

Plant Science 289 (2019) 110278

A.H. Ahkami, et al.

3.2. Metabolic responses to sustained drought

Green PCR Master Mix in a 96-well plate format (Applied Biosystems, Foster City, CA, USA). Relative quantification (RQ) for transcripts were calculated using the comparative CT (2−ΔΔCT) method and normalized to the control of the Pre-C sample. The B. distachyon gene S-adenosylmethionine decarboxylase (SamDC) (BRADI_5g14640) was used as internal control, since it has been ranked as the most stable reference gene for qRT-PCR in Brachypodium grown under various environmental stresses, including drought [54]. Cycle thresholds of amplification were determined by the StepOne Software v2.3 (Applied Biosystems, Foster City, USA). All samples were run in triplicate. The averages and standard deviations of RQs for the same condition of all sets were calculated and plotted. Primers used in this study are listed in Supplementary Table S3. If more than one putative paralogue appeared to exist, the gene copy (or in some cases two copies) with the higher expression level in leaves, based on the Brachypodium gene expression data hosted by Phytozome (https://phytozome.jgi.doe.gov) was selected.

A non-targeted metabolomics approach was employed to assess the effects of drought stress on the pools of intermediates involved in primary metabolism. We compared GC–MS profiles (total ion chromatograms) of samples types directly, to determine if differences existed across samples types. Polar extracts contained, in order of elution from the GC column, short-chain organic and amino acids, aromatic amino acids, polyhydroxy organic acids and monosaccharides, functionalized phenylpropanoid acids and uronic acids, and disaccharides. Substantial drought-induced changes (mostly increases) were evident in the retention time region of organic and amino acids (Fig. S1A). The intensity of peaks corresponding to monosaccharides increased substantially in both controls and drought-treated samples over the course of the experimental period (Fig. S1B). Non-polar extracts were separated in the elution order of fatty acids and prenyl alcohols, α-tocopherol, and phytosterols. The composition of the dominant free fatty acids changed in drought-treated plants (compared to all control samples), with a relative increase in the proportion of saturated fatty acids (Fig. S1C). The abundance of the peak corresponding to α-tocopherol increased significantly over the course of the experiment in controls, but dropped to very low levels in drought-treated samples (Fig. S1D). To further interrogate our data sets statistically, log10-transformed data were subjected to Principal Component Analysis (PCA), a multivariate analysis approach that can reveal the internal structure of data sets in a way that captures the variance observed between sample types. To reduce the dimensionality of our data set (time course of droughttreated samples and appropriate controls), the first three principal components (PCs), which accounted for 51.1% of the variance, were plotted (Fig. 3A-C; full data available in Supplementary Tables S1 and S2). Controls had mostly negative values in PC1. The data points for Pre-C, ED and LD controls were intermixed (mostly negative PC2 values), while the PD data points of controls clustered separately at positive PC2 values (Fig. 3A). The data points for drought treatment samples had exclusively positive values in PC1, which separated them from most controls. Among the samples representing the drought treatment, data points for the ED and LD time points clustered together (mostly negative PC2 values), while PD clustered separately (positive PC2 values) (Fig. 3A). In a two-dimensional plot of PC1 and PC3, the sample types were mostly separated in PC1 (controls are at values of ≤ 2.4, while drought-treated samples at values of ≥ 3.2) (Fig. 3B). Data points for the Pre-C samples clustered together (but mostly separately from others) at negative PC2 values and positive PC3 values (the exception is one replicate with a less negative PC2 value) (Fig. 3C). The PC2/PC3 plot also indicated a separation of the PD controls (PC2 ≥ 4.6) (Fig. 3C). To assess the implications of our PCA for metabolism under drought stress, we evaluated component loading scores. By simultaneously displaying data points (samples) and vectors for original variables (molecular features) in Biplots, we were able to determine which metabolites contributed significantly to the observed separation of sample types in PCA plots (Fig. 3D, E). We will focus our description of results on a few metabolite classes with particularly high correlation coefficients (a ranking of all molecular features by PC scores is provided in Supplementary Table S2). A combination of positive PC1 and negative PC2 scores in drought-treated samples (ED and LD) correlated with a trisaccharide (raffinose), amino acids (e.g., L-alanine, L-threonine, Lglutamate/L-glutamine (detected as pyroglutamic acid), and L-proline), free fatty acids (in particular, palmitic and stearic acid), and organic acids (e.g., malic and fumaric acid). Monosaccharides (glucose and fructose) and prenyl lipids (phytol and α-tocopherol) correlated prominently with positive PC1 and positive PC2 scores (drought-treated samples of the PD time point) (Fig. 3D). High PC2 scores (> 4) combined with moderate PC3 scores (-5 to +2) (characteristic of PD controls) were highly correlated with monosaccharides (glucose and fructose) and prenyl lipids (phytol and α-tocopherol) (Fig. 3E). Negative

3. Results 3.1. Developmental and physiological responses to sustained drought A gradual increase in drought stress was applied to 5-week-old Brachypodium plants as described in Table 1. Reduced watering led to mild visible symptoms such as partial leaf color change at 42 DAS (Early-Drought or ED time point) (Fig. 1). Additional symptoms, including leaf rolling and yellow-brownish leaf coloring, were observed at 50 DAS (Late-Drought or LD time point). Severe drought damage, such as leaf desiccation and senescence, was noted after prolonged water withholding at 63 DAS (Post-Drought or PD time point) (Fig. 1). Stressed plants showed > 95% leaf area discoloration at 58 DAS. Fresh biomass was reduced up to six-fold in LD and PD samples compared to control (Fig. 2A), and the number of leaves, panicles, and tillers was also dramatically lower in plants exposed to drought (Fig. 2B-D). The experimental treatment had large impacts on mid-day water potential levels. While the mid-day water potential of control plants remained unchanged over the course of experimental period, the corresponding values in drought-treated plants dropped dramatically at ED, and proceeded to drop to -8.0 MPa at LD (Fig. 2E). We also monitored the flowering status in our experiment and did not notice any significant changes between controls and treated plants. To evaluate whether the accumulation of carbon-containing metabolites was reflected by a change of carbon to nitrogen ratios during drought treatment, and in particular to evaluate the possible relationship between C/N ratios with drought-induced senescence under severe water deficiency, total concentrations of carbon and nitrogen were measured. There was a significant increase in the carbon to nitrogen ratio up to 2-fold in drought-treated plants at PD, while the C/N ratio of controls did not change significantly over the time course of the experiment (Fig. 2F). The total protein content decreased in controls over the course of the experimental period from 17.0 mg/g DW (Pre-C) to 9.2 mg/g DW (PD). The protein concentration in drought-treated plants decreased at a lower rate and stayed above control levels for the duration of the experiment (12.9 mg/g DW; P-value of 0.01 for comparison with corresponding controls at PD) (Fig. 2G). The chlorophyll concentration in controls decreased significantly during the experimental period from 44.1 mg/g DW (Pre-C) to 6.1 mg/g DW (PD). In drought-treated plants, this decrease occurred much faster, with chlorophyll being near the detection limit in the PD sample (P-value < 10−6) (Fig. 2H). In controls, the chlorophyll a/b ratio decreased slightly from 4.1 (Pre-C) to 3.2 (PD). The chlorophyll a/b ratio increased initially in droughttreated plants compared to controls (4.4 at ED) and then decreased rapidly (2.7 in LD) (Fig. 2I).

