Ecotoxicology and Environmental Safety 100 (2014) 178–187

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Ultraviolet-B induced changes in morphological, physiological and biochemical parameters of two cultivars of pea (Pisum sativum L.) Krishna Kumar Choudhary, S.B. Agrawal n Laboratory of Air Pollution and Global Climate Change, Department of Botany, Banaras Hindu University, Varanasi 221005, India

art ic l e i nf o

a b s t r a c t

Article history: Received 30 July 2013 Received in revised form 26 October 2013 Accepted 28 October 2013 Available online 21 November 2013

Increase in perception of solar ultraviolet-B (UV-B) radiation on Earth’s surface due to anthropogenic activities has potential in causing detrimental effects on plants. The present study was performed to evaluate the effect of elevated UV-B on Pisum sativum L., a leguminous plant with emphasis on nitrogen metabolism, flavonoids and hormonal changes. Elevated UV-B (ambient þ7.2 kJ m  2 day  1) negatively affected the growth, biomass, yield and its quality by generating oxidative stress directly or due to elevation of salicylic acid in two cultivars with higher magnitude being observed in HUP-2 as compared to HUDP-15. The increased accumulation of flavonoids (quercetin and kaempferol) under elevated UV-B neither provided sufficient protection to the photosynthetic machinery nor helped in elevation of biological nitrogen fixation. Nitrogen fixation and its assimilation were negatively affected under elevated UV-B as observed by the decline in nitrogenase, nitrate reductase, nitrite reductase activities and leghaemoglobin contents. Higher accumulation of salicylic acid in HUP-2 might be associated with its higher degree of sensitivity against UV-B, while higher induction of jasmonic acid and antioxidative enzymes (superoxide dismutase, catalase and ascorbate peroxidase activities) provided resistance to HUDP-15 against applied stress vis-a-vis exhibited less reduction in biomass, yield and quality of produce. & 2013 Elsevier Inc. All rights reserved.

Keywords: Antioxidant enzymes Carbohydrate content Yield Flavonoids Jasmonic and salicylic acid Nitrogen metabolism

1. Introduction Stratospheric ozone (O3) layer absorbs ultraviolet-B (UV-B) radiation and checks its penetration to the Earth’s surface. The thinning of this layer caused by contamination with man-made chlorofluorocarbons, nitrogen oxides and methyl bromide leads to an increase in solar UV-B radiation (280–315 nm) having potential deleterious consequences on agricultural production and natural plant ecosystems (Caldwell et al., 1995). UV-B radiation causes changes in morphological traits (Correia et al., 1998; Gonzalez et al., 1998; Yang et al., 2004), physiological characteristics (Feng et al., 2003; Sullivan et al., 2003; Yang et al., 2005) and modifies the activities of antioxidant enzymes (Agrawal et al., 2009; Kumari et al., 2009). Damage to genetic material has also been reported under elevated UV-B radiation (Hidema and Kumagai, 1998; Mazza et al., 1999; Tripathi et al., 2011). Plants develop a wide range of defensive strategies such as DNA repair, synthesis of UV-B

Abbreviations: DAG, days after germination; NSPP, number of seeds plant  1: NSPPo: number of seeds pod-1; SC, starch content; SP, seed protein; TFAA, total free amino acids; TSS, total soluble sugars; WSPP, weight of seeds plant-1; WSPPo, weight of seeds pod-1 n Corresponding author. Fax: þ91 542 2368174. E-mail addresses: [email protected] (K.K. Choudhary), [email protected] (S.B. Agrawal). 0147-6513/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2013.10.032

absorbing compounds, thickening of leaves, etc. to compensate the damage caused by UV-B radiation (Filella and Peñuelas (1999); Jansen et al., 1998; Schumaker et al., 1997). The amount of UV-B radiation reaching a particular place on the Earth’s surface mainly depends upon its latitude as well as on the O3 profile above it. The equatorial and tropical region is predicted to receive more UV-B radiation owing to its narrow solar zenith angle. Anderson et al. (2012) observed that the presence of water vapors can fundamentally change the catalytic chlorine/bromine free radical chemistry of the lower stratosphere and would led to increased risk of ozone depletion with more penetration of solar ultraviolet-B on the Earth’s surface. Numerous stations lying in the northern India showed significant declining trend in total ozone column suggesting the potential vulnerability of terrestrial plants to increased UV-B under field conditions (Sahoo et al., 2005). Although, UV-B represents only a small part of the solar radiation reaching the surface of the Earth, its impact on the biological processes can be very important. Elevated UV-B (eUV-B) induces the synthesis and activity of two important enzymes of phenylpropanoid pathway i.e., chalcone synthase and phenyl ammonia lyase which are essential for flavonoids synthesis (Meijkamp et al., 1999). These enzymes might be transported from shoot to root and after reaching to root they may influence the composition of root exudates (Van de Staaij et al. (1999)). Changes in root exudates due to eUV-B are mainly

