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EFFECTS OF SALINITY ON OXYGEN CONSUMPTION AND BLOOD PROPERTIES OF YOUNG GREY MULLETS Mugil cephalus

Oxygen consumption (OC) is one of important factors in aquaculture activities, as the oxygen is a vital condition for all the organisms living in the water and having an aerobic type of respiration. OC is the preferred method for measuring and reporting the metabolic rate in fish. The aims of this study were to evaluate the effects of salinity on OC and blood properties of grey mullets. Five experimental groups were conducted to measure OC and blood properties of grey mullets Mugil cephalus (BW: 187.9 ± 45.8 g) according to salinity (30→0 psu, 0→30 psu) changes; SDS: fish reared in seawater (SW, 30 psu) directly shifted to SW, SGF: SW fish gradually shifted to freshwater (FW, 0 psu), SDF: SW fish directly shifted to FW, FDF: FW fish directly shifted to FW, and FDS: FW fish directly shifted to SW. The result showed that OC tended to decrease in the groups of SW fish shifted to FW showing 194.5 mg O 2 /kg/h at 25°C in SDS to 82.4 mg O 2 /kg/h at 15°C in SGF. On the contrary, OC increased in the groups of FW fish shifted to SW showing 80.5 mg O 2 /kg/h at 15°C in FDF to 184.0 mg O 2 /kg/h at 25°C in FDS. Cortisol levels at the end of experiments were rapidly increased with the lowering salinities in SW fish shifted to FW showing 20.6 ng/mL in SDS to 316.2 ng/mL in SDF, while those were decreased with the increasing salinities in FW fish shifted to SW showing 40.2 ng/mL in FDF to 10.3 ng/mL in FDS. However, glucose levels showed no significant differences among all experimental groups. Based on the information from this study, aquaculture of grey mullet might be applied or developed in freshwater due to its osmotic adaptation ability.

INTRODUCTION
Oxygen consumption (OC) is one of a vital parameter for all the organisms living in the water and having an aerobic type of respiration. OC is the preferred method for measuring and reporting the metabolic rate in fish. OC rate by fish is useful in determining carrying capacity of fish in culture system and in predicting aeration needs and flow rates in various aquaculture environments (Lovell, 1998).
Related with salinities, grey mullet Mugil cephalus is one of interesting species to be observed for aquaculture development, especially for freshwater aquaculture development. Grey mullet is a good osmoregulator species which can be found in coastal marine, brackish waters, and freshwater. They move between marine and freshwater environments of rivers and flooded rice fields (Saleh, 2008). Therefore, basic information on OC and blood properties of grey mullets is needed for development of grey mullets' freshwater aquaculture. The aims of this study were to evaluate the effects of salinity on OC and blood properties of grey mullets.

MATERIALS AND METHODS
Thirty one grey mullets Mugil cephalus (TL: 27.3 ± 2.1 cm, BW: 187.9 ± 45.8 g) which were collected from Suncheon Bay and reared in culture tanks were used for the experiments. Before the experiments, grey mullets were divided and acclimated into two different rearing environments, which were seawater (SW) and freshwater (FW). The fish were reared in recirculating tanks and fed two times a day at 2% of their body weight with commercial feed. No food was given to any experimental fish for 24 hours until experiments.
Five experiments were divided into two groups, the groups of SW fish shifted to FW (SDS, SGF, and SDF), and the groups of FW fish shifted to SW (FDF and FDS). SDS: fish reared in SW directly transferred to SW, SGF: fish reared in SW gradually transferred to FW, SDF: fish reared in SW directly transferred to FW, FDF: fish reared in FW directly transferred to FW, FDS: fish reared in FW directly transferred to SW.
The experiments were conducted to observe the effect of different salinity level on OC of grey mullets, from 0 psu (Practical Salinity Unit) up to 30 psu (Table 1). In each experiment, acclimation to exact salinity level was carried out 2 days before fish OC measurement in respiratory chamber running for six days, except in SDF (direct transfer without acclimation).
To measure the OC according to salinity and temperature changes, OC measurement system and OC calculation methods were adapted to Chang et al. (2005). Three grey mullets were put into respiratory chamber (dimension= 20 cm × 30 cm × 20 cm) inside the closed recirculating system with photope-riod of 12 hours light (07:00-19:00) : 12 hours dark (19:00-07:00). DO of inflow water was maintained above 7.0 mg/L in each experiment. During experiment, water temperature inside the OC measurement system was increased slowly from 15°C to the target temperature (15°C, 20C, and 25°C) at a rate of 0.5°C/ h to minimize any thermal shock to the fish.
In addition to measuring the OC, the behavior of the fish was observed during experiments, including their movements in the water and breathing frequency per minute. Behavioral index was used to evaluate the fish activity in each experiment inside the respiratory chamber (Table 2). At the end of each experiment of OC measurement according to salinity and temperature changes, blood samples were collected from three grey mullets from respiratory chamber (Exp.) and three grey mullets from rearing tank (Con.). In lethal DO experiments, blood samples were collected from three of five fish just after the fish died. Fish were anesthetized using 2-phenoxyethanol and blood samples were collected using heparinized syringes, centrifuged (12,000 rpm, 5 min.), and stored in deep freezer until analyzing blood properties of hemoglobin (Hb) and hematocrit (Ht) as the oxygen binding factor. Na + , K + , Cl -, Ca, Mg, and osmolality as the osmoregulation factor. Furthermore, cortisol, glucose, and total protein were analyzed for the stress factor. Hematocrit (Ht) was analyzed by using microhematocrit reader (Micro Hematocrit Reader, Hawksley). Hemoglobin (Hb), Na + , K + , Cl -, Ca, Mg, glucose, and total protein were analyzed by using Chemical Analyzer (Fujifilm Dri-Chem 3500i, Japan). Plasma cortisol was analyzed by Enzyme Immonoassay (EIA) using Cortisol EIA kit (Oxford Biomedical Research, USA). Plasma osmolality was examined with Vapor Pressure Osmometer (Vapro 5520; Wescor Co., USA).

SDS SGF SDF
The data were analyzed by using PASW Statistics 18 software. One-way ANOVA was conducted to analyze OC and physio-chemical properties of blood according to different salinity experiment, two-way ANOVA was conducted to analyze the interaction between salinity and temperature on OC. and t-test was conducted to analyze the differences of OC according to different photoperiod and compare the physio-chemical properties of blood between control and experiment.

Oxygen Consumption According To Salinity
The grey mullets from the groups of SW fish shifted to FW and the groups of FW fish shifted to SW showed various rhythm in closed recirculating system at each water temperature from the continuous OC measurements with stepwise rising temperature from 15°C to 25°C. As shown in Figure 1 and 2, the changes in OC with water temperature for each experiment showed a linear increase in line with water temperature, except the SDF. The OC of grey mullets which determined every hour clearly showed various type of OC fluctuations. Sudden increase of OC occured in each experiment during the beginning of light period and dark period with the various amount in each experiment.
The OC of grey mullets in SDS during the light period was higher than dark period at each temperature ( Figure 1). Same patterns with SDS were also found in SDF and SGF, while grey mullets in FDF showed a reversed day/night OC rhythm and consumed less oxygen during the day period. The behavioral change also occured in FDS. In FDS, the grey mullets shifted to consume oxygen higher in light period start from 20°C.

FDF FDS
In the groups of SW fish shifted to FW, grey mullets consumed 112.4, 136.0, and 194.5 mg O 2 /kg/h at 15°C, 20°C, and 25°C, respectively in SDS, showing a linear increase in OC with water temperature, which was significantly different at each temperature (P<0.05). In SGF, grey mullets acclimated in freshwater by gradual salinity change consumed lower amount of oxygen compare to SDS in each temperature. The value was 82.4, 124.1, 164.6 mg O 2 /kg/h at 15°C, 20°C, and 25°C, respectively (P<0.05). These values were also lower compare to FDF and FDS, showing clearly different response due to the different salinity. Another result showed that grey mullets consumed 95.5 mg O 2 /kg/h at 15°C in SDF. Meanwhile, OC data from SDF experiment during 20°C and 25°C cannot be observed due to fish mortality during experiment caused by the effect of abrupt salinity changes from SW to FW (Table 3).
OC of fish increased linearly with the temperature rise in this study. The OC of fish reflected the activity of fish itself (Beamish & Mookherjii, 1964). This seems in this present study the activity of fish including their breath frequency increased by the rising of temperature.
The grey mullets in SDS showed that the energy demand of grey mullets reared in seawater elevated on active at the morning. This result was opposite Figure 2. OC of grey mullets Mugil cephalus by water temperature in the groups of FW fish shifted to SW; FDF and FDS are the same abbreviations as shown in Table 1 Table 3 Note: All abbreviations are the same as shown in Table 1. Each values represent means ± SD (n = 24). Different letters indicate significant differences between water temperatures in each experiment, respectively. Asterisks indicate significant differences within groups of seawater to freshwater and within the groups of FW fish shifted to SW (P<0.05, one-way ANOVA) to FDF where grey mullets reared was active at night. Another pattern occured in some experimental groups, which was slightly different. The OC behavior shifted from consumed lower amount at dark period to lower amount at light period or the vice versa. Lyytikäinen & Jobling (1998) concluded that daily variations in OC had an influence on the water requirements of fish in aquaculture, water requirements should be estimated according to peak OC rates rather than the daily average OC.
OC tended to decrease in the groups of SW fish shifted to FW showing the highest value of 194.5 mg O 2 /kg/h at 25°C in SDS and the lowest value of 82.4 mg O 2 /kg/h at 15°C in SGF. On the contrary, OC increased in the groups of FW fish shifted to SW showing the highest value of 184.0 mg O 2 /kg/h at 25°C in FDS.
A two-way ANOVA was conducted that examined the effect of salinity and temperature on OC. There was a significant interaction between the effects of salinity and temperature on OC within the groups of FW fish shifted to SW, while the interaction between the effects of salinity and temperature within the groups of SW fish shifted to FW was not significant (P≥0.05) ( Table 4).
The fish in each experiment showed various pattern on OC according to salinity in both of group during light and dark period. In the groups of SW fish shifted to FW, the average of OC in SDS during the dark period was 81.2%, 91.5%, and 90.7% lower than light period at 15°C, 20°C, and 25°C, respectively. Same pattern was found in SGF and SDF. In SGF, the average of OC during the dark period was 97.8%, 90.9%, and 95.0% lower than light period at 15°C, 20°C, and 25°C, respectively. Meanwhile, the average of OC in SDF during the dark period was 51.2% lower than light period only at 15°C. There was no record of OC at 20°C and 25°C due to fish mortality.
In the groups of FW fish shifted to SW, another different pattern was found in FDF, the average of OC during the dark period in FDF was 111.9%, 120.6%, and 115.7% higher than light period at 15C, 20C, and 25°C, respectively. Which means the OC pattern was all higher in dark period than light period. Meanwhile, the average of OC in FDS during the dark period was 127.0% and 104.1% higher than light period at 15°C and 20°C, and it was 82.7% lower than light period at 25°C. The change of tendency on OC due to salinity changes during light and dark period was found in these experiments (Table 5).
The slope (b) of linear regression in the groups of SW fish shifted to FW during the light period was significantly higher than that of the groups of FW fish shifted to SW. Same pattern was also occured during the dark period. The slope (b) of linear regression in the groups of SW fish shifted to FW during the dark period was significantly higher than that of the groups of FW fish shifted to SW. These conditions indicated that increment of OC of grey mullets were faster in the groups of SW fish shifted to FW than the groups of FW fish shifted to SW (Table 5).
Increasing or decreasing of OC clearly related with salinity changes due to stress response reflecting in immune-related parameters. It caused physiological disturbances and change photoperiodical activity and metabolism of fish. Fish mortality could be occured by the abrupt salinity changes from seawater to freshwater. In contrary, Arnason et al. (2013) on Atlantic cod Gadus morhua that were reared in salinity and abrupt salinity changes have limited or no effects on stress and immune-related parameters, and there were no indications of ion regulatory disturbances at low salinities. Meanwhile, the effect of salinity change from freshwater to seawater will not cause the mortality due to grey mullets' osmoregulation ability to tolerate seawater as its natural habitat.