4

Plant Science 289 (2019) 110278

A.H. Ahkami, et al.

Fig. 2. Morphological (A–D) and physiological (E–I) changes in control (white circles or bars) and drought-stressed (gray triangles or bars) plants. Each value is represented by the mean of at least five independent replicates ± SE. Asterisks indicate significant differences between drought-exposed and well-irrigated plants (Student’s t-test, * for P < 0.01; ** for P < 0.001; and *** for P < 0.0001). Abbreviations as in Fig. 1.

DW), citric acid (9.9 mg/g DW) and fumaric acid (0.9 mg/g DW); concentrations given for Pre-C time point) decreased in controls during the experimental period (Fig. 4B). The patterns in drought-treated samples were quite different. The concentration of malic acid increased gradually to 3.8-fold of control levels at the PD time point (40.8 vs. 10.9 mg/g DW, respectively). The concentration of fumaric acid increased transiently as a response to the drought treatment but remained higher than in controls (3.8-fold difference in drought-treated samples (0.8 mg/g DW) compared to controls at the PD time point (0.2 mg/g DW)). No significant differences in citric acid content were determined (decrease from 9.9 to 3.9 mg/g DW in controls and to 4.1 mg/g DW in drought-treated samples) (Fig. 4B). The concentrations of three amino acids (L-alanine (0.07 mg/g DW), L-threonine (0.13 mg/g DW), and L-glutamate/L-glutamine (0.1 mg/g DW); concentrations listed for Pre-C time point) decreased in controls during the experimental period, while L-proline remained at extremely low levels in all control samples (< 0.0008 mg/g DW). Drought treatment resulted in gradual increases of L-threonine (to 0.2 mg/g DW) and L-glutamate/L-glutamine (0.4 mg/g DW) at the PD time point. LAlanine and L-proline increased dramatically to fairly high concentrations at LD (2.3 and 4.3 mg/g DW, respectively), before declining slightly (L-alanine to 2.2 mg/g DW) or significantly (L-proline to

PC2 paired with positive PC3 scores (characteristic of Pre-C samples) correlated with raffinose and citric acid.

3.3. Sustained drought leads to increases in compatible solutes, shifts in fatty acid composition, and dramatic decreases in prenyl lipids To further explore the relevance of the PCA findings, absolute quantitation was performed for representative metabolites across the experimental period (controls and drought-treated samples). As part of these efforts, two monosaccharides (glucose and fructose), a disaccharide (sucrose), and a trisaccharide (raffinose) were quantified. In Pre-C samples, sucrose had the highest concentration (23.9 mg/g dry weight (DW)), followed by glucose (5.3 mg/g DW), fructose (2.6 mg/g DW), and raffinose (0.9 mg/g DW) (Fig. 4A). The concentrations of monosaccharides in controls increased during the latter part of the experimental period (onset of senescence between LD to PD time points), while those of di- and trisaccharides decreased (with a gradual decline of sucrose and a more dramatic drop (from LD to PD) of the raffinose concentration). In comparison to controls, the concentrations of glucose, fructose, sucrose and raffinose were higher in droughttreated samples throughout the experimental period (Fig. 4A). The levels of three selected organic acids (malic acid (28.2 mg/g 5

Plant Science 289 (2019) 110278

A.H. Ahkami, et al.

Fig. 3. Principal Component Analysis of GC–MS data obtained with Brachypodium plants at different time points during a drought treatment and appropriate controls. (A) Principal Components (PCs) 1 and 2, (B) PCs 1 and 3, and (C) PCs 2 and 3. Symbols: white circles, Pre-Control; yellow circles, Early Drought (Controls); yellow triangles, Early Drought (Treatment); green circles, Late Drought (Controls); green triangles, Late Drought (Treatment); blue circles, Post Drought (Controls); blue tringles, Post Drought (Treatment). (D) Biplot for PCs 1 and 2. (E) Biplot for PCs 2 and 3. Names of selected metabolites are given and signature metabolites are indicated with larger font (color code: green, carbohydrate; red, fatty acid; blue, amino acid or derivative; purple, prenyl lipid). For details see text. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

chlorophylls, phylloquinone (vitamin K1) and α-tocopherol, also increased in controls from the Pre-C to the PD time points. In extracts of drought-treated plants, a transient increase of phytol was determined, with a sharp increase from 0.2 mg/g DW at Pre-C to 0.9 mg/g DW at LD, followed by a dramatic drop to 0.08 mg/g DW at PD (Fig. 4F). To help with contextualizing the results presented here, we also calculated fold-change differences between all detected metabolites of drought-treated and control plants and visualized these with a color code on a metabolic map (Fig. 5). This analysis indicated an extensive induction of metabolite accumulation across multiple pathways in drought-treated samples, with decreases occurring primarily during late senescence and affecting mainly chlorophylls, antioxidants (ascorbic acid and α-tocopherol) and a membrane sterol (stigmasterol) (Fig. 5).

2.5 mg/g DW) at the PD time point (Fig. 4C). The levels of free saturated fatty acids (not incorporated into lipids) increased in samples of drought-treated plants (stearic acid (18:0) from 0.2 mg/g DW (Pre-C) to 0.3 mg/g DW (PD) and palmitic acid (16:0) from 0.3 mg/g DW (Pre-C) to 0.8 mg/g DW (PD) (Fig. 4D). The concentration of linolenic acid (18:3) as the major free unsaturated fatty acid increased slightly in controls (from 0.18 (Pre-C) to 0.28 mg/g DW (PD)), and showed a transient surge in drought-treated samples (with a maximum of 0.38 mg/g DW at the ED time point). Linoleic acid (18:2) was much less abundant and showed a similar pattern (slight increase in controls from 0.013 to 0.021 mg/g DW in the Pre-C and PD samples; transient increase to 0.028 mg/g DW at ED in drought-treated samples). Because of these dynamic experimental responses, the total amount of free fatty acids was consistently higher in drought-treated plants (compared to appropriate controls) (Fig. 4E). The concentration of the α-tocopherol, a prenyl lipid also referred to as vitamin E, increased in controls over the course of the experimental period, reaching 1.4 mg/g DW at the PD time point (Fig. 4F). The same pattern was observed early during drought treatment but was followed by a sharp decrease to 0.02 mg/g DW at PD. Free phytol, which constitutes the prenyl side chain of several prenyl lipids, including

3.4. Sustained drought affects transcript levels of senescence-associated genes To determine potential transcript markers for the status of naturaland drought-induced senescence, we selected putative Brachypodium orthologs of genes that had previously been characterized as being associated with senescence in other species and performed qRT-PCR 6

Plant Science 289 (2019) 110278

A.H. Ahkami, et al.