K.K. Choudhary, S.B. Agrawal / Ecotoxicology and Environmental Safety 100 (2014) 178–187

responsible for the down regulation of nod genes and thus affecting the activity of root nodules in biological nitrogen fixation (Tok et al., 1997). Singh (1997) reported reduced nitrogenase activity in three tropical legume crops, while Choudhary et al. (2013) found reduction in number of root nodules and their fresh weight in Vigna radiata, due to eUV-B. Jasmonic acid (JA) and salicylic acid (SA) are the important central components of signaling pathways leading to the activation of defense responses (Creelman and Mullet, 1997; Durner et al., 1997). Earlier studies have shown the accumulation of SA during abiotic stresses as in O3 and UV treated tobacco plants (Sharma et al., 1996), while concentrations of JA increased in tomato (Zadra et al., 2006) and Arabidopsis (Rao et al., 2000) under O3 exposure. Studies related to effects of eUV-B on leguminous crops with emphasis on nitrogen metabolism and hormonal changes are still limited. Therefore, a field study was conducted with two cultivars of pea (Pisum sativum L. cultivar HUP-2 and HUDP-15) under natural field conditions to evaluate (1) cultivar specific response on growth, biomass, nitrogen metabolism, yield and its quality (2) accumulation of contents of JA, SA and flavonoids and their role in plant protection and nitrogen metabolism and (3) interactive effects of various studied parameters in two cultivars. Ascertain of cultivars would definitely help in selecting a suitable cultivar for cultivation in areas under higher influx of UV-B. In the present study, an effort was made to test whether differences in UV-B sensitivity exhibited by test cultivars of pea are related with differential accumulation of H2O2, flavonoids, SA and JA.

2. Materials and methods 2.1. Experimental site, plant material and treatments The study was performed at the Botanical Garden of Banaras Hindu University (November, 2010 to March, 2011). The site is situated in the Eastern Indo-Gangetic plains of India located at 251 14′ N, 821 3′ E and 76.1 m above mean sea level. The soil of the experimental plot was alluvial, pale brown and sandy loam in texture (sand 45 percent, silt 28 percent and clay 27 percent) with pH slightly alkaline (7.3). Soil had organic carbon content 0.7 percent, total nitrogen content 0.12 percent and available phosphorus content 0.55 mg g  1. Exchangeable calcium and potassium contents were 0.77 and 0.19 mg g  1, respectively. The pea (Pisum sativum L.) cultivars Malviya Matar-2 (HUP-2) and Malviya Matar-15 (HUDP-15) were selected for the experiment. These cultivars have been developed by the Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, India. HUP-2 was developed from the cross F3(Afila Knudk  B 5064)  S 143 and HUDP-15 through a cross involving [PG 3  F 3(PG 3  S 143)]  FC 1. Both the cultivars are widely grown in the Northern region of India and are resistant to powdery mildew, rust, tolerant to frost and are less affected by birds scaring. Yield potential of HUP-2 is 25–28 q ha  1 however it is 32–60 q ha  1 for HUDP-15. HUP-2 matures within 115–118 days while HUDP-15 in 120 days after germination and the growing season ranged from November to March for both the cultivars. The experimental design was random block consisted of two treatments, ambient (A; 5.8 kJ m  2 day  1 biologically effective UV-B) and elevated (E; 7.2 kJ m  2 day  1 above ambient). Elevated level of UV-B (eUV-B) was artificially provided by Q panel UV-B 313 40 W fluorescent lamps (Q panel Inc. Cleveland, OH, USA). Three lamps (120 cm long) per bank fitted on the adjustable steel frame were suspended perpendicular to the planted rows of each plot. The lamps were covered by either 0.13 mm cellulose diacetate filter (transmission 137 down to 280 nm) for elevated UV-B (eUV-B) radiation or 0.13 mm polyster filter (absorbed radiation below 320 nm) for the control. The filters were replaced weekly to maintain uniform optical properties. The control plants received only ambient levels of UV-B. Lamps in frames were adjusted weekly to a distance of 45 cm from plant canopy. This provided a mean eUV-B having ambient þ7.2 kJ m  2 day  1 to plant apices for 3 h daily over the middle of photoperiod. The UV-B fluence at plant apices under the lamps was measured with the help of spectro power meter (Scientech, Boulder, USA). In total, twelve plots of 1.5  1.5 m2 each were prepared for maintaining three replicates per treatment for each cultivar. Recommended doses of NPK for pea is 20, 80 and 30 kg ha  1, respectively and was applied as urea, single-super phosphate and muriate of potash. Whole of P and K were applied as basal dressing, while urea was splitted into basal dressing (2/3rd) and top dressing (1/3rd). Seeds were sown on the next day of land preparation. After germination, thinning was done to maintain one plant at every 15 cm. Regular watering was done in each plot to

