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Role of external proline on enhancing defence mechanisms of Vicia Faba L. against ultraviolet radiation.

INTRODUCTION

Plants differ substantially in their reaction to ultraviolet radiations dependent on experimental settings, treatment doses and exposure times [1-3]. Regarding of these factors, several published research have revealed evidences for plant resistance to ultraviolet radiation possibly through constitutive or induced plant protection against and/ or repair of UV-ruin [4-5]. Rahimzadeh and others showed that UV exposure decreased plant length, fresh weight and biomass; this decrease was significant in the UV-C-exposed plants [6]. The growth of many species is reduced in response to UV treatment. Similar changes have been observed in Capsicum longum [7], Fagopyrom tataricum [8], Pisum sativum [9], and sweet flag [10]. Shoot fresh weight and length did not change significantly after 14-days of low regimes of UV-B and low regimes of UV-C, but decreased considerably after irradiation with moderate to high doses of both UV-B and UV-C [11]. Other study revealed that changes in root growth were not significant, but shoots growth decreased in UV-R-exposed plants and decreased significantly in UV-C-exposed plants [7].

A decrease in leaf photosynthetic pigments was obvious during irradiations to enhanced levels of UVB radiation doses in most of studied crops [12-17]. Moreover, carotenoids have an essential role against UV-B radiations damage in plants. Carotenoids, scavengers of the singlet oxygen species created under intense light radiation, are involved in harvesting of light and protecting chlorophylls from destruction by photo-oxidation. Substantial reduction in leaf carotenoid level was detected in UV-B irradiated bean plants [14]. Tasgin and Nadaroglu stated that chlorophyll content was greatly reduced in both treatments with UV-B and then butylated hydroxanisol (BHA) in winter wheat leaves [18]. In a field experiment with Vigna radiate [19], and Phyllanthus amarus [20] an initial increase and subsequent decrease in chlorophyll content was observed. Also, UV irradiance caused the reduction of the contents of chlorophyll and carotenoid of savory and Capsicum annuum leaves [6].

Ultraviolet radiations can cause oxidative damage consequences in plant cells; however, plants acquire various UV-protection mechanisms [21-22]. One of the most significant mechanisms is anti-oxidative defence systems either enzymatic or non-enzymatic machineries that could alleviate UV-induced damage due to the production of active oxygen species (AOS). The magnitude to which each of these defence mechanisms is functional or whether there is a balance between the screening mechanisms and mitigation mechanisms such as antioxidants is poorly understood [21]. The impact of other environmental consequences on carbon and nitrogen limitations could also interact to alter the balance or consequences of a balance between these defence mechanisms. However, a detailed understanding of the AOS production and scavenging is required before this interaction can be fully understood. The AOS induced by ultraviolet radiations includes not only free radicals (e.g. superoxide; [O.sub.2.sup.-], and hydroxyl radicals; *OH), but also hydrogen peroxide ([H.sub.2][O.sub.2]) and singlet oxygen ([sup.1][O.sub.2]). The UV-induced AOS can cause oxidative destruction to membrane lipids, proteins and nucleic acids [23]. Plants exhibit various enzymatic and non-enzymatic anti-oxidant defence systems to minimize this oxidative damage. Among these anti-oxidative defence systems, are carotenoids, flavonoids, glutathione (GSH) and ascorbic acid (AsA) [21,24]. Enzymatic antioxidants include enzymes such as superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX; EC1.11.1.11), and others. Superoxide dismutase (SOD) instantly converts [O.sub.2.sup.-] to [H.sub.2][O.sub.2], which can then be converted to water and oxygen by catalase (CAT) [25]. However, catalase is found predominantly in the peroxisome and has low substrate affinity. An alternative mode of [H.sub.2][O.sub.2] destruction is via peroxidase (APX), which is found throughout the cell [26]. APX uses two molecules of AsA to reduce [H.sub.2][O.sub.2] to water, with the production of two molecules of monodehydroascorbate (MDHA); MDHA can be reduced to AsA, catalyzed by MDHAR, and AsA can also be non-enzymatically regenerated from MDHA. Dehydroascorbate (DHA) is always generated during the rapid disproportionation of the MDHA radical and DHA is then reduced to AsA by the action of DHAR using GSH as the reducing substrate. This results in the production of glutathione disulphide (GSSG), which is reduced to GSH. The removal of [H.sub.2][O.sub.2] through this series of reactions is known as the ascorbate-glutathione cycle [25].

Few studies were carried out to intensively evaluate the oxidative stress and anti-oxidative mechanisms. Two studies were carried out on Soybean exposed to elevated doses of ultraviolet (UV-B and [UV.sub.A+B]) radiations [21-22]. It is of valuable to carry out intensive investigation on various antioxidants status after exposure to elevated levels of ultraviolet radiations especially UV-A and UV-B so as to evaluate their contribution to plant defense machinery. This work was carried out to study the behaviour of the antioxidant system of three broad bean genotypes to different levels of [UV.sub.A+B] radiation and assessment of the role of proline pretreatment to alleviate deleterious effects of [UV.sub.A+B].

MATERIALS AND METHODS

1. Plant materials and proline treatment:

Broad bean (Vicia faba L.) seeds of three genotypes (Sakha-1, Sakha-2, and Giza-716) were obtained from Agricultural Research Centre, Ismailia, Egypt. Surface sterilisation was performed to the broad bean seeds with 2.5% sodium hypochlorite solution for fifteen minutes and was then washed with distilled water. Seeds were sown in plastic pots (25 cm height, 20 cm diameter) and were equally filled with a mixture of pre-sieved sandy loam soil. Each genotype's seeds were divided into two groups; the first group were soaked in water for 6 h, and the second were soaked in 0.01 M proline for 6 h. All pots were irrigated up to field capacity, kept in the open air until UV-treatments. Broad bean seedlings were grown under 12h daylight photoperiod throughout the experiment (Table 1).

2. UV-irradiation Experiment:

Twenty days post germination; plants were randomly divided into two main groups: (1) control group (FL) exposed to fluorescent lamp light giving light intensity of 1100 Lux; (2) [UV.sub.A+B] Irradiated plants with 4.16, 8.32 and 12.48 kJ.[m.sup.-2].[d.sup.-1] dose after irradiation with [UV.sub.A+B] radiation for 4, 8, and 12 h/day beside (FL) fluorescent light. Distance between [UV.sub.A+B] lamps and apical bud of broad bean was fixed to 45 cm and were periodically monitored and reset as broad bean grows. Plants were subjected to [UV.sub.A+B] radiation for seven days. Plants were harvested after 27 days from germination. Dry weight of shoots and roots was determined after drying the freshly harvested organs in an aerated oven at 80[degrees] C to constant weight.

