Assessment of Salinity Tolerance Deploying Antioxidant Defense Systems in Gerbera Jamesonii

Inconsistency in the environment exposes plants to various abiotic stresses. This results in damage of a plant’s cellular components due to excessive accumulation of unstable reactive oxygen. Besides, it also disrupts enzymatic/non-enzymatic detoxification mechanisms in plants making them more sensitive. Salinity is one such abiotic stress which disrupts regular physiological mechanisms in plants. In this study, we examined the effects of salinity using NaCl in four different genotypes of Gerbera jamesonii cv Bolus, an important ornamental plant of family Compositae. We hypothesized that, upon treatment with NaCl (50, 100, 150 200mM concentration), alterations in the morphological features along with elevated levels of H2O2, MDA, proline, and degradation of chlorophyll will be observed. The enzymatic antioxidant defenses were also hypothesized to differ among genotypes based on their level of tolerance. These parameters were monitored on the 5th and 20th day of NaCl treatment and results were recorded. The observations suggest that 1. the Lattara genotype of Gerbera is sensitive to NaCl and 2. Faith is tolerant, while 3. Alcatras and Basic are moderately tolerant. These findings accompanied by further research on the physiological parameters responsible for attaining salinity tolerance may help in developing salt-tolerant varieties in Gerbera. Future studies on decoding molecular networks associated with the antioxidative defense system in Gerbera can help improve breeding and create novel germplasm in various ornamentally important plants besides Gerbera.

to absorb water gets badly affected by salinity, resulting in reduced plant growth, causing a suite of physiological adaptations in plants 43,14,15 . Under salinity stress, increased stomatal closure via transpiration results in an imbalance in the carbon dioxide to oxygen content leading to ROS generation 21 . The generated ROS content triggers cellular reactions such as lipid peroxidation, DNA denaturation and protein deprivation causing reduced plant growth, yield loss and eventual death 24,42,58 . Minimizing soil salinization and/or increasing salinity stress tolerance of crops have become serious issues which are needed to be resolved immediately 43 .
Daisies or Gerbera (Gerbera jamesonii) are important ornamental plants of Compositae/ Asteraceae family widely known for their attractive flowers and long shelf/vase life. Gerbera being sensitive to abiotic/biotic stresses are commercially grown under protected and controlled cultivation (net/poly house) 11 . Gerbera is cultivated throughout the year and ranks fifth after Rose, Carnation, Chrysanthemum, and Tulip in cut flower trade 09, 45 . Several Gerbera cultivars were being grown throughout the world promoting employability and sustainability in the agribusiness sector.
Despite being polyhouse cultivated, Gerbera has a high risk of salinity-induced oxidative stress due to excessive fertigation on the soil surface in a polyhouse. The accumulated chemicals present in fertilizers may cause a decline in the plant's productivity causing a threat to its existence 6 . Therefore the development of salt tolerance in Gerbera varieties may protect its commercial significance with regard to its aesthetic value and productivity. To date, little to no studies was attempted on the effect of salinity in several commercially important cultivars of Gerbera. Therefore, in this study, we examined the effect of salinity using NaCl and its influence on potential stress indicators to understand the stress-induced changes in four commercially important genotypes of Gerbera.

Plant Material
Plantlets of Gerbera jamesonii cv Bolus L., (Terraregina Latara -white colored flower variety, Basic -pink colored flower variety, Alcatras -red colored flower variety and Faith -yellow colored flower variety) aged one month were procured from Virat Agri Biotech company. The plants were potted in sand: cocopeat: soil (1:1:1) mixture and raised in a greenhouse by maintaining controlled conditions (27-29°C day and 23-25°C night temperatures and relative humidity of 45-51%, respectively for the entire growth period). optimization of naCl concentrations based on the morphology of leaves Preliminary morphological examinations were done for a period of 30 days and the concentration of NaCl to be applied for salinity treatments were assessed using 100-300mM of NaCl concentration. The response to salt treatment was initiated on the 5 th day which was aggravated on the 20 th day of treatment (data not shown).

salinity treatments
For initiating salinity treatments, 45 days old plants with at least four true leaves were selected. The experiment was planned as a complete randomized block which included four treatments and a control (1mM NaCl) each comprising of three replicates. The desired NaCl concentrations of 1mM, 50mM, 100mM, 150mM, and 200mM NaCl were supplemented every third day by dissolving in water in their respective concentrations to the whole plant for twenty days. Sampling was done to these treated plants on the 5 th and 20 th day of treatment. Secondary leaves were harvested and stored at -20°C after freezing in liquid nitrogen until experiments were carried out. The samples were used for further biochemical studies and Spectrophotometrical assays using UV 3000+ spectrophotometer (Shimadzu).