Fig. 4. Metabolite quantitation in control (white circles) and drought-stressed (gray triangles) plants. Each value is represented by the mean of five independent replicates ± SE. Asterisks indicate significant differences between drought-exposed and well-irrigated samples (Student’s t-test, (*) for P < 0.05; * for P < 0.01; ** for P < 0.001; and *** for P < 0.0001). Time point abbreviations as in Fig. 1..

7

Plant Science 289 (2019) 110278

A.H. Ahkami, et al.

Fig. 5. Metabolic pathway outline with fold-change difference between control and drought-treated plants for three time points (from left to right ED, Early Drought; LD, Late Drought; and PD, Post Drought).

period (Fig. 6E), which is also consistent with previously published data on drought-responsive NACs in Brachypodium [62]. Galactinol synthase (GolS) is involved in the biosynthesis of raffinose family oligosaccharides, which serve as osmolytes during drought stress [64], and GolS overexpression in transgenic Brachypodium has been demonstrated to increase drought tolerance [65]. Notably, transcript levels of a gene putatively encoding Brachypodium GolS (Bradi1g17200) increased transiently in drought-treated Brachypodium plants (Fig. 6F).

assays. SAG12 was recognized as a drought-responsive cysteine protease in Arabidopsis [55], with the subsequent characterization of drought-inducible orthologs of rice (SAG12-2, SAG39) [56,57] and maize [58]. The expression levels of the two most closely related genes (based on sequence identity) of Brachypodium (Bradi1g74227 and Bradi4g40647) increased steadily in controls during the experimental period (Fig. 6A, B). Transcript abundance for both of these genes remained fairly constant in drought-treated plants at the ED and LD time points, but then increased significantly at the PD time point. Among the Early Responsive to Dehydration (ERD) class of genes, ERD7 of Arabidopsis was recognized earlier as a potential marker for drought-induced senescence [59]. The expression levels of the putative Brachypodium ERD7 ortholog (Bradi1g30150) increased slightly in controls during the experimental period (Fig. 6C). Transcript abundance for this gene followed a similar pattern in drought-treated plants until the LD time point, but then rose more significantly at the PD time point. Members of the NAC family of transcription factors play vital roles in the integration of developmental signals during senescence [60]. The putative Brachypodium ortholog (Bradi1g63600; also referred to as BdNAC022) of the wheat NAC transcription factor NAM-B1, which has been demonstrated to function in regulating senescence [61], increased early in drought-treated plants (Fig. 6D), which was consistent with previously published data on drought-induced NACs in Brachypodium [62]. In contrast, transcript abundance for a second NAC, Bradi5g12407 or BdNAC092, a putative ortholog of ORE1, a well-characterized senescence regulatory NAC in Arabidopsis [63], remained below control levels in drought-treated Brachypodium throughout the experimental

4. Discussion 4.1. Drought causes an early onset of senescence, as indicated by both physiological and metabolic measurements We employed an integrated physiological and biochemical approach to investigate the responses of Brachypodium to slowly increasing water deficiency. This drought treatment resulted in a decrease of all measured growth characteristics (Fig. 2), and affected several physiological parameters including chlorophyll content, phytol and antioxidants (Figs. 2H and 4 F). Free fatty acids increased in leaves of drought-treated plants when compared to controls (up to 72% increase at the LD time point), with a transient (18:0, 18:2 and 18:3) or linear plateau (16:0) induction time course (Fig. 4). Decreases in membrane lipid content, with concomitant increases in free fatty acid pools, have been reported repeatedly to occur under drought conditions (reviewed in [66]). We hypothesize that the increase of the pool of free fatty acids in our experiment is an 8

Plant Science 289 (2019) 110278

A.H. Ahkami, et al.

Fig. 6. Gene expression (relative quantification) of selected genes by qRT-PCR in control (white circles) and drought-stressed (gray triangles) plants. Each value is represented by the mean of four independent replicates ± SE. Asterisks indicate significant differences between drought-exposed and well-irrigated samples (Student’s t-test, * for P < 0.05). Time point abbreviations as in Fig. 1. For details see text.

Brachypodium, a drought-induced increase in the concentrations of mono-, di- and trisaccharides (e.g., glucose, fructose, sucrose and raffinose) was observed in leaves (Fig. 4), which is in agreement with previous reports [76–79]. Our results revealed that GolS (Bradi1g17200), involved in the biosynthesis of raffinose, was significantly up-regulated in drought-treated plants (showed a sharp transient increase at LD) compared to controls (Fig. 6). The relative transcript levels of this gene remained almost unchanged in plants undergoing natural senescence. Interestingly, raffinose levels exhibited higher concentration in drought-treated samples compared to wellwatered plants throughout the experimental period (Fig. 4). As a response to drought stress in Brachypodium leaves, we also observed significantly increased accumulation of free amino acids and derived metabolites, in particular L-alanine, L-threonine, L-glutamate/ L-glutamine (detected collectively as pyroglutamic acid), L-proline, βalanine, and γ-aminobutyric acid (Figs. 4 and 5), which is again consistent with findings reported previously [17,25]. Under drought conditions, soluble nitrogen-containing metabolites are broken down, ultimately releasing their nitrogen in the form of ammonia. Ammonia in higher cellular concentrations is toxic and can be removed through the formation of amino acids [80], which could be one explanation for the increased accumulation of soluble amino acids in our drought experiment. Some free amino acids, such as L-alanine and L-proline, have also been implicated in stabilizing proteins under high osmolytic pressure [81,82]. The observed increase in certain amino acids is of significance because the soluble protein content in leaves of drought-stressed Brachypodium plants decreased in drought-treated samples, but this decrease was significantly less than in the control plants, a novel result of this study (Fig. 2). Interestingly, the decrease in protein content in drought-treated plants (minus 4.17 mg/g DW from Pre-C to PD) was roughly the same as the increase in measured free amino acids (4.97 mg/g DW), but the time course was substantially different (Figs. 2 and 4). We therefore hypothesize that a combination of increased amino acid biosynthesis and/or protein breakdown might be responsible for the induced soluble amino acid levels under drought