Fish Behavior and Breath Frequency
As shown in Table 6, the fish in each experiment showed six subsequent responses based on their swimming activity and breath frequency. All experimental groups were in normal behavior during experiments, showing stable activities with behavioral index from I to III, except for SDF. SDF showed abnormal behavior with index from IV to VI because of abrupt salinity change. The highest index was found in SDS, SGF, and FDS, while the lowest index was found in SDF. The breath frequency of grey mullets from whole experiments at 15°C, 20°C, and 25°C were shown in Figure 3. The slope of linear regression of breath frequency according to different water temperature in grey mullets from the groups of SW fish shifted to FW which consisting of SDS and SGF was 3.07 and 1.81, respectively, while the slope of linear regression of breath frequency according to different water temperature in grey mullets from the groups of FW fish shifted to SW which consisting of FDF and FDS was 0.99 and 2.18, respectively. These values were indicating the breath frequency was higher at SW condition. The slope of linear regression of breath frequency from the results might be related with fish metabolism. Grey mullets had higher metabolic rates during SW rearing condition, while the result was vice versa during FW rearing condition. Morgan & Iwama (1991) suggested that low metabolic rates were the most often associated to the water salinity, which was in species that was commonly found and adapted at a particular life stage. This condition seems to be related with the natural environment of grey mullets. Grey mullets occured in seawater as their natural habitat. The grey mullets Mugil cephalus, migrates to feed, moving from seawater to brackish as it grew from fry to adult (Kim et al., 2004).

Blood Properties
The salinity change from SW to FW tended to decrease Ht and Hb values in the groups of SW fish shifted to FW. The highest value of Ht and Hb was 30.6% and 7.3 g/dL in SDS, respectively. Whereas, Ht and Hb tended to increase in the groups of FW fish shifted to SW.   I  II  II  I  I  SGF  II  II  II  I  I  SDF  IV  V  VI  --FDF  II  II  II  II  II  FDS  II  II  I  I  I  47.9 ± 0.2% and 8.5 ± 0.6 in FDS, respectively (Table  7).
Na + values were decreased within the groups of SW fish shifted to FW from 163.5 mEq/L in SDS to Figure 3. Breath frequency per minute and OC per breath in grey mullets Mugil cephalus from the groups of SW fish shifted to FW and the groups of FW fish shifted to SW; All abbreviations are the same as shown in Table 1 134.5 mEq/L in SDF, while those values were increased within groups of FW fish shifted to SW from 136.5 mEq/L in FDF to 162.0 mEq/L in FDS. Cland Ca values also showed the same pattern. Clvalues decreased showing 155.5 mEq/L in SDS to 134.5 mEq/L in SDF,  while those increased from 152.0 mEq/L in FDF to 158.0 mEq/L in FDS. Furthermore, Ca values decreased from 12.6 mg/dL in SDS to 8.4 mg/dL in SDF. Those increased from 9.4 mg/dL in FDF to 12.0 mg/dL in FDS. Other factors such as K + and osmolality were not significantly different. The highest value of Mg was found at 3.2 mg/dL in SDF, and the lowest value was found at 1.3 mg/dL in SGF (P<0.05) ( Table 8).
Cortisol levels at the end of experiments increased with the lowering salinities in SW fish shifted to FW showing 20.6 ng/mL in SDS to 316.2 ng/mL in SDF, while those decreased with the increasing salinities in FW fish shifted to SW showing 40.2 ng/mL in FDF to 10.3 ng/mL in FDS. However, the glucose levels among all the experimental groups were not significantly different, while total protein values were different only in the groups of FW fish to SW with the highest value of 3.6 g/dL in FDS, while the lowest value was 2.7 g/dL in FDF (P<0.05).
The results relating to the blood properties showed that salinity changes affected to the oxygen binding factors, and its clearly related to the OC rate. Changes in Ht and Hb according to salinity found somewhat similarity in Ht from other study (Chang & Hur, 1999;Chen et al., 1995;Houston & Rupert, 1976). Ht and Hb values from this study were also compared to grey mullets (30.5 ± 2.9% and 8.0 ± 1.5 g/dL) which was reported by Min et al. (2010). Some experimental groups had somewhat similar in Ht and Hb factors, but some experimental groups had lower results. Changes in blood factors caused by salinity on fish Channa punctatus was also reported by Dheer et al. (1986).
The results of biochemical properties of grey mullets' blood plasma also indicated that salinity changes in grey mullets clearly affected their blood factors. Some ion contents on grey mullets in FW environments were lower amount than that of grey mullets in SW. Almost 77% of the salts in blood were sodium and chloride. The remainder was made up primarily of bicarbonate, potassium, and calcium. Sodium (Na + ), potassium (K + ), and calcium (Ca) salts were critical for the normal function of heart, nerve, and muscle tissue (Wurts, 1998).
Salinity changes from SW to BW or FW which was lower amount of ions than that of SW caused the grey mullets became weaker and lower in their metabolism than before. Khodabandeh et al. (2009) observed golden grey mullets and concluded that the main ion content such as Na + , Cl -, and K + were lower in line with the decreasing salinity. This result could be the main reason of the difference behavior in activity and OC according to salinity changes. To inhabit at low salinity or freshwater, fish need to replace salts lost through diffusion to the water and eliminate excess water absorbed from the environment (Wurts, 1998).
The results of OC and blood properties also showed relationship between OC and other blood properties parameters such as osmolality, cortisol, and glucose. The correlation can be found between OC and cortisol, while the osmolality and glucose were not clearly correlated to OC since the values were not significantly different (Table 9).
Salinity changes from SW to FW tended to decrease the OC of grey mullet and increased the cortisol levels inside the blood. However, salinity changes from FW to SW tended to increase the OC and decreased the cortisol levels. These results suggested that stress factors clearly affected to OC of grey mullets. At the stress conditions by salinity changes, these salinity differences result in modifications in OC and energy demands (Morgan & Iwama, 1991). Cortisol was assigned to stimulate the liver gluconeogenesis and elevating blood sugar levels (Wedemeyer et al., 1990), which was related to the Table 9. OC, osmolality, cortisol, and glucose levels of grey mullets Mugil cephalus in each experiment Note: SDS, SGF, SDF, FDF, and FDS are the same abbreviations as in Table 1. Letters indicate significant differences between each experiment (P<0.05, one-way ANOVA). fish osmoregulation ability. In other results, grey mullets exposed in low salinity did not change the osmolality levels, indicating that grey mullets was a good osmoregulator species. These results were similar with gulf killifish (Boily et al., 2007) and genus Fundulus (Griffith, 1974).

CONCLUSION
Salinity clearly affected the oxygen consumption and blood properties of grey mullets Mugil cephalus. Lowering salinity from seawater to freshwater decreased the oxygen consumption and causing mortality if the grey mullets transferred directly from seawater to freshwater without gradual salinity changes. Meanwhile, salinity change from freshwater to seawater increased the oxygen consumption of grey mullets. Furthermore, lowering salinity tend to increase the stress level of grey mullets. It mainly identified by the increase of cortisol levels during low salinity environment. Meanwhile, the stress level of grey mullets decreased with the increase of salinity. According to the information from this study, grey mullet rearing in freshwater might be applied for aquaculture development due to its osmotic adaptation ability.


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Effect of Harvest Time on Yield, Chemical Composition, Antimicrobial and Antioxidant Activities of Thymus vulgaris and Mentha pulegium Essential Oils

Department of Food Technology, Research Unit of Nuclear Technology, Environment and Quality Laboratory of Food Technology, National Institute of Agronomic Research (INRA), Tangier, Morocco. Department of Animal Production and Food Science, Veterinary Faculty, Universidad de Zaragoza, Spain. 3 Department of Chemistry, Faculty of Science and Technology, Université Abdelmalek Esaâdi, Morocco. 4 Department of Biology, Faculty of Science and Technology, Université Abdelmalek Esaâdi, Morocco. Department of Biology, Polydisciplinary Faculty of Larache, Université Abdelmalek Esaâdi, Morocco.