Investigations of salt affected parameters determination of lipid peroxidation by measuring Mda levels
Using the standard protocol of Heath and Packer (1968) 22 with slight modifications, the content of malondialdehyde (MDA) was determined which gives us the extent of peroxidation of lipids in cellular membranes. In brief, 1 gram of frozen leaf material was ground to a fine paste in a mortar and pestle by adding 1 mL of 0.5% TCA and the collected sample was centrifuged at 19000 g for 20min. The supernatant is used as a crude extract. TBA (thiobarbutiric acid) reagent was prepared by adding 15% trichloroacetic acid (w/v) and 0.375% TBA(w/v) in 0.25 M HCl. Equal volumes (0.4 mL) of crude extract and TBA reagent in a microfuge tube were heated for 15 min at 95 o C, immediately shifted to an ice bath. Following this, the mixture is centrifuged for 15 min at 15000 g. The supernatant obtained is read for absorbance at 532 nm by subtracting turbidity at 600 nm. The quantity of MDA was calculated from the extinction coefficient of 155 mM "1 cm "1 . Data were expressed as ìmol per g FW.

determination of Content of Free Proline
Using the method of Ninhydrin, the content of free proline was determined as per Bates et al. (1973) 07 . In a spectrophotometer, absorbance was measured at 540nm. Data were expressed as ìmol per g FW.

determination of Chlorophyll Content
The content of chlorophyll was determined as per the standard protocol of Lichtenthaler and Wellburn (1983) 33 . Absorbance was read at 646nm and turbidity obtained at 663 nm was subtracted from it. In brief, leaf samples were extracted using 80% (v/v) acetone and the quantities of chlorophyll a, b, and total chlorophyll were calculated. Data were expressed as mg per g FW.

determination of antioxidant enzyme assays
For enzyme assays, 100mg of sample was thoroughly ground to a paste in a mortar and pestle by adding 1mL of ice cold extraction buffer. The extraction buffer consists of 100mM potassium phosphate buffer (pH 7.0) with 1mM EDTA. The obtained homogenate was centrifuged at 5,000 rpm for 15 min (Eppendorf Centrifuge 5430 R, Hamburg, Germany) and the supernatant was used to analyze enzyme activities.
The quantity of total soluble protein was determined as per the Lowry's method (1951) 36 using bovine serum albumin (BSA) as a standard at 640nm. In brief, leaf samples of 100 mg were ground in 10 mM ice cold potassium phosphate buffer of pH 6.8 and centrifuged for 20 min at 15 000 g. The obtained supernatant was used to determine soluble protein content. Values were expressed as mg per g fresh weight (FW).
Superoxide dismutase activity, SOD of (EC 1.15.1.1) was done by the method of Beyer and Fridovich (1987) 08 . Leaves were extracted using extraction buffer supplemented with PVP (1% (w/v) polyvinyl-pyrrolidone) and 0.5% (v/v) Triton X. The photochemical reduction of nitro blue tetrazolium (NBT) was recorded at 560 nm. The amount of SOD used up in the reaction to inhibit 50% of NBT gives us one unit of SOD activity.
Catalase activity, CAT (1.11.1.6) was measured as per Aebi et al. 1974 3 . Briefly, the decrease in the substrate H 2 O 2 incubated with Leaf Extract was monitored at 240 nm and quantified by its molar extinction coefficient of 36 M -1 cm -1 . The activity of catalase was expressed as ìmoles of H 2 O 2 decreased min -1 mg -1 protein.

statistical analysis
The data presented are the average values (±SE) of results from three experiments conducted on different time points including four Gerbera species and five salinity levels. The data were subjected to further statistical analysis by one-way ANOVA (Holm-Sidak method) using SigmaPlot Version 14.0.

