Introduction

Since higher plants are immobile, they are inevitably exposed to solar UV-B radiation. Natural variations in UV-B irradiances at the ground are produced due to natural latitudinal gradient in total atmospheric ozone column thickness, prevailing solar angles, elevation above sea level and optical amplification effect (Cabrera et al. 1995). The amount of UV-B radiation reaching tropical latitudes is higher than in temperate because the lower solar zenith angle leads to a less atmospheric UV-B absorption in tropics. Numerous stations lying in the northern India showed significant declining trend in total ozone column (TOC), suggesting the potential vulnerability of plants to increased UV-B under field conditions (Sahoo et al. 2005).

Some plants are sensitive to even ambient fluences (Krizek et al. 1997), while many plants appear quite tolerant to even high fluences of UV-B (Allen et al. 1998). UV-B photons cause cellular damage by generating DNA photoproducts and through direct damage to proteins, lipids and RNA (Casati and Walbot 2003). Enhanced UV-B exposure to plants can impair all major photosynthesis processes including photochemical reaction in thylakoid membrane, enzymatic processes in the Calvin cycle and stomatal limitation to CO2 (Allen et al. 1998). Cell membranes are regarded as one of the potential targets to UV-B (Björn 1996). Studies have shown that enhanced UV-B irradiation decreased plasma membrane ATPase (Murphy 1983), increased membrane permeability (Imbrie and Murphy 1982), caused damage to unsaturated fatty acids of membrane lipids (Predieri et al. 1995) and even changed the properties of thylakoid membrane (Murphy 1983).

Previous studies have shown that the plant’s response to UV-B can be modified by other environmental factors like photosynthetic photon flux density (PPFD) (Adamse and Britz 1992), atmospheric CO2 concentration (Visser et al. 1997), water availability (Balakumar et al. 1993) and nutrient supply (Singh et al. 2010). Studies on the combined effects of UV-B and other environmental factors on plant species are of importance for the accurate assessment of potential consequences of stratospheric ozone layer reduction. C4 plants are reported to be better adapted to water stress than C3 plants (Uzilday et al. 2012). C3 and C4 plant species have evolved in different climates and consequently differ from each other both structurally and functionally, and for their climatic requirements as well (Ward et al. 1999; Nayyar 2003). Such differences may allow different defence strategies including antioxidative potential, UV-B screening capacity and nutrient use efficiency among the plant species. Hence, the interactive effects of enhanced UV-B and nutrients were assessed on spinach (Spinacia oleraceae L. cv All green), a C3 and amaranthus (Amaranthus tricolor L. cv Pusa Badi Chaulai), a C4 plant species under natural field conditions.

The main objectives of the study were to compare the responses of both the species at ambient UV-B (5.8 kJ m−2 day−1 biologically effective UV-B) and enhanced UV-B over ambient (7.2 kJ m−2 day−1 biologically effective UV-B above ambient; Caldwell 1971) with respect to (i) photosynthesis, stomatal conductance, water use efficiency (WUE), chlorophyll fluorescence, photosynthetic pigments, UV-B-absorbing compounds, lipid peroxidation (LPO), antioxidative enzymes, metabolites, total biomass, root shoot ratio (RSR) and yield, (ii) nitrogen use efficiency (NUE) and (iii) the modification in above responses at different doses of nutrients and their combinations. We hypothesize that C3 and C4 plant species will respond differently under enhanced UV-B at varying nutrient levels due to physiological and metabolic differences. The combination of 1.5× NPK will help the plant species more efficiently under enhanced UV-B by adapting different defence strategies compared with other combinations. The plant species which will have better NUE will show higher tolerance to enhanced UV-B.

Materials and methods

Experimental site and plant material

The field experiment was conducted from March 2010 to April 2010 at the Botanical Garden of the Banaras Hindu University, Varanasi, Uttar Pradesh (25°81′N, 83°1′E and about 76 m above mean sea level) situated in the eastern gangetic plains of India. Soil of the study site was sandy loam in texture (sand 45 %, silt 28 % and clay 27 %) and had a pH close to neutral (7.2–7.4). Variations in climatological data during the study period are shown in Fig. 1. Mean, minimum and maximum temperatures and PPFD were higher during April compared with March, while relative humidity showed a contrasting trend (Fig. 1).

Fig. 1
figure 1

Variations in the climatological conditions during the study period

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The test plant species were spinach (S. oleraceae L. cv All green) and amaranthus (A. tricolor L. cv Pusa Badi Chaulai). Both the varieties are high-yielding varieties developed by Indian Agricultural Research Institute (IARI), New Delhi and are widely grown in northern Indian conditions.

