**4. Osmoregulatory effects of proline**

*2.4.1. PCR confirmation and gus histochemical staining*

68 Water Stress in Plants

performed as described previously [24].

ascorbic acid as a specific electron donor [29–31].

ROS in plant cells [28].

**3. Tall fescue biochemical response to short drought stress**

The presence of *uidA* gene in embryogenic calli was confirmed by PCR amplification of tall fescue-calli genomic DNA with gene-specific primers: *gus*-F (GCTGTGCCAGGCAGTTT‐ TAAC) and *gus*-R (ATATCGTCCACCCAGGTGTTC). The predicted size of the amplified DNA fragments of *uidA* was 425 bp. DNA amplifications were performed in a total volume of 20 μl containing 1 μl of 10 μM forward pimer, 1 μl of 10 μM reverse primer, 2 μl of 10× Ex Taq Buffer, 0.5 μl of dNTP mixture (2.5 mM each), 0.1 μl of TakaRa Ex Taq enzyme (5 unit/μl) (Takara, Shuzo, Kyoto, Japan), by Thermocycler (Bio-Rad, United States). PCR was carried out for screening of regenerated transformed plantlets with an initial denaturation at 94°C for 5 min, followed by 30 cycles of 94°C, 30 min; 58°C, 30 min; 72°C, 30 s; and a final extension, 75°C for 10 min. The PCR products were separated in 1% agarose gel containing 0.5 μg/ml ethidium bromide. The size of the amplification products was estimated using a 100 bp DNA ladder (GeneRuler DNA Ladder Mix # SM 0331, Fermentas). GUS histochemical staining was

In response to water stress, plants exert adaptive modifications in their morphological, physiological, and biochemical properties. Photosystem II (PSII) is more vulnerable than PSI during drought stress in which inhibition of CO2 assimilation, coupled with the changes in photosystem activities and photosynthetic electron transport capacity, results in accelerated production of reactive oxygen species (ROS). The damaged targets by ROS attack are recovered by repair or by replacement via de novo biosynthesis. However, under severe drought stress, cell death would be inevitable. The fate of stressed cells is determined by the duration of stress as well as the protective capacity of the plant. Reactive oxygen species play a crucial role in causing cellular damage under drought stress [25]. Reactive oxygen species not only play a signaling role in coordinating nuclear gene expression in order to protect cells during biotic and abiotic stress responses, but can also cause lipid peroxidation and consequently membrane injury, protein degradation, and enzyme inactivation [26, 27]. All plants have ROS detoxifica‐ tion mechanisms, which are enzymatic, with superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), peroxidase (POD), glutathione reductase (GR), and monodehy‐ dro ascorbate reductase (MDAR), and nonenzymatic detoxification mechanisms through flavanones, anthocyanins, carotenoids, and ascorbic acid (AA). The activity of antioxidant enzymes under drought stress will be enormously flexible among several plant species and even between two cultivars of one species. It has been demonstrated that enzymatic break‐ down of ROS is one of the major processes the plant uses to scavenge this signaling molecule [28]. Superoxide dismutase, the first enzyme in the detoxifying process, converts superoxide anion radicals (O2?) to hydrogen peroxide (H2O2), and APX reduces H2O2 to water using

It has been stated that ROS not only cause damage to membrane, but also disturb the correct functions of DNA and proteins. Enzyme-mediated disruption of ROS effectively quenches Proline is a very specific amino acid essential for primary metabolism, which can accumulate in eubacteria, protozoa, marine invertebrates, and plants under stress condition. First proline accumulation report has been addressed in welting perennial ryegrass [46]. Soon after that, numerous reports have shown proline accumulation under drought [47], high salinity [48], high light and ultraviolet (UV) irradiation [49], heavy metal [50], oxidative stress [32], and biotic stress [51], in which proline metabolism by far has mainly been studied in response to osmotic stress [52]. Proline is synthesized in cytosol mainly from glutamate, which can convert to proline in two steps. First, glutamate is reduced to glutamate-semialdehyde (GSA) and then spontaneously converted to pyrroline-5-carboxylate (P5C) by the pyrroline-5-carboxylate synthetase (P5CS) and P5C reductase (P5CR), respectively [53, 54]. Proline is eventually catabolized to P5C in mitochondria by proline dehydrogenase (PDH) or proline oxidase, and then P5C dehydrogenase (P5CDH) converts P5C to glutamate [55]. Kumar et al. [56] evaluated T1 transgenic plants of *indica* rice overexpressing a *P5CS* gene. Their research indicated a better growth performance, biomass production, higher proline accumulation, and lower rate of lipid peroxidation in transgenic plants compared to nontransgenic plants under 150 mM NaCl stress. Furthermore, the ameliorating effects of proline on heavy metal stress have been reported on *Chlamydomonas reinhardtii*, which was able to express *P5CS* gene 80% higher than wild type [57].