results effect of naCl treatment on phenotypic characteristics in four different genotypes of Gerbera
In this study, we treated all the four genotypes of Gerbera (Red, Yellow, Pink, and White) with a concentration of 200 mM NaCl for 20 days (Figure 1). Upon morphological observation, bleaching was observed which was more pronounced in White followed by Red and Pink genotypes combined with a decrease in leaf size and shape. NaCl stress also inhibited the emergence of young leaves showing stunted growth. In contrast the leaves of the Yellow genotype exhibited little to no effect.

levels of h 2 o 2 in four different genotypes of Gerbera on exposure to salinity stress
In the given study, the four different genotypes of Gerbera were treated with a 200 mM concentration of NaCl and the H 2 O 2 levels were monitored at two different time points (5 th and 20 th DOT). In the initial days of treatment, i.e., on the 5 th day we observed a rise in H 2 O 2 levels upon treatment with 200 mM NaCl particularly in Pink and White Gerbera genotypes ( Fig.2A), while there was a considerable decrease in the levels of H 2 O 2 in Yellow and Red Gerbera genotypes compared to control ( Fig.2A). At 20 th DOT, the levels of H 2 O 2 were in significant increment in the Pink and White genotype of Gerbera when compared to control ( Fig.2A) whereas no such increase was observed in other genotypes ( Fig. 2A).

Levels of MDA in four different genotypes of Gerbera on exposure to salinity stress
In the current study, after treating the four different genotypes of Gerbera with 200 mM NaCl, the MDA levels were monitored at two different time points (5 th and 20 th DOT). Initial days of treatment, i.e., 5 th day we observed a significant rise in MDA levels upon treatment with NaCl particularly in Red, Pink, and White Gerbera genotypes (Fig.2B), while there was no considerable change in MDA levels in Yellow genotype (Fig.2B). Similar to the 5 th day even on 20 th day of DOT, we observed a significant increment in MDA levels in all genotypes of Gerbera when compared to control, except Yellow which also exhibited an increase but was not significant (Fig.2B).

Content of total chlorophyll in four genotypes of Gerbera on exposure to salinity stress
In the given study, we determined the content of total chlorophyll in four Gerbera genotypes after giving a treatment of 200 mM NaCl for twenty days and measured the content of chlorophyll at two time points (5 th and 20 th DOT). At 5 th day, we observed no considerable change in the chlorophyll content in Yellow/ Pink genotypes of Gerbera whereas in White and Red genotypes a non-significant decrease in the chlorophyll content was seen (Fig.2C). Similarly, at 20 th DOT we observed a drop in total chlorophyll content in NaCl treated plants particularly in Pink, Red and White genotypes of Gerbera (while in Yellow we did not noticed any change in chlorophyll content when compared to control (Fig.2C).