179

maintain similar moisture regime. Manual weeding was performed four times during the entire course of experiment. Two border rows were also sown around each plot in order to minimize heterogeneity in microclimate. 2.2. Meteorological parameters Meteorological parameters, like maximum and minimum temperature, relative humidity, total rainfall and sunshine hours of the experimental site were periodically recorded from the Indian Meteorological Division, BHU station, Varanasi, India. During the experimental period (November 2010 and March 2011), mean temperature ranged from 7.0 1C to 33.9 1C, total rainfall was 7.83 mm, relative humidity ranged from 33.9 to 88.5 percent and sunshine hour averaged 7.3. 2.3. Plant sampling and analysis Random sampling in triplicates was done at 30, 60 and 90 days after germination (DAG) from each plot and sampled leaves were stored at  20 1C for various analysis. For estimating the nitrogenase activity and leghaemoglobin contents, plants were taken out with intact soil cores with utmost care and brought to the lab. Roots were washed carefully to remove all the soil particles adhering to the root surfaces and after drying on the blotting paper, root nodules were taken out for further analysis. 2.3.1. Analysis of morphological parameters Morphological characteristics were analyzed with respect to root and shoot lengths, number of leaves, number of nodules and pods. Total leaf area was calculated by using the portable leaf area meter (Model Li-3100, Li-COR, Inc., USA). For the estimation of total biomass, plants were oven dried at 80 1C for 24 h and then weighed. Root- shoot ratio (RSR) was calculated by using the dry weights of root and shoot. 2.3.2. Analysis of biochemical parameters Two major types of flavonoids, quercetin and kaempferol were estimated using the method provided by Sigma Aldrich through HPLC (Waters, USA) with Waters 600 controller, 717 plus autosampler and 2998 photodiode array detector having Nova-pak (Waters spherisorb, 5 mm, ODS2, 4.6  50 mm) analytical column. For the analysis of quercetin gradient of acetonitrile (A) and ammonium dihydrogen phosphate (B; 25 mM, pH ¼ 3) as a solvent at the flow rate of 1.5 ml min  1 was used. The pecentage of A:B was 25:75 (at 0 min) reaching to 65:35 (at 20 min) then finally 100:0 (at 25 min) and monitoring was done at 258 nm. For the kaempferol gradient solvent system of acetonitrile (with 0.1 percent phosphoric acid) and HPLC grade water (with 0.1 percent phosphoric acid) was used in the ratios 40:60 (at 0 min) reaching finally to 75:25(at 20 min). Flow rate of solvent was 1.5 ml min  1 and the monitoring was done at 267 nm. Superoxide dismutase (SOD) was isolated from homogenized leaves in 100 mM EDTA–phosphate buffer (pH 7.8), filtered and centrifuged at 10,000 g for 15 min. SOD activity was determined in the supernatant by inhibition of the photochemical reduction of nitro-blue tetrazolium (NBT) at 560 nm, following a modified method of Beauchamp and Fridovich (1971). The reaction mixture consisted of 50 mM sodium-potassium phosphate buffer (pH 7.8), 13 mM methionine, 2 mM riboflavin, 75 mM NBT, 100 nM EDTA and 20 ml enzyme extract. Ascorbate peroxidase (APX) activity was determined in a 3 ml volume at 290 nm, as ascorbate oxidized in 50 mM phosphate buffer (pH 7.6), 0.1 mM EDTA, 0.5 mM ascorbate and 0.1 mM H2O2, according to the method of Nakano and Asada (1981). Catalase (CAT) was extracted by homogenizing 0.1 g fresh leaves at 4 1C in 100 mM cold phosphate buffer (pH 7.2) containing 0.5 percent Triton-X. Activity of CAT was assessed in 100 ml phosphate buffer, 400 ml 200 mM H2O2 and 100 ml of enzyme extract. A decrease in H2O2 was measured at 240 nm, and activity of CAT was measured according to the method of Aebi (1984). Hydrogen peroxide (H2O2) content was determined by Alexieva et al. (2001) by homogenizing the leaf in 0.1 percent TCA. The supernatant was kept in dark for 1 h after mixing with phosphate buffer and potassium iodide and finally absorbance was recorded at 390 nm. 2.3.3. Analysis of physiological parameters Nitrate reductase (NR) and nitrite reductase (NiR) activities were estimated according to the methods of Nicholas and Nason (1957). Enzyme was extracted in 0.1 M phosphate buffer (pH 7.5) containing cysteine and EDTA. Extract was incubated with KNO3 for NR and KNO2 for NiR with NADH for 15 min at room temperature and the reaction was stopped by adding sulfanilamide. The mixture was then incubated for 5 min with 1- (napthyl) ethylenediamine (NNED) and absorbance of pink color was measured at 540 nm. Appearance of pink color was measured for NR while the degradation in color was measured for NiR and potassium nitrite (KNO2) was used as standard. For estimating nitrogenase activity acetylene reduction assay was followed as described by Stewart et al. (1967). Gas chromatograph (Model-3800, Varian Inc., USA) was used having column Porapak-Q and flame ionization detector (FID). Leghaemoglobin content in root nodules was

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K.K. Choudhary, S.B. Agrawal / Ecotoxicology and Environmental Safety 100 (2014) 178–187