Experimental design and nutrient application

The experimental design was a split plot with a UV-B treatment as main plot and nutrient treatments and plant species as subplots randomized within the whole plots. Each treatment had three replicate plots. In total there were 48 plots of 1 × 1 m2 each. The experiment had two factors: (i) UV-B treatment, and (ii) N, P and K amendment. The four NPK amendments were recommended dose of NPK (F0), 1.5× recommended dose of NPK (F1), 1.5× recommended dose of N (F2) and 1.5× recommended dose of K (F3). Recommended dose of NPK is the optimum nutrient required by the plants and 1.5× NPK was chosen to test the plant’s response to UV-B at higher than required nutrient level. Higher than recommended dose of N is used to test the plant’s response to single nutrient under enhanced UV-B, and 1.5× K was used to test the role of K in membrane damage protection under enhanced UV-B.

For convenience, control plants grown at ambient level of UV-B were designated as F0C, F1C, F2C and F3C with corresponding UV-B treated plants as F0T, F1T, F2T and F3T, respectively. Recommended dose of NPK for spinach and amaranthus is declared by IARI. The recommended dose of NPK was 80:40:40 kg ha−1 for spinach and 75:50:40 kg ha−1, respectively, for amaranthus. N, P and K were given in the form of urea, single super phosphate and muriate of potash, respectively. A half dose of N and full doses of P and K were given as basal dressing and another half dose of N was given as top dressing after 7 days after germination (DAG).

Genetically uniform seeds of spinach and amaranthus procured from Indian Institute of Vegetable Research, Varanasi were sown in rows (30 cm apart) in each plot. There were three rows in each plot. There were 18 plants in each plot with 6 plants in each row. After germination, plants were thinned to one plant every 15 cm for uniformity in growth. Plants were watered every alternate day with same amount of water through channel irrigation.

Enhanced UV-B treatment

Enhanced UV-B was artificially provided by Q-panel UV-B 313 40 W fluorescent lamps (Q panel Inc. Cleveland, OH, USA). For each treatment, three replicate plots were maintained with a separate frame. Each steel frame contained three lamps (120 cm long) fitted 30 cm apart and were suspended perpendicular to the planted rows (3) of each plot. The lamps were covered by either 0.13 mm cellulose diacetate filter (transmission down to 280 nm) for enhanced UV-B radiation or 0.13 mm polyester filter (absorbed radiation <320 nm) for the control.

Ultraviolet irradiance was measured with a double monochromator spectroradiometer (Scientech, Boulder, CO). Plants under polyester filter lamps received only ambient UV-B (5.8 kJ m−2 day−1 biologically effective UV-B) on the summer solstice weighted against generalized plant response action spectrum of Caldwell (1971). The plants beneath cellulose diacetate film received ambient + enhanced UV-B (7.2 kJ m−2 day−1 biologically effective UV-B; Caldwell 1971) that mimicked 20 % reduction in stratospheric ozone at Varanasi during clear sky condition (Green et al. 1980) normalized at 300 nm, at 0 albedo and 1.0 scatter. Lamps in frames were adjusted weekly to a distance of 45 cm above plant canopy to provide a mean enhanced UV-B of 7.2 kJ m−2 day−1 (weighted; Caldwell 1971) for 3 h daily over the middle of photoperiod (10.00 am to 1.00 pm).

Gas exchange and chlorophyll fluorescence

Photosynthetic rate (Ps) and stomatal conductance (gs) were measured using portable photosynthetic system (Model LI-6200, LI-COR, USA) at 20 days after germination (DAG). The measurements were made on the third fully expanded leaf from the top of the plant species on cloud free days between 08.00 and 10.00 h local time on three randomly selected plants in each plot. During the measurements, PPFD ranged between 1100 and 1500 μmol m−2 s−1. The system was calibrated using a known CO2 source of 509 ppm concentration. During measurements of photosynthesis, the mean temperature ranged between 29.8 and 31.8 °C, relative humidity ranged between 37.3 and 38.1 %. Intrinsic WUE was calculated as a ratio of photosynthesis to stomatal conductance.

Chlorophyll fluorescence characteristics such as initial fluorescence (F0), maximum fluorescence (Fm), variable fluorescence (Fv = Fm − F0) and Fv/Fm ratio were measured between 8.00 and 10.00 h using portable plant efficiency analyzer (PEA, Hansatech Instruments Ltd., UK) on the same leaves where Ps was measured. Leaf clips for dark adaptation were placed on the adaxial side of the leaves for 20 min before measurement at saturating flash of 3000 μmol m−2 s−1.

Pigments, antioxidants and metabolites

Random samplings of plants were performed in triplicate at 20 DAG from each plot and leaves were cut and stored at −20 °C before analyses of photosynthetic pigments, antioxidants and metabolites.