3. Photosynthetic pigments

The photosynthetic pigments (Chlorophylla+b, and Carotenoids) were determined for the three broad bean cultivars by the spectrophotometric method recommended by Metzner and others [27]. The absorbance was measured against a blank of pure 85% aqueous acetone at 3 wavelengths of [A.sub.452.5], [A.sub.644] and [A.sub.663] (nm). The pigment fractions (chlorophylla+b and carotenoids) were expressed as mg/g-DW.

4. Oxidative damage

4.1. Electrolyte leakage

Electrolyte leakage was determined after Dionisio-Sese and Torbita [28] using 100 mg fresh plant leaf, cut into 5mm length and placed in test tubes containing 10 ml deionized water. All tubes were placed in water bath maintained at temperature of 32[degrees]C, initial electrical conductivity was measured using a Chemitrix type 700 portable conductivity meter after 2 hours. Samples were then autoclaved afterwards at 121[degrees]C for 20 min and cooled to 25[degrees]C and the final electrical conductivity were measured, and the electrolyte leakage (EL) percent was calculated [28].

4.2. Determination of Lipid Peroxidation

Lipid peroxidation was estimated by spectrophotometric method using Thiobarbituric acid (TBA)Malondialdehyde (TBA-MDA assay). Extraction of lipid peroxides were carried out using 500 mg fresh shoot tissues with 0.3 ml of 1% metaphosphoric acid of pH=2.0 plus 1 ml of 0.6% TBA (Thiobarbituric acid). The TBA-chromogen colour was measured spectrophotometrically at 532 nm [29].

4.3. Determination of hydrogen peroxide

Leaf [H.sub.2][O.sub.2] concentration following irradiation with UV was measured by the FOX method [30]. 500[micro]g of fresh leaves were extracted in trichloro-acetic acid (TCA). 500 pL of the extraction solution was added to 500 [micro]L of assay reagent (500 [micro]M ammonium ferrous sulphate, 50 mM [H.sub.2][SO.sub.4], 200 [micro]M xylenol orange, and 200 mM sorbitol). Absorbance of the [Fe.sup.3+]-xylenol orange complex was detected after 45 min at 560nm. The standard curves of [H.sub.2][O.sub.2] were performed using different dilution of [H.sub.2][O.sub.2]. Data were expressed as [micro]M [H.sub.2][O.sub.2] per gram of fresh weight of explants. Each data point was the average of three independent samples.

5. Antioxidant enzymes activity

The enzymes extracts were prepared by homogenizing broad bean plant in a previously chilled mortar in 20 ml chilled phosphate buffer (pH= 7.5). Centrifugation of the obtained enzyme extract was carried out at 6000 rpm for 20 minutes at 5[degrees]C. Enzyme assays were conducted immediately following extraction.

5.1. Catalase activity

Activity of catalase enzyme was assessed by method following Aebi [31]. Catalase activity was assayed spectrophotometrically by following the hydrogen peroxide decomposition at 240 nm. The absorbance was recorded versus a control cuvette including enzyme solution plus [H.sub.2][O.sub.2]-free-[PO.sub.4] buffer (M/15). 3 ml of [H.sub.2][O.sub.2]-[PO.sub.4] were transferred into the experimental cuvette, and mixed with the sample. At for absorbance decrease from 0.45 to 0.40 was recorded, At was used in calculations.

5.2. Peroxidase (POX) activity

The activity of peroxidase antioxidant enzyme was assayed by following the guaiacol dehydrogenation spectrophotometrically at 436 nanometres [32]. Pipette out 3 mL of buffer solution (phosphate buffer 0.1 M and pH= 7), 0.05 ml of guaiacol solution, 0.1 ml of enzyme extract and 0.03 ml of hydrogen peroxide in a cuvette. Mixture was will shaken and placed in the spectrophotometer. At for absorbance increase by 0.1 were recorded and used in calculations. Peroxidase enzyme activity was expressed as Peroxidase activity/ g-protein.

5.3. Ascorbic acid peroxidase (APX) activity

Ascorbic acid peroxidase (APX) activities were assessed by using Nakano and Asada (1987) method

[33]. The APX activity in broad bean following the pretreatment with proline and irradiation with [UV.sub.A+B] radiations was assayed by following the hydrogen peroxide-dependent dissociation of ascorbate at 290 nm, one millilitre of the reaction mixture contained 50 mM potassium phosphate (pH=7), 0.5 mM ascorbate, 0.1 mM EDTA and 0.1 mM hydrogen peroxide. The reaction was initiated by addition of hydrogen peroxide, and oxidation of ascorbate was followed by the decrease in absorbance at 290 nm at 30 seconds interval for 5 min. One unit of ascorbic acid peroxidase enzyme activity is expressed as the amount of APX enzyme that oxidizes 1 pmol of ascorbate per min at room temperature.

6. Statistical analyses

Proline pre-treatment and [UV.sub.A+B] irradiation experiments were performed using completely randomized design based on three repetitions. The effect of seed pretreatment with proline and doses of ultraviolet radiation of different genotypes were assessed using multivariate test statistics followed by post-hoc analysis. Statistical analyses were performed using SPSS version 22 software in probability level of 0.05 and 0.01 and with the help of Microsoft excel 2016.

RESULTS

Morphological and growth parameters

Leaf area expressed as [cm.sup.2] were measured and recorded for the three Vicia faba genotypes irradiated with enhanced levels of [UV.sub.A+B] radiations both control and presowing treatment with 0.01 proline for 6 hours. Figure (1) represent the leaf area in [cm.sup.2] of the three V. faba genotypes; (A) Sakha-1, (B) Sakha-2 and (3) Giza716. The genotype Giza-716 showed no major difference under the effect of [UV.sub.A+B] when compared to the control plants, while for genotypes Sakha-1 and Sakha-2, external proline treatment of Vicia faba plants under [UV.sub.A+B] exhibited a general improvement in leaf area (Figure, 1). The three genotypes showed a significant difference in leaf area ([cm.sup.2]) between the external proline soaking treatment (+) and water soaked plants.

The interactive effect of [UV.sub.A+B] exposure and proline induced a marked stimulatory effect in dry matter production of shoots of genotype sakha-2. Surprisingly and interestingly the highest stimulatory effect in shoot dry matter production was recorded at the highest [UV.sub.A+B] dose (12.48 KJ.[m.sup.-2].[d.sup.-1]) pretreated with 0.01 mole proline, where the percent increase in dry matter production was more than 100% in relation to absolute control. A marked stimulatory effect in dry matter production was recorded as a result of the interaction of UV radiations and proline pretreatment.

[FIGURE 1 OMITTED]

Photosynthetic Pigments

Chlorophyll-a, chlorophyll-b, total chlorophyll (Chla+b) and carotenoids contents in broad bean genotypes were estimated spectrophotometrically. Data of pigments revealed that proline treatment greatly increased the amount of photosynthetic pigments as compared with those of plants subjected only to [UV.sub.A+B] radiations. The external proline presowing treatment induced a marked stimulatory effect in the biosynthesis of photosynthetic pigments especially at lower and moderate irradiation dose of [UV.sub.A+B] (4.16 and 8.32 KJ.[m.sup.-2].[d.sup.-1]).