extracted and heated with saturated solution of oxalic acid at 120 1C. The emission of supernatant was measured at 600 nm using the fluorescence spectrophotometer (Model- F-2500, Hitachi, Japan). Leghaemoglobin content was estimated by adopting the procedure of LaRue and Child (1979). 2.3.4. Analysis of jasmonic and salicylic acids JA was estimated by the method of Meyer et al. (1984) and for the quantification of SA the method of O'Donnell et al. (2001) was followed. Analysis was performed through HPLC (Waters, USA) with Waters 600 controller, 717 plus autosampler and 2998 photodiode array detector having Nova-pak (Waters spherisorb, 5 mm, ODS2, 4.6  50 mm) analytical column. Mobile phase used for JA was isocratic with 70 percent methanol and 0.1 percent phosphoric acid at the flow rate of 1 ml/min and monitored at 228 nm, however isocratic system of 100 percent methanol at the flow rate of 1 ml/min was used for SA and the monitoring was done at 254 nm. 2.3.5. Analysis of yield and its quality After final harvesting at 120 DAG, yield attributes such as weight of seeds pod  1 (WSPPo), weight of seeds plant  1 (WSPP), number of seeds pod  1 (NSPPo), number of seeds plant  1 (NSPP), test weight (TW; weight of 1000 seeds) and harvest index (HI) were recorded. HI was calculated as follows: HI ð%Þ ¼ ðEconomic yield=Biological yieldÞ  100 where, economic yield is referred to the seed yield (g plant  1) and biological yield in the form of above ground biomass (g plant  1). Seeds were oven dried and grinded in a stainless steel grinder and passed through a 2.0 mm sieve. Three replicates were taken from each treatment. For extracting sugars and starch, 50 mg powdered grain sample was boiled with 5 ml 80 percent ethanol (v/v) and then centrifuged. The pellets were successively washed with 80 percent ethanol for four times and centrifuged after each washing. Finally the pellets were washed with distilled water and centrifuged again. The supernatant collected after each washing was used for estimating total soluble sugars (TSS) and pellets for estimating starch content (SC). Estimation of total soluble sugar and starch was conducted by following phenol/H2SO4 colorimetric assay (Dubois et al., 1956). Total free amino acids (TFAA) were estimated by the methodology given by Moore and Stein (1948). The method consists of extraction of amino acids in 80 percent ethanol and its colorimetric estimation using ninhydrin reagent at 570 nm wavelengths. Soluble protein (SP) content in grains was estimated by following the method of Lowry et al. (1951). 2.4. Statistical analysis All the statistical analysis was done using the SPSS software (SPSS Inc.,Version 16.0). The means and standard errors of treatments were calculated. The significance of difference between treatments was calculated by paired sample t-test. Two-way ANOVA was performed to test the individual and interactive effects of treatment (T) and cultivar (Cv) for the yield attributes and seed quality parameters while other parameters were analyzed by three-way ANOVA to test the individual and interactive effects of treatment (T), age (A) and cultivar (Cv). Pearson’s correlation test was done to explore the correlation among changes in various parameters.

as compared to HUP-2 (eleven percent) at 60 DAG due to eUV-B (Fig. 1d). 3.2. Effects of elevated UV-B on biochemical parameters Increment of quercetin was higher in response to eUV-B as compared to kaempferol in both the cultivars. Accumulation of both the compounds was maximum at 60 DAG (Fig. 2a and b). Increase in kaempferol was observed by 55 and 72 percent, however quercetin increased by 88 and 114 percent in HUP-2 and HUDP-15, respectively. Both the flavonoids were significantly affected due to all the individual factors and by their interactions in three-way ANOVA (Table 1). H2O2 content increased 56 percent in HUP-2 and by 33 percent in HUDP-15 at 60 DAG due to eUV-B (Fig. 3a). Three-way ANOVA revealed that H2O2 content was significant due to all individual factors as well as by their interactions (Table 1). Activities of APX and CAT increased by twelve and eleven percent in HUP-2 and by 22 and 19 percent in HUDP-1, respectively due to eUV-B (Fig. 3b and c). CAT was significant due to T, A and also due to the interaction of A  Cv. SOD activity also increased significantly by 29 and 55 percent in HUP-2 and HUDP-15, respectively at 60 DAG (Fig. 3d). SOD and APX activities were affected significantly by all the individual factors in three-way ANOVA test (Table 1). 3.3. Effects of elevated UV-B on physiological parameters eUV-B negatively affected the nitrogen metabolism of test plants. There was significant reduction in all the parameters of nitrogen metabolism viz. NR, NiR, nitrogenase activities and leghaemoglobin content (Fig. 2 e–h). Nitrogen metabolism of HUP-2 was affected most due to eUV-B as compared to HUDP15. Activities of NR, NiR and nitrogenase as well as leghaemoglobin content were found to be maximal at 60 DAG and reduced at a later stage. Maximum reduction in NR and NiR was found by 31 and 50 percent in HUP-2 where as it was 23 and 38 percent in HUDP 15, respectively. Nitrogenase activity decreased by 42 and 29 percent, while leghaemoglobin content reduced by 58 and 44 percent in HUP-2 and HUDP-15, respectively due to eUV-B. Leghaemoglobin content, NR, NiR and nitrogenase activities were significantly affected due to all the individual factors and their interactions except nitrogenase activity which was not significant due to interaction of T  A (Table 1). 3.4. Effects of elevated UV-B on jasmonic and salicylic acids

3. Results 3.1. Effects of elevated UV-B on morphological parameters Growth of pea plants was negatively affected as leaf area, number of root nodules, pods and total biomass were significantly decreased in both the cultivars due to eUV-B (Fig. 1a–c and e). HUP-2 showed higher reduction in leaf area by twenty percent as compared to HUDP-15 at 60 DAG, due to eUV-B. Leaf area was affected significantly by all the factors except A  T  Cv interaction (Table 1). Reduction in number of root nodules at 60 DAG was more in HUP-2 (36 percent) whereas it was less for HUDP-15 (27 percent) due to eUV-B and was affected by T, A and Cv individually and due to interaction of T  A. Reduction in number of pods was 30 and 21 percent in HUP-2 and HUDP-15, respectively at 90 DAG (Fig. 1b). Total biomass was also reduced significantly and it was 29 and 19 percent in HUP-2 and HUDP-15, respectively at 60 DAG (Fig. 1e). Three-way ANOVA analysis showed that both, pod number and total biomass were affected significantly by all the individual factors and due to interaction of T  A and A  Cv (Table 1). Reduction in RSR was maximal in HUDP-15 (22 percent)