Pigments

For pigment determination, 500 mg of leaf samples were homogenized in 20 ml of 80 % acetone and centrifuged at 6000× rpm for 15 min. The optical densities (O.D.) of supernatant were measured at 480 and 510 nm wavelengths for carotenoids and 645 and 663 nm for chlorophylls on a UV–Vis spectrophotometer (Systronics Model 119, India). The amounts of chlorophyll a and b and carotenoids were calculated by using the formulae given by Maclachlan and Zalik (1963) and Duxbury and Yentsch (1956), respectively. For determination of UV-B-absorbing compounds, 0.1 g of leaf discs of 100 mm2 were taken and extracted in 10 ml of acidified methanol (79:30:1 v/v, methanol, water, HCl) according to the procedure of Mirecki and Teramura (1984). Extract absorbance at 305 nm wavelength was measured on a UV–Vis spectrophotometer (Model 119, Systronics, India), as a measure of UV-B-absorbing compounds.

Lipid peroxidation

Lipid peroxidation in the leaf tissues was determined in terms of malondialdehyde (MDA, a product of LPO) content by trichloroacetic acid (TCA) reaction as described by Heath and Packer (1968).

Antioxidants and metabolites

Peroxidase (POX) activity was determined by using the method of Britton and Mehley (1955). Superoxide dismutase (SOD) activity was assayed according to the method of Fridovich (1974). For protein extraction, fresh leaves were homogenized in tris buffer (0.1 M) followed by mixing of TCA (10 %) and then dissolved into 0.1 N NaOH. Estimation of protein was performed by the method of Lowry et al. (1951). For ascorbic acid, leaf samples were homogenized in oxalic acid and NaEDTA extraction solution. 2,6-dichlorophenol-indophenol dye was used to develop colour and the absorbance was taken at 520 nm. Ascorbic acid content was quantified using the method of Keller and Schwager (1977). Phenol content was estimated by homogenizing the leaf sample in acetone and then using Folin–Ciocalteu reagent and Na2CO3 (Bray and Thorpe 1954). From the standard curve prepared from catechol, different concentrations of phenols in test samples were calculated in mg g−1.

Total biomass and yield

For biomass estimation, root and shoot portions of plants were oven dried separately at 80 °C till constant weight was achieved. Dry weights of component samples were measured separately. Fresh weights of leaves (consumed portion) of five different plants of each plot were taken for yield estimation of amaranthus and spinach plants. RSR was calculated from biomass data using formula given by Hunt (1982).

Nitrogen and nitrogen use efficiency

For nitrogen analysis, oven-dried samples of leaves were ground in a stainless steel grinder and passed through 2-mm sieve. Total N was quantified using the micro-Kjeldahl technique in a Gerhardt Automatic N Analyzer (Automatic Analyzer, Model KB8S, Bonn, Germany). NUE was determined as dry matter accumulation production per unit nitrogen content (Moll et al. 1982) at the level of edible part, i.e. leaves.

Statistical analyses

The statistical significance of the data for biochemical and physiological parameters were tested through two way analysis of variance (ANOVA) test to examine the individual and combined effects of nutrient (N) and treatment (T). Significantly, different means between control and its respective enhanced UV-B treated plants were calculated using the ‘Students t test’. All the statistical tests were performed using SPSS software (SPSS Inc., version 14.0). The entire data set of 48 plots was subjected to a principal component analysis (PCA) using the Varimax method. This analysis allows the identification of interrelated variables.

Results

The results of two-way ANOVA showed that all the measured parameters in amaranthus were significantly affected by nutrients, treatment except NUE, initial fluorescence and Fm, and their interactions except NUE, POX and biomass (Table 1). In spinach, all the parameters except biomass under N × T interaction varied significantly with individual factors and their interactions (Table 1). Photosynthesis reduced significantly in both the plant species under enhanced UV-B with minimum in F1T treatment (amaranthus; 18.4 % and spinach; 30.9 %) and maximum in F0T (37.1 %) amaranthus and F2T (41.8 %) spinach (Fig. 2). Stomatal conductance declined significantly under enhanced UV-B with maximum decline in F0T (53.4 %) and minimum in F1T (5.7 %) amaranthus, whereas spinach exhibited maximum decline in F1T (81.6 %) and minimum in F3T (40.5 %) (Fig. 2). Intrinsic WUE increased in F1C amaranthus (4.04-folds), and F0T (1.47-folds) and F1T (3.77-folds) spinach under enhanced UV-B compared with their controls (Fig. 2).