Oxidative damage

Electrolyte leakage expressed as cell death (%) was monitored in plants under enhanced levels of [UV.sub.A+B] both proline presowing treatment and the control (Figure, 2). Electrolyte leakage has increased markedly in genotype Sakha-1 and marginally in Giza-716 as a result of [UV.sub.A+B] exposure. The percent of increase in the two genotypes varied noticeably. At the highest level of [UV.sub.A+B] doses (12.48 KJ.[m.sup.-2].[d.sup.-1]), the percent increase of electrolyte leakage was 32.45% and 18.61% in genotype Sakha-1 and Giza-716; respectively. The genotype Sakha-2 mainly retained the values of electrolyte leakage around those of control plants. The interactive effect of proline and [UV.sub.A+B] exposure resulted in progressive dropping of the values of the electrolyte leakage whatever the genotypes tested and even the dose of [UV.sub.A+B] exposure (Table, 2).

[FIGURE 2 OMITTED]

Hydrogen peroxide accumulated significantly under the treatment with [UV.sub.A+B] radiations. A significant and progressive increasing trend in [H.sub.2][O.sub.2] accumulation in genotype in all genotypes as the dose of [UV.sub.A+B] radiation increased (r=0.53, p>-2-tailed=0.000*) and were statistically assessed using Spearman's rank correlation followed by two-tailed significance test. Sakha-2 showed the marked increase in hydrogen peroxide accumulation with increasing does of [UV.sub.A+B] (Figure, 3).

[FIGURE 3 OMITTED]

Consequently, the highest increase in [H.sub.2][O.sub.2] content was recorded at the highest dose of [UV.sub.A+B] radiation (12.48 KJ.[m.sup.-2].[d.sup.-1]). At the level of 12.48 KJ.[m.sup.-2].[d.sup.-1], the percent increase in [H.sub.2][O.sub.2] was about 122% over the absolute control (Figure, 3a-c). The interaction effect of proline and [UV.sub.A+B] radiation resulted in a marked increase in [H.sub.2][O.sub.2] content as compared with those treated only with [UV.sub.A+B], and the interaction assessed to highly significant using multivariate test (F=80.37; p>=0.000*; Table 4). In broad bean genotype Giza-716; [H.sub.2][O.sub.2] content increased progressively and quickly by UV treatments. The magnitude of this effect was much pronounced at the highest dose of [UV.sub.A+B]. At the level of 12.48 KJ.[m.sup.-2].[d.sup.-1], the percent increase of [H.sub.2][O.sub.2] content was 171% in relation to the absolute control values.

Proline treatment greatly offset the accumulation of [H.sub.2][O.sub.2] whatever the dose of [UV.sub.A+B] radiation in relation to [UV.sub.A+B] radiation treated plants (Table 4). Interestingly, the amount of [H.sub.2][O.sub.2] in plants treated with 12.48 KJ.[m.sup.-2].[d.sup.-1] and proline become half fold those of plants treated with only 12.48 KJ.[m.sup.- 2].[d.sup.-1] [UV.sub.A+B] dose. Accordingly, proline treatment successes in a great reduction in [H.sub.2][O.sub.2] content especially in Sakha-1, because the amount of [H.sub.2][O.sub.2] in this genotype maintained mainly around those of control value. In broad bean genotype Sakha-1; hydrogen peroxide content increased significantly by increasing the irradiation dose of [UV.sub.A+B]; at irradiation dose of 12.48 KJ.[m.sup.-2].[d.sup.-1] [H.sub.2][O.sub.2] were increased by 44%. Proline pretreatment retained the amount of [H.sub.2][O.sub.2] around those of absolute control values at the lowest doses of [UV.sub.A+B] radiation.

Free radicals generate the lipid peroxidation process in cell of the stressed organisms. Malondialdehyde (MDA) is one of the final products of polyunsaturated fatty acids peroxidation in cells as a result of ultraviolet radiation [UV.sub.A+B]. Lipid peroxidation as MDA were monitored and data in table (3) and figures ([4.sub.A-C], 5) represented the different responses of the three genotypes to [UV.sub.A+B] radiation stress. The general trend of lipid peroxidation (MDA) was significant increase in response to enhanced levels of [UV.sub.A+B] radiations (Spearman's r= 0.63; p=0.000*). Enhanced doses of ultraviolet radiations significantly induced lipid peroxidation in V. faba (Table 4; F=1793.7, p=0.000*) tested by multivariate analysis. Moreover interaction between proline and [UV.sub.A+B] were also significant revealing that external proline pretreatment could improve the cellular response to ultraviolet radiation and decrease lipid peroxidation level (Table 4; external proline versus [UV.sub.A+B]; F= 62.67, p=0.000*).

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

The genotype Sakha-2: Ultraviolet radiation exhibited insignificant changes in the values of MDA content whatever the [UV.sub.A+B] dose tested. Proline treatment induced reduction in MDA values even below the absolute control. In broad bean genotype Giza-716: [UV.sub.A+B] radiation induced smooth and gradual accumulation of MDA content which was more observed at the highest [UV.sub.A+B] dose 12.48 KJ.[m.sup.-2].[d.sup.-1] where the percent increase was only 17.97% in relation to absolute control. Proline treatment induced significant reduction in MDA values whatever the UV dose experimented with. In broad bean genotype Sakha-1: MDA content remained unchanged up to 4.16KJ.[m.sup.2].[d.sup.-1] dose, the slight increase was reported at the level of 8.32 KJ.[m.sup.-2].[d.sup.-1], and then marked and progressive accumulation of MDA was reported at the level of 12.48 KJ.[m.sup.-2].[d.sup.-1] where the percent increase was 38.23% in relation to the absolute control. Proline treatment induced significant reduction in the values of MDA content in relation to corresponding [UV.sub.A+B] dose only.

Antioxidant Enzyme activity

Catalase as one of the protective enzymes in plants were evaluated of V. faba plants under ultraviolet radiations [UV.sub.A+B] following proline pretreatment (Figure, 6). Generally, the trend of catalase activity was nonsignificant ranged between increase in sakha-2 and giza-716 and decrease in sakha-1 (r=0.17, p=.15). Elevated doses of ultraviolet radiations significantly induced changes in catalase activity V. faba (Table 4; F=10.45, p=0.000*) tested by multivariate analysis. The interaction between proline and [UV.sub.A+B] were also significant revealing that external proline pretreatment could improve the cellular response to ultraviolet radiation and changed the catalase activities (Table 4; external proline vs. [UV.sub.A+B]; F= 9.09, p=.000*). Figure (5) represented that the activity of catalase enzyme varied considerably among the three studied broad bean genotypes and different treatments. That whilst the activity of catalase enzyme decreased markedly in Sakha-1, on the other hand it elevated considerably in Sakha-2 and Giza-716 due to [UV.sub.A+B] exposure. However the percent increase varied considerably between the two genotypes, which were 58.82% and 400% over those of control plants (-ve proline) at the level of 12.48 KJ.[m.sup.-2].[d.sup.-1]. It's worthy to mention that the pretreatment with proline of the [UV.sub.A+B] stressed plants mostly retained the activity of catalase enzyme around those of control plants whatever the broad bean genotype tested.