Content of free proline in four Gerbera genotypes upon exposure to salinity stress
Further, we determined the content of free proline in four Gerbera genotypes treated with 200 mM concentration of NaCl at two time points (5 th and 20 th DOT). At 5 th day, we observed increased accumulation of free proline in Red and White genotypes of Gerbera in NaCl treated plants. While there was little to no enhancement in proline accumulation in Yellow and Pink genotypes of Gerbera compared to control (Fig.2D).Whereas, at 20 th DOT we observed significant shoot up in proline levels in White, Pink and Yellow genotypes of Gerbera (in Yellow this increase was not significant). In contrast, the Red genotype of Gerbera did not exhibit much alteration upon treatment with 200 mM of NaCl concentration (Fig.  2D). enzymatic activity in four Gerbera genotypes on exposure to salinity stress In the present study, we monitored the effect of salinity on SOD in four different genotypes of Gerbera after treating with 200 mM NaCl at two time-points (5 th and 20 th DOT). On the 5 th day, except in the White genotype, a significant increase in the SOD activity was noticed in NaCl treated plants of all genotypes of Gerbera compared Reductase in Yellow, Red, Pink and White genotypes of Gerbera respectively at 5th DOT and 20th DOT to control (Fig. 3A). Similarly at 20 th DOT we observed the same pattern of results when studying the activity of SOD in Gerbera (Fig.3A). Thereafter, we monitored the activity of CAT in four different genotypes of Gerbera treated with 200 mM NaCl at two time-points (5 th and 20 th DOT). At 5 th day, a noticeable increase in the CAT activity on treatment with 200 mM NaCl concentration was seen in Yellow and Pink and a slight decreased activity was seen in White and Red genotypes of Gerbera (Fig.3B). Whereas, at 20 th DOT we observed a noteworthy increase in the activity of CAT in Yellow, Red, Pink genotypes, in contrast CAT activity in White genotype showed little to no change in NaCl treated plants when compared to control.
We also examined the activity of APX in all four genotypes of Gerbera upon treatment with 200 mM NaCl at two time points (5 th and 20 th DOT). On the 5 th day, we observed a gradual increase in APX activity compared to control upon treatment with 200 mM NaCl concentration in all genotypes of Gerbera (particularly significant in Yellow and Pink) (Fig.3C). Whereas, at 20 th DOT we observed an increase in APX activity with increasing concentrations of NaCl particularly in Yellow, Pink and Red genotypes of Gerbera which was significant, in contrast, the APX activity in the white genotype showed a slight decrease at 200mM NaCl compared to control (Fig. 4(G)).
Finally, we studied the activity of GR in all four genotypes of Gerbera after subjecting it to salt stress by treating it with 200 mM NaCl for a period of twenty days. The observations were recorded at two different time points (5 th and 20 th DOT). On the 5 th day, we observed a significant shoot in the activity of GR compared to control in Yellow and Red genotypes of Gerbera, while in both Pink and White genotypes we observed a decline in GR activity (Fig.3D). At 20 th DOT a noticeable decrease in the activity of GR in all genotypes except Yellow was observed (Fig.3D).

disCussion
Salinity is a major concern in crop and ornamental plant species negatively affecting growth and productivity 19 . Although much research has been conducted in crop plants, salinity studies in ornamentals are still at a budding stage urging stress biologists to focus their scientific research and studies on salt tolerance 18 . In the given study we chose Gerbera as a research model in ornamental plants to study salt tolerance across four different genotypes. As a part of our study, we investigated the intensity of oxidative damage caused by salinity stress in Gerbera and the mechanisms to mitigate it. Earlier, we carried out some preliminary studies on the white genotype of Gerbera to assess its sensitivity towards NaCl using leaf disc culture system and found the White genotype to be sensitive (data not shown). This provoked us to move to a further level in determining the ability of Gerbera to confer salt tolerance across its other genotypes which have huge market demand in the floriculture sector (red, pink and yellow Gerbera varieties).
In this regard, we initially performed preliminary phenotypic experiments in four genotypes of Gerbera by treating them with increasing NaCl concentrations (50 mM, 100 mM, 150 mM, and 200 mM) for a period of 30 days. Based on phenotypic/morphological observations, the toxicity of NaCl was evident from5 th day onwards which aggravated in 20 days of salt treatment (symptoms like wilting, disfiguration of leaves were seen after 20 th day) (data not shown) which made us plan further biochemical studies at 5 th day and 20 th day of NaCl treatment.

Salinity effect on plant growth and morphology in four different genotypes of Gerbera
In this study, we characterized salt-tolerant ability in four different genotypes of Gerbera based on their morphological observations upon NaCl treatment. We observed varied responses in plant height, leaf size, and color with increasing concentrations of NaCl, particularly in white genotype with conspicuous yellowing/bleaching of leaves signifying the intensity of oxidative damage. These findings are in line with the previous reports 4, 38,43 which show a similar response. The results obtained in our study reports a detrimental effect of salinity on plant growth, causing a gradual reduction of leaf area.