Table 1 F-ratios and levels of significance of three-way ANOVA test for different parameters of amaranthus and spinach plants
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Fig. 2
figure 2

Effects of enhanced UV-B treatment on photosynthesis, stomatal conductance and water use efficiency of amaranthus and spinach plants under varying nutrient levels. Values are mean ± 1SE. Level of significance between control and enhanced UV-B treated plants: ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. F0: recommended dose of NPK, F1: 1.5× recommended dose of NPK, F2: 1.5× recommended dose of N, F3: 1.5× recommended dose of K

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Under enhanced UV-B, initial fluorescence increased significantly in F0T (6.6 %), whereas decreased in F1T (4.9 %) amaranthus; however, in spinach, it increased in F0T (6.2 %), F1T (3.4 %), F2T (8.6 %) and F3T (17.3 %) (Table 2). Fm increased in F0T (11.9 %) and F3T (1.2 %), whereas decreased in F1T (1.2 %) and F2T (1.7 %) amaranthus under enhanced UV-B exposure (Table 2). Spinach, however, showed reductions in Fm values in F0T (1.3 %), F1T (8.9 %), F2T (5.1 %) and F3T (5.3 %) under enhanced UV-B (Table 2). Fv increased significantly in F0T (14.5 %) amaranthus and decreased significantly by 5.1, 15.8, 15.4 and 29.6 %, respectively, in F0T, F1T, F2T, F3T spinach compared with their controls (Table 2). (Table 2). Fv/Fm ratio reduced significantly at all nutrient levels in spinach with maximum reduction in F3T (25.6 %) and minimum in F0T (3.9 %), and in F0T and F1T amaranthus (Table 2).

Table 2 Effects of enhanced UV-B on chlorophyll fluorescence yields of amaranthus and spinach plants under varying nutrients
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Malondialdehyde content increased significantly under enhanced UV-B with highest percent increment in F3T (54.8 and 57.7) and lowest in F1T (16.8 and 25.9) amaranthus and spinach, respectively, compared with their controls (Fig. 3). Enhanced UV-B exposure led to significant reductions in chlorophyll content in F0T (9.9 %), F1T (5.4 %), F2T (10.1 %) and F3T (25.4 %) amaranthus, and F2T (47.1 %) and F3T (56.5 %) spinach, respectively (Fig. 3). Carotenoids increased under enhanced UV-B in amaranthus at all NPK levels, while spinach showed increments in F0T and F1T and reductions in F2T and F3T (Fig. 3). Enhanced UV-B exposure induced increments in UV-B-absorbing compounds in F0T (86.3 %), F1T (57.3 %), F2T (19.6 %) and F3T (129.1 %) amaranthus, while spinach exhibited increments in F0T (54.4 %) and F1T (33.5 %) and reduction in F3T (41.1 %) (Fig. 4).

Fig. 3
figure 3

Effects of enhanced UV-B treatment on MDA, total chlorophyll and carotenoid content of amaranthus and spinach plants under varying nutrient levels. Values are mean ± 1SE. Level of significance between control and enhanced UV-B treated plants: ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. F0: recommended dose of NPK, F1: 1.5× recommended dose of NPK, F2: 1.5× recommended dose of N, F3: 1.5× recommended dose of K

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Fig. 4
figure 4

Effects of enhanced UV-B treatment on UV-B-absorbing compounds of amaranthus and spinach plants under varying nutrient levels. Values are mean ± 1SE. Level of significance between control and enhanced UV-B treated plants: ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. F0: recommended dose of NPK, F1: 1.5× recommended dose of NPK, F2: 1.5× recommended dose of N, F3: 1.5× recommended dose of K

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Ascorbic acid content increased under enhanced UV-B radiation with higher increase in amaranthus compared with spinach; however, spinach exhibited significant increments in F0T (32.4 %), F1T (8.0 %), F2T (33.8 %), but insignificant in F3T (13.3 %) plants (Fig. 5). Protein content declined under enhanced UV-B with maximum reduction in F3T (64.6 and 67.2 %) and minimum in F2T (30.9 and 39.8 %) amaranthus and spinach plants, respectively (Fig. 5). Total phenol content increased in F1T (7.8 %) and F3T (10.1 %) amaranthus under enhanced UV-B and reduced in spinach at all NPK levels with maximum in F2T (44.7 %) (Fig. 5).