[FIGURE 6 OMITTED]

Although the activity of peroxidase enzyme increased in the three selected broad bean genotypes as a result of [UV.sub.A+B] exposure, the percent increase of peroxidase activity differed greatly (Figure, 7). Elevated doses of ultraviolet radiations significantly induced changes in peroxidase activities of V. faba (Table 4; F=203.3, p=0.000*) tested by multivariate analysis.

[FIGURE 7 OMITTED]

The interaction between the effect of proline pretreatment and different irradiation doses of [UV.sub.A+B] were also significant revealing that external proline pretreatment could improve the cellular response to ultraviolet radiation and changed the catalase activities (Table 4; external proline vs. [UV.sub.A+B]; F=166.2, p=0.000*). At the level of 12.48 KJ.[m.sup.-2].[d.sup.-1], the percent increase in the activity of peroxidase was 280 %, 37.76% and 62.22% over the control values in genotypes Sakha-1, Sakha-2 and Giza-716; respectively. This means that the most sensitive genotype recorded the highest activity of peroxidase activity, followed by Giza-716 (the intermediately responded genotype) and the activity of peroxidase increased marginally in the most tolerant genotype (only 37.76% over the absolute control). Therefore the criteria of peroxidase went opposite to the degree of tolerance among the [UV.sub.A+B] stressed genotypes. Consequently the differences in the activity of peroxidase can be used as suitable marker in the genotypic variation under stress. In confirmatory, exogenous application of proline greatly dropped the activity of peroxidase enzyme in the three studied broad bean genotypes in relation to those treated with only [UV.sub.A+B] radiation, accompanied with a marked improvement in plant growth. Elevated doses of ultraviolet radiations significantly induced changes in ascorbic peroxidase (APX) activities of V. faba (Table 4; F=39.3, p=0.000*) tested by test of between-subject multivariate analysis. The interaction between the effect of proline pretreatment and different irradiation doses of [UV.sub.A+B] were also significant revealing that external proline seed pretreatment could improve the cellular response to ultraviolet radiation and changed the catalase activities (Table 4; external proline vs. [UV.sub.A+B]; F=57.23, p=.000*).

[FIGURE 8 OMITTED]

The activity of ascorbic acid peroxidase remained unchanged even at the highest doses of [UV.sub.A+B] radiation in Sakha-1, and increased by 50% over the control in Sakha-2 (Figure, 8). In Giza-716 there is a marked and progressive increasing trend in the activity of ascorbic acid peroxidase whatever the [UV.sub.A+B] dose applied. At the level of 12.48 KJ.[m.sup.-2].[d.sup.-1], the percent increase in ascorbic acid peroxidase was 233% over the control. It's worthy to mention that the activity of ascorbate peroxidase retarded under the interactive effect of [UV.sub.A+B] doses and seed pretreatment with proline as compared the corresponding to [UV.sub.A+B] treated plants.

DISCUSSION

Ozone layer is continuously being damaged resulting in increasing the levels of UV radiation, which can be harmful for all life forms especially higher plants. UV radiation often causes different changes in physiological parameters especially in antioxidant system among plant species and genotypes.

The study showed the genotype Sakha-1 exhibited dropping in the photosynthetic pigments (chla+b) especially at the highest [UV.sub.A+B] doses (12.48 KJ.[m.sup.-2].[d.sup.-1]). While, the genotype Sakha-2 recorded stimulation trend in photosynthetic pigments as the [UV.sub.A+B] dose increased. In addition, the genotype Giza-716 mainly equilibrated the values of photosynthetic pigments round the control values. Additionally, the stimulatory effect of proline pre-soaking of [UV.sub.A+B] stressed plants, greatly confirmed this positive correlation.

Insignificant changes in the superior genotype Sakha-2 may be explained as that the genotype already did not expose to reduction in pigmentation thus there is no deleterious effect of [UV.sub.A+B] stress on the biosynthesis of photosynthetic pigments. Thus there is no need to the increase in carotenoids, which play a role as accessory light harvesting pigments and dissipating excess of excitation solar energy. In contrast the sensitive genotype Sakha-1 exhibited a significant reduction in photosynthetic pigments, thus the need to increase carotenoids is important to decrease the deleterious effects of [UV.sub.A+B] radiation on [UV.sub.A+B] stressed plants. This was greatly confirmed by proline treatment, which greatly enhanced the biosynthesis of photosynthetic pigments chlorophyll ([Chl.sub.a+b]) and induced insignificant changes in carotenoids, thus there is no need for further increase in carotenoids as a protecting agent.

According to the present data, it seems that the accumulation of carotenoids was found to be associated with the harmful effects of [UV.sub.A+B] radiation in plant growth as well as the efficiency of photosynthetic apparatus. Therefore, photosynthetically active pigments (Chla+b) but not carotenoid can be used as a suitable selection criterion for the various responses to [UV.sub.A+B] radiation among the three studied broad bean genotypes. The conclusion was greatly confirmed by the observable enhancement of exogenous proline on the all studied growth parameters as well as the photosynthetic pigments without further increase in carotenoids.

In some cases, plants exposed to various stresses can increase [H.sub.2][O.sub.2] content as a strategy to trigger the activity of multiple functional enzymes as well as many metabolic pathways [34]. Generation of ROS (such as [H.sub.2][O.sub.2], OH and [O.sup.*]) causes rapid cell damage by triggering a chain of reactions to protect themselves from the harmful effects of oxidative stress, plants develop ROS- scavenging mechanism that involve detoxification process carried out by an integrated system of the non-enzymatic molecules and the enzymatic antioxidant [34-36]. Exogenous application of proline ameliorated the inhibitory effect of [UV.sub.A+B] radiation via offset the damaging effect via maintaining MDA content mainly around the control plants and moreover, proline treatment resulted in some reduction in MDA content in comparison to absolute control accompanied with the great reduction in [H.sub.2][O.sub.2] content in relation to the corresponding [UV.sub.A+B] treatment only. Therefore, there is no need for enhancing the activity of antioxidant system in plants treated with proline. [H.sub.2][O.sub.2] received much attention as a signal molecule in response to different stresses [37-42]. [H.sub.2][O.sub.2] mediated the regulation of transcription in response to UV-B exposure as an important early upstream signal 39 Activation of endogenous protective mechanisms can in turn tolerate or delete excess ROS burst. They found that the enhanced [H.sub.2][O.sub.2] level under the stresses was followed by the up-regulation of the enzyme activities. This suggests that [H.sub.2][O.sub.2] may act more as a signal molecule than directly inducing oxidative damage.