altered levels of h 2 o 2 content in four different genotypes of Gerbera upon salt stress
In plants, the accumulation of H 2 O 2 signifies an intercellular physiological breakdown and signals the plant defense system to activate genes involved in stress responses, such as catalase, peroxidase, etc., 31 . We attempted to look into it by estimating the accumulation of H 2 O 2 levels in NaCl treated leaves of four Gerbera genotypes. We observed that on 5 th and 20 th DOT, when compared with its respective controls, in Pink and White genotypes NaCl treatment showed a considerable rise in H 2 O 2 levels. Whereas, H 2 O 2 levels in the Red genotype remained unaltered. In contrast, the yellow genotype exhibited a lower accumulation of H 2 O 2 content. These findings give us a hint that a rise in H 2 O 2 levels in Pink and White Gerbera was also may be due to the induction of SOD in plants to defend itself against oxidative stress as a part of H 2 O 2 defense signaling 28 . Our findings are similar to the previous reports in Dianthus superbus 37 and Rice 29 which suggested that there is an increased content of H 2 O 2 under salt stress.

Altered levels of MDA content in four different genotypes of Gerbera upon salt stress
Accumulation of malondialdehyde, produced due to peroxidation of lipids in cellular membranes signals oxidative damage in plants subjected to stress conditions. We attempted to look into it by estimating the accumulation of MDA levels in NaCl treated leaves of four Gerbera genotypes. We observed that on 5 th DOT, when compared with its respective control, Red and White genotypes exhibited high levels of MDA, whereas the MDA levels in the Pink genotype remained unaltered. In contrast, the Yellow genotype exhibited a lower accumulation of MDA content. These findings signify that the White genotype is the first and foremost one to show the stress response. In addition to this, the MDA content on the 20 th day was also significantly higher in White followed by Pink, Red and Yellow indicating the hyper sensitivity of White genotype when compared to others. However, in all genotypes, MDA content was found to be higher compared to their respective control in which Yellow genotype exhibited negligible increase (1 fold) suggesting the ability of Yellow genotype to withstand stress.
Our findings are similar to the previous reports in Sunflower (Helianthus anuus L), which suggested that there is an increase in lipid peroxidation under salt stress 13 . Apart from this, the salt sensitive cultivar Wuxi of Jerusalem Artichoke (Helianthus tuberosus) has also exhibited significant enhancement in MDA levels upon NaCl stress 58 .

Modulation of Free Proline content in four different genotypes of Gerbera upon salt stress
Further, we investigated free proline content as it is a major organic osmolyte that is known to accumulate in plants under stressful conditions 40 . Particularly, in salt-induced oxidative stress proline protects cellular membrane by enhancing activities of various antioxidants 47 . In our study also, at 5 th DOT we noticed a high accumulation of proline in all genotypes except Yellow with respect to their control. Even at 20 th DOT, proline accumulation was highly significant in pink genotype followed by Yellow and White genotypes suggesting the role of proline in minimizing ROS production. While in the Red genotype, we observed a slight increase in proline content which was not significant. Among all genotypes, both at 5 th and 20 th DOT the Yellow genotype of Gerbera accumulated significantly higher proline content under saline conditions.
The findings of this present study are concurrent with earlier published data which clearly demonstrated that salt stresses markedly enhanced free proline content upon treatment with NaCl in eight sunflower cultivars 51 and in roots of Chrysanthemum 25 .

degradation of total chlorophyll content in different genotypes of Gerbera on treatment with NaCl
It is a well-known fact that any oxidative stress would definitely cause either a decrease in chlorophyll synthesis or degradation of its complexes 39,50 . This is evident by previous reports which suggest that salt sensitive cultivar Wuxi of Jerusalem artichoke; an ornamental plant exhibited a dramatic decrease in chlorophyll content with increasing salt concentrations 58 . Similarly, in Helianthus anuus L. plants also salt treatment exhibited a decreased production of chlorophyll 51 . In corroboration with these reports, in our study particularly at 20 th DOT, the total chlorophyll content was decreased to a larger extent in all genotypes except yellow.