Fig. 5
figure 5

Effects of enhanced UV-B treatment on protein, phenol and ascorbic acid content of amaranthus and spinach plants under varying nutrient levels. Values are mean ± 1SE. Level of significance between control and enhanced UV-B treated plants: ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. F0: recommended dose of NPK, F1: 1.5× recommended dose of NPK, F2: 1.5× recommended dose of N, F3: 1.5× recommended dose of K

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Superoxide dismutase activity increased in F3T (215.9 %), but reduced in F1T (12.2 %) and F2T (32.7 %) amaranthus under enhanced UV-B exposure (Fig. 6). Percent reductions in SOD activities of F0T, F1T, F2T and F3T were 63.5, 44.9, 37.7 and 50.2, respectively, in spinach compared with their controls (Fig. 6). Enhanced UV-B led to increase in POX activity at all nutrient levels with maximum in F2T and minimum in F0T amaranthus plants (Fig. 6). POX activity increased in F3T (43.5 %), but reduced in F0T (30.1 %) and F1T (64.4 %) spinach under enhanced UV-B (Fig. 6).

Fig. 6
figure 6

Effects of enhanced UV-B treatment on SOD and POX activities of amaranthus and spinach plants under varying nutrient levels. Values are mean ± 1SE. Level of significance between control and enhanced UV-B treated plants: ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. F0: recommended dose of NPK, F1: 1.5× recommended dose of NPK, F2: 1.5× recommended dose of N, F3: 1.5× recommended dose of K

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Both amaranthus and spinach showed reductions in total biomass with maximum in F3T (47.9 and 43.2 %) and minimum in F1T (19.2 and 23.9 %) (Fig. 7). RSR was lower in F0T (32.2 and 32.8 %), F1T (25.2 and 62.9 %), F2T (52.9 and 66.7 %) amaranthus and spinach under enhanced UV-B, while increased in F3T amaranthus (33.8 %) and decreased in F3T spinach (19.2 %) (Fig. 7). Enhanced UV-B exposure led to reduction in yield with maximum in F3T (41.2 %) of amaranthus and F2T (28.2 %) of spinach (Fig. 8). Minimum reduction in yield was observed in F1T (8.1 %) for both the species (Fig. 8).

Fig. 7
figure 7

Effects of enhanced UV-B treatment on total biomass and RSR of amaranthus and spinach plants under varying nutrient levels. Values are mean ± 1SE. Level of significance between control and enhanced UV-B treated plants: ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. F0: recommended dose of NPK, F1: 1.5× recommended dose of NPK, F2: 1.5× recommended dose of N, F3: 1.5× recommended dose of K

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Fig. 8
figure 8

Effects of enhanced UV-B treatment on yield and nitrogen use efficiency of amaranthus and spinach plants under varying nutrient levels. Values are mean ± 1SE. Level of significance between control and enhanced UV-B treated plants: ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. F0: recommended dose of NPK, F1: 1.5× recommended dose of NPK, F2: 1.5× recommended dose of N, F3: 1.5× recommended dose of K

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Enhanced UV-B radiation led to insignificant reduction in NUE in amaranthus plants (Fig. 7), while significant reductions in F0T (44.2 %), F1T (9.6 %) and F2T (31.4 %) spinach compared with their controls (Fig. 8).

The control and enhanced UV-B treated values of various parameters under different NPK levels of spinach and amaranthus were subjected to a principal component analysis (Fig. 9). In both the species, first two components accounted for the majority of variations in the data set. In spinach, 39.6 % variations could be explained by the first principal component (PC1) and 23.6 % by the second principal component (PC2). Similarly in amaranthus, 47.1 % variations could be explained by PC1 and 21.2 % by the PC2. It is clear from Fig. 9 that spinach with high values of chlorophyll, SOD and phenol, and amaranthus with high values of chlorophyll, SOD and phenol are located towards the positive end of the PC1 axis, and spinach with high values of protein, gs and ascorbic acid and amaranthus with high values of protein and gs are located towards the positive end of PC2 axis. Membrane damage, carotenoid content, Fv/Fm and UV-B-absorbing compounds were most commonly affected parameters in both the species, while specific sensitivity was shown by ascorbic acid content in amaranthus. Protein, chlorophyll and gs were moderately affected by enhanced UV-B under different NPK levels in both the species. SOD and phenol in spinach are the other parameters moderately affected by enhanced UV-B. Phenol and SOD in amaranthus are the least affected parameters.

Fig. 9
figure 9

The principal component analysis (PCA) of spinach and amaranthus. The scatter plot of both the species on the PC1 and PC2 side. Open (white circle) and closed (black circle) symbols denote amaranthus and spinach, respectively, under ambient and enhanced UV-B radiation at varying NPK levels. Each symbol represents the mean values for the PC scores of the three replicates of each parameter. For the convenience, the parameters are denoted by numbers, carotenoid (1), MDA content (2), Fv/Fm (3), UV-B-absorbing compounds (4), ascorbic acid (5), chlorophyll (6), protein (7), gs (8), SOD (9), phenol (10)