JA and SA contents increased significantly in both the cultivars after eUV-B exposure. At 60 DAG of sampling, accumulation of JA was higher in HUDP-15 (91 percent), while it was lower in HUP-2 (71 percent) (Fig. 2c). Accumulation of JA increased from 30 DAG to 60 DAG but declined afterwards as noticed at 90 DAG. Threeway ANOVA results revealed that JA was affected significantly by all the individual and interactive factors (Table 1). Increase in accumulation of SA was 59 and 33 percent in HUP-2 and HUDP-15, respectively and it increased continuously from 30 DAG to 90 DAG (Fig. 2d). SA was affected by all the individual factors and their interactions except A  Cv and T  A  Cv (Table 1). 3.5. Effects of elevated UV-B on yield and its quality Yield of pea decreased significantly due to eUV-B. NSPPo and NSPP were significantly reduced by 35 and 34 percent, respectively in HUP-2 while it reduced by 18 and 21 percent, respectively in HUDP-15 (Fig. 4a and b). NSPPo and NSPP were significant due to T and Cv but not due to their interaction in two-way ANOVA results (Supplementary Table S1). Significant reduction in WSPPo was 44

K.K. Choudhary, S.B. Agrawal / Ecotoxicology and Environmental Safety 100 (2014) 178–187

A

250

E 30

200

** 100

*

*

* 150

*

**

*

*

Number of pods

Leaf area (cm2)

181

20

*

*

10 50

0

25

0.4 ns

*

20

ns

*

*

ns *

15

*

*

0.3

*

ns

ns

ns

RSR

Number of root nodules

0

0.2

*

10

0.1

*

5 *

0

Total Biomass (g plant-1)

30 10

60 90 DAG

30

60 90 DAG

0.0

HUDP 15

HUP 2 **

8

** **

6 **

4 2 *

**

0

30

60 DAG

90

30

60 90 DAG

HUDP 15

HUP 2

Fig. 1. Effect of elevated UV-B on (a) leaf area, (b) number of pods, (c) number of root nodules, (d) RSR and (e) total biomass of pea cultivars at different sampling ages (mean7 1 SE). np o 0.05; nnp o0.01;nnnp o 0.001; ns, not significant; A, ambient UV-B; E, elevated UV-B.

Table 1 Results of three way ANOVA tests showing level of significance for selected parameters of pea cultivars grown in ambient and elevated UV-B. Parameters

T

A

Cv

TA

T  Cv

A  Cv

T  A  Cv

SA JA Kaempferol Quercetin Leghaemoglobin NR NiR Nitrogenase Leaf area Number of pods Number of root nodules Total biomass SOD APX CAT H2O2

nnn

nnn

nnn

nn

nn

ns

ns

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

n

nnn

nnn

nnn

nnn

nnn

nnn

ns

ns

nnn

nnn

nnn

nnn

n

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

nnn

ns

nnn

n

nnn

nn

n

nnn

ns

ns

ns ns ns ns ns ns

ns ns ns ns ns ns

nnn

nnn

nnn

nnn

nnn

Note: T, treatment; A, age; C, cultivar. n

po 0.05. p o0.01. nnn p o 0.001; ns, not significant. nn

ns nnn n nnn

nn

nn

ns

nnn

n

182

K.K. Choudhary, S.B. Agrawal / Ecotoxicology and Environmental Safety 100 (2014) 178–187

A

2.4

E ***

2.2

-1

Quercetin (µg g FW)

1.8 1.6

7 ***

***

***

1.4

***

1.2

6 5

***

1.0 0.8

***

***

***

**

4

**

0.6

Kaempferol (µg g-1 FW)

2.0

8

3

0.4 0.2

***

1.8 1.6 *

***

4

**

**

1.2

3

*

** 2

1.4

1.0

**

SA (µg g-1 FW)

JA (µg g-1 FW)

**

***

5

2 2.0

*

0.8

*

0.6 **

3

**

0.7

*

*

0.6 *

**

2

0.5 0.4

1

*

*

0.3 *

* 0

**

**

0.2 0.1 1.2

***

1.0

***

60

0.8 ** 0.6

**

40

0.4 20

0

30

*

*

*

*

* 60 DAG

HUP 2

90

30

*

60 DAG

90

HUDP 15

0.2

Leghaemoglobin -1 (mg g root nodule)

Nitrogenase (nmol h-1 g-1 ethylene formed)

0.4

**

**

NiR (µmol of KNO2 removed h-1 g-1 FW)

NR (µmol of KNO2 formed h-1 g-1 FW)

1

0.0 30

60 DAG

90

HUP 2

30

60 DAG

90

HUDP 15

Fig. 2. Effect of elevated UV-B on (a) quercetin, (b) kaempferol, (c) JA, (d) SA, (e) NR, (f) NiR, (g) nitrogenase and (h) leghaemoglobin contents of pea cultivars at different sampling ages (mean 71 SE). np o0.05; nnp o 0.01;nnnpo 0.001; ns, not significant; A, ambient UV-B; E, elevated UV-B.