INTRODUCTION
Essential oils of medicinal and aromatic plants are subjected to a high demand. This demand is justified by the biological effects exhibited by some active compounds contained in these oils and by their condition of natural products as an alternative for replacing chemical substances in the pharmaceutical, cosmetic and food industries [1].
Among biological properties investigated in EOs, antimicrobial and antioxidant activities have been widely demonstrated [2][3][4][5][6]. For this reason, they have been proposed as potential agents for food preservation.
Nowadays, the major concern for EOs producers is to obtain oils with better quality and quantity, and with known and stable biological properties. However, the quantity (yield) and the quality (chemical composition) of EOs are influenced by the interaction among several factors such as genotype, environment, plant age, season and time of harvest [7][8][9][10][11].
While the effect of the vegetative cycle on yield and chemical composition of EOs has been well demonstrated [7,8,[12][13][14][15], its implication on biological properties has scarcely been studied. Among the few published data in this sense, it can be deduced that the plant vegetative cycle is of great importance in determining EOs biological properties [16,17].
Due to its geographical position, Morocco offers a wide range of Mediterranean bioclimates allowing the growth of rich flora, which is constituted by more than 4200 species. Aromatic and medicinal plants are estimated by 500 to 600 species, and most of them are endemic [18].
Morocco is a traditional supplier of medicinal and aromatic plants to Europe. More than twenty plants are used for EOs or other aromatic extracts production to be used in perfumery and cosmetic industry, as hygiene products, etc.
T. vulgaris and M. pulegium are two spontaneous aromatic and medicinal plants belonging to the Lamiaceae family that grow in several regions of Morocco. Both aromatic plants have an undeniable commercial interest because of the diverse biological activities of their extracts which have been widely demonstrated [19][20][21][22].
Despite the great interest of both plants, to the best of our knowledge, there are no studies about the effect of the vegetative cycle (preflowering and full-flowering stages) on their antioxidant or antimicrobial activities, which makes difficult the adequate exploitation of these natural resources.
This study aims to investigate the effect of the vegetative cycle (pre-fowering and full flowering stages) on the yield, chemical composition, antimicrobial and antioxidant activity of the EOs of T. vulgaris and M. pulegium harvested in the north and north east of Morocco, respectively.

Plant Material
The studied plants (T. vulgaris and M. pulegium) were collected during the period of April 2011 (Pre-flowering) to June 2011 (Full-flowering). T. vulgaris plants were harvested in the region of Beni Idder (391m, 35°23'59"N, 5°30'35"W) in the Northeast of Morocco and M. pulegium plants from Bougedour in the North (35m, 35°39'35"N, 5°50'59"W). The taxonomic identification was performed following the procedure described by Fennane et al. [23]. Subsequently, plants were dried for 48 h at 40ºC under ventilation. Leaves and flowers were used for the extraction of EOs.

Essential Oils Extraction
The EOs were extracted via steam distillation for 2 h using a Clevenger-type apparatus. The supernatant was separated by decantation after adding 50% of NaCl. The EOs were stored in sealed glass vials at 4ºC prior to analysis. The yield was based on the dry weight of the samples (% w/w). EOs were collected and stored at 4ºC in dark bottles in order to protect them from heat and light radiation.

Gas Chromatography/Mass Spectrometry (GC/MS) Analysis
The GC/MS analyses were carried out using a gas chromatograph coupled with Mass Identification of the components was made by the determination of the their retention indices (KI) relative to those of a homologous series of nalkanes (C8-C26) (Fluka, Buchs/sg, Switzerland) and by matching their recorded mass spectra with those stored in the spectrometer data base (NIST MS Library v. 2.0) and the bibliography [24].
Quantification was computed as the percentage contribution of each compound to the total amount present.

Antimicrobial Activity
EO antimicrobial activity was determined by paper disc agar plates. Microbial strains were provided by the Spanish Collection of Type Cultures (CECT). Two Gram negative bacteria, Escherichia coli (SCTC 471) and Salmonella Senftenberg 775W (ATCC 43845), and two Gram-positive, Listeria monocytogenes (SCTC 4031) and Staphylococcus aureus (SCTC 976), were used. A volume of 10 mL of Muller Hinton Agar medium (Liofilchem, Roseto degli Abruzzi, Italy) (MHA) was poured into Petri dishes. One hundred μL of culture bacteria were plated at a final concentration of 10 6 cells/mL approximately.
After 15 min, a paper disc Whatman No. 1 of 6 mm (Whatman International L7d Maidstone, England), impregnated with 10 μL of the EO, was placed on the agar surface. The plates were incubated for 24 h at 37ºC (E. coli, Salmonella Senftenberg, S. aureus) or 48 h at 30ºC (L. monocytogenes). The diameters of the resulting zones of inhibition were measured including the diameter of the paper disc. An inhibition zone (disk diameter included) lower than 9 mm was considered as non inhibitory. An average zone of inhibition was calculated after carrying out three independent replicates.

Radical scavenging activity assay
Following the method described by Blois [25], for each EO, 1 mL from a 1 mM methanol solution of 2,2-diphenyl-1-picrylhydrazyl (Sigma-Aldrich) (DPPH) was added to 3 mL of diluted EO in ethanol (1,15,30 uL/mL), butylated hydroxytoluene (Sigma-Aldrich) (BHT) (reference) or ethanol (control). Mixtures were shaken and incubated (room temperature/dark). After 30 min, absorbance at 517 nm was measured. Scavenging activity of DPPH radical was calculated as follows: where A blank is the absorbance of the control reaction (containing all reagents except the test compound), and A sample is the absorbance in presence of the test compound.

Statistical Analysis
Analyses of variance were performed by Statgraphics Plus 4.0 statistical software. All experiments were performed in triplicate and the results were presented as the mean with its standard deviation (n = 3) in each case. Differences were considered significant at P <0.05.

Yield and Chemical Composition
The yields of EO extraction for T. vulgaris and M. pulegium plants, as a function of the vegetative cycle, are illustrated in Table 1. For T. vulgaris EO, average yield at pre-flowering period was 1.9% while it was 3.6% at full-flowering stage. These percentages are relatively high when compared to some published data that found values ranging from 0.5 to 1.2% [7,18,26]. Table  1 also shows that yield values for M. pulegium EO were 0.9% and 3.5% for pre and fullflowering period respectively. These percentages were similar to those obtained by Hmiri et al. [20]. Therefore, these results demonstrate that the highest yield was obtained during fullflowering period. Therefore, it seems advisable to control the plants vegetative period in order to optimize oil production and obtain the maximum yield per plant. Similar trend has been previously reported by other authors [27,28]. Verma et al. [29] reported that EOs high yield at this stage is probably due to its ecological role in attracting pollinators and in being an antifungal defense mechanism.
The EOs obtained at pre-and full-flowering period were analyzed by GC-MS and results are included in Tables 2 and 3 for T. vulgaris and M. pulegium respectively. Chromatographic analysis highlighted the occurrence of six main components for T. vulgaris EO at higher levels than 0.5% of the content. Carvacrol was the major component with a content ranging from 70.97 to 74.10% accompanied with other components at relatively low levels: δ-terpinene (1.05 to 1,09%), p-cymene (5.17 to 8.74%), terpinene (4.08 to 5.89%), β -humulene (2.40 to 3.04%). Our results are in accordance with those obtained by Bouhdid [30] who found that carvacrol was the major compound. However, they differ from those obtained by Imelouane et al. [26] in eastern Morocco and Jordan et al. [8] from Murcia region in Spain. For Imelouane et al [26], camphore and camphene were the major compounds, while 1,8-cineole (29.2 to 36.5 %) and terpenyl acetate (18.2 to 25%) were identified as major compounds for Jordan et al. [8]. Such variations between countries could be due to numerous factors, such as differences in climatic conditions, geographical location, season at the time of collection, stage of development, processing of plant materials before extraction of the oils, and occurrence of different chemotypes. Table 2 also shows that vegetative cycle affects significantly the chemical composition of T. vulgaris EO. The differences consist of the variation of carvacrol percentage that increased from 70.97 to 74.10% and the decrease in the content of p-cymene from 8.74 to 5.17 and terpinene from 5.89 to 4.08%. Our results are in accordance with those obtained by Hudaib et al. [7] who found that the chemical composition of EOs varied markedly with vegetative cycle. These authors concluded that the end of vegetative cycle was the best period to obtain EOs with better quality and quantity. We also noted that when the content of carvacrol increased, the content of p-cymene and terpinene decreased. This might be explained by the simultaneous bioconversion of p-cymene and -terpinene to carvacrol [31].
Our results are similar to those reported by Sutour [32] who found that pulegone was the major compound, but differ from those of Mahboubi and Haghi [33] and Verma et al. [29] who identified piperitenone and menthol as the major constituents. As in T. vulgaris EO, mostly quantitative rather than qualitative variations were observed with respect to chemical composition as affected by vegetative cycle stages. The differences were reflected in the variation of pepiritenone percentage that increased from 1.71 to 6.54%, and the decrease of menthone from 6.25 to 1.37%. Nevertheless, the content of the major component (pulegone) was not affected. Similarly, Verma et al. [29] described considerable variation in the chemical composition of Mentha arvensis and Mentha piperita EOs at different stages of plants growth.