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Discussion

Photosynthetic rate reduced significantly at all concentrations and combinations of NPK under enhanced UV-B, with more reduction in spinach compared with amaranthus. Allen et al. (1998) reported that the lowering in photosynthetic rate under enhanced UV-B is related to its negative effects on photosynthetic apparatus and/or on Calvin cycle enzymes, CO2 diffusion changes, etc. An amendment of 1.5× NPK provided best protection to photosynthetic system of both the species under enhanced UV-B stress, and the magnitude of protection was higher in amaranthus compared with spinach. Better performance of amaranthus, a C4 species under enhanced UV-B at 1.5× NPK is related to its capability to capitalize N, when abundant. C4 plants achieve higher photosynthetic capacity than C3 plants due to lack of photorespiration, thus enabling the plant to achieve high NUE (Furbank et al. 2000). The photosynthetic capacity of leaves is related to the nitrogen content primarily due to the proteins of the Calvin cycle, which represent the majority of leaf nitrogen (Evans 1989). In the present study, amaranthus showed higher NUE and photosynthesis rate compared with spinach at all NPK levels.

Variation in photosynthesis is also dependent on the ability of a leaf to draw down CO2 within the leaf. Lower stomatal conductance in both the species suggests a direct impact of UV-B on stomatal closure (Day and Vogelmann 1995) and stomatal limitation accounting for the decline in photosynthesis. A non-linear relationship between stomatal conductance and photosynthetic capacity in spinach observed at 1.5× NPK under enhanced UV-B suggests that decline in photosynthesis is not fully accounted due to stomatal closure. Conner and Zangori (1998) showed that plants supplied with more than optimum nitrogen, compensated for, or protected themselves from detrimental effects of UV-B on photosynthesis and stomatal conductance. F2C and F3C plants of both the species with low photosynthesis and stomatal conductance had higher intrinsic WUE than other treatments with proportionally higher Ps and gs, which further varied insignificantly under enhanced UV-B. F1T amaranthus and F0T and F1T spinach plants with higher intrinsic WUE had the capacity to compensate the photosynthetic loss associated with reduced stomatal conductance (Gilbert et al. 2011). Chlorophyll fluorescence has been widely used to detect stress-induced perturbations in the photosynthetic apparatus. Light dependent inactivation of PSII reaction centres is associated with a decline in Fm and Fv/Fm and with an increase of initial fluorescence yield as observed in spinach under enhanced UV-B at all NPK levels and in amaranthus at recommended NPK. Increase in Fv of F0T amaranthus may be ascribed to the large increase in Fm. Reductions in Fm values of F1T and F2T amaranthus and at all NPK levels in spinach denote reduced electron transfer capacity under UV-B stress. An increase in the rate constant of non-radiative energy dissipation leads to a decrease in both initial fluorescence and Fm as is observed in F1T amaranthus. Such an increase in thermal de-excitation could function as an overflow valve to allow for nondestructive dissipation of excess excitation energy, and may therefore be viewed as a potentially protective process rather than as an indication of damage (Kyle et al. 1984). Pattern of change in Fv/Fm was different between the two species with amaranthus having higher average values than spinach at all nutrient levels. Reductions in Fv/Fm can be due to slow quenching and photodamage to PSII reaction centres, which may reduce the maximum quantum efficiency of PSII photochemistry (Baker and Rosenqvist 2004). It is probable that the linear flow of electron beyond PSII is also decreased under enhanced UV-B radiation, leading to a reduced proportion of e−(s) available for the Calvin cycle. The reduction in maximum e− transport rate leads to decrease in RuBP regeneration (Onoda et al. 2005), and thus, decreases the maximum rate of Rubisco carboxylation, thereby lowering the photosynthetic performance even more. Additional soil NPK supply increased the photosynthetic capacity manifested by increase in the light saturated rate of photosynthesis and the apparent quantum yield of PSII as recorded in the present study. Also, insignificant variation in Fv/Fm of F2T and F3T amaranthus suggests that the primary damaging effect of UV-B on photosynthesis may have resulted from the decreased Calvin cycle enzyme activity or content or transcription of photosynthetic genes and not due to PSII damage. Maximum reduction in Fv/Fm ratio was observed in F3T spinach grown in K-amended soil.

Enhanced UV-B radiation induced oxidative stress in both the species as observed by increments in MDA content, which was recorded maximum in F3T plants grown at 1.5× K and minimum in 1.5× NPK, suggesting that NPK at higher dose reduced the oxidative stress more than any other amendments. This observation proves our proposed hypothesis that the oxidative stress induced in plants under enhanced UV-B will be reduced maximally at 1.5× NPK. Changes in the photochemistry of the chloroplasts under enhanced UV-B result in dissipation of excess light energy, resulting in the generation of reactive oxygen species (ROS) (Peltzer et al. 2002). Spinach showed higher MDA content than amaranthus as ROS production and oxidative damage are expected to be lower in C4 plants, mostly due to an absence of photorespiration (Uzilday et al. 2012). Huang et al. (2004) also reported an increase in MDA content of N-deficient rice plants. Reduction in CO2 assimilation capacity under enhanced UV-B was further enhanced by low N leading to an over reduction of photosynthetic electron chain and leakage of electron thus enhancing the ROS formation.