Modulation of antioxidant enzyme activities by NaCl treatment in four different genotypes of Gerbera
Generally in plants exposed to stress, the first line of defense which are the earliest to respond are the antioxidant enzymes like SOD, CAT, APX, GR, POX, GPX etc 30,54 . There are several reports which demonstrated the role of antioxidant enzymes under salinity stress of different ornamental plants such as Helianthus anuus 51 , Helainthus tuberosus 58 , Artemesia 32 , Catharanthus 27 . In the given study we mainly focused and examined SOD, CAT, APX and GR activities at 5 th and 20 th DOT by applying salt stress using 200 mM NaCl concentration in four different genotypes of Gerbera. The activity of SOD increased in all the NaCl treated genotypes except in the White genotype on both 5 th and 20 th DOT which are in compliance with the results obtained in Cotton 41 , where the tolerant cultivar (Pora) showed higher activity of SOD activity upon exposure to NaCl stress when compared to sensitive cultivar (Guazuncho). Similar results were also observed in Kandelia candel 56 and sensitive (cv Challis) and tolerant cultivar (cv Granada) of Pisum sativum, where the activity of SOD was found to be more intolerant cultivar upon treatment with NaCl when compared to sensitive cultivar 23 . These results leave us a clue that the White genotype may be sensitive to NaCl stress when compared to Red, Pink and Yellow genotypes. The activity of CAT at 5 th and 20 th DOT increased considerably in Pink and Yellow genotypes, whereas this increase in Red was not seen. In contrast the activity of CAT slightly decreased which was not significant upon exposure to 200 mM NaCl in White genotype. The white genotype exhibited contrasting CAT activity by showing decreasing pattern which may be due to extreme sensitivity of white genotype towards higher NaCl levels. Our findings are in line with previous reports which suggested that there was a reduction in the CAT activity in salt sensitive cultivars of various plant species such as Echinea purpuria 48 , Jerusalem artichoke 58 , Amsonia orientalis 2 . Besides, there are also reports which suggested increased CAT activity in salt tolerant cultivars of plant species 12,1,32 .
In continuation with the previous studies on SOD and CAT, we have monitored APX activity at 5 th and 20 th DOT. At 5 th DOT, we observed a considerable increase in APX activity in all four genotypes, wherein the activity of APX was more significant in Yellow and Pink genotypes. While in Red and White genotypes, the activity of APX slightly increased even after treatment with 200 mM NaCl. Whereas at 20 th DOT, the activity of APX is significantly high in Pink, Yellow and Red genotype, in contrast the White genotype exhibited decreased APX activity in NaCl treated plants indicating its sensitivity towards NaCl stress. We noticed that our present observations correlate with previous reports which demonstrated that there was a reduction in the APX activity in Helianthus annuus 51 . There are also a few reports from which it is evident that enhanced APX activity in Artemesia annua 46 , Echinea purpuria 48 , Calendula officianalis 12 , Stevia roubidiana 10 .
Finally, we studied the activity of GR by applying NaCl stress at 5 th and 20 th DOT. At 5 th DOT, we noticed a considerable increase in GR activity in Yellow genotype, in anomaly we noticed a significant decreasing pattern in White and Pink genotype with increasing concentrations of NaCl. Whereas at 20 th DOT, the activity of GR is significantly high in Pink and Yellow genotypes, in contrast the Red and White genotypes exhibited decreased GR activities with increasing NaCl levels. Our results also support previous evidence in several plants species belonging to Asteraceae family like Chrysanthemum 25 , Artemesia annua 46 , Calendula officianalis 12 which exhibited enhanced GR activity upon salt stress. Contradictory to it, in certain plant species like Helianthus annuus 51 , Stevia roubidiana 10 there was a decline in GR activity at higher saline conditions.
As per Abogadallah et al. 1 plants subjected to salinity show elevated activities of antioxidant enzymes in tolerant species than the sensitive ones. This relates with our study which has shown that SOD, APX, CAT and GR were highest in Yellow and all enzyme activities are lowest in White. These findings imply that Yellow genotype can tolerate the negative effects of salinity better than other studied genotypes.

ConClusions
To conclude, our study is the first of its kind in members of the Compositae family which deals with the antioxidative defense response of different genotypes of Gerbera to salinity stress. A clear disparity was observed in the defense action of four genotypes of Gerbera employing enzymatic and non-enzymatic components. This kind of study may pave a way for further understanding of varied defense responses and their associated key metabolic activities across different genotypes of Gerbera. Our findings may leave a clue to ornamental plant researchers in the development of salt-tolerant transgenic plants and incite them to do further research in the area of abiotic stress physiology in ornamentals. This may have a global impact on the floral market with improved and large-scale productivity of Gerbera species.