percent in HUP-2 and 26 percent in HUDP-15 whereas reduction in WSPP was 45 and 31 percent in HUP-2 and HUDP-15, respectively due to eUV-B (Fig. 4c and d). WSPPo and WSPP were also affected significantly by all the individual factors (Supplementary Table S1). Higher reduction in TW (Fig. 4e) was observed in HUP-2 (21 percent) as compared to HUDP-15 (thirteen percent) and was significantly affected due to all the individual factors and their interactions in twoway ANOVA. eUV-B also led to the reduction in HI by 24 and 15 percent in HUP-2 and HUDP-15, respectively (Fig. 4f) and it was significant only due to T and Cv (Supplementary Table S1). eUV-B deteriorated the seed quality of pea cultivars significantly (Fig. 4g–j) and the effect was cultivar specific. SP decreased in HUP-2 by eighteen percent and by twelve percent in HUDP-15.

Higher reduction in TFAA (26 percent) was observed in HUP-2. TSS decreased significantly by seventeen and twelve percent in HUP-2 and HUDP-15, respectively. Reduction in SC was more in HUP-2 as compared to HUDP-15. SP and TSS were significantly affected by T and Cv individually while TFAA and SC were significant due to T and Cv individually and by their interaction in two-way ANOVA (Supplementary Table S1).

4. Discussion In the present investigation eUV-B negatively affected the overall performance of both the test cultivars. Reduction in growth

K.K. Choudhary, S.B. Agrawal / Ecotoxicology and Environmental Safety 100 (2014) 178–187

0.4

A

E *

*** 0.3

0.8

** **

**

0.2

1.0

*

***

***

1.2

ns

ns ns

ns

0.6 0.4

0.1

CAT (µmol min-1 g-1 FW)

H2O2 (µmol g-1 FW)

183

0.2 0.0 ns

0.0 *

***

ns

ns

*** **

0.015

*** ***

2.5 2.0 1.5

ns 0.010

1.0

0.005

0.5

0.000

30

60 DAG

HUP 2

90

30

60 90 DAG

HUDP 15

30

60 90 DAG

HUP 2

30

60 90 DAG

SOD (unit g-1 FW)

APX (µmol min-1 g-1 FW)

0.025 0.020

*

*

0.0

HUDP 15

Fig. 3. Effect of elevated UV-B on activities of (a) H2O2, (b) CAT, (c) APX and (d) SOD of pea cultivars at different sampling ages (mean 71 SE ). np o 0.05; nnn p o 0.001; ns, not significant; A, ambient UV-B; E, elevated UV-B.

as observed in the present study due to eUV-B has clearly been shown in large number of plant species at different developmental stages due to changes in metabolic or developmental processes (Agrawal and Rathore, 2007; Mishra and Agrawal, 2006; Sharma et al., 1998; Kakani et al., 2003). Lower growth of irradiated plants points to a negative effect of UV-B as well as activation of protective mechanisms, leading to a reduction in leaf surface area and subsequent lower radiation absorbance. Our results have been consistent with the earlier studies as reduction in leaf area has also been found in both the cultivars. Decrease in pod number was partly due to enhanced abscission of pods during the final stage of plant growth. Reduction in growth parameters of pea was more drastic in HUP-2 as compared to HUDP-15 proving it to be more prone to eUV-B. An increase in flavonoids and total phenol content is also responsible for the decrease in the number of root nodules (Choudhary et al., 2013). As a matter of fact, nodulation and plant growth are related symbiotically and any stress on one process affects the other negatively. According to available literature, reduction in nodulation may also be due to reduction in the photosynthesis and chlorophyll content due to eUV-B (Singh, 1997). Reduction in nodulation is expected to create an extra nutrient stress on the pea plants. A reduction in total biomass further confirmed that eUV-B directly interferes with various fundamental plant processes. Our results are consistent with Agrawal and Rathore (2007) as they have also observed reductions in total biomass of different cultivars of wheat and mungbean under eUV-B. Positive significant correlation was found between total biomass, leaf area and pod number in both the cultivars (Supplementary Table S2). It is suggested that, all of these parameters contributed significantly to the reduction of total biomass. Lower RSR in HUP-2 as compared to HUDP-15 might be due to lower availability of photosynthates to the roots as compared to shoots of HUDP-15 under eUV-B. The accumulation of flavonoids occurs mainly in the epidermal region of leaves and it protects the photosynthetic tissue through

nn

p o0.01;

