Growth, photosynthesis, and antioxidant responses of Vigna unguiculata L. treated with hydrogen peroxide

Cowpea (Vigna unguiculata L.) is an important legume well grown in semiarid and arid environment. Hydrogen peroxide solutions (0.1, 0.5, 1.0, and 1.5 mM) have been used to optimize growth and photosynthetic performance of cowpea plant at two growth stages [30 and 45 DAS (days of sowing)]. Foliar application of H2O2 at 0.5 > 1.0 mM solution at 29 DAS optimally promoted the photosynthetic attributes [leaf chlorophyll content, net photosynthetic rate (PN), water use efficiency, and maximum quantum yield of PSII (Fv/Fm)] and growth performance [root and shoot length; fresh and dry weight] of plants where the responses were more significant at the later growth stage. It was favored by activity of enzymes as carbonic anhydrase [CA; E.C. 4.2.1.1] and nitrate reductase [NR, E.C. 1.6.6.1] and those of antioxidant enzymes viz. peroxidase [POX; EC 1.11.1.7], catalase [CAT; EC 1.11.1.6], and superoxide dismutase [SOD; EC 1.15.1.1] and leaf proline content. Strengthened root system and antioxidant activity, particularly leaf proline level appeared to be the key factor for efficient photosynthesis and growth responses. Subjects: Agriculture & Environmental Sciences; Botany; Nutrition

ABOUT THE AUTHOR Dr Mohd. Irfan obtained his doctoral degree from Department of Botany, Aligarh Muslim University, India. His research group at Aligarh, India, is working on plant stress physiological responses of crop plants and their amelioration using different plant growth regulators including recently recognized phytohormones. The thrust area of Dr Irfan is individual and interactive effects of heavy metals, plant growth regulators, and soil microbes in crop plants.

PUBLIC INTEREST STATEMENT
Vigna unguiculata or cowpeas are widely grown legume in arid and semiarid regions with good heat and drought resistance, therefore, is used as food vegetable, fodder, and in improving soil fertility. The legumes and seeds have high nutritive value and palatability. The performance of crop though varies depending upon edaphic and climatic conditions, genotype performance manifested as physiological outcome. Plant growth regulators are chemical signals which regulate plant growth and development under changing regimes of edaphoclimatic conditions to unleash genotypic potential to its optimum. Hydrogen peroxide (H 2 O 2 ), a well known secondary signal molecule has its dual effects depending upon its internal tissue level or externally applied concentration of H 2 O 2 . The effective dose of this plant growth regulator was tested as foliar spray for the cowpea crop in terms of growth, photosynthesis, enzymes activity of carbonic anhydrase, nitrate reductase, and those of antioxidant system at two stages of growth.

Introduction
Legumes are the members of family Leguminaceae, which are important grain yielding plants after Gramineae. Besides most important staple food crop worldwide, legumes are ecologically important due to their ability to fix nitrogen in soil. Herbaceous legume crops are important source of nitrogenrich vegetables and pulses in diet. Cowpea (Vigna unguiculata L.; Walp) is an important leafy summer vegetable used for both grains and leaves. It is tolerant to drought and heat conditions, therefore are successful under arid and semiarid conditions. However, grains production of legumes, including cowpeas are greatly sensitized by the native abiotic stresses including heavy metal stress, salinity, and high temperature, registering yield output below the genetic potential of the plants.
Phytohormones are important regulators of plant growth both under non-stress and stressed conditions to unleash the genetic potential of plants in terms of yield output. Among non-classical plant growth regulators several natural molecules have been discovered amongst them hydrogen peroxide is important (Li, Qiu, Zhang, & Wang, 2011;Zelinová, Bočová, Huttová, Mistrík, & Tamás, 2013). It counteract biotic stress (Małolepsza & Różalska, 2005;Orozco-Cardenas, Narváez-Vásquez, & Ryan, 2001) re-establishing the cellular redox status (Kumar, Goswami, Singh, Rai, & Rai, 2013) for growth and development. It interact with certain other plant hormones as abscissic acid, nitric oxide, brassinosteroids, etc. (Jiang et al., 2012;Kumar, Sirhindi, Bhardwaj, Kumar, & Jain