The activity of antioxidant system revealed that a huge activation of catalase and ascorbic acid peroxidase in genotype Giza-716 followed be genotype Sakha-2. On the other hand, genotype Sakha-1 decreased the activity of both enzymes. A surprising observation recorded in the peroxidase activity that the highest peroxidase activity was recorded in genotype Sakha-1 followed by Giza-716 and marginal enhancement was recorded in genotype Sakha-2, indicating that the criteria of peroxidase was opposite to the degree of tolerance. Thus high peroxidase accumulation was sign sensitivity to [UV.sub.A+B] radiation. This was confirmed by proline treatment, which reduced partially or completely the stimulatory effect of [UV.sub.A+B] stress on the activity of peroxidase enzyme.

Increasing levels of ROS due to enhancing of UV trigger the activity of several antioxidant enzymes such as superoxide dismutase, catalase and peroxidase [44]. Hollosy stated that the overall UV-B sensitive of the cells is determined by the balance of damage that occurs and the efficiency of the repair processes that can restore the impaired functions protection against oxidative stress caused by UV exposure is complex process and includes both enzymatic and non-enzymatic antioxidant [45].

The activity of catalase, ascorbic acid peroxidase and peroxidase varied considerably among the three tested broad bean genotypes. (1) The activity of catalase and ascorbic acid peroxidase increased by 400% and 233% in genotype Giza-716. (2) Their activity increased by about 50% in genotype Sakha-2. (3) On other hand, in Sakha-1 (the most sensitive genotype), the activity of catalase enzyme decreased significantly and that of ascorbic acid peroxidase reduced marginally by about 10% which confirmed the triggering force of [H.sub.2][O.sub.2] on the activity of these enzymes in the tolerant genotypes (Sakha-2 and Giza-716). In Giza-716, which was the highest [H.sub.2][O.sub.2] producing genotype under UV stress, it exhibited the highest antioxidant enzymes activities of catalase and ascorbic acid peroxidase.

Surprisingly the opposite cut held was recorded in the most sensitive genotype (Sakha-1). The genotype Sakha-1 greatly lowered the two processes ([H.sub.2][O.sub.2] content and catalase as well as ascorbic acid peroxidase in relation to the other two genotypes (Sakha-2 and Giza-716). Thus the two processes were greatly interdependent. Other situation was recorded in Sakha-2, this genotype elevated the two enzymes (catalase and ascorbic acid peroxidase) by about 50 % (much less than genotype Giza-716).

Although the activity of peroxidase enzyme increased in the three selected broad bean genotypes as a result of [UV.sub.A+B] exposure, the percent increase of peroxidase activity differed greatly. At the level of 12.48 KJ.[m.sup.-2].[d.sup.-1], the percent increase in the activity of peroxidase was 280%, 37.76% and 62.22% over the control values in genotypes Sakha-1, Sakha-2 and Giza-716 respectively. This means that the most sensitive genotype recorded the highest activity of peroxidase activity, followed by Giza-716 (the intermediately responded genotype) and the activity of peroxidase increased marginally in the most tolerant genotype (only 37.76% over the absolute control). Therefore the criteria of peroxidase went opposite to the degree of tolerance among the [UV.sub.A+B] stressed genotypes. Consequently the differences in the activity of peroxidase can be used as suitable marker in the genotypic variation under stress, indicating sensitivity rather than tolerance of genotypes or varieties under different stresses.

In confirmatory, proline treatment reduced partially or completely the stimulatory effect of [UV.sub.A+B] stress on the activity of peroxidase enzyme. This reduction in the activity of peroxidase under the interaction of [UV.sub.A+B] stress and proline treatment was found to be associated with the general stimulatory effect of proline treatment on the dry matter yield of broad bean genotypes. This means that the increase in peroxidase activity of [UV.sub.A+B] stressed plants may be considered as a sign of [UV.sub.A+B] injury. Thus the retarded activity of peroxidase in [UV.sub.A+B] -proline treated broad bean genotypes may be one aspect of the role of proline in equilibrating certain metabolic pathways which in turn up-regulated the production of toxic free radicals, such proline-induced traits in peroxidase activity needs further studies to be more clarified. The excess UV-B radiation could promote and stimulate the generation of ROS leading to increase in the activities of antioxidant enzymes as a defence system induced antioxidant defences protecting plant against major fatal effects of ROS [18,21,44].

REFERENCES

[1] Tevini, M., 1994. Physiological changes in plants related to UV-B radiation: An overview. In: Biggs R.H., Joiner MEB (eds) Stratospheric Ozone Depletion/ UV-B Radiation in the Biosphere. NATO ASI Series I, Springer-Verlag, Berlin, Heidelberg, 18: 37-56.

[2] Middleton, E.M. and A.H. Teramura, 1994. Understanding photosynthesis, pigment and growth responses induced by UV-B and UV-A irradiances. Photochem. Photobiol, 60: 38-45.

[3] Weih, M., U. Johanson, D. Gwynn-Jones, 1998. Growth and nitrogen utilization in seedlings of mountain birch (Betula pubescens ssp. tortuosa) as affected by ultraviolet radiation (UV-A and UV-B) under laboratory and outdoor conditions trees. Struct. Funct., 12: 201-204

[4] Rozema J., Staaij J.vd., L.O. Bjorn, M. Caldwell, 1997. UV-B as an environmental factor in plant life: stress and regulation. Trends Ecol. Evol., 12: 22-28.

[5] Gwynn-Jones, D., J.A. Lee, T.V. Callaghan, 1997. The effects of UV-B radiation and elevated carbon dioxide on subarctic forest ecosystems. Plant Ecol, 128: 242-249.

[6] Rahimzadeh, P., S. Hosseini, K. Dilmaghani, 2011. Effects of UV-A and UV-C radiation on some morphological and physiological parameters in Savory (Satureja hortensis L.). Ann. Biol. Research, 5: 164-171.

[7] Hosseini, S., C. Jirair, K. Jalil, 2010. The effects of UV radiation on some structural and ultrastructural parameters in pepper (Capsicum longum). Turk J. Biol, 35: 69-77.

[8] Yao, Y., Z. Xuana, Y. Li, 2006. Effects of ultraviolet-B radiation on crop growth, development, yield and leaf pigment concentration of tartary buck wheat (Fagopyrum tataricum) under field conditions. Eur. J. Agron, 25: 215-222.

[9] Nogues, S., D.J. Allen, J.I.L. Morison, 1998. Ultraviolet-B radiation effects on water relations, leaf development, and photosynthesis in droughted pea plants. Plant Physiolm, 117: 173-181.

[10] Kumari, R., S. Singh, S.B. Agrawal, 2009. Responses of ultraviolet-B induced antioxidant defense system in a medicinal plant Acorus calamus L. J. Environ. Biol, 31: 907-911.