The decline in chlorophyll content of both the species under enhanced UV-B at least in part, can be due to LPO in the chloroplast membranes. The photoreduction of protochlorophyllide to chlorophyllide by protochlorophyllide oxidoreductase is one of the possible targets of UV-B (Marwood and Greenberg 1996). Because this reaction is light driven, it is possible that UV-B can damage this enzyme resulting in lower rate of chlorophyll accumulation (Marwood and Greenberg 1996). Also, chlorophyll is stabilized by association with proteins. UV-B radiation causes a downregulation of the expression of genes, which encode for chlorophyll a/b binding proteins (Casati and Walbot 2003). Degradation of chlorophyll occurred maximally in F3T spinach and minimum in F1T plants. In general, chlorophyll content increases with increasing N supply, but at higher than recommended NPK chlorophyll content was minimum in spinach both in control and enhanced UV-B treatment. Moreover, if Liebig’s law of the minimum, which states that ‘the environmental resource present in the least amount will determine plant growth’, applies, then positive response to higher N may not necessarily occur. Carotenoids increased at all NPK levels in amaranthus, but only in F0T and F1T spinach under enhanced UV-B radiation. The decreased carotenoid levels in F2T and F3T imply increased sensitivity of spinach to photo-oxidative damage by UV-B (Rau et al. 1991). Ravindran et al. (2001) reported reduction in carotenoids by 13.3 % in Suaeda maritima under 12.2 kJ m−2 day−1 UV-B radiations. Becatti et al. (2009), however, reported accumulation of carotenoids (52.6 %) in tomato plants under UV-B radiation.

Ascorbic acid increased in both the species under enhanced UV-B (except F3T spinach) and the increment was more pronounced in amaranthus. Increase in ascorbic acid in response to enhanced UV-B was reported earlier (Galatro et al. 2001). Higher nutrient availability did not change the magnitude of increase in ascorbic acid under enhanced UV-B stress. Ascorbic acid reacts directly with ROS and also reduces the oxidized form of α-tocopherol (Xu et al. 2008), thus prevents oxidative stress. SOD activity decreased under enhanced UV-B (except F3T amaranthus), while POX increased in amaranthus at all NPK levels, but decreased in spinach except F3T. This trend clearly shows variations in POX activities under enhanced UV-B between the species and nutrient treatment. Higher induction in POX in amaranthus suggests greater scavenging of H2O2 which make these plants less sensitive to UV-B compared with spinach.

Enhanced UV-B radiation led to reduction in protein content with higher reduction in spinach. Despite maximum reduction in total protein in F3T plants, the biochemical evaluations showed that the measured antioxidative enzymes increased. This suggests that the reduction in protein content is mainly related to primary metabolism or structural protein. Higher reductions in protein content were observed in F1T plants of both the species. Higher N availability to plants grown in 1.5× NPK-amended soil prompted the plants to invest a greater proportion of carbon in protein such as different enzymes which have been identified as a target of UV-B. Even then, the average protein values of control and enhanced UV-B treated plants at 1.5× NPK were higher than the other treatments. Phenol content either did not change (F1T) or reduced in spinach while increased in amaranthus plants under enhanced UV-B (F1T and F3T). As an adaptation to UV-B, phenols can reduce the amount of UV-B reaching the photosynthetic tissues of the leaves (mesophyll), where UV-B can be damaging. Phenylalanine ammonia lyase (PAL) mediating the initial step of phenylpropanoid metabolism can liberate ammonium ions from phenylalanine and skeletons of t-cinnamate could contribute to increase in different phenolic metabolites under low N availability (Stewart et al. 2001), but excess of NPK also led to a similar response under enhanced UV-B in the present study. In F1T and F3T amaranthus plants, reductions in protein were followed by increments in total phenolics, suggesting that there exist an inverse correlation between phenol and protein. Ilvessalo and Tuomi (1989) proposed that relative abundance of carbon and mineral nutrients especially N influences the rate at which the substrate, i.e. phenylalanine is diverted to protein synthesis. It seems that higher nutrient also increased carbon, which led to N dilution, and hence, protein declined with consequent increase in total phenolics as is observed in F1T amaranthus.