indication of increased lipid turnover, but we are aware that free fatty acids represent only a minor fraction of the total pool of this metabolite class, while the vast majority is incorporated into membrane and storage lipids [67]. For the interpretation of these results, it should be noted that control plants entered senescence toward the end of the experimental period, as indicated by their phenotypic appearance (Fig. 1), loss of fresh weight, and decrease in chlorophyll concentration during the LD to PD transition (Fig. 2). However, all physiological and metabolic measurements described above for drought-treated plants (arrested growth, loss of chlorophyll and transient appearance of phytol, loss of water, increase in free fatty acids, and early increase and then loss of antioxidants) occurred earlier and/or in a more severe fashion when compared to controls. In other words, our data are consistent with the notion that drought causes an early onset of senescence [44,68–71]. Accelerated leaf senescence in response to drought is considered to confer adaptive advantages because it reduces the water demand at the whole-plant level, with examples across the plant kingdom and also including grasses [72–74]. The expression patterns of putative orthologs of genes with established roles in drought responses in other plants, such as those belonging to the ERD, NAC and raffinose oligosaccharides families (Fig. 6), were generally in agreement with expectations based on the available literature, and possibly with the interpretation of an early onset of senescence [59–65]. 4.2. Drought-induced senescence triggered accumulation of metabolites implicated in osmotic protection and stomatal closure One of the most commonly described responses of higher plants to drought stress is the accumulation of osmolytes, also known as compatible solutes [75]. These are non-toxic, metabolically inactive, small molecules that are not structurally or metabolically related, but are thought to have common functional roles in protecting cellular components from dehydration injury. The accumulation of compatible solutes increases cellular osmolarity, which in turn increases water influx (or reduces efflux), thus maintaining cell turgor. In our experiment with 9

Plant Science 289 (2019) 110278

A.H. Ahkami, et al.

directly tied to cellular disintegration. It is intriguing to speculate that in Brachypodium, a grass usually considered as being fairly drought tolerant [16], the metabolic adjustments described here enable an ordered breakdown of valuable C, N, P and S resources, despite the dramatically low water content of leaf cells. Future research to test this hypothesis will be highly informative to better understand mechanisms of drought tolerance. It will also be informative to expand the metabolite coverage of Brachypodium drought experiments beyond those accessible through the methods employed in the current study, for example, by using a series of extractions with different solvents and performing downstream analyses using high performance liquid chromatography coupled to mass spectrometry [94].

stress, as recently demonstrated in Arabidopsis thaliana [83]. For specific amino acids whose concentrations increased dramatically from ED to LD, in particular L-alanine and L-proline, metabolic upregulation (rather than protein breakdown) would appear a more likely explanation. Following up on these observations with biochemical studies and comparisons across Brachypodium genetic diversity will be an important future goal to develop a better understanding of mechanisms of drought tolerance in grasses and enable a more informed model-to-crop translation. Our experimental results indicated a significant and sustained increase of malic acid under drought conditions. Malic acid was previously shown to increase as a response to drought-treatment in Brachypodium [25] but its physiological potential deserves a more indepth discussion. This organic acid has been reported to play a role in osmotic adjustments under drought stress (reviewed in [30]). Metabolites from various realms of cellular metabolism, including hormones, inorganic ions, amino acids, sugars and organic acids (including malic acid), have been demonstrated to occur in xylem sap as possible chemical signals that are transported from roots to shoots [84]. However, among the organic acids found in the xylem sap at higher concentrations, only malate has been directly implicated in stomatal closure and therefore reduced water loss through transpiration [85–90]. We hypothesize that malic acid, likely in conjunction with other signaling molecules, may cause stomatal closure as a response to drought in Brachypodium. Our experimental design does not allow us to evaluate if root-to-shoot transport plays a role in the induced accumulation of malic acid, but this would be an interesting focus for follow-up research. It would also be informative to acquire metabolomic data for the epidermal cell layer, separately from the remainder of the leaf, to test if the accumulation of malate and other metabolites occurs in a cell type-specific manner.

Declaration of Competing Interest The authors declare no competing interests. Acknowledgments We acknowledge support through the Laboratory Directed Research and Development (LDRD) SEED Program funding at Pacific Northwest National Laboratory (PNNL). A portion of this research is part of the PREMIS Initiative at PNNL. It was conducted under the Laboratory Directed Research and Development Program at PNNL, a multi-program national laboratory operated by Battelle for the U.S. Department of Energy under Contract DE-AC05-76RL01830. A portion of this research was performed using EMSL, a DOE Office of Science user facility sponsored by the Department of Energy's Office of Biological and Environmental Research and located at PNNL. This work was also supported in part by seed funds from the USDA National Institute of Food and Agriculture, Hatch umbrella project #1015621. We thank Gabriel L. Myers for his technical support and taking care of plants.

4.3. Evidence for continued metabolic activity during sustained drought Appendix A. Supplementary data An important goal of this study was to shed light onto processes that occur during sustained drought. The majority of studies on this area focus on short and sudden severe drought stress at early plant vegetative stages, coupling with the rates of plant survival or recovery as a tool to indicate plant stress tolerance [49]. These studies have identified various stress-responsive genes and proteins; however, the extension of the outcomes into field studies has had limited success [91]. In this work, by the completion of the vegetative stages (just before heading and flowering stages), plants were progressively exposed to a prolonged drought stress by withholding irrigation. During the transition from the LD to PD time points, there was no detectable growth in droughttreated plants, the water potential stayed at a critically low level, and any remaining color in leaves disappeared (Figs. 1 and 2). The C:N ratio increased dramatically in drought-treated plants from LD to PD, concomitant with increases in the concentrations of abundant, small-molecule carbohydrates, in particular fructose and sucrose, and malic acid (Fig. 4). Safeguarding the carbohydrate reserve for the synthesis of compatible solutes under drought was reported and associated with starch degradation [92]. During the same time period, the total protein content decreased slightly in drought-treated plants. These results would indicate a remobilization of nitrogen, possibly to support grain filling [93]. Nevertheless, while the concentrations of some metabolites dropped close to the detection limit at the PD time point (e.g., chlorophylls, phytol and α-tocopherol), the majority of detected metabolites remained at surprisingly high levels (Figs. 4 and 5), substantially above those in controls (which were still partially green) (Fig. 1). Metabolites whose concentration increased from LD to PD, in addition to those mentioned above, include β-alanine, γ-aminobutyric acid, L-glycine, Lthreonine, and L-glutamate/L-glutamine (Fig. 5). This suggests that, despite a full discoloration that is generally assumed to coincide with the remobilization of cellular constituents, leaves of plants subjected to sustained drought continued to support metabolic reactions that are not