Antimicrobial Activity
The average diameters of the inhibition zone observed around the disks impregnated with the EOs of both plants studied are summarized in Table 4. According to these results, T. vulgaris EO showed a strong activity on all tested bacterial strains based on the inhibition diameters obtained between 30.7 and 36.7 mm. The largest antimicrobial activity was observed against L. monocytogenes (36.7 mm) and the weakest against E. coli (30.7 mm). Our results also demonstrated that Gram-positive bacteria were more sensitive to T. vulgaris EO than Gram-negative bacteria. Similar trend was observed by Rota et al. [3]. The resistance of Gram-negative bacteria to EOs is partly due to the complexity of the cell wall of these microorganisms which contains an outer membrane, unlike the simpler cell wall structure of the Gram-positive ones [1].
Despite the chemical variation of T. vulgaris EO between the two vegetative stages investigated (pre-and full-flowering period), this variation did not statistically (P <0.05) affected its antimicrobial activity against E. coli, Salmonella Senftenberg and S. aureus. Only L. monocytogenes was more sensitive to the EO from the full-flowering period. The difference in sensitivity of L. monocytogenes between preand full-flowering periods might be due to the higher concentration of carvacrol at this stage, which is a very effective antimicrobial agent, especially against Gram positive bacteria [5,34,35].
M. pulegium EO showed a lower antibacterial activity compared to the EO of T. vulgaris (Table  4); the inhibition diameters ranged from 9 mm to 12.7 mm. The largest antimicrobial activity was obtained against E. coli (12.7 mm) and the weakest against Salmonella Senftenberg (9 mm). Our results differ from those described by Ait-Ouazzou et al. [36] and Teixeira et al. [37] that demonstrated a higher antimicrobial activity of M. pulegium EOs.
Like in T. vulgaris, the chemical variation of M. pulegium EO during the vegetative cycle did not affect its antimicrobial activity against E. coli, Salmonella Senftenberg and S. aureus. In fact, the concentration of the major compound (pulegone) did not vary with the vegetative cycle. Again, only L. monocytogenes was more sensitive to the EO obtained during the preflowering period. The difference in sensitivity of L. monocytogenes between pre-and fullflowering periods could be due to the main differences found: the concentrations of menthone that changes from 6.25 to 1.37% and pepiritenone from 1.71 to 6.54% Therefore, these results point out the fullflowering period as the most appropriate time to harvest T. vulgaris EO and the pre-flowering for the M. pulegium EO in order to guaranty their greatest antimicrobial effect.

Antioxidant Activity
The effect of the vegetative cycle on the scavenging activity of DPPH radical of M. pulegium and T. vulgaris EOs is given in Table 5. As we can see, T. vulgaris EO showed a greater antioxidant activity than M. pulegium EO. Antioxidant activity of T. vulgaris increased with increasing concentration and it was comparable to the antioxidant effect of BHT at a concentration of 15 µL/mL. EOs from fullflowering period exhibited significantly higher antioxidant activity (P<0.05). This reduction capacity of free radicals of T. vulgaris EO is mainly due to its chemical profile, rich in carvacrol that has already been proved to possess a strong antioxidant activity [38][39][40].
Opposed to the results obtained with T. vulgaris EO, M. pulegium EO exhibited a higher antioxidant activity at pre-flowering than fullflowering period. Probably, the difference between the antioxidant activities from the two periods studied was related to the proportion of the present active components, especially the  content of menthone that decreased from 6.25 (pre-flowering) to 1.37% (full-flowering). Our results are in accordance with those obtained by Zhen et al. [41] who observed that the antioxidant activity of Citrus medica EOs decreased with increasing maturity at harvest. Also, Hussain et al. [16] showed that the antioxidant activity of Ocimum basilicum EOs varied significantly with the season.
So, our results demonstrate that full-flowering is not always the best period to harvest aromatic and medicinal plants with better antioxidant activity, but that depends on the type of plant.

CONCLUSION
As far as we know, this is the first time that the effect of vegetative cycle on the yield, chemical composition, antimicrobial and antioxidant activity of T. vulgaris and M. pulegium EO from Morocco has been studied.
According to our results, in general, full-flowering period is the best period to harvest T. vulgaris plants in order to produce EOs with better yield, antimicrobial and antioxidant activity. In contrast, pre-flowering stage is recommended to obtain M. pulegium EO with higher antioxidant and antimicrobial activity. The optimization of the harvest time will facilitate the exploitation of these natural resources and will allow producers to obtain EOs with better quality and quantity.

CONSENT
It is not applicable.


text:
Nutrient-rich versus nutrient-poor foods for depressed patients based on Iranian Traditional Medicine resources.

Objectives: Considering the positive effects of certain nutrients on depression, increasingly prevalent in the contemporary societies, we investigated the nutritional content of prescribed and prohibited foodstuffs for depressed patients in Iranian Traditional Medicine resources. Materials and Methods: In order to conduct the study, credible sources of Iranian Traditional Medicine were primarily reviewed for the prescribed and prohibited foodstuffs for depressed patients. USDA database, as a well-known and valuable source, was then visited to determine the amount of effective nutrients in each foodstuff. Finally, the obtained amounts were compared with each other in three food groups, namely vegetables, fruits and nuts and also high protein products. Results: In Iranian Traditional Medicine texts, the following are prescribed for depression management: basil, coriander, spinach, lettuce, squash, peppermint, dill, chicory, celery, chard, quince, cucumber, watermelon, grape, peach, pomegranate, banana, apple, currant, pistachio, dried fig, almond, egg, chicken, lamb, and trout; cabbage, eggplant, onion, garlic, broad beans, lentils, and beef, meanwhile, are prohibited. In this regard, the effective nutritional content of these foodstuffs was obtained and then compared in the three food groups. Conclusion: This study revealed that spinach, lettuce, chicory, and squash (vegetables), pomegranate and almond (fruits and nuts) and ultimately trout (high protein products) are the best effective foodstuffs on depressed patients from nutritional content aspect.

Introduction
Major depressive disorder (unipolar major depression), dysthymic disorder, and minor depressive disorder represent depressive syndromes which are distinguished by their type and number of symptoms as well as their duration.
Depressive symptoms may include depressed mood states, loss of interest or pleasure in most or all activities, insomnia or hypersomnia, changes in appetite or weight, psychomotor retardation or agitation, low energy, poor concentration, thoughts of worthlessness or guilt, and recurrent thoughts about death or suicide (American Psychiatric Association, 2000). Accordingly, depression is a major public health issue associated with increased functional disability (Donohue and Pincus, 2007). Furthermore, depressive disorders (Richards, 2011) and some of their significant complications such as cardiovascular diseases (Nemeroff and Goldschmidt-Clermont, 2012), Alzheimer (Wint, 2011), and diabetes (Silva et al., 2012) are becoming more prevalent in modern societies. Nowadays, pharmacotherapy and psychotherapy are the more widespread treatments for depressive disorders. In the scope of pharmacotherapy, long-term drug therapy leads to impairments in the intellectual and cognitive functions alongside other considerable side effects. To this end, public tendency for alternative or complementary treatments with fewer side effects has been recently intensified. (Sadock et al., 2007).
Iranian Traditional Medicine (ITM) as a Complementary and Alternative Medicine (CAM) involves several nonpharmacological methods of treating melancholia (a psychotic type of depression) like food therapy, aromatherapy, color therapy, phototherapy, voice therapy, exercise, massage, sleep strategies, bathroom strategies, anointment, sexual intercourse, puking, feces laxation, induced menstruation, game therapy, narrative therapy, and traveling. One of the most notable of these methods is food therapy which includes singular and combined foodstuffs, and nutrition rules (AhwaziArjani, 1973;Ibn-e Sina, 2005;JorJani, 1976).

Materials and Methods
In this study, three authoritative ITM references, namely Kamel al-Sanaat al-Tibbyyah, Al-Qanun fi al-Tibb, and Zakhireh Kharazmshahi were carefully reviewed to extract prohibited and prescribed foodstuffs concerning depression management. Based on these resources, melancholia can be considered as a psychotic type of depression.  Arjani, 1973;Ibn-e Sina, 2005;JorJani, 1976). It should be noted that the scientific names of the vegetarian foodstuffs were retrieved from USDA database (United States Department of Agriculture, 2011).
It is worth mentioning that, considering the positive effect of polyunsaturated fatty acids on depression (Kraguljac, 2009), the omega-3 content of the target foodstuffs has also been investigated in another study . However due to the importance of this topic, we referred to the findings of mentioned study; wherever it is necessary in the present research.
If a specific nutrient amount of a foodstuff was not specified in USDA database, we would try to approximate the intended amount based on the information given for an alternative foodstuff in USDA database. The needed data, however, were extracted from a supplementary reference (Dorosti and Tabatabai, 2007) in the case where USDA method was not feasible. It was impossible to obtain the amount of coriander, pomegranate, quince amino acids and quince vitamin E through these two methods, hence their exclusion from calculation and analysis.
In the second step, each nutrient's amount in 100 g of each foodstuff was divided into the necessary daily value of each nutrient determined based on the specified values for an adult male (Mahan and Escott-Stump, 2008). After that, the result was multiplied by 100 to determine the percentage of each nutrient's necessary daily value provided by 100 g of each foodstuff. In this way, the percentage of "provided nutrient per 100 g" was calculated for every nutrient in each foodstuff.
In the third step, the percentage of "provided nutrient per 100 g" of each foodstuff for every nutrient was divided into 100. The results were then multiplied by specified serving sizes (g) for each foodstuff (Clínica Mayo, 2002;Ghaffarpoor et al., 2001) so as to determine the percentage of necessary daily value of each nutrient provided by a serving size of each foodstuff. In this way, the percentage of "Provided Nutrient per Serving size" (PNS) was obtained for every nutrient contained in each foodstuff.
Next, PNS values were separately calculated in five groups of amino acids, fat soluble vitamins, water soluble vitamins, minerals and carbohydrates. for each foodstuff.

Statistical analysis
In the vegetables group, the PNS values of amino acids, fat soluble vitamins, water soluble vitamins, minerals, and carbohydrates were compared in both prescribed and prohibited foodstuffs using t-test (SigmaPlot12), where P-value <0.05 was considered as statistically significant.

Results
Since it was necessary to separately compare the nutritional content of the prescribed and prohibited foodstuffs, the results were divided to three groups of 1) vegetables, 2) fruits, dry fruits and nuts, and 3) high protein products.