Production of UV-B-absorbing compounds is a known defence strategy against the deleterious effects of UV-B radiation (Meijkamp et al. 1999). Amaranthus showed greater ability to synthesize UV-B-absorbing compounds than spinach, resulting in a higher UV-B screening capacity. The improved screening capacity ought to result in a stress release on photosynthetic performance. Although, F0T of both the species having higher induction of UV-B-absorbing compounds than F1T, showed more decline in photosynthesis than F1T. Higher increase in specific leaf weight of F1T amaranthus (10.2 %; data not shown) might have reduced transmittance of UV-B towards photosynthesizing cells, thus effectively protected the photosynthetic machinery. Maximum increase in UV-B-absorbing compounds was recorded for F3T amaranthus plants. But despite this increase, the negative effects of UV-B on the photosynthetic apparatus could not be prevented. UV-B-absorbing compounds reduced under enhanced UV-B in F3T spinach showed highest sensitivity. Plants that do not show induction in UV-B-absorbing compounds under UV-B are sensitive (Xu et al. 2010).

Reductions in total biomass and yield were observed in both the species under enhanced UV-B exposure at all NPK levels with minimum at 1.5× NPK. Smith et al. (2000) suggested that reduced biomass accumulation is often a reliable indication of plant’s sensitivity to UV-B radiation, as it represents the cumulative effects of damaged or inhibited physiological functions. Amaranthus may have invested more N into new leaf production under 1.5× NPK than spinach, and therefore has a higher whole plant carbon gain compared with later species. Alleviation of UV-B stress under higher availability of NPK was more in amaranthus than in spinach. Reduction in biomass was accompanied by substantial modification in the partitioning of biomass in above and below ground plant parts. Reduction in RSR values of all the spinach plants, and F0T, F1T and F2T amaranthus plants under enhanced UV-B showed that UV-B treated plants retained more carbon for repair of above ground parts under different NPK combination compared with individual nutrient. The observed increase in RSR of F3T amaranthus was due to increase in partitioning of resources to roots to stimulate uptake of nutrients under low N and P availability.

In the present study, plant species differed in their NUE, which was higher in amaranthus compared with spinach under enhanced UV-B exposure. Amaranthus did not show significant difference in NUE, but spinach showed significant decline under enhanced UV-B except in F3T. This suggests that amaranthus maintained high NUE, and hence, showed greater protection under enhanced UV-B exposure compared with spinach. NUE is related to inclusion of N in the enzymes of carboxylation and metabolism (Brown 1978). It appears that the ability of amaranthus to maximize photosynthetic carbon uptake for a given unit investment of N is higher compared with spinach. This observation is consistent with the hypothesis that the plant species showing higher NUE will show lower sensitivity to enhanced UV-B.

In our study, the important physiological and biochemical trait variances were extracted into two principal components by a PCA approach. The presence of most sensitive and moderately sensitive parameters was recorded for both the species, but least sensitive parameter was only observed for amaranthus. The analysis of moderately sensitive parameters suggest that impairment in the protection provided by UV-B-absorbing compounds, carotenoid and ascorbic acid content led to membrane damage and inhibition of PSII reaction centre. Amaranthus showed lower sensitivity due to higher phenol accumulation and SOD activity.

Conclusions

The present investigation revealed that the sensitivity of spinach and amaranthus plants to enhanced UV-B radiation varied with the availability of different concentrations and combinations of NPK. The varying sensitivity of both the plants depends on their ability to counter the oxidative stress generated under enhanced UV-B exposure. Photosynthetic performance of amaranthus was better than spinach under enhanced UV-B radiation at all NPK levels. Quantum yield declined in both the species under enhanced UV-B stress, with maximum decline under low N and P availability and minimum at 1.5× NPK. Enhanced UV-B led to increase in MDA content at all NPK levels with minimum at 1.5× NPK. Spinach showed larger increase in MDA content compared with amaranthus. Correspondingly, higher ascorbic acid content and reduction in POX activity were recorded in later compared with former species. Amaranthus showed higher induction in UV-B-absorbing compounds denoting higher UV-B screening capability compared with spinach. Amaranthus also showed higher NUE compared with spinach at all NPK levels. It may be concluded that spinach is more sensitive compared with amaranthus due to lower antioxidative capacity, UV-B screening capability and more disturbances in photosynthetic performances. Both the species showed lowest sensitivity against UV-B at 1.5× NPK. PCA score also revealed that the sensitivity of both the species to UV-B is mainly ascribed to membrane damage and inhibition of PSII reaction centre. Lower sensitivity of amaranthus to UV-B at all NPK levels was due to more accumulation of phenol and SOD activity. More studies having mechanistic approach are required to evaluate the inter species response to UV-B at varying soil NPK levels.