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.plantsci.2019.110278. References [1] C.D. Allen, D.D. Breshears, N.G. McDowell, On underestimation of global vulnerability to tree mortality and forest die-off from hotter drought in the Anthropocene, Ecosphere 6 (2015). [2] D.B. Lobell, W. Schlenker, J. Costa-Roberts, Climate trends and global crop production since 1980, Science 333 (2011) 616–620. [3] S.P. Long, E.A. Ainsworth, A.D.B. Leakey, J. Nosberger, D.R. Ort, Food for thought: lower-than-expected crop yield stimulation with rising CO2 concentrations, Science 312 (2006) 1918–1921. [4] A.J. Challinor, J. Watson, D.B. Lobell, S.M. Howden, D.R. Smith, N. Chhetri, A meta-analysis of crop yield under climate change and adaptation, Nat. Clim. Change 4 (2014) 287–291. [5] D. Deryng, D. Conway, N. Ramankutty, J. Price, R. Warren, Global crop yield response to extreme heat stress under multiple climate change futures, Environ. Res. Lett. 9 (2014). [6] E. Steudle, C.A. Peterson, How does water get through roots? J. Exp. Bot. 49 (1998) 775–788. [7] Y.J. Zhao, B.Q. Weng, Y.X. Wang, G.Z. Xu, Plant physio-ecological responses to drought stress and its research progress, Fujian Sci. Technol. Rice Wheat 27 (2009) 45–50. [8] IPCC, Part a: global and sectoral aspects. Contribution of working group II to the Fifth assessment report of the intergovernmental panel on climate change, Climate Change 2014: Impacts, Adaptation, and Vulnerability, Cambridge University Press, 2014. [9] N.G. McDowell, S.T. Michaletz, K.E. Bennett, K.C. Solander, C.G. Xu, R.M. Maxwell, R.S. Middleton, Predicting chronic climate-driven disturbances and their mitigation, Trends Ecol. Evol. 33 (2018) 15–27. [10] T.P. Brutnell, J.L. Bennetzen, J.P. Vogel, Brachypodium distachyon and setaria viridis: model genetic systems for the grasses, Annu. Rev. Plant Biol. 66 (2015) 465–485. [11] C. Jansson, J. Vogel, S. Hazen, T. Brutnell, T. Mockler, Climate-smart crops with enhanced photosynthesis, J. Exp. Bot. 69 (2018) 3801–3809. [12] J.P. Vogel, D.F. Garvin, T.C. Mockler, J. Schmutz, D. Rokhsar, M.W. Bevan, K. Barry, S. Lucas, M. Harmon-Smith, K. Lail, H. Tice, J. Grimwood, N. McKenzie,

10

Plant Science 289 (2019) 110278

A.H. Ahkami, et al.

[13] [14] [15]

[16] [17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28] [29]

[30] [31]

[32]

[33]

[34]