Vegetables
Tables 1 and 2 indicate the PNS values of the five groups of amino acids, fat soluble vitamins, water soluble vitamins, minerals, and carbohydrates along with total nutrients contained in both prescribed and prohibited vegetables.
In addition, table 3 shows the results of the comparisons made among the PNS averages of amino acids, fat soluble vitamins, water soluble vitamins, minerals, carbohydrates, and total nutrients between prescribed and prohibited vegetables. According to the obtained results, the prescribed vegetables group surpassed the prohibited vegetables group in minerals and fat-soluble vitamins tracks (Table 3).
Additionally, investigations without using software in the cases of statistically insignificant were also carried out as follows: In the case of amino acids, the difference between the two groups was in favor of the prescribed group, but it was not significant. On the other hand, PNS values of amino acids in spinach, chicory, and lettuce were more than eggplant, onion, garlic, and cabbage (Table 1 and 2).
In the case of water soluble vitamins, the difference between the two groups was in favor of the prescribed group, but it was not significant. On the other hand, PNS values of water soluble vitamins in spinach, chicory, lettuce, squash, and dill were more than eggplant, onion, garlic and cabbage (Table 1 and 2).
In the case of carbohydrates, the difference between the two groups was in favor of the prohibited group but it was not significant. On the other hand, PNS values of carbohydrates in chicory and squash were more than eggplant, onion, garlic, and cabbage (Table 1 and 2).
Regarding omega-3 content, the ratio of omega-6 to omega-3 in mint, spinach, basil, lettuce, and squash was less than all the prohibited vegetables. Nevertheless, coriander, dill, chicory, celery and chard, which are actually prescribed in this group, do not have the same properties. It is worth mentioning that the fewer the amount of this ratio in a foodstuff the more desirable it will be; and the best defined ratio of omega-6 to omega-3 in one foodstuff was 2:1 or 3:1 (Mahan and Escott-Stump, 2008).
The results showed that spinach and lettuce, with higher PNS values of amino acids and water soluble vitamins and also lower ratio of omega-6 to omega-3, surpassed all the prohibited vegetables in these three tracks. Furthermore, chicory contained higher PNS values of amino acids, water soluble vitamins, and total carbohydrates, hence surpassing all of the prohibited vegetables in these three tracks.  Squash with higher PNS amounts of water soluble vitamins and total carbohydrates and also lower omega-6 to omega-3 ratio, surpassing all the prohibited vegetables in these three tracks. Dill, with higher PNS values of water soluble vitamins exceeded all of the prohibited vegetables in this particular track. Also, peppermint and basil with lower omega-6 to omega-3 ratio surpassed all of the prohibited vegetables in this track. Consequently, spinach, lettuce, chicory, and squash were more effective than all of the prescribed vegetables through three other tracks except for minerals and fat soluble vitamins (spinach and lettuce: amino acids, water soluble vitamins, omega-6 to omega-3 ratio chicory: amino acids, water soluble vitamins, total carbohydrates Squash: water soluble vitamins, total carbohydrates, omega-6 to omega-3 ratio).

Fruits, dry fruits, and nuts
Among the fruits, the total PNS decreases in pomegranate, peach, grape, banana, watermelon, quince, cucumber, and apple respectively. But it should be considered that the amount of amino acids in pomegranate and quince and also the amount of vitamin E in quince were not specified. Among the dried fruits and nuts, the total PNS decreased in almond, pistachio, currant, and dried fig respectively (Table 4 and 5). Nevertheless, in this section, it was assumed that the more the total PNS value of one foodstuff is, the more effective that foodstuff would be on depressed patients.  It should be noted that according to the study of the prescribed fruits, dry fruits and nuts considering their omega-3 content, among the prescribed fruits, the ratio of omega-6 to omega-3 in cucumber is less than other prescribed fruits. Also, among the prescribed dried fruits and nuts the ratio of omega-6 to omega-3 in pistachio is less than other prescribed dried fruits and nuts.

High protein products
In this group, PNS values of fat soluble vitamins in chicken and egg were more than in broad beans, lentils, and beef. Moreover, lamb had higher PNS amounts of water soluble vitamins than broad beans, lentils, and beef. Also, PNS values of amino acids, fat soluble vitamins, and water soluble vitamins were more in trout than in broad beans, lentils, and beef (Table 6).
Regarding omega-3 content, lamb had a lower omega-6 to omega-3 ratio than broad beans and lentils, yet a higher ratio than beef. Egg, chicken, and trout, also prescribed in this group, did not have the same properties.
The results showed that trout by its amino acids, fat soluble and water soluble vitamins content, lamb, with its water soluble vitamins content and finally chicken and egg with their fat soluble vitamins content, were more effective on depression than broad beans, lentils and beef.

Discussion
The main finding of this study is as follows: in the vegetables group, spinach, lettuce, chicory and squash, in the fruits and nuts group, pomegranate and almond and ultimately in the high protein products group, trout were the best effective foodstuffs on depressed patients considering their nutrients content. But it should be noted that cucumber and pistachio would be more effective than other fruits and nuts if only the omega-6 to omega-3 ratio was to be considered.
This research also revealed that not all the ITM-prescribed foodstuffs for depressed patients are nutrient-rich and not all the ITM-prohibited foodstuffs are nutrient-poor, suggesting that other active ingredients are to be analyzed in the aforementioned foodstuffs. There are several limitations in this study that may affect the generalizability of the findings. One of the limitations is that only USDA database was used in determining the nutritional amounts. USDA database, however, is one of the most valuable and well-known databases in this domain; therefore, the odds that the obtained results were affected by this factor are low. The other point concerning factors affecting generalization is that the assumption in the research was that all nutrients would have a same effect on depression while, in fact, this is not exactly the case.
In addition, previous studies investigated the interaction among certain nutrients (Novitskala, 1950;Reeves and DeMars, 2004) that were not actually considered in the present study. Moreover, considering the strong effect of insoluble fibers on depression, it was necessary to study the values of this case in the prohibited and prescribed vegetables, fruits, and nuts; the USDA database, however, only contained the values of total fiber. The mentioned points would be the unavoidable limitations of this research work.
Many studies have been conducted regarding the nutrition of patients suffering from depression. These studies can be classified in two groups: the first group suggests helpful nutrients while the second proposes appropriate diets or foods.
For example when the amount of a desirable nutrient in a diet is low and the incidence of depression is high, the implication is that the paucity of that nutrient leads to the increasing of depression. On the other hand, when the amount of a desired nutrient in a diet is adequate and the incidence of depression is low, it is concluded that the adequate supplying of that nutrient leads to decreasing of depression. It is noticeable that, the aim of these studies is to determine the helpful nutrients, not the particular foodstuffs providing them helpful nutrients.
The second group of the studies introduces appropriate diets or foodstuffs for these patients. In this group the effect of a specific diet or foodstuff has been investigated on depressed patients. For instance, it has been shown that Mediterranean diet (including vegetables, fruits, grains, frijoles, nuts, dairy, olive oil, and aquatic) has certain positive effects on depression (Sánchez-Villegas, 2006). What seems to be lacking in such studies, however, is the investigation of the effective compounds and their nutritional content in the prescribed foodstuffs.
Unlike the aforementioned groups, the present research introduced prescribed vegetables, fruits, dry fruits, nuts, and high protein products alongside investigating their nutritional content. Prohibited vegetables and frijoles were also proposed along with their nutritive content. In the second group of studies, however, all of the vegetables and frijoles were prescribed without mentioning any exceptions in the framework of the Mediterranean diet (Sánchez-Villegas, 2006). These can be considered as the strong points of the present study.
Certain studies were done in the second group regarding the effectiveness of fish as a providing source of omega-3 fatty acids (Bountziouka, 2009). According to the obtained results, the content of amino acids, fat soluble vitamins, and water soluble vitamins in trout is more than beef (a prohibited foodstuff). Nonetheless, the omega-6 to omega-3 ratio in beef (2:1) is higher than it is in trout (8:1). Since the approximate ratio of omega-6 to omega-3 in wild trout is 2:1, this contradiction could be related to the selection of the framed trout instead of the wild trout in the calculations (Tavakkoli et al., 2014b).
The present study offers spinach, lettuce, chicory, squash, pomegranate, almond and wild trout as foodstuffs having the highest effect on patients suffering from depression, a fact that can be considered by clinicians.
Since in this research work only the effective nutrients on depressed patients have been investigated; study on the possible impacts of other effective compounds in the intended foodstuffs would lead to more accurate results. Moreover, it would be remarkably useful to design observational or interventional studies based on the main findings of the present research.


extfieldsofstudy: Biology Medicine

text:
Identification of genomic regions involved in tolerance to drought stress and drought stress induced leaf senescence in juvenile barley

Background Premature leaf senescence induced by external stress conditions, e.g. drought stress, is a main factor for yield losses in barley. Research in drought stress tolerance has become more important as due to climate change the number of drought periods will increase and tolerance to drought stress has become a goal of high interest in barley breeding. Therefore, the aim is to identify quantitative trait loci (QTL) involved in drought stress induced leaf senescence and drought stress tolerance in early developmental stages of barley (Hordeum vulgare L.) by applying genome wide association studies (GWAS) on a set of 156 winter barley genotypes. Results After a four weeks stress period (BBCH 33) leaf colour as an indicator of leaf senescence, electron transport rate at photosystem II, content of free proline, content of soluble sugars, osmolality and the aboveground biomass indicative for drought stress response were determined in the control and stress variant in greenhouse pot experiments. Significant phenotypic variation was observed for all traits analysed. Heritabilities ranged between 0.27 for osmolality and 0.61 for leaf colour in stress treatment and significant effects of genotype, treatment and genotype x treatment were estimated for most traits analysed. Based on these phenotypic data and 3,212 polymorphic single nucleotide polymorphisms (SNP) with a minor allele frequency >5 % derived from the Illumina 9 k iSelect SNP Chip, 181 QTL were detected for all traits analysed. Major QTLs for drought stress and leaf senescence were located on chromosome 5H and 2H. BlastX search for associated marker sequences revealed that respective SNPs are in some cases located in proteins related to drought stress or leaf senescence, e.g. nucleotide pyrophosphatase (AVP1) or serine/ threonin protein kinase (SAPK9). Conclusions GWAS resulted in the identification of many QTLs involved in drought stress and leaf senescence of which two major QTLs for drought stress and leaf senescence were located on chromosome 5H and 2H. Results may be the basis to incorporate breeding for tolerance to drought stress or leaf senescence in barley breeding via marker based selection procedures. Electronic supplementary material The online version of this article (doi:10.1186/s12870-015-0524-3) contains supplementary material, which is available to authorized users.