[11] Katerova, Z., S. Ivanov, S. Mapelli, V. Alexieva, 2009. Phenols, proline and low-molecular thiol levels in pea (Pisum sativum) plants respond differently toward prolonged exposure to ultraviolet-B and ultraviolet C radiations. Acta. Physiol. Plant, 31: 111-117.

[12] Kakani, V.G., K.R. Reddy, D. Zhao, K. Sailaja, 2003. Field crop responses to ultraviolet-B radiation: a review. Agric. For. Meteorol, 120: 191-218.

[13] Agrawal, S.B., D. Rathore, 2007. Changes in oxidative stress defence in wheat (Triticum aestivum L.) and mung bean (Vigna radiate L.) cultivars grown with or without mineral nutrients and irradiated by supplemental ultraviolet-B. Environ. Exp. Bot., 59: 21-27.

[14] Singh, R., S. Singh, R. Tripathi, S.B. Agrawal, 2011. Supplemental UV-B radiation induced changes in growth, pigments and antioxidant pool of bean (Dolichos lablab) under field conditions. J. Environ. Biol. 32: 139-145.

[15] Zlatev, S.Z., J.C. Fernando, Lidon and M. Kaimakanova, 2012. Plant physiological responses to UV-B radiation. Emir. J. Food Agric, 24(6): 481-501.

[16] Kataria, S., A. Jajoo, K.N. Guruprasad, 2014. Impact of increasing ultraviolet-B (UV-B) radiation on photosynthetic processes. Journal of Photochemistry and Photobiology. B, Biology, 137: 55-66.

[17] Aphalo, Pedro, J., A.K. Marcel, Jansen, R. Andy, Mcleod, Otmar Urban, 2015. "Ultraviolet Radiation Research: From the Field to the Laboratory and Back." Plant, Cell & Environment, 38(5): 853-55. doi:10.1111/pce.12537.

[18] Tasgin, E., H. Nadaroglu, 2013. Effects of UV-B radiation and butylated hydroxyanisole (BHA) on the response of antioxidant defense systems in winter wheat (Triticum aestivum L.) seedlings. African J. of Agric. Research, 8(4): 398-404.

[19] Pal, M., U.K. Sengupta, A.C. Srivastava, V. Jain, R.C. Meena, 1999. Changes in growth and photosynthesis of mung bean induced by UV-B radiation. Ind. J. Plant. Physiol, 4(5): 79-84.

[20] Indrajith, A. and K.C. Ravindran, 2009. Antioxidant Potential of Indian Medicinal Plant Phyllanthus Amarus L. Under Supplementary UV-B Radiation. Recent Res. Sci. Technol, 1(1): 034-042.

[21] Xu, C., S. Natarajan, J.H. Sullivan, 2008. Impact of solar ultraviolet-B radiation on the antioxidant defense system in soybean lines differing in flavonoid contents. Environ. Exp. Bot., 63: 39-48.

[22] Abdel-Kader, D., A.H.S. Saleh, A. Abu-Elsaoud, 2007. Enhanced UV A+B induced oxidative damage and antioxidant defence system in Glycine max L. cultivars, Acta Botanica Hungarica, 49(3-4): 233-250.

[23] Foyer, C.H., M. Lelandais, K.J. Kunert, 1994. Photooxidative stress in plants. Physiol. Plant, 92: 696-717.

[24] Larson, R.A., 1988. The antioxidants of higher plants. Phytochemistry, 24: 889-896.

[25] Noctor, G., C.H. Foyer, 1998. Ascorbate and glutathione: keeping active oxygen under control. Annu Rev Plant Physiol Plant Mol Biol, 49: 249-79; PMID:15012235; http://dx.doi.org/10.1146/ annurevarplant, 49.1.249.

[26] Jimenez, A., J.A. Hernandez, L.A. del Rio, F. Sevilla, 1997. Evidence for the presence of the ascorbateglutathione cycle in mitochondria and peroxisomes of pea leaves. Plant Physiol, 114: 275-284.

[27] Metzner, H., H. Rau, H. Senger, 1965. Untersuchungen zur synchronisier-barkeit einzelner pigment Mangei. Mutanten von Chlorella Planta, 65: 186-194.

[28] Dionisio-Sese, M.L. and S. Tobita, 1998. 'Antioxidant responses of rice seedlings to salinity stress', Plant Science, 135(1): 1-9.

[29] Hodges, D.M., J.M. DeLong, C.F. Forney, R.K. Prange, 1999. Improving the thiobarbituric acid- reactivesubstances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta, 207: 604-611.

[30] Jiang, Z.Y., A.C.S. Woollard, S.P. Wolff, 1990. Hydrogen peroxide production during experimental protein glycation. FEBS Lett, 268: 69-71.

[31] Aebi, H.E., 1983. Catalase. In: Bergmeyer HU, ed. Methods of enzymatic analysis, Vol. 3.Weinhem: Verlag Chemie, 273-286.

[32] Malik, C.P. and M.B. Singh, 1980. Plant Enzymology and Histo Enzymology. Kalyani Publishers, New Delhi, 286.

[33] Nakano, Y. and K. Asada 1987. Purification of ascorbate peroxidase in spinach chloroplasts; Its inactivation in ascorbate-depleted medium and reactivation by monodehydroascorbate radical. Plant Cell Physiol, 28: 131-140.

[34] Mittova V., M. Tal, M. Volokita, M. Guy, 2003. Up-regulation of the leaf mitochondrial and peroxisomal antioxidative system in response to salt induced oxidative stress in the wild salt-tolerant tomato species Lycopersion pennellii, Plant Cell Environ, 26: 845-856.

[35] Kojo, S., 2004. Vitamin C: basic metabolism and its function as an index of oxidative stress. Curr. Med. Chem, 11(8): 1041-64

[36] Jaleel, C.A., K. Riadh, R. Gopi, P. Manivannan, J. Ines, H.J. Al-Juburi, Z.S. Hong-Bo, R. Panneerselvam, 2009. Antioxidant defense responses: physiological plasticity in higher plants under abiotic constraints. Acta Physiol Plant, 31: 427-436.

[37] Gong, L., K. Takayama, S. Kjelleberg, 2001. Near-ultraviolet resistance and carbon starvation survival in cool white light-exposed cells of Escherichia coli and Vibrio angustum S14. In 101th General Meeting of the American Society for Microbiology. ASM Press, Orlando. Abstract Q, 111.

[38] Aroca, R., P. Vernieri, J.J. Irigoyen, M. Sanchez-Diaz, F. Tognoni, A. Pardossi, 2003. Involvement of abscisic acid in leaf and root of maize (Zea mays L.) in avoiding 7 chilling-induced water stresses. Plant Sci., 165: 671-679.