N.X. Huo, Y.Q. Gu, G.R. Lazo, O.D. Anderson, F.M. You, M.C. Luo, J. Dvorak, J. Wright, M. Febrer, D. Idziak, R. Hasterok, E. Lindquist, M. Wang, S.E. Fox, H.D. Priest, S.A. Filichkin, S.A. Givan, D.W. Bryant, J.H. Chang, H.Y. Wu, W. Wu, A.P. Hsia, P.S. Schnable, A. Kalyanaraman, B. Barbazuk, T.P. Michael, S.P. Hazen, J.N. Bragg, D. Laudencia-Chingcuanco, Y.Q. Weng, G. Haberer, M. Spannagl, K. Mayer, T. Rattei, T. Mitros, S.J. Lee, J.K.C. Rose, L.A. Mueller, T.L. York, T. Wicker, J.P. Buchmann, J. Tanskanen, A.H. Schulman, H. Gundlach, A.C. de Oliveira, L.D. Maia, W. Belknap, N. Jiang, J.S. Lai, L.C. Zhu, J.X. Ma, C. Sun, E. Pritham, J. Salse, F. Murat, M. Abrouk, R. Bruggmann, J. Messing, N. Fahlgren, C.M. Sullivan, J.C. Carrington, E.J. Chapman, G.D. May, J.X. Zhai, M. Ganssmann, S.G.R. Gurazada, M. German, B.C. Meyers, P.J. Green, L. Tyler, J.J. Wu, J. Thomson, S. Chen, H.V. Scheller, J. Harholt, P. Ulvskov, J.A. Kimbrel, L.E. Bartley, P.J. Cao, K.H. Jung, M.K. Sharma, M. Vega-Sanchez, P. Ronald, C.D. Dardick, S. De Bodt, W. Verelst, D. Inze, M. Heese, A. Schnittger, X.H. Yang, U.C. Kalluri, G.A. Tuskan, Z.H. Hua, R.D. Vierstra, Y. Cui, S.H. Ouyang, Q.X. Sun, Z.Y. Liu, A. Yilmaz, E. Grotewold, R. Sibout, K. Hematy, G. Mouille, H. Hofte, J. Pelloux, D. O’Connor, J. Schnable, S. Rowe, F. Harmon, C.L. Cass, J.C. Sedbrook, M.E. Byrne, S. Walsh, J. Higgins, P.H. Li, T. Brutnell, T. Unver, H. Budak, H. Belcram, M. Charles, B. Chalhoub, I. Baxter, I.B. Initiative, Genome sequencing and analysis of the model grass Brachypodium distachyon, Nature 463 (2010) 763–768. M. Opanowicz, P. Vain, J. Draper, D. Parker, J.H. Doonan, Brachypodium distachyon: making hay with a wild grass, Trends Plant Sci. 13 (2008) 172–177. A. Sharma, M.B. Singh, P.L. Bhalla, Cytochemistry of pollen development in Brachypodium distachyon, Plant Syst. Evol. 300 (2014) 1639–1648. S.P. Gordon, B. Contreras-Moreira, D.P. Woods, D.L.D. Marais, D. Burgess, S.Q. Shu, C. Stritt, A.C. Roulin, W. Schackwitz, L. Tyler, J. Martin, A. Lipzen, N. Dochy, J. Phillips, K. Barry, K. Geuten, H. Budak, T.E. Juenger, R. Amasino, A.L. Caicedo, D. Goodstein, P. Davidson, L.A.J. Mur, M. Figueroa, M. Freeling, P. Catalan, J.P. Vogel, Extensive gene content variation in the Brachypodium distachyon pangenome correlates with population structure, Nat. Commun. 8 (2017). N. Luo, J.X. Liu, X.Q. Yu, Y.W. Jiang, Natural variation of drought response in Brachypodium distachyon, Physiol. Plant. 141 (2011) 19–29. W. Verelst, E. Bertolini, S. De Bodt, K. Vandepoele, M. Demeulenaere, M.E. Pe, D. Inze, Molecular and physiological analysis of growth-limiting drought stress in Brachypodium distachyon leaves, Mol. Plant 6 (2013) 311–322. A. Harb, A. Krishnan, M.M.R. Ambavaram, A. Pereira, Molecular and physiological analysis of drought stress in Arabidopsis Reveals early responses leading to acclimation in plant growth, Plant Physiol. 154 (2010) 1254–1271. F. Qin, K. Shinozaki, K. Yamaguchi-Shinozaki, Achievements and challenges in understanding plant abiotic stress responses and tolerance, Plant Cell Physiol. 52 (2011) 1569–1582. M.M. Vaughan, S. Christensen, E.A. Schmelz, A. Huffaker, H.J. Mcauslane, H.T. Alborn, M. Romero, L.H. Allen, P.E.A. Teal, Accumulation of terpenoid phytoalexins in maize roots is associated with drought tolerance, Plant Cell Environ. 38 (2015) 2195–2207. E. Meyer, M.J. Aspinwall, D.B. Lowry, J.D. Palacio-Mejia, T.L. Logan, P.A. Fay, T.E. Juenger, Integrating transcriptional, metabolomic, and physiological responses to drought stress and recovery in switchgrass (Panicum virgatum L.), BMC Genomics 15 (2014). H.T. Shi, T.T. Ye, B. Song, X.Q. Qi, Z.L. Chan, Comparative physiological and metabolomic responses of four Brachypodium distachyon varieties contrasting in drought stress resistance, Acta Physiol. Plant. 37 (2015). Y.W. Bian, X. Deng, X. Yan, J.X. Zhou, L.L. Yuan, Y.M. Yan, Integrated proteomic analysis of Brachypodium distachyon roots and leaves reveals a synergistic network in the response to drought stress and recovery, Sci. Rep.-Uk 7 (2017). P.P. Handakumbura, B. Stanfill, A. Rivas-Ubach, D. Fortin, J. Vogel, C. Jansson, Metabotyping as a stopover in genome-to-phenome mapping, Sci. Rep. (2019) in Press. L.H.C. Fisher, J.W. Han, F.M.K. Corke, A. Akinyemi, T. Didion, K.K. Nielsen, J.H. Doonan, L.A.J. Mur, M. Bosch, Linking dynamic phenotyping with metabolite analysis to study natural variation in drought responses of Brachypodium distachyon, Front. Plant Sci. 7 (2016). L. Shu, Q. Lou, C. Ma, W. Ding, J. Zhou, J. Wu, F. Feng, X. Lu, L. Luo, G. Xu, H. Mei, Genetic, proteomic and metabolic analysis of the regulation of energy storage in rice seedlings in response to drought, Proteomics 11 (2011) 4122–4138. C.M. Spickett, N. Smirnoff, R.G. Ratcliffe, Metabolic response of maize roots to hyperosmotic shock - an invivo P-31 nuclear-magnetic-Resonance study, Plant Physiol. 99 (1992) 856–863. P.D. Hare, W.A. Cress, J. Van Staden, Dissecting the roles of osmolyte accumulation during stress, Plant Cell Environ. 21 (1998) 535–553. K.L. Dennison, W.R. Robertson, B.D. Lewis, R.E. Hirsch, M.R. Sussman, E.P. Spalding, Functions of AKT1 and AKT2 potassium channels determined by studies of single and double mutants of arabidopsis, Plant Physiol. 127 (2001) 1012–1019. J. Krasensky, C. Jonak, Drought, salt, and temperature stress-induced metabolic rearrangements and regulatory networks, J. Exp. Bot. 63 (2012) 1593–1608. A. Michaletti, M.R. Naghavi, M. Toorchi, L. Zolla, S. Rinalducci, Metabolomics and proteomics reveal drought-stress responses of leaf tissues from spring-wheat, Sci. Rep. 8 (2018) 5710. R. Guo, L.X. Shi, Y. Jiao, M.X. Li, X.L. Zhong, F.X. Gu, Q. Liu, X. Xia, H.R. Li, Metabolic responses to drought stress in the tissues of drought-tolerant and drought-sensitive wheat genotype seedlings, AoB Plants 10 (2018). X.J. Wu, K.F. Cai, G.P. Zhang, F.R. Zeng, Metabolite profiling of barley grains subjected to water stress: to explain the genotypic difference in drought-induced impacts on malting quality, Front. Plant Sci. 8 (2017). N. Ullah, M. Yuce, Z.N.O. Gokce, H. Budak, Comparative metabolite profiling of

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48] [49]

[50]

[51]

[52]

[53]

[54]

[55] [56] [57]

[58]

[59]

[60] [61]

[62]