the screening of UV-B radiation. Higher content of flavonoids against eUV-B may be due to the increased activity of PAL, a key enzyme catalyzing the first reaction of flavonoid biosynthetic pathway (Liu et al., 2002). Kaempferol and quercetin flavonols responded differently in test pea cultivars under eUV-B exposure. A relatively higher increase of quercetin than kaempferol concentration in UV-B exposed plants was also reported in earlier studies (Reifenrath and Muller (2007); Winter and Rostas (2008)). It has been suggested that quercetin flavonols have a better ability for scavenging free radicals than kaempferol flavonols (Harborne and Williams, 2000). So, higher increment of quercetin at initial stage of growth is certainly beneficial to screen out the UV-B radiation and preventing the plants from its harmful effects. Significant positive correlation exists between eUV-B with quercetin (HUP-2: r ¼0.70 and HUDP-15: r ¼0.71, p r0.001) and kaempferol (HUP-2: r ¼0.69 and HUDP-15: r ¼ 0.76, p r0.001) (Supplementary Table S2). However, flavonoids also serve as plant signals to symbiotic bacteria in the Rhizobiaceae (Phillips, 2000) and their accumulation in root tissues has been shown to promote nodule formation (Muofhe and Dakora, 1999). Thus, an increase in the concentration of flavonoids in roots of eUV-B exposed plants might affect the nodulation (Choudhary et al., 2013) and nitrogen fixation process in legumes. Our results clearly showed that higher accumulation of flavonoids certainly interfered in the nodulation process as less number of root nodules were observed due to exposure of eUV-B (Supplementary Fig. S1). In fact, Nod genes are responsible for nodulation process and its effective stimulus depends on specific concentration of flavonoids (Tok et al., 1997). Rajendiran and Ramanujam (2006) reported the reduction in nodule number (49 percent) and NR (33 percent) and nitrogenase (67 percent) activities in Vigna radiata exposed with 12.2 kJ m  2 day  1 UV-B. Exposure of plants to eUV-B reduces the net photosynthesis and alters the allocation of photosynthates to different plant organs (Adamse and Britz, 1992) with a consequent decrease in the release of root exudates compounds into the rhizosphere. This, in turn, could affect nodulation process and ultimately biological N2-fixation. Thus, in the present study, net effect of eUV-B was observed as reduction in the

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3

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Fig. 4. Effect of elevated UV-B on (a) number of seeds pod  1, (b) number of seeds plant  1, (c) weight of seeds pod  1 (d) weight of seeds plant  1, (e) test weight, (f) harvest index, (g) total soluble sugar, (h) starch content, (i) seed protein, (j) total free amino acids of pea cultivars at different sampling ages (mean 71 SE).np o 0.05; nnp o 0.01; nnnp o 0.001; ns, not significant; A, ambient UV-B; E, elevated UV-B.

activities of nitrogenase, NR and NiR as well as leghaemoglobin content depicting that UV-B is certainly detrimental for processes involved in nitrogen metabolism. Singh (1997) and Van de Staaij et al. (1999) also found significant decrease in nitrogenase activities in symbiotic legumes exposed to eUV-B. Reduction of various enzymes related to nitrogen metabolism vis-a-vis number of root nodules was greater in HUP-2 as compared to HUDP-15 showing genotypic

differences for cultivar sensitivity. Significant positive correlation was also seen in nitrogenase activity with leghaemoglobin content (HUP-2: r¼0.84 and HUDP-15: r¼0.92, pr0.001) and number of root nodules (HUP-2: r¼0. 87 and HUDP-15: r¼0.84, pr0.001) in both the cultivars in Pearson’s correlation test (Supplementary Table S2). The reduction in N-fixing ability could also be due to reduction in leghaemoglobin, which was reduced under eUV-B as observed in

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the present study. Higher the content of leghaemoglobin more will be the efficiency of the plants in terms of its capacity to fix atmospheric nitrogen (Gurumoorthi et al., 2003). Balakumar et al. (1999a, b) established that UV-B radiation has several target sites in the nitrate assimilation pathway of crop plants which is catalyzed by the two metalloproteins, NR and NiR. Present study showed reduction in activities of NiR as well as NR under eUV-B, though it was more for NiR. However, Balakumar et al. (1999a, b) revealed that NR is more sensitive to UV-B than NiR. Assay of JA and SA showed significant increment in both the cultivars due to eUV-B. UV-B stimulates accumulation of these signaling molecules that mediate N-fixation as well. Nod genes are inducible by JA as well as by flavonoids (Rosas et al., 1998). JA have been shown to regulate the activation of genes that encode plant defencins (Penninckx et al., 1996, 1998) and enzymes involved in systemically induced defense responses (Farmer and Ryan, 1992; Penninckx et al., 1996, 1998). JA was positively correlated with eUV-B in both the cultivars but more extensively with HUDP-15 and it showed a positive correlation with number of pods (r ¼ 0.48, p r0.05) and total biomass (r ¼0.47, p r0.05) (Supplementary Table S2). More accumulation of JA in HUDP-15 might provide better tolerance against UV-B stress as compared to HUP-2. Contrary to this, SA accumulation was more in HUP-2 and helps plants in making more susceptible to UV-B. Higher concentrations of SA caused a higher level of oxidative stress which the plants are unable to overcome and may result in the death of the plant (Horvath et al., 2007). The oxidative stress caused to the plants due to higher accumulation of SA might be due to its inhibitory effects on catalase and ascorbate peroxidase activity (Durner and Klessig, 1995; Rao et al., 1997; Vicente and Plasencia, 2011). This led to higher accumulation of H2O2 in plants as also observed in the present study. Higher accumulation of H2O2 was seen in HUP-2 with concomitant increase of SA level. Beside this, the activities of CAT and APX are also reduced in HUP-2 as compared to HUDP-15, suggesting the inhibition of enzyme activities in HUP-2 due to higher accumulation of SA. However, higher activity of SOD due to eUV-B in both the cultivars are mainly responsible for more generation of H2O2 but its final accumulation was less in HUDP15 due to increased activities of APX and CAT. Lee et al. (2004) has shown the inverse relation between JA and SA response to wounding in rice plants. Consistent results were observed cultivar wise in present experiment also, as cultivar HUDP-15 showed more increment in JA while HUP-2 showed higher contents of SA under eUV-B. This specific response of test cultivars suggests that higher accumulation of SA in HUP-2 might be one of the important factors for the sensitivity of HUP-2 against UV-B as significant positive correlation (r ¼0.52, p r0.05) was found with UV-B, while it was not significant for HUDP-15 (Supplementary Table S2). The yield of any cash crop is always a central point of discussion in assessing the impact of any abiotic stress factor. JA and flavonoids accumulation assumed to provide better protection in cultivar HUDP-15 while SA led to more oxidative stress on HUP-2 resulting in greater loss of yield. Reduction in yield represents the cumulative effects of damaged or inhibited physiological functions (Agrawal and Mishra, 2009). Significant reductions in grain yield of Triticum aestivum L. (Rathore et al., 2003) and of Pisum sativum L. (Agrawal and Mishra, 2009) resulting from UV-B exposure have already been reported. Reduction in yield reflects the alteration of photosynthetic allocation and the adverse effect could be due to reduction of photosynthesis in both the cultivars (Agrawal et al., 2004). Another possibility of reduction in yield may be due to reductions in quantity of pollen grains vis-à-vis their viability under enhanced UV-B radiation (Demchik and Day, 1996). In the present field study, plants were also exposed to UV-B throughout the anthesis and the pod filling stage, so the penetration of UV-B might be more effective in reducing seed set/yield (Agrawal et al., 2004). Since, the number

