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To cope with drought: two forage grass species – Festuca arundinacea and F. glaucescens can activate similar survival strategies although they differ with molecular response


 Background: Photosynthesis is among the primary processes affected by drought and its disturbances result with the reduction of growth, reactive oxygen species (ROS) overproduction, and alterations in antioxidant system activity. Our study was performed on two closely related forage grasses: Festuca arundinacea and F. glaucescens. Two genotypes within each species significantly differing with the potential of drought resistance: high drought resistant (HDR) and low drought resistant (LDR), were used. The research involved: (i) the analysis of gene expression at transcript and protein levels for the selected enzymes of Calvin cycle (fructose-1,6-bisphosphate aldolase (pFBA), phosphoglycerate kinase (PGK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) and (ii) the activity of pFBA, as a protein marker of the Calvin cycle, (iii) the analysis of gene expression at protein level for the selected antioxidant enzymes (glutathione reductase (GR), glutathione peroxidase (GPX), L-ascorbate peroxidase (APX), catalase (CAT), and superoxide dismutase (SOD)), (iv) the measurements of physiological parameters describing a plant’s physiological status under control, drought and re-watering conditions (relative water content (RWC), electrolyte leakage (EL), lipid peroxidation, chlorophyll fluorescence, gas exchange, and ROS level).Results: Our analysis clearly showed that physiological reactions to water deficit were similar in both HDR and both LDR genotypes of Festuca arundinacea and F. glaucescens but the species revealed significant differences in the potential to tolerate tissue dehydration, what was correlated with distinct expression of the Calvin cycle enzymes under drought stress.Conclusions: The maintenance of stable efficiency of dark phase of photosynthesis seems to be crucial for drought tolerance and recovery in F. glaucescnes, whereas acquisition of drought tolerance in F. arundinacea and F. glaucescens does not involve marked changes at the protein level in the enzymatic antioxidant system.

at the protein level in the enzymatic antioxidant system. Background A lack of water is one of the main environmental factors that affects plant growth and development, and significantly reduces the yield of many crop species with economic importance. Considering rapid climate changes with global warming being the most noticeable, we can expect that periods of drought will occur more often than previously, even in temperate regions (1). Thus, the recognition of cellular mechanisms conferring plant drought resistance seems to be crucial.
Grasslands cover around 70% of the world's agricultural area and are mainly used for fodder purposes (2). Forage grasses belonging to Lolium and Festuca genera are one of the most important in the temperate regions. Our previous studies revealed that Festuca species could be successfully used as models to precisely dissect mechanisms of resistance to a wide range of abiotic stresses in a group of forage grasses (e.g. 3,4,5,6,7,8,9).
Allohexaploid Festuca arundinacea Schreb. (2n = 6x = 42) and tetraploid F. arundinacea var. glaucescens Boiss. (hereafter recognized as F. glaucescens) (2n = 4x = 28) are closely related cool-season forage grasses (10) with a relatively high potential to withstand periods of water shortage. In F. arundinacea, drought resistance is mostly manifested by a drought avoidance strategy which relies on the development of deep root system, leaf rolling and a rapid stomatal closure (5,11). However, our earlier experiments revealed that F. arundinacea could also develop a drought tolerance strategy mainly associated with the adjustment of leaf metabolism to water deficit in plant tissues (5,6).
On the other hand, F. glaucescens was described earlier as a species characterized mainly by a metabolism slowdown and a reduction of growth associated with a quiescence under drought conditions, followed by a further recovery after stress cessation, which enables it to survive and to resume the growth following irrigation (12,13). The knowledge about molecular basis of drought resistance is strongly limited in this species.
Photosynthesis is among the primary processes to be affected by drought. Its efficiency can be restricted by both stomatal and non-stomatal mechanisms. Stomata closing declines CO 2 availability, whereas among the non-stomatal mechanism, a reduction of Calvin cycle efficiency was indicated to be crucial (8,14,15,16).
Drought, especially the initial phase of stress duration, can also result in the oxidative stress manifested by the reactive oxygen species (ROS) overproduction (17,18), with hydrogen peroxide (H 2 O 2 ), hydroxyl radical (OH) and superoxide anions (O 2 •-) being the most severe (19). Although these molecules serve as signals in the numerous processes associated with plant growth and development, ROS are also recognized as key players in stress signaling (20,21). However, a stress-induced overproduction of ROS can lead to the damage of photosynthetic apparatus and the disturbance of different metabolic pathways (21,22,23,24). Furthermore, the enhanced production of ROS during drought supports lipid peroxidation, and finally contributes to the damage of cellular membranes (25). The ROS-scavenging enzymatic system was proved to be one of the most crucial components of drought resistance in many plant species (26,27). Moreover, the enzymes contribute to the maintenance of cellular redox balance, particularly during stress response (28,29,30).  (30,31,32).
Herein, we hypothesize that under water deficit in specific environmental conditions, especially those preventing the development of deep root system F. arundinacea and F. glaucescens could cope with this stress activating a set of similar survival strategies, involving mostly drought tolerance and recovery after stress cessation. We also assume that these strategies will be manifested at the levels of both photosynthetic performance and cellular antioxidant system. Thus, the comprehensive research presented in this paper

Results
Genomic structure of F. arundinacea and F. increased significantly in all the genotypes during the stress treatment. After further rewatering, a reduction of this parameter was observed in all the genotypes, except the Fg-HDR genotype (Fig. 4D).
In the Fa-HDR genotype, the majority of parameters of chlorophyll fluorescence remained stable or decreased during prolonged drought. Values of the following parameters:   No relevant changes in the pFBA accumulation level in the Fa-LDR genotype during the whole experiment, were observed. In the Fa-HDR genotype, a slight increase was noticed on the 3 rd and 11 th day of water deficit. However, that accumulation level dropped again after rehydration to the values observed in the control conditions. The accumulation level of pFBA protein in the Fg-HDR genotype increased from the initial time-points of drought duration (D1-D2) to the rehydration time-point (RH). Interestingly, its level was constant during the whole experiment in the Fg-LDR genotype (Fig. 6B).
The amount of GAPDH protein was more or less constant during drought treatment in two F. arundinacea genotypes, and it dropped after rehydration. The similar tendency was observed for F. glaucescens genotypes, however, after re-watering, the amount of GAPDH decreased only in the Fg-LDR genotype (Fig. 6C).

Chloroplast aldolase activity
The activity of pFBA decreased in both F. arundinacea genotypes in response to drought.
In the Fa-HDR genotype lowered activity was observed during the whole stress period, whereas in the Fa-LDR genotype only on the 11 th (D3) day of drought. In the recovery phase pFBA activity increased to the control level only in the Fa-LDR genotype. In F.
glaucescens genotypes, a significant decline of pFBA activity was observed between the 6 th (D2) and 11 th (D3) day of drought, compared to the control. After re-watering an increase of pFBA activity comparing to the advanced water deficit (D3) was revealed for both F. glaucescens genotypes, but it was lower than in the control. The genotypes differed with pFBA activity during the whole stress period, that was higher in the Fg-HDR genotype ( Fig. 7).

Expression of genes coding antioxidant enzymes at the protein level
The protein accumulation of Cu/Zn-SOD was rather stable during the whole experiment in both Festuca species. Its slight reduction in the initial phase of stress duration (D1) and higher accumulation, compared to control at D3, were observed in the Fa-HDR genotype and Fg-LDR genotypes, respectively. The lowering of its level was also noticeable in both F. arundinacea genotypes after re-hydration. In the control conditions the protein level of Mn-SOD was about twofold higher in the Fa-HDR genotype than in the Fa-LDR genotype.
During the whole drought period, its level was elevated in the Fa-LDR, and then it dropped and returned to the control level after stress cessation (RH). In the Fa-HDR genotype, the reduced protein level of Mn-SOD in D2 and after re-watering, was noticed. A significant Mn-SOD accumulation was observed between the 6 th and 11 th day (D2-D3) of stress duration in the Fg-HDR genotype. In the Fg-LDR genotype a slow declined of Mn-SOD protein level was remarked during the whole experiment in relation to control. The slight increase of Fe-SOD protein amount was observed only at the beginning of stress treatment (D1) in the Fa-HDR genotype. In the Fa-LDR genotype, a significant reduction in the amount of Fe-SOD during water deficit, was observed. After re-watering it returned to the control level. In the Fg-LDR genotype the Fe-SOD protein was highly accumulated during the stress treatment as well as after regeneration ( Fig. 8A, B, C).
Stress-induced changes in APX protein accumulation were noticed in the Fa-LDR genotype.
The protein level was reduced comparing to the control during the whole drought period and after re-watering. Lowered APX protein level was also observed at D2 in the Fg-HDR genotype and at D2 and D3 time-points in the Fg-LDR genotype (Fig. 9A). In the Fa-HDR genotype a statistically significant decrease of GPX, was observed during the treatment Furthermore, a significant reduction in H 2 O 2 generation was noticed for the two genotypes but was more deeper for the Fg-LDR genotype, after stress treatment (RH) (Fig. 10C).

Discussion
The plants response to water deficiency is a combination of biological mechanisms that involve morphological, physiological and molecular adaptations. They use different strategies to survive under stress conditions that trigger several metabolic pathways at the same time (14). Acquired strategy of majority of the plant species strongly depends on the environmental conditions. Certainly, the efficient photosynthesis and the activation of enzymatic antioxidant system under drought are among important goals.
Our earlier research performed on F. arundinacea allowed to recognize crucial components of leaf metabolisms as well as roots performance, including its architecture and metabolism, involved in drought resistance of this species (5,33). On the other hand, it has been here first time when general response to drought in F. glaucescens at the physiological as well as molecular levels was more deeply analysed. The species identities were confirmed by using FISH with rDNA probes. Both species presented the karyotypes precisely characterized earlier by Thomas et al. (1997) (34).

Physiological response to drought
The reduction of RWC could be a good indicator of cellular dehydration in plants, including grass species (5,35). Our results clearly indicate that both genotypes of F. arundinacea and F. glaucescens were suffered from water deficit. However, the higher reduction of leaf RWC in the stress conditions were observed in both LDR genotypes, what could be partially due to differences in root metabolism and more efficient water uptake in case of HDR plants at the early stage of the stress (36). Our recent study proved that in case of F. arundinacea, deep root system is not sufficient to fully avoid cellular damage, caused by drought. In the experiment performed in tubes, thus enabling undisturbed development of root system, it was demonstrated that not only architecture but also metabolic performance of roots were crucial to cope with negative effect of water deficit by using different survival strategies (33).
The chlorophyll fluorescence parameters provides the information about the efficiency of flow of energy from antenna to the electron transport chain components through the reaction centre of photosystem II (PSII) (37). Photochemical processes were more affected in the Fa-LDR and Fg-LDR genotypes, compared to the HDR genotypes. However, both LDR genotypes showed a high capacity of regeneration after stress cessation, which resulted in a return to the levels similar to the control conditions. Interestingly, in the Fg-HDR genotype no significant changes during drought treatment were observed in numerous parameters, while their significant decrease after re-watering, was visible. ROS production and performance of enzymatic

antioxidant system
Reactive oxygen species are involved in plant development processes. The balance between their production and scavenging plays a crucial role in a proper functioning of plants. Abiotic stresses occurrence in the environment lead to elevated ROS generation that causes cellular damage (42). However, the increase of cellular antioxidant activity could enhance the protection against oxidative damage caused by stresses, including drought (43,44,45). Our results show that water deficit did not generate the high level of ROS favouring oxidative stress in the analyzed species. An initial decrease in superoxide anion radical and hydrogen peroxide release may be caused by effective functioning of antioxidant system in response to early water deficit. The prolongation of stress duration contributed to a generation of significantly higher amount of superoxide anion radical in both F. glaucescens genotypes. After further re-watering, the abundance of ROS dropped to levels in the control conditions or it was even lower.
The protein levels of Mn-SOD, APX, GPX, GR and CAT were significantly higher in the Fg-HDR genotype than in the Fg-LDR genotype. These results indicate that the Fg-HDR genotype exhibited higher antioxidant protein accumulation against ROS-induced oxidative stress damage than did the Fg-LDR genotype.
The superoxide dismutases with different metal co-factor are the enzymes that catalyze the dismutation of superoxide anion radical into molecular oxygen (O 2 ) or hydrogen peroxide (H 2 O 2 ). In plant cells, Fe-SOD are located in chloroplasts, Mn-SOD in mitochondria and peroxisomes, and Cu/Zn-SOD in chloroplasts, cytosol, and possibly also in extracellular space (46). Accumulation of Fe-SOD and Mn-SOD in the Fg-LDR and Fg-HDR genotype, respectively may be associated with the compensatory mechanisms to counteract enhanced superoxide anion radical production in the response to drought stress in F. glaucescens.
Noteworthily, O 2 ·− amount raised 2.5-fold in succulent purslane under heat and combined stress, but not in plants exposed to drought (40). In cotton cultivars, no significant differences in H 2 O 2 levels were observed for drought and combined drought/heat stress (42). It is well known that catalase, which is involved in the degradation of H 2 O 2 into water and oxygen, is the major H 2 O 2 -scavenging enzyme in plants (47

Conclusions
Our analyses clearly shows that physiological reactions to water deficit were similar in both HDR and LDR genotypes of Festuca species. The HDR genotypes were able to maintain water homeostasis and membrane stability during stress treatment, whereas the LDR genotypes revealed higher recovery capacity after stress cessation. This clearly shows that physiological reaction to drought was similar in both species. However, F. The genotypes of both Festuca species were exposed for short-term drought in the pot- (D3) day of watering cessation, and 10 days after subsequent re-watering (RH) (Fig. 12).

Fluorescent in situ hybridization (FISH) analysis
To verify the genomic status of F. arundinacea and F. glaucescens plants, FISH experiment with two highly conserved rDNA sequences (5S and 35S rDNA) as probes was applied. The wheat clone pTa794 containing 5S rDNA was labeled by PCR with tetramethyl-rhodamine-

Physiological parameters
A relative water content (RWC), electrolyte leakage (EL), chlorophyll 'a' fluorescence and gas exchange (net photosynthesis (CO 2 assimilation), transpiration, stomatal conductance) were measured as described previously in detail by Kosmala

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Competing interests
The authors declare that they have no competing interests.         The activity of fructose-1,6-bisphosphate aldolase (pFBA) in two genotypes of F. arundinacea (Fa-HDR, Fa-LDR) and F. glaucescens (Fg-HDR, Fg-LDR) before stress treatment (C), on the 3rd (D1), 6th (D2) and 11th (D3) day of water deficit and 10 days after subsequent re-watering (RH). Error bars represent the standard errors (SE) of three biological and two technical replicates. Homogeneity groups according to Fischer LSD test (P = 0.01) are denoted by the same letters.    after re-hydration (RH) in relation to D3.

Figure 12
The scheme of short-term drought experiment performed with F. arundinacea and

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