Background
Barley (Hordeum vulgare L.) is one of the first cereals domesticated in the Fertile Crescent [1] and today it is the fourth most important crop species concerning acreage next to wheat, maize, and rice [2]. Worldwide, barley is mainly used for animal feed and malting and only a very small amount is used for direct human consumption and bakery. Average yield of barley on the worldwide level is 2.9 t/ha but in some European countries, e.g. Germany average yield is up to 6.5 t/ha [2]. Barley yield in many parts of the world is reduced by biotic stress but also by abiotic stress e.g. heat, salt, deficits in nitrogen nutrition and drought [3][4][5][6]. Especially, in the juvenile stages from sowing to tillering, drought can severely influence barley development already reducing the potential yield [7]. Research on drought stress tolerance has become more important worldwide as due to climate change the number of drought periods will increase in the future [8,9]. Up to now, most studies conducted in barley focused on effects of terminal drought stress whereas drought in juvenile stages is less well documented [10].
Drought tolerance is a complex quantitative trait, that is controlled by various mechanisms [11,12]. Abscisic acid (ABA) is a key phytohormone involved in adaption to environmental stresses and regulation of plant development. It promotes the closure of stomata under drought stress conditions initiated by a loss of turgor [13]. Furthermore, it increases the hydraulic conductivity of water, promotes chlorophyll breakdown and leads to leaf senescence [14]. Another relevant protein is ubiquitin which regulates the degradation of proteins [15,16]. Moreover, late embryo abundant (LEA) proteins and heat shock proteins that are involved in the protection of functional proteins are induced in response to various abiotic stresses [17][18][19]. For example protein kinases and protein phosphatases which activate or deactivate proteins by phosphorylation and dephosphorilation [20], as well as the LEA protein dehydrin which is described to have different functions in different stresses [21] is often present along with drought stress [22]. A lot of parameters indicative for drought stress influenced by these and additional genes were analysed in different crops [23,24]. For example, biomass production [25], yield [26], photosynthesis rate [27], as well as the content of free proline [28], total content of soluble sugars [29], or osmolality [30] are parameters which are affected by drought stress in barley.
Another factor relevant for yield improvement is leaf senescence [31], which is a natural degradation process at the final stage of the development of organs and plants. This process is divided into three steps and starts with reprogramming of gene expression to turn on senescence activating genes. Before programmed cell death in the terminal phase occurs, the second step in which nutrients and metabolites are transported from source (e.g. roots, leaves) to sink (e.g. fruits, seed) is important for yield and quality of the seeds harvested [32]. During this reorganization phase, degradation of chlorophyll and a decrease in photosynthesis is observed [33,34]. Because of degradation of chlorophyll, yellowing of the leaves is a symptom of leaf senescence [35], which in many studies is rated visually [36,37], but can be more precisely determined by Soil Plant Analysis Development (SPAD) readings which estimate leaf greenness [38]. Degradation of chlorophyll is regulated by chlorophyllase, pheophorbide α oxygenase and red chlorophyll catabolite reductase among others [34], but so far for regulation of leaf senescence only a few genes are known [39,40]. Leaf senescence is a process which is influenced by a lot of external stress conditions e.g. drought stress [24,41]. Stress often results in premature induction of leaf senescence and therefore leads to an inefficient recycling of resources and a massive yield loss [42][43][44]. In contrast, plants showing delayed leaf senescence under stress, represented by a "stay green effect", minimize yield loss [45].
Genome wide association studies (GWAS) are a powerful tool to subdivide such complex pathways as drought stress and leaf senescence by the detection of quantitative trait loci (QTL) out of the regression analysis of genotypic and phenotypic data [19,[46][47][48]. Up to now, some QTLs involved in drought stress response were published in barley [49][50][51][52], whereas for leaf senescence only few QTLs are known [42,53]. Besides this, QTLs which are involved in the response to other abiotic stresses, e.g. salt stress were identified in barley [6,54]. Molecular markers, such as single nucleotide polymorphisms (SNP) flanking QTLs having a significant influence on the respective trait can be used for efficient marker assisted selection and smart breeding procedures [55].
The aim of the present study is therefore the identification of QTLs for drought stress and drought stress induced leaf senescence in early developmental stages of barley suited to be used in future barley breeding programs using GWAS followed by the identification of the function of these QTLs associated to respective traits.

Plant material and experimental setup
A set of 156 winter barley genotypes (Additional file 1) consisting of 113 German winter barley cultivars [49 two-rowed and 64 six-rowed, [56] and 43 accessions of the Spanish barley core collection (SBCC) [57] were used to investigate drought stress induced leaf senescence in juvenile barley plants. Drought stress was applied in greenhouses of the Julius Kühn-Institut in Groß Lüsewitz, Germany according to Honsdorf et al. [51]. Trials were conducted in a split plot design with three replications per genotype and variant (control, drought stress). Ten seeds of each accession were sown per plastic pot (16x16x16 cm) containing 1,500 g of a mixed clay soil ED73 (H. Nitsch & Sohn GmbH & Co. KG, Dorsten Germany). After germination, seedlings were reduced to seven plants per pot. Plants were grown under semicontrolled long day conditions in a temperature range from 20 to 22°C at day (16 h) and 17 to 19°C at night (8 h). If natural radiation was below 20 klx, additional light was applied from 6 a.m. to 10 p.m. Drought stress started at the primary leaf stage (BBCH 10) seven days after sowing (das). At this time watering of the stress variant was stopped till the soil reached 20 % of the maximal soil water capacity, and then this level was kept by weighting each pot and re-watering. Control plants were continuously watered to 70 % of the maximal soil water capacity. Water capacity was calculated of the saturated soil weight and drought weight according to DIN ISO 11465 1996-12 [58]. At the end of a four weeks stress period (BBCH 33) physiological traits were determined and above ground biomass was harvested (experimental setup A). Experimental setup A was repeated in three years. A modified experimental setup was conducted to optimize lightening conditions of the primary leaves for the measurement of leaf colour (SPAD) and electron transport rate at photosystem II (ETR) (experimental setup B). General settings were the same as in setup A, but only four plants were grown in smaller pots (12x12x12 cm) to allow wider spacing of pots and all leaves except the primary leaves were tied up to reduce shading. Experimental setup B was repeated in two years.

Physiological parameters determined
Six physiological traits, i.e. leaf colour (SPAD), electron transport rate at photosystem II (ETR), content of free proline (CFP), content of soluble sugars (CSS), osmolality (OA) and the aboveground biomass yield (BY) were determined in the control and stress treatment. Measurement and sampling respectively were conducted on primary leaves.
Chlorophyll content which was used as the main indicator for drought stress induced leaf senescence was measured 33-34 das by Minolta SPAD readings (Konica Minolta Chlorophyll Meter SPAD-502 Plus, Osaka Japan), which gives a value for leaf colour. Three primary leaves of three plants for each pot were measured at five positions per leaf. These SPAD readings turned out to be correlated to the chlorophyll content analysed photometrically [59,60]. Because of this relation, the chlorophyll content can be indirectly measured by SPAD [61].
At 34-35 das chlorophyll fluorescence was measured in all genotypes using light adapted plants with the OS1P-Chlorophyll Fluorometer (OPTI-SCIENCE, Hudson USA) in the middle of three primary leaves per pot at one position per leaf. The relative electron transport rate at photosystem II (PSII) (ETR = Y(II) * PAR * 0, 84 * 0, 5) was calculated including the photosynthetically active radiation (PAR), as well as the quantum photosynthetic yield of PSII (Y(II)) and constants representing light which is absorbed by the leaf (0.84) and light which is equally absorbed by PSI and PSII (0.5) [62].
At 36 das five primary leaves per pot were harvested and cut in pieces of 1 cm length for the analysis of CFP, CSS and OA. These samples were frozen in liquid nitrogen immediately and samples for CFP and CSS measurement were freeze dried. For CFP measurement the ninhydrin method [63] was applied, and for CSS measurement the anthron method [64] was used. Both traits were measured photometrical using a spectrophotometer. The concentration of these ingredients was determined with a standard curve calculated on a dry weight basis. To assess OA, frozen leaf samples were grinded in a swing mill (30/s for 3 min), filled up with 200 μl water and centrifuged at 15,000 rpm for 15 min to get cell sap for the measurement of osmolality with a freezing-point osmometer (Osmomat O-30 Gonotec, Berlin Germany). Osmolality was corrected for the water content of fresh and dry weight. Above ground biomass was harvested 36 das, too. Leaf material was dried in a compartment dryer at 105°C and weighted.
For all traits an outlier test was calculated to exclude extreme deviations [65]. To get information on the stability of all analysed traits in response to drought stress compared to the control, the drought susceptibility index (DSI) was calculated [66] for each trait and across treatments according to the formula: DSI is a relative value estimated for each genotype and trait. According to the formula genotypes revealing a DSI close to one are highly susceptible to drought and those close to zero or showing a negative value are tolerant.

Statistical analysis of phenotypic data
Statistical analyses were performed with SAS 9.3 [67]. Least square means (LSMeans) were calculated with GLM procedure for the replications of each genotype in the respective years and for both control and drought conditions. Descriptive statistics was calculated out of LSMeans by PROC UNIVARIATE. Analysis of variance (ANOVA) was calculated using PROC MIXED with genotypes (G), drought stress treatment (T) and GxT as fixed effects. Replication (R), year (Y) and row type are chosen as random factors. The heritability (h 2 ) was calculated with SAS in two steps. First, the variance components for the genotypes (V G ), variance associated with the genotype by year interaction (V GY ) and V E which is the error variance were calculated with PROC VARCOMP. Next, h 2 was calculated with the following formula: VG/(VG + VGY/Y + VE/RY) for both well watered and drought stress conditions. Furthermore, the coefficient of correlation (PROC CORR) by Pearson was calculated with SAS based on LSMeans.