Proline can be encoded by *P5CS1* and *P5CS2* in cytosol. While *P5CS1* translational fusion of GFP normally localizes in cytosol, but right after osmotic stress, the *P5CS1* signal was detectable in chloroplast but not *P5CS2*. Székely et al. [58] and Strizhov et al. [59] reported the augmentation of proline under osmotic stress in chloroplast, which was induced by *P5CS*. This report sheds light on different subcellular compartments of proline biosynthesis, which is pretty different based on the environmental conditions. A proline uniporter, which facilitates proline transport into the mitochondrial matrix, and a proline/glutamate antiporter, which appears to have an important role in the Pro/Glu shuttle between the mitochondrial matrix and the cytosol, have been identified in *Triticum durum* Desf. mitochondria [60]. In *Arabidopsis*, *P5CS2* is a house‐ keeping gene, whereas *Arabidopsis P5CS1* is induced by osmotic and salt stresses [48]. Proline has been shown to function as a molecular chaperone able to protect protein integrity and enhance the activities of different enzymes. Proline acts as a singlet oxygen quencher and has a ROS scavenging activity. Hien et al. [61] reported that proline accumulation in *Oryza sativa* was related to induction of proline biosynthesis by osmotic stress and not proteolysis. In *Opuntia* spp. exposed to heat, salinity, and water-deficit stress, an increase in the proline content was reported, but this accumulation was not necessarily correlated with P5CS enzyme activity, whereas the transcript level of *P5CS* was correlated with proline accumulation in the report of Silva-Ortega et al. [62]. We evaluated the proline content of the tall fescue leaf during the course of drought stress, because predominately proline accumulation is a physiological plant response to biotic and abiotic stress. The research results indicated that proline content in all tested genotypes increased significantly, especially at the highest deficit irrigation treatment. For example, Pixie showed over 50-fold increase in proline content than control plants, while this was about twofold increase in h–d genotype. We concluded that the substantial increase in proline content in Pixie and a lesser extent in Mini-mustang likely are because of their dependency on proline, so as to maintain cell homeostasis during water stress. However, other drought-tolerance mechanisms may be responsible for slight changes of proline in h–d and Jaguar. Our research results collectively suggest that proline is an important compatible osmolyte which serves as a protectant for enzymes and cellular structures of tall fescue under severe drought stress.

### **5. Overexpression of** *Arabidopsis P5CS* **in** *F. arundinacea* **calli**

Particle bombardment–genetic transformation of *F. rubra* L. was one of the preliminary reports by Ref. [63]. However, 12 years later, the first successful *F. arundinacea Agrobacterium-*mediated transformation was published [64]. Since the success of *Agrobacterium*-mediated transforma‐ tion of rice in the early 1990s, transformations of some other monocotyledonous species occurred routinely. *Agrobacterium*-mediated transformation has several advantages over the biolistic method, not the least of which is the stable integration and expression of the target gene in offspring, mostly due to lower copy number, and fewer rearrangements in genome [47, 65]. Bettany et al. [64] were able to regenerate only one transgenic *F. arundinacea* expressing *uidA* gene after establishment in soil, even though they failed to produce seeds. The *Agrobac‐ terium* LBA4404 was the strain that had been used in their research. They reported a huge variability in their transgenic events, which in most cases did not follow Mendelian segregation [64].

catabolized to P5C in mitochondria by proline dehydrogenase (PDH) or proline oxidase, and then P5C dehydrogenase (P5CDH) converts P5C to glutamate [55]. Kumar et al. [56] evaluated T1 transgenic plants of *indica* rice overexpressing a *P5CS* gene. Their research indicated a better growth performance, biomass production, higher proline accumulation, and lower rate of lipid peroxidation in transgenic plants compared to nontransgenic plants under 150 mM NaCl stress. Furthermore, the ameliorating effects of proline on heavy metal stress have been reported on *Chlamydomonas reinhardtii*, which was able to express *P5CS* gene 80% higher than