11

drought stress in roots and leaves of seven Triticeae species, BMC Genomics 18 (2017). M.J.M. Ebskamp, I.M. Vandermeer, B.A. Spronk, P.J. Weisbeek, S.C.M. Smeekens, Accumulation of fructose polymers in transgenic tobacco, Bio-Technol. 12 (1994) 272–275. J.N. Egilla, F.T. Davies, M.C. Drew, Effect of potassium on drought resistance of Hibiscus rosa-sinensis cv. Leprechaun: plant growth, leaf macro- and micronutrient content and root longevity, Plant Soil 229 (2001) 213–224. S.B. Chemikosova, N.V. Pavlencheva, O.P. Gur’yanov, T.A. Gorshkova, The effect of soil drought on the phloem fiber development in long-fiber flax, Russ. J. Plant. Physl. 53 (2006) 656–662. T. Taji, C. Ohsumi, S. Iuchi, M. Seki, M. Kasuga, M. Kobayashi, K. YamaguchiShinozaki, K. Shinozaki, Important roles of drought- and cold-inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana, Plant J. 29 (2002) 417–426. M.G. Selvaraj, T. Ishizaki, M. Valencia, S. Ogawa, B. Dedicova, T. Ogata, K. Yoshiwara, K. Maruyama, M. Kusano, K. Saito, F. Takahashi, K. Shinozaki, K. Nakashima, M. Ishitani, Overexpression of an Arabidopsis thaliana galactinol synthase gene improves drought tolerance in transgenic rice and increased grain yield in the field, Plant Biotechnol. J. 15 (2017) 1465–1477. T.H.H. Chen, N. Murata, Enhancement of tolerance of abiotic stress by metabolic engineering of betaines and other compatible solutes, Curr. Opin. Plant Biol. 5 (2002) 250–257. C.J. Wang, W. Yang, C. Wang, C. Gu, D.D. Niu, H.X. Liu, Y.P. Wang, J.H. Guo, Induction of drought tolerance in cucumber plants by a consortium of three plant growth-promoting rhizobacterium strains, PLoS One 7 (2012). J.B. Bowne, T.A. Erwin, J. Juttner, T. Schnurbusch, P. Langridge, A. Bacic, U. Roessner, Drought responses of leaf tissues from wheat cultivars of differing drought tolerance at the metabolite level, Mol. Plant 5 (2012) 418–429. I. Hummel, F. Pantin, R. Sulpice, M. Piques, G. Rolland, M. Dauzat, A. Christophe, M. Pervent, M. Bouteille, M. Stitt, Y. Gibon, B. Muller, Arabidopsis plants acclimate to water deficit at low cost through changes of carbon usage: an integrated perspective using growth, metabolite, enzyme, and gene expression analysis, Plant Physiol. 154 (2010) 357–372. S. Munne-Bosch, T. Jubany-Mari, L. Alegre, Drought-induced senescence is characterized by a loss of antioxidant defences in chloroplasts, Plant Cell Environ. 24 (2001) 1319–1327. H.S. Chen, P.D. Ruiz, W.M. McKimpson, L. Novikov, R.N. Kitsis, M.J. Gamble, MacroH2A1 and ATM play opposing roles in paracrine senescence and the senescence-associated secretory phenotype, Mol. Cell 59 (2015) 719–731. S. Gepstein, G. Sabehi, M.J. Carp, T. Hajouj, M.F.O. Nesher, I. Yariv, C. Dor, M. Bassani, Large-scale identification of leaf senescence-associated genes, Plant J. 36 (2003) 629–642. R.M. Rivero, M. Kojima, A. Gepstein, H. Sakakibara, R. Mittler, S. Gepstein, E. Blumwald, Delayed leaf senescence induces extreme drought tolerance in a flowering plant, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 19631–19636. R. Mittler, E. Blumwald, Genetic engineering for modern agriculture: challenges and perspectives, Annu. Rev. Plant Biol. 61 (61) (2010) 443–462. L. Shaar-Moshe, E. Blumwald, Z. Peleg, Unique physiological and transcriptional shifts under combinations of salinity, drought, and heat, Plant Physiol. 174 (2017) 421. S.Y. Hong, J.H. Park, S.H. Cho, M.S. Yang, C.M. Park, Phenological growth stages of Brachypodium distachyon: codification and description, Weed Res. 51 (2011) 612–620. M.L. Gaylord, T.E. Kolb, W.T. Pockman, J.A. Plaut, E.A. Yepez, A.K. Macalady, R.E. Pangle, N.G. McDowell, Drought predisposes pinon-juniper woodlands to insect attacks and mortality, New Phytol. 198 (2013) 567–578. A. Ahkami, Molecular physiology of adventitious root formation (ARF) in Petunia hybrida cuttings: involvement of primary metabolism in root formation, MLU of Halle-Wittenberg, halle, Germany. (2010), p. 150. R.J. Bino, R.D. Hall, O. Fiehn, J. Kopka, K. Saito, J. Draper, B.J. Nikolau, P. Mendes, U. Roessner-Tunali, M.H. Beale, R.N. Trethewey, B.M. Lange, E.S. Wurtele, L.W. Sumner, Potential of metabolomics as a functional genomics tool, Trends Plant Sci. 9 (2004) 418–425. S.Y. Hong, P.J. Seo, M.S. Yang, F. Xiang, C.M. Park, Exploring valid reference genes for gene expression studies in Brachypodium distachyon by real-time PCR, BMC Plant Biol. 8 (2008). K.N. Lohman, S. Gan, M.C. John, R.M. Amasino, Molecular analysis of natural leaf senescence in Arabidpsi thalina, Physiol. Plant 92 (1994) 322–328. L. Liu, Y. Zhou, M.W. Szczerba, X.H. Li, Y.J. Lin, Identification and application of a rice senescence-associated promoter, Plant Physiol. 153 (2010) 1239–1249. S. Singh, A. Singh, A.K. Nandi, The rice OsSAG12-2 gene codes for a functional protease that negatively regulates stress-induced cell death, J. Biosci. 41 (2016) 445–453. R.S. Sekhon, K.L. Childs, N. Santoro, C.E. Foster, C.R. Buell, N. de Leon, S.M. Kaeppler, Transcriptional and metabolic analysis of senescence induced by preventing pollination in maize, Plant Physiol. 159 (2012) 1730–1744. T. Kiyosue, K. Yamaguchi-Shinozaki, K. Shinozaki, Cloning of cDNAs for genes that are early-responsive to dehydration stress (ERDs) in Arabidopsis thaliana L.: identification of three ERDs as HSP cognate genes, Plant Mol. Biol. 25 (1994) 791–798. H.J. Kim, H.G. Nam, P.O. Lim, Regulatory network of NAC transcription factors in leaf senescence, Curr. Opin. Plant. Biol. 33 (2016) 48–56. C. Uauy, A. Distelfeld, T. Fahima, A. Blechl, J. Dubcovsky, A NAC gene regulating senescence improves grain protein, zinc, and iron content in wheat, Science 314 (2006) 1298–1301. J. You, L. Zhang, B. Song, X. Qi, Z. Chan, Systematic analysis and identification of

Plant Science 289 (2019) 110278

A.H. Ahkami, et al.