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of pods was also decreased due to eUV-B in both the cultivars, thus contributing to the loss in final yield. Reduction in TW of both the cultivars is directly related with the smaller size of seeds due to eUV-B. TW positively correlated (HUP-2: r¼ 0.88, pr0.05 and HUDP-15: r¼ 0.93, pr0.01) with HI (Supplementary Table S3). HI indicates the partitioning of dry matter between seeds (economic yield) and above ground biomass (biological yield) and reduction in HI of plants under eUV-B revealed less dry matter partitioning toward seeds as compared to the control ones in both the cultivars. Seed quality of both the cultivars was also deteriorated in the form of SP, TFAA, TSS and SC as observed in some earlier investigations (Gao et al., 2004; Teramura et al., 1990) under eUV-B and is critical to pea quality. Reduction in SP may be correlated to generation of ROS which causes oxidative damage to proteins and also responsible for induced expression of various new stress proteins (Sharma et al., 2012). Another reason for the degradation of seed quality might be that UV radiation causes modification and destruction of amino acids and disulfide groups strongly absorbs UV radiation (Hollosy, 2002). Changes in various storage proteins such as albumin–globulin, glutelin and prolamin have been reported in UV-B treated rice grains (Hidema et al., 2005). TSS and SC were decreased against eUV-B might be the effect of decline in rate of photosynthesis, more allocation of photosynthates for repair and altered translocation of solute and nutrients to the seeds of pea plants. It is widely known that H2O2 triggers the expression of a set of genes including various antioxidant enzymes (SOD, APX, GR, CAT) related to plant defense (Neill et al., 2002). The induction of antioxidant enzymes, in general protect the plants for maintaining the rate of photosynthesis and keeping high values of TSS and SC in seeds. Higher reduction in seed quality of HUP-2 may be directly correlated with the higher accumulation of SA, resulting in more oxidative stress to it. Also, less accumulation of flavonoids in HUP-2 as compared to HUDP-15 made it more susceptible against eUV-B, resulting in deterioration of seed quality. Seed quality parameters are negatively correlated with the UV-B treatment significantly (Supplementary Table S3). Recently, Tripathi and Agrawal (2013) observed deterioration in the seed quality of linseed due to sUV-B exposure.

5. Conclusions This study reports the growth, nitrogen metabolism, yield and seed quality responses of two cultivars of pea in relation to the accumulation of H2O2, JA, SA and flavonoids against eUV-B under natural field conditions. Results of the present study showed that pea plants were adversely influenced under eUV-B conditions in tropical environment, but the response was cultivar specific. Higher accumulation of JA proved to be an important factor in providing the better protection to HUDP-15 against eUV-B in the form of better growth, nitrogen metabolism, biomass, yield and its quality as compared to HUP-2. However, induction of flavonoids (quercetin and kaempferol) protected the plants up to some extent but it was neither helpful for providing complete protection against eUV-B nor it helped in improving nitrogen metabolism of test cultivars. More accumulation of SA, possibly led to higher accumulation of H2O2 in HUP-2 and thus made it more vulnerable against eUV-B. So, the resistant varieties must be developed through genetic manipulations which have the capacity for higher accumulation of JA compared to SA with better ability of biological nitrogen fixation and productivity.

Acknowledgments We thank, Head, Department of Botany and to Coordinator, Centre of Advanced Study, Department of Botany, Banaras Hindu

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University, India for providing all the necessary laboratory facilities and to U.G.C., Government of India, New Delhi for financial assistance in the form of RGN-JRF and RGN-SRF. Authors are also grateful to Prof. C. P. Srivastava, Institute of Agricultural Sciences, Banaras Hindu University, India for providing the seeds of pea.

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