Genotyping and genome wide association study (GWAS)
For genotyping the whole set of genotypes was analysed with the barley Illumina 9 k iSelect SNP-chip [68]. Population structure was calculated with STRUCTURE 2.3.4 [69] based on 51 simple sequence repeat (SSR) markers covering the whole genome. The STRUCTURE programme was run 20 times for pre-defined k (the number of population groups) from 1 to 5 each. To get the number of calculated subpopulations (k) with highest likelihood the procedure of Evanno et al. [70] was applied. An independent run with 500,000 iterations of a Monte Carlo Markov Chain with a length of the burn in period of 500,000 was conducted for the k with the highest likelihood to obtain the q-matrix. Kinship was calculated with SPAGeDi 1.3d [71] based on 51 SSRs and allele size correlation coefficient [72] with 5,000 permutations.
Out of 3,886 genetically mapped SNP markers (398 at 1H, 690 at 2H, 583 at 3H, 342 at 4H, 781 at 5H, 546 at 6H and 546 at 7H) [68], 3,212 polymorphic markers with minor allele frequencies higher than 5 % were taken into account. Based on these data and respective phenotypic data (LSMeans) GWAS was conducted applying a mixed linear model (MLM) using TASSEL 3.0 [73]. All results with p values <0.001 were considered as significant marker trait associations. Linkage disequilibrium (LD) was calculated on mapped polymorphic SNPs with R [74] by an estimate of the average decay [75] over all barley chromosomes.
Sequences of significantly associated SNP-markers (p <0.001) were downloaded from the James Hutton Institute [http://bioinf.hutton.ac.uk/iselect/app] and respective sequences were compared against the plant proteome in the UniProtKB/Swiss-Prot protein database by BlastX (Basic Local Alignment Search Tool, p <10 −5 or query cover of minimum 80 % in NCBI [https://www.ncbi.nlm.nih.gov] accessed Oct 2014) to get information on the proteins coded by these sequences [76]. Using UniProt [77] the involvement of respective proteins in drought stress and leaf senescence processes was analysed. In a last step a genetic map with all significantly associated SNPs in genes coding for proteins known to be involved in drought stress tolerance was generated using MapChart 2.2 [78].

Phenotyping
The experiments revealed variability for genotype and treatment in all analysed traits as shown in Table 1. For the traits biomass yield (BY), leaf colour (SPAD) and the electron transport rate (ETR) the mean values for the stress treatment were lower than in the well watered variant. An exception are some genotypes of the SBCC (SBCC 3, 12, 14, 76, 80, 138 and 140) showing no decrease in SPAD and ETR or even an increase, represented by negative values across treatments (DSI). In contrast to the above mentioned traits, osmolality (OA), content of free proline (CFP) and total content of soluble sugars (CSS) increased under drought stress.
The coefficient of variation (CV) was comparable for control and drought stress treatment (Table 1) for all six traits. Heritabilities (h 2 ) estimated ranged between 0 for OA to 0.80 for BY in the control treatment and 0.27 for OA and 0.61 for SPAD in the stress treatment. Generally, h 2 was higher for the stress treatment except for BY and SPAD. Analysis of variance (ANOVA) revealed significant (p <0.001) genotype and treatment effects for all investigated traits and genotype x treatment interactions for BY, CFP and CSS (Table 2). To get information on the influence of the physiological parameters estimated on biomass yield as the indicator for drought stress and SPAD as the indicator for drought stress induced leaf senescence, correlations to these traits were calculated (Table 3). For control and stress treatment BY is significantly correlated to SPAD with r = 0.39 and r = 0.36, respectively. A significant correlation was also determined for CSS to SPAD with r = 0.42 and for CFP with r = 0.42 in the drought stress treatment whereas for the control treatment significantly negative correlations were found. Low but nevertheless significant correlations to SPAD were also detected for ETR and OA in the control treatment. Similar correlations were detected for BY. High and significant correlations were found between BY and CSS (r = 0.36) and CFP (r = 0.31) for the drought stress treatment. Under control conditions the SBCC being a sub-population of its own, influences the correlation by producing less BY which results in reduced shading of the primary leaves and a negative correlation especially to CFP and CSS. By correlating only the German cultivars, these effects are excluded and no correlations (r = −0.16 for CFP and r = −0.03 for CSS) were observed. ETR and OA were not significantly correlated to BY.

Genotyping
The set of genotypes was analysed with the 9 k iSelect SNP-chip available for barley. In summary 6,807 SNPs turned out to be polymorphic. Out of these, 3,212 SNPs are mapped on the seven barley chromosomes [68], showing a minor allele frequency (MAF) >5 %. This set of SNPs was used for the calculation of the linkage disequilibrium decay (LD), which turned out be on average 2.52 cM for this set of genotypes. The number of subpopulations was estimated at k = 4 ( Fig. 1).

Genome wide association study (GWAS)
Results of GWAS are shown in detail in Additional file 2 and summarized in Tables 4 and 5. 191 SNPs significantly (p <0.001) associated to traits estimated in the control variant, 70 significantly associated SNPs in the stress treatment and 92 significantly associated SNPs across treatments (DSI), were detected using the MLM analysis in TASSEL. Significant associations were found on all barley chromosomes. Most of the significant   marker trait associations were located on barley chromosome 2H and 5H. A large number of SNPs on chromosome 5H around 45 cM turned out to be associated to SPAD and BY in the stress treatment (Fig. 2). Most significant associations for these traits were detected on chromosome 5H. The highest number of marker trait associations was detected for BY. No associations were observed for CFP in control and stress treatment, whereas across treatments one significant association was found. For ETR two significant associations and for CSS one significant association in the stress treatment explaining 5.5 % (ETR) and 1.6 % (CSS) of the phenotypic variance respectively were detected. The strongest association was observed on chromosome 1H for BY across treatments with a (−log p) value of 7.57 explaining 7.1 % of the phenotypic variance. For OA 29 significant associations were detected in the stress treatment located over all barley chromosomes, whereas in the control treatment no associations and across treatments only one association was found on chromosome 4H. For SPAD as an indicator of leaf senescence, twelve significant (p <0.001) marker trait associations in the control treatment, six under stress conditions and two across treatments were detected. For the drought stress indicator biomass yield 179 significant marker trait associations were found in the control treatment, 32 in the stress treatment and 87 across treatments. Significant marker trait associations for BY were evenly distributed over all chromosomes. Out of these, eight were identical in the stress treatment, control treatment and across treatments and a high number of 65 marker trait associations were identical in control treatment and across treatments.
In total the 191 significant associations estimated in the control treatment account for 80 genomic regions (LD = 2.52 cM), the 70 significant associations determined in the stress treatment represent 47 genomic regions and the 92 significant associations across treatments (DSI) account for 54 genomic regions. By comparing the localisation of marker trait associations detected, it turned out that significant associations were found for different traits at same positions, e.g. on chromosome 2H at 50 cM for SPAD and OA in the stress treatment and at 120 cM for BY and SPAD in the control treatment. Furthermore, on chromosome 5H at 45 cM significant marker trait     Fischer & Maurer (1978) associations for BY, SPAD and OA were detected in the stress treatment, and at 95 cM significant associations for BY and SPAD in the control treatment. Summarizing, overlapping of QTL was found across treatments and for different traits, especially for BY and SPAD, which are also significantly correlated (Table 3). One interesting QTL was observed on chromosome 5H at 45 cM where a significant association to BY and SPAD in the drought stress treatment was found, which is also within the LD of a significant association to OA (Table 4). Therefore, at this position a putative major QTL for drought stress and leaf senescence may be located.
From the 353 significantly associated SNPs detected in the control treatment, stress treatment and across treatments (DSI), 127 proteins were identified by an NCBI Blast of the marker sequences. Out of these 19 proteins turned out to be related to drought stress, 10 proteins related to leaf senescence and 98 proteins turned out to be not related to drought stress or leaf senescence. Out of the 29 proteins for drought stress and leaf senescence (Table 5), 16 revealed associations under drought stress conditions. These were in a next step assigned to the barley chromosomes by the known genetic localization of respective SNPs (Fig. 3). Most of these were located at barley chromosome 2H and 5H, none were mapped on chromosome 6H.