[39] Veal, E.A., A.M. Day, B.A. Morgan, 2007. 'Hydrogen peroxide sensing and signaling', Mol. Cell, 26(1): 1-- 14.

[40] Veal, E.A., A. Day, 2011. Hydrogen peroxide as a signalling molecule, Antioxid. Redox Signal., 15(1): 147--151.

[41] Zentgraf, U., P. Zimmermann, A. Smykowski, 2012. Role of intracellular hydrogen peroxide as signaling molecule for plant senescence, In-tech open access publishing, In: Senescence, Tetsuji Nagata (Ed.), ISBN: 978-953-51-0144-4,

[42] Hossain, M.A., S. Bhattacharjee, S.M. Armin, P. Qian, W. Xin, H.Y. Li, D.J. Burritt, M. Fujita, L.S.P. Tran, 2015. Hydrogen peroxide priming modulates abiotic oxidative stress tolerance: insights from ROS detoxification and scavenging', Frontiers in Plant Science, 6.

[43] Brosche, M., A. Strid, 2003. 'Molecular events following perception of ultraviolet-B radiation by plants', Physiologia Plantarum, 117(1): 1--10.

[44] Mishra, V., P. Mishra, G. Srivastava, S.M. Prasad, 2011. Effect of dimethoate and UV-B irradiation on the response of antioxidant defense systems in cowpea (Vigna unguiculata L.) seedlings. Pestic. Biochem. Phys, 100: 118--123.

[45] Hollosy, F., 2002. Effects of ultraviolet radiation on plant cell. Micron, 33: 179--197.

(1) Amal A.H. Saleh, (1) Abdelghafar M. Abu-Elsaoud, (1) Amr A. Elkelish, (2) Mohamed A. Sahadad and (3) Eman M. Abdelrazek

(1) Deparment of Botany, Faculty of Science, Suez Canal University, Ismailia 41522, Egypt

(2) Deparment of Botany, Faculty of Science, Assiut University, 71515 Assiut, Egypt

(3) Deparment of Botany, Faculty of Science, Suez University, Suez, Egypt

Received 23 September 2015; Accepted 25 November 2015

Address For Correspondence:

Abdelghafar M. Abu-Elsaoud, Department of Botany, Faculty of Science, Suez Canal University, Ismailia 41522, Egypt Tel: +201203332514; E-mail: Abuelsaoud@Science.suez.edu.eg
Table 1: Experimental conditions and UV-lamp specifications
for UV/proline treatment of three Vicia faba genotypes.

Experimental
conditions              Details

Radiation type          Ultraviolet radiations ([UV.sub.A+B])
UV Lamp Model           Turbo Black Light Blue FL20T8/BLB
UV lamp                 Diameter: 38.10 mm, Length: 609.60 mm
  dimensions/Specs
Wavelengths             300-410 nm
Maximum wavelength      350 nm
  ([lambda].sub.max])
UV-Exposure time/day    0, 4, 8 and 12 hours/day
UV-Irradiation dose     0, 4.16, 8.32, and 12.48
                          KJ.[m.sup.-2] x [d.sup.-1]
Proline treatment       6 hours pre-sowing
                          soaking water/proline
Genotypes treated       Vicia faba L. genotype Sakha-1
                        Vicia faba L. genotype Sakha-2
                        Vicia faba L. genotype Giza-716

Table 2: Enhanced levels of UV-radiations on electrolyte leakage
(%-fresh wt.) in three broad bean genotypes (-proline=not
treated with proline, +proline: soaked in 0.01 proline).

                  Electrolyte leakage
Treatment      (% fresh wt.) in Genotypes
UVA+B (kJ x
[m.sup.-2] x         Sakha-1
[d.sup.-1])      Mean [+ or -] SE      %

(-) proline
  0            114.8a [+ or -] 0.46   100
  4.16         117.4 [+ or -] 0.58    102
  8.32         132.6 [+ or -] 0.35    116
  12.48        152.0 [+ or -] 0.58    132
(+) proline
  0            103.0 [+ or -] 0.58    90
  4.2          106.4 [+ or -] 0.23    93
  8.32         108.6 [+ or -] 0.35    95
  12.48        119.4 [+ or -] 0.58    104

One Way Analysis of Variance (ANOVA)

F-ratio              1130.38
p-value              0.000 *

                     Electrolyte leakage
                (% fresh wt.) in Genotypes
Treatment
UVA+B (kJ x
[m.sup.-2] x         Sakha-2
[d.sup.-1])      Mean [+ or -] SE      %

(-) proline
  0            123.5a [+ or -] 0.29   100
  4.16         124.0a [+ or -] 0.58   100
  8.32         126.6 [+ or -] 0.35    102
  12.48        130.0 [+ or -] 0.58    105
(+) proline
  0            105.0 [+ or -] 0.29    85
  4.2          113.0 [+ or -] 0.58    92
  8.32         114.0 [+ or -] 0.58    92
  12.48        117.0 [+ or -] 0.29    95

One Way Analysis of Variance (ANOVA)

F-ratio               325.32
p-value              0.000 *

                     Electrolyte leakage
Treatment        (% fresh wt.) in Genotypes
UVA+B (kJ x
[m.sup.-2] x         Giza-716
[d.sup.-1])      Mean [+ or -] SE       %

(-) proline
  0            109.6a [+ or -] 0.35    100
  4.16         121.8 [+ or -] 0.46    111.1
  8.32         127.4 [+ or -] 0.58    116.2
  12.48        130.0 [+ or -] 0.58    118.6
(+) proline
  0            102.0 [+ or -] 0.29    93.1
  4.2          115.0 [+ or -] 0.58    104.9
  8.32         113.0 [+ or -] 0.58    103.1
  12.48        106.5 [+ or -] 0.29    97.2

One Way Analysis of Variance (ANOVA)

F-ratio               433.81
p-value              0.000 *

Means not labelled with letter (a) are significantly different from
control level mean, * significant at p [less than or equal to] 0.05

Table 3: Enhanced levels of UV-radiations on Lipid peroxidation
([??]mole / g-fresh wt.) in three broad bean genotypes
(-proline = not treatedwith proline, +proline: soaked in
0.01 proline).