[63]

[64]

[65]

[66] [67] [68] [69] [70]

[71] [72] [73] [74] [75] [76] [77]

[78]

[79] H. Chen, J.G. Jiang, Osmotic adjustment and plant adaptation to environmental changes related to drought and salinity, Environ. Rev. 18 (2010) 309–319. [80] Y. Liu, N. von Wiren, Ammonium as a signal for physiological and morphological responses in plants, J. Exp. Bot. 68 (2017) 2581–2592. [81] L.G. Paleg, G.R. Stewart, J.W. Bradbeer, Proline and Glycine Betaine Influence Protein Solvation, Plant Physiol. 75 (1984) 974–978. [82] T. Arakawa, S.N. Timasheff, The stabilization of proteins by osmolytes, Biophys. J. 47 (1985) 411–414. [83] T.F. Huang, G. Jander, Abscisic acid-regulated protein degradation causes osmotic stress-induced accumulation of branched-chain amino acids in Arabidopsis thaliana, Planta 246 (2017) 737–747. [84] D.P. Schachtman, J.Q. Goodger, Chemical root to shoot signaling under drought, Trends Plant Sci. 13 (2008) 281–287. [85] R. Hedrich, I. Marten, Malate-induced feedback-regulation of plasma-membrane anion channels could provide a Co2 sensor to guard-cells, EMBO J. 12 (1993) 897–901. [86] M.P. Patonnier, J.P. Peltier, G. Marigo, Drought-induced increase in xylem malate and mannitol concentrations and closure of Fraxinus excelsior L-stomata, J. Exp. Bot. 50 (1999) 1223–1229. [87] N. Asai, N. Nakajima, M. Tamaoki, H. Kamada, N. Kondo, Role of malate synthesis mediated by phosphoenolpyruvate carboxylase in guard cells in the regulation of stomatal movement, Plant Cell Physiol. 41 (2000) 10–15. [88] S. Penfield, S. Clements, K.J. Bailey, A.D. Gilday, R.C. Leegood, J.E. Gray, I.A. Graham, Expression and manipulation of PHOSPHOENOLPYRUVATE CARBOXYKINASE 1 identifies a role for malate metabolism in stomatal closure, Plant J. 69 (2012) 679–688. [89] A. De Angeli, J.B. Zhang, S. Meyer, E. Martinoia, AtALMT9 is a malate-activated vacuolar chloride channel required for stomatal opening in Arabidopsis, Nat. Commun. 4 (2013). [90] L.B. Wang, M. Ma, Y.R. Zhang, Z.F. Wu, L. Guo, W.Q. Luo, L. Wang, Z. Zhang, S.L. Zhang, Characterization of the genes involved in malic acid metabolism from pear fruit and their expression profile after postharvest 1-MCP/Ethrel treatment, J. Agric. Food Chem. 66 (2018) 8772–8782. [91] M. Reguera, Z. Peleg, E. Blumwald, Targeting, metabolic pathways for genetic engineering abiotic stress-tolerance in crops, Bba-Gene Regul. Mech. 1819 (2012) 186–194. [92] S.A. Hosseini, M.R. Hajirezaei, C. Seiler, N. Sreenivasulu, N. von Wiren, A potential role of flag leaf potassium in conferring tolerance to drought-induced leaf senescence in barley, Front. Plant Sci. 7 (2016). [93] T. Girin, L.C. David, C. Chardin, R. Sibout, A. Krapp, S. Ferrario-Mery, F. DanielVedele, Brachypodium: a promising hub between model species and cereals, J. Exp. Bot. 65 (2014) 5683–5696. [94] T.F. Jorge, J.A. Rodrigues, C. Caldana, R. Schmidt, J.T. van Dongen, J. ThomasOates, C. Antonio, Mass spectrometry-based plant metabolomics: metabolite responses to abiotic stress, Mass Spectrom. Rev. 35 (2016) 620–649.

stress-responsive genes of the NAC gene family in Brachypodium distachyon, PLoS One 10 (2015) e0122027. J.H. Kim, H.R. Woo, J. Kim, P.O. Lim, I.C. Lee, S.H. Choi, D. Hwang, H.G. Nam, Trifurcate feed-forward regulation of age-dependent cell death involving miR164 in Arabidopsis, Science 323 (2009) 1053–1057. T. Taji, C. Ohsumi, S. Iuchi, M. Seki, M. Kasuga, M. Kobayashi, K. YamaguchiShinozaki, K. Shinozaki, Important roles of drought- and cold-inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana, Plant J. 29 (2002) 417–426. Y. Himuro, K. Ishiyama, F. Mori, T. Gondo, F. Takahashi, K. Shinozaki, M. Kobayashi, R. Akashi, Arabidopsis galactinol synthase AtGolS2 improves drought tolerance in the monocot model Brachypodium distachyon, J. Plant Physiol. 171 (2014) 1127–1131. I. Yordanov, V. Velikova, T. Tsonev, Plant responses to drought, acclimation, and stress tolerance, Photosynthetica 38 (2000) 171–186. H. Tjellstrom, M. Strawsine, J.B. Ohlrogge, Tracking synthesis and turnover of triacylglycerol in leaves, J. Exp. Bot. 66 (2015) 1453–1461. C. Peisker, T. Duggelin, D. Rentsch, P. Matile, Phytol and the breakdown of chlorophyll in senescent leaves, J. Plant Physiol. 135 (1989) 428–432. J.E. Thompson, C.D. Froese, E. Madey, M.D. Smith, Y.W. Hong, Lipid metabolism during plant senescence, Prog. Lipid Res. 37 (1998) 119–141. B. Chrost, J. Falk, B. Kernebeck, H. Mölleken, K. K, Tocopherol biosynthesis in senescing chloroplasts - a mechanism to protect envelope membranes against oxidative stress and a prerequisite for lipid remobilization? in: J.H. ArgyroudiAkoyunoglou, H. Senger (Eds.), The Chloroplast: from Molecular Biology to Biotechnology, Kluwer Academic Publishers, Dordrecht, NL, 1999, pp. 171–176. S. Hortensteiner, U. Feller, Nitrogen metabolism and remobilization during senescence, J. Exp. Bot. 53 (2002) 927–937. H. Thomas, Senescence, ageing and death of the whole plant, New Phytol. 197 (2013) 696–711. J.H.M. Schippers, R. Schmidt, C. Wagstaff, H.C. Jing, Living to die and dying to live: the survival strategy behind leaf senescence, Plant Physiol. 169 (2015) 914–930. N. Sade, M.D. Rubio-Wilhelmi, K. Umnajkitikorn, E. Blumwald, Stress-induced senescence and plant tolerance to abiotic stress, J. Exp. Bot. 69 (2018) 845–853. R. Serraj, T.R. Sinclair, Osmolyte accumulation: can it really help increase crop yield under drought conditions? Plant Cell Environ. 25 (2002) 333–341. M. Tester, A. Bacic, Abiotic stress tolerance in grasses. From model plants to crop plants, Plant Physiol. 137 (2005) 791–793. P. Valentovic, M. Luxova, L. Kolarovic, O. Gasparikova, Effect of osmotic stress on compatible solutes content, membrane stability and water relations in two maize cultivars, Plant Soil Environ. 52 (2006) 186–191. A. Mostajeran, V. Rahimi-Eichi, Effects of drought stress on growth and yield of Rice (Oryza sativa L.) cultivars and accumulation of proline and soluble sugars in sheath and blades of their different ages leaves, American-Eurasian J. Agric. Environ. Sci. 5 (2009) 264–272.

12