Discussion
Using the experimental design described above a clear cut influence of drought stress on biomass yield and physiological parameters was observed (Table 1). This effect of reduced biomass under drought conditions in juvenile stages was also reported in Honsdorf et al. [51] with an even higher reduction due to drought in early developmental stages of barley by Jamieson et al. [25]. In experiments on terminal drought stress application in barley this effect was not so pronounced [25,79,80] giving hint that barley is most susceptible to drought stress in early developmental stages. Furthermore, a reduction of the chlorophyll content under drought stress conditions in barley has been observed [80,81], but there are also reports on adverse effects, e.g. for rapeseed or potato [82,83], which may be due to a reduced leaf growth under drought stress conditions resulting in a reduced cell expansion leading to a relatively higher chlorophyll density in the leaves. In the present study measurement was done on primary leaves, which were fully expanded at the initiation of drought stress, so that this effect was excluded. For biomass yield a significant correlation to the leaf senescence parameter chlorophyll content was observed (Table 3). This correlation may be based on a true genetic relationship between these parameters, as it is also reported in drought stress field studies on wheat [59,84] and in glasshouse experiments [85].
The electron transport rate at PS II as a parameter for the chlorophyll fluorescence decreased under drought stress indicating the degradation of chlorophyll during drought stress induced leaf senescence (Table 1), as already shown by Fang et al. [86], Li et al. [27], Netto et al. [87] and Silva et al. [88].
As expected, osmolality increased under drought stress thereby protecting cells against a turgor loss [30]. This is also reported in other drought stress studies on barley [29], but is more often detected in barley under salt stress conditions [89,90]. For OA no correlation was found to BY, as also shown in a drought stress study on spring barley [91].
The amino acid proline has been described as an osmoprotectant [92] and is accumulated along with several abiotic stresses, such as drought stress, as seen in the present study (Table 1). This effect was also found in other drought stress studies, for example on barley in pot experiments [28], in greenhouse [93] and on Arabidopsis thaliana in climate chamber experiments [94]. The role of proline accumulation is still controversially discussed as it is described to function as a radical scavenger, antioxidant and is involved in the regulation of apoptosis and in seed development [95,96,97]. High correlations were detected for CFP to SPAD and BY giving hint that this trait is involved in drought stress tolerance and leaf senescence ( Table 3). The correlation of the proline content to SPAD was also found in winter survival studies of barley [98] and in studies on salt stress tolerance in Trigonella foenum-graecum [99]. Up to now no correlations of CFP to BY under drought stress have been described for barley, but positive correlations to yield in wheat were observed under drought stress [100].
Soluble sugars are acting also as osmo-protectants and consequently like in our study an increase was detected in several drought stress experiments on barley [29], wheat [101], potato [102] and also pea [103]. Furthermore, studies showed that an increase of soluble sugars occurs along with leaf senescence [104] and that CSS was correlated to leaf senescence and biomass production [105,106]. Interactions between sugar and ABA signalling may be responsible for the induction of senescence during drought stress [107].
Quite high values for the heritability of respective traits estimated under drought stress conditions ( Table 1) give hint that such an experimental design is suited together with a set of diverse genotypes and the respective number of SNP-markers to detect QTLs using a genome wide association approach (GWAS). Like in other studies [49,108,109], the highest number of associations was detected for the traits with the highest heritability. In the present study these were SPAD and BY. Most associations were found on barley chromosomes 2H and 5H on which QTLs were located at 50 cM and at 45 cM, respectively. Also in other GWAS studies of barley significant QTLs for SPAD and BY were located on these chromosomes. Close to the QTL for SPAD located on chromosome 2H (50 cM) a QTL for SPAD under drought stress was also mapped by Li et al. [50]. Moreover, on chromosome 2H at 115 cM a QTL for SPAD was identified in a pot experiment with post-flowering drought stress [53]. QTLs for SPAD were also located on chromosome 2H at 102.7 cM and on chromosome 5H at 165.2 cM in Mediterranean dry land experiments (110), but no significant marker trait associations were detected at these positions in our experiments. The same holds true for a QTL for SPAD on chromosome 5H at 139 cM [49]. Varshney et al. [49] also detected a QTL for biomass yield on chromosome 5H at 95 cM and 156 cM, which is near to associations, which were found in our study on chromosome 5H at 152 cM and 167 cM. In addition, in the present study a QTL for SPAD and BY under drought stress treatment was detected on chromosome 5H at 45 cM, which has not been described before. Furthermore, a lot of significant marker trait associations were observed for osmolality under stress treatment distributed over all barley chromosomes. This was also reported for barley based on growth chamber drought experiments [29].
Proteins involved in drought stress and leaf senescence were detected by a blast of SNP marker sequences and it turned out that they are distributed over all barley chromosomes, except 6H with a focus on chromosomes 2H and 5H (Table 5). Most interesting proteins detected in the drought stress treatment (Fig. 3) are discussed in detail.
On chromosome 1H an ATP-dependent zinc metalloprotease (FTSH3) which is a regulator of heat shock proteins turned out to be associated to BY under drought stress. This protein is involved in the thylakoid formation and in the removal of damaged D1 in the photosystem II, preventing cell death under high-intensity light conditions. In interaction with heat shock proteins it reduces chlorophyll a/b ratios in heat tolerance regulation in Arabidopsis thaliana [111]. Heat stress often occurs simultaneously with drought and also leads to leaf senescence. Besides this, on chromosome 1H the pectin methylesterase 49 (PME49) was found by sequence alignment to be associated to CSS. This protein acts in the modification of cell walls via demethylesterification of pectin and turned out to be up-regulated by drought stress in rice [112]. It influences the mechanical stability of cell walls and thereby also of leaves.
On chromosome 2H a sucrose synthase 4 (SUS4) turned out to be associated with SPAD. This is a sucrose-cleaving enzyme that provides UDP-glucose and fructose for various metabolic pathways and is involved in nucleic acid break down during leaf senescence as revealed by expression analysis e.g. in cucumber and rapeseed [113]. The SNP marker with the homolog sequences to this protein was associated to the leaf senescence parameter SPAD at 49.2 cM. So there may be a direct relationship between the SPAD values and the activation of this enzyme, especially because this SNP marker explains 3.8 % of the phenotypic variance. Furthermore, a probable glutamate carboxypeptidase (AMP) revealed an association to OA which plays an important role in shoot apical meristem development and phytohormone homeostasis. By microarray analysis it turned out that AMP mediates ABA production and is involved in abiotic stress response such as drought stress in Arabidopsis thaliana [114]. Moreover, a marker with a sequence homologue to a phospholipid hydroperoxide glutathione peroxidase (GPX1) associated to BY was found which protects cells and enzymes from oxidative damage. Photometrical analyses of protein quantity and activity showed that the expression of GPX1 and GPX3 is reduced under drought stress and restored after recovery in winter wheat [115].
On chromosome 3H an F-box protein was detected (FBL21) associated to OA. These proteins are ubiquitin related and negatively regulate ABA mediated drought stress response in Arabidopsis thaliana [116]. Furthermore, an association of OA to 1-aminocyclopropane-1carboxylate oxidase (ACO1) was detected which limits leaf growth by inhibiting the ethylene biosynthesis and so leads to drought tolerance. This was figured out in barley by expression analyses of protein related genes [117]. Surprisingly, in the control treatment associated to BY, a well known drought stress related protein, i.e. dehydrin (DHN) [118] was found which was also in another study located on chromosome 3H [119]. This protein belongs to the family of late embryogenesis abundant (LEA) proteins and is reported to be up-regulated in the protection mechanisms activated by plants in response to drought stress in wheat [120].
On chromosome 4H an ABA receptor (PYL5) was located and associated to OA that activates ABA signalling and ABA-mediated responses such as stomatal closure and germination inhibition. Immuno-detection experiments of protein extracts revealed that ABA signalling is involved in several stresses for example drought stress in Arabidopsis thaliana [121].
On chromosome 5H nucleotide pyrophosphatase/ phosphodiesterase (AVP1) was found associated to BY which facilitates auxin transport by modulating apoplastic pH and regulating auxin-mediated developmental processes. Increased expression of protein related genes in transgenic barley confers tolerance to NaCl and to drought by increasing ion retention [122]. Furthermore, three protein kinases associated to SPAD were located on this chromosome regulating protein activity by phosphorylation. First a serine/threonine-protein kinase (ATM) which leads to stress induced programmed cell death, shown in Arabidopsis thaliana by expression profiles of protein related genes [123], second a serine/ threonine-protein kinase (SAPK9) which is activated by hyperosmotic stress in rice [124] and third the abscisic acid-inducible protein kinase (TRIUR3) observed in wheat, which is also involved in dehydration stress response [125]. Moreover, an anthocyanin regulatory protein (R-S) was detected associated to BY. Anthocyanin is often accumulated in abiotic stress response, among others in drought stress with a photoprotective function as shown in Arabidopsis thaliana [94,126]. All of these proteins showed homologies to sequences of SNPs at chromosome 5H around 45 cM and were associated significantly to BY or SPAD, representing an interesting candidate QTL for drought stress and leaf senescence. In addition, a dehydration-responsive element-binding protein (DREB1A) was found associated to OA, which delays water stress symptoms and promotes expression of drought tolerance genes in transgenic wheat [127].
On chromosome 7H another ethylene responsive protein was found to be associated to BY under drought stress. The transcription factor ERF062 is involved in the regulation of gene expression by stress factors (transcriptional repressors) and progression of leaf senescence in Arabidopsis thaliana [128]. Besides, on chromosome 7H the cation/H(+) antiporter 2 (CHX) was detected associated to ETR, which is important for pH gradients in the cell. This protein plays a vital role in maintaining both cellular and intercellular ionic balances under stresses such as drought stress as observed in Arabidopsis thaliana [129].
In summary the blast of the associated SNPs to protein data bases revealed many proteins which are known to be involved in drought stress response or leaf senescence, respectively giving hint that the GWAS approach is well suited for the genetic dissection of these traits in barley. Out of the QTL detected, the ones on chromosome 2H at 50 cM and chromosome 5H at 45 cM are of prime importance and may be involved in breeding barley for drought tolerance in the future due to the quite high amount of phenotypic variance explained.

Conclusions
By GWAS marker trait associations for above ground biomass and physiological traits involved in drought stress tolerance and leaf senescence in early developmental stages of barley were detected. Major QTL for BY and SPAD under drought stress were located at chromosome 2H at 50 cM and chromosome 5H at 45 cM, giving hint that in these regions putative major QTLs for drought stress and leaf senescence are located. With respect to the QTL on chromosome 2H, QTLs for drought stress and leaf senescence were located at comparable positions in other GWAS studies while the one on chromosome 5H was detected for the first time. By BlastX of respective SNP carrying sequences, 29 proteins were identified being involved in drought stress or leaf senescence, respectively. Respective QTLs may be the starting point for marker based selection in barley for drought stress tolerance in the juvenile stage.

Additional files
Additional file 1: Overview of the 156 analysed genotypes. a SBCC: Spanish Barley Core Collection.
Additional file 2: Significant associations (p <0.001) of the genome wide association study and blasted proteins out of the marker sequences. a BY: biomass yield, CSS: total content of soluble sugars, ETR: electron transport rate at PSII, CFP: content of free proline, OA: osmolality, SPAD: leaf colour. b C: control treatment, S: stress treatment, DSI: drought susceptibility index across treatments by Fischer & Maurer (1978).

Competing interests
The authors declare that they have no competing interests.
Authors' contributions GW conducted all experiments, including statistical and bioinformatics analyses, as well as the sequence alignment and mainly wrote the manuscript. ME participated in the GWAS and LD calculation. CB, KH and FO designed the research, supervised the experimental design, data analysis and participated in writing the manuscript. All authors approved the final manuscript.