                   Lipid peroxidation
                  (Mmole/g fresh wt.)
                        Genotypes

                         Sakha-1
UVa+b Treatment
(kJ.m-2.d-1)        Mean [+ or -] SE       %

(-)         0     9.6 (a) [+ or -] 0.06   100
proline   4.16    10.0 [+ or -] 0.12      104
          8.32    12.8 [+ or -] 0.06      134
          12.48   13.3 [+ or -] 0.06      138

(+)         0     9.4a [+ or -] 0.06      98
proline   4.16    10.0 [+ or -] 0.06      104
          8.32    10.4 [+ or -] 0.06      108
          12.48   10.4 [+ or -] 0.06      108

One Way Analysis of Variance (ANOVA)

F-ratio                  475.59
p-value                  0.000*

                   Lipid peroxidation
                   (Mmole/g fresh wt.)
                        Genotypes

                         Sakha-2
UVa+b Treatment
(kJ.m-2.d-1)         Mean [+ or -] SE       %

(-)         0     11.5 (a) [+ or -] 0.01   100
proline   4.16    11.8 [+ or -] 0.02       103
          8.32    12.0 [+ or -] 0.01       105
          12.48   12.3 [+ or -] 0.06       107

(+)         0      9.4 [+ or -] 0.06       82
proline   4.16     9.4 [+ or -] 0.01       82
          8.32    10.3 [+ or -] 0.06       90
          12.48   10.9 [+ or -] 0.01       95

One Way Analysis of Variance (ANOVA)

F-ratio                   995.3
p-value                  0.000 *

                   Lipid peroxidation
                   (Mmole/g fresh wt.)
                        Genotypes

                         Giza-716
UVa+b Treatment
(kJ.m-2.d-1)         Mean [+ or -] SE       %

(-)         0     11.0 (a) [+ or -] 0.01   100
proline   4.16    11.5 [+ or -] 0.02       105
          8.32    12.5 [+ or -] 0.06       113
          12.48   13.0 [+ or -] 0.12       118

(+)         0      8.4 [+ or -] 0.06       76
proline   4.16    10.7 [+ or -] 0.06       97
          8.32    10.8 (a) [+ or -] 0.02   98
          12.48   12.2 [+ or -] 0.01       111

One Way Analysis of Variance (ANOVA)

F-ratio                   666.9
p-value                   0.000*

Means not labelled with letter (a) are significantly different
from control level mean; *significant at p [less than or
equal to] 0.05

Table 4: Multivariate analysis assessing the effect of
external proline on V. faba under elevated levels of [UV.sub.A+B].
Model carried out include dependent parameters (water
content, total chlorophyll, leaf area, lipid peroxidation,
proline, hydrogen peroxide, catalase, ascorbic peroxidase
and peroxidase), and [UV.sub.A+B] and genotypes as covariates.

Multivariate Test: Pillai's Trace test

                   Pillai's
                     Trace                Hypothesis   Error
Effect               Value     F-ratio        df        df      Sign.

1. External          1.00      18256.00     17.00      32.0    0.000 *
proline
pretreatment

2. Genotypes         2.00      10241.00     34.00      66.0    0.000 *

3. Ultraviolet       2.98       235.00      51.00      102.0   0.000 *
([UV.sub.A+B])
doses

4. External          2.00      1403.00      34.00      66.0    0.000 *
proline vs.
genotypes

5. External          2.98       292.00      51.00      102.0   0.000 *
proline vs.
[UV.sub.A+B]

6. Genotypes         5.84       77.00       102.00     222.0   0.000 *
vs. [UV.sub.A+B]

7. External          5.82       69.00       102.00     222.0   0.000 *
proline vs.
genotypes vs.
[UV.sub.A+B]

Tests of Between-Subjects Effects

Source                        df   WC        [Chl.sub.a+b]   L.Area

1. External         F-ratio   1    4.00      76050           5934
proline             sign.          0.051     0.000 *         0.000 *
pretreatment
(n=2)

2. Genotypes        F-ratio   2    4.05      200718          2855
(n=3)               sign.          0.024 *   0.000 *         0.000 *

3. [UV.sub.A+B]     F-ratio   3    1.67      33138.8         533.9
(n=4)               sign.          0.187     0.000 *         0.000 *

4. External         F-ratio   2    0.21      6919.1          154.7
proline vs.         sign.          0.809     0.000 *         0.000 *
genotypes
(n=6)

5. External         F-ratio   3    0.74      2882.3          70.4
proline vs.         sign.          0.533     0.000 *         0.000 *
[UV.sub.A+B]
(n=8)

6. Genotypes        F-ratio   6    0.94      74788.7         904.25
vs. [UV.sub.A+B]    sign.          0.478     0.000 *         0.000 *
(n=12)

7. Proline tr.      F-ratio   6    4.92      464.96          345.07
vs. genotypes vs.   sign.          0.001 *   0.000 *         0.000 *
[UV.sub.A+B]
(n=24, df=23)

Source                        MDA       Proline   H2o2      CAT

1. External         F-ratio   5118      39480     130.8     41.1
proline             sign.     0.000 *   0.000 *   0.000 *   0.000 *
pretreatment
(n=2)

2. Genotypes        F-ratio   200       25234     47.9      62.6
(n=3)               sign.     0.000 *   0.000 *   0.000 *   0.000 *

3. [UV.sub.A+B]     F-ratio   1793.7    14635.5   157.1     10.45
(n=4)               sign.     0.000 *   0.000 *   0.000 *   0.000 *

4. External         F-ratio   49.1      12069.9   56.47     59.62
proline vs.         sign.     0.000 *   0.000 *   0.000 *   0.000 *
genotypes
(n=6)

5. External         F-ratio   62.7      1178.8    80.4      9.1
proline vs.         sign.     0.000 *   0.000 *   0.000 *   0.000 *
[UV.sub.A+B]
(n=8)

6. Genotypes        F-ratio   134.76    3885.88   46.66     9.74
vs. [UV.sub.A+B]    sign.     0.000 *   0.000 *   0.000 *   0.000 *
(n=12)

7. Proline tr.      F-ratio   230       2475.21   3.46      17.58
vs. genotypes vs.   sign.     0.000 *   0.000 *   0.000 *   0.000 *
[UV.sub.A+B]
(n=24, df=23)

Source                        ASPX      POD

1. External         F-ratio   9.4       583
proline             sign.     0.004 *   0.000 *
pretreatment
(n=2)

2. Genotypes        F-ratio   30.29     228
(n=3)               sign.     0.000 *   0.000 *

3. [UV.sub.A+B]     F-ratio   39.62     203.27
(n=4)               sign.     0.000 *   0.000 *

4. External         F-ratio   8.37      234.36
proline vs.         sign.     0.000 *   0.000 *
genotypes
(n=6)

5. External         F-ratio   57.2      166.2
proline vs.         sign.     0.000 *   0.000 *
[UV.sub.A+B]
(n=8)

6. Genotypes        F-ratio   46.56     40.72
vs. [UV.sub.A+B]    sign.     0.000 *   0.000 *
(n=12)

7. Proline tr.      F-ratio   20.82     33.3
vs. genotypes vs.   sign.     0.000 *   0.000 *
[UV.sub.A+B]
(n=24, df=23)

Additive information; df(error) = 48; dftotal) = 72;
df(corrected model) = 23; df(corrected model) = 71
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Author:Saleh, Amal A.H.; Abu-Elsaoud, Abdelghafar M.; Elkelish, Amr A.; Sahadad, Mohamed A.; Abdelrazek, Em
Publication:American-Eurasian Journal of Sustainable Agriculture
Article Type:Report
Date:Dec 1, 2015
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