Proline can be encoded by *P5CS1* and *P5CS2* in cytosol. While *P5CS1* translational fusion of GFP normally localizes in cytosol, but right after osmotic stress, the *P5CS1* signal was detectable in chloroplast but not *P5CS2*. Székely et al. [58] and Strizhov et al. [59] reported the augmentation of proline under osmotic stress in chloroplast, which was induced by *P5CS*. This report sheds light on different subcellular compartments of proline biosynthesis, which is pretty different based on the environmental conditions. A proline uniporter, which facilitates proline transport into the mitochondrial matrix, and a proline/glutamate antiporter, which appears to have an important role in the Pro/Glu shuttle between the mitochondrial matrix and the cytosol, have been identified in *Triticum durum* Desf. mitochondria [60]. In *Arabidopsis*, *P5CS2* is a house‐ keeping gene, whereas *Arabidopsis P5CS1* is induced by osmotic and salt stresses [48]. Proline has been shown to function as a molecular chaperone able to protect protein integrity and enhance the activities of different enzymes. Proline acts as a singlet oxygen quencher and has a ROS scavenging activity. Hien et al. [61] reported that proline accumulation in *Oryza sativa* was related to induction of proline biosynthesis by osmotic stress and not proteolysis. In *Opuntia* spp. exposed to heat, salinity, and water-deficit stress, an increase in the proline content was reported, but this accumulation was not necessarily correlated with P5CS enzyme activity, whereas the transcript level of *P5CS* was correlated with proline accumulation in the report of Silva-Ortega et al. [62]. We evaluated the proline content of the tall fescue leaf during the course of drought stress, because predominately proline accumulation is a physiological plant response to biotic and abiotic stress. The research results indicated that proline content in all tested genotypes increased significantly, especially at the highest deficit irrigation treatment. For example, Pixie showed over 50-fold increase in proline content than control plants, while this was about twofold increase in h–d genotype. We concluded that the substantial increase in proline content in Pixie and a lesser extent in Mini-mustang likely are because of their dependency on proline, so as to maintain cell homeostasis during water stress. However, other drought-tolerance mechanisms may be responsible for slight changes of proline in h–d and Jaguar. Our research results collectively suggest that proline is an important compatible osmolyte which serves as a protectant for enzymes and cellular structures of tall

wild type [57].

70 Water Stress in Plants

fescue under severe drought stress.

**5. Overexpression of** *Arabidopsis P5CS* **in** *F. arundinacea* **calli**

Particle bombardment–genetic transformation of *F. rubra* L. was one of the preliminary reports by Ref. [63]. However, 12 years later, the first successful *F. arundinacea Agrobacterium-*mediated Soon after the last aforementioned report, Wang and Gee [66] developed a much more efficient *Agrobacterium*-mediated transformation procedure aimed at regenerating transgenic tall fescue fertile plants. They reported that the number of hygromycin-resistant calli obtained per dish was in the range of 2.0–5.8; the number of transgenic plants recovered per dish was in the range of 0.4–1.7, and a 1:1 segregation ratio of the transgenes was found in the progenies. Gao et al. [67] gained 10.5% average transformation efficiency across the four callus lines of *F. arundinacea*. In their report, *Agrobacterium*-mediated transformation appears to be the preferred method for producing transgenic tall fescue plants. Zhao et al. [68] examined whether salt tolerance can be improved stably by overexpressing vacuolar Na+/H+ (*AtNHX1*) antiporters in tall fescue. Their research leads to identifying a single-copy inheritance of *AtNHX*1—perform‐ ing better in the presence of 200 mM NaCl than control plants—in most of the T1 and T2 lines of tall fescue after *Agrobacterium*-mediated transformation, with a near 1:1 segregation ratio which subsequently has been approved for release by the Chinese Department of Agriculture [68]. Dong and Qu [69] reported that *Agrobacterium*-mediated transformation of tall fescue yields 34% *hyg* B-resistant calli and had 8% overall transformation efficiency. Hu et al. [53] had improved the cold resistance of tall fescue through *Agrobacterium* transformation of *ipt* gene. Transgenic plants have had higher chlorophyll content and stayed greener, besides having a higher tillering ability as in contrast to that of control plant, which is of great economic importance to improve the tolerance of this plant to environmental stress. To the best of our knowledge, the *Agrobacterium*-mediated transformation of *F. arundinacea* by *P5CS* gene has not yet been reported.

The finding that abiotic stress is involved in chloroplast–stroma protein degradation under drought stress in numerous plant species, together with the demonstration of enhanced osmotic stress tolerance in plants by proline overproduction, spurred us on applying *P5CS* gene encoding proline for improving drought stress in *F. arundinacea*. The preliminary work on callus production resulted in a high amount of callus during a year and so, with different types of calli. Soft, yellowish calli were chosen as a primary sample for agrotransformation. Whereas the callus regeneration rate was astonishing, plant regeneration was far behind callus regeneration. An increase in regeneration efficiency was observed in media supplemented with 8 mg/l 2,4-D, which was almost six times greater than 2 mg/l 2,4-D treatment by dehusked, longitudinally sliced seeds. Apart from 2,4-D concentration, dehusked *Festuca* mature seeds showed a further callus induction efficiently as opposed to intact seeds. The callus regeneration frequency from small *Festuca* callus explants was also enhanced in dark as compared with light situation. Acetosyringone in cocultivation media greatly improved *Agrobacterium* growth, while acetosyringone-free MS media delayed *Agrobacterium* growth and subsequently, most probably, could decrease *Agrobacterium*-mediated transformation efficiencies. The heterolo‐ gous transformation of *P5CS* in *F. arundinacea* background was confirmed by PCR and transient *GUS* assay, which most probably can improve tall fescue tolerance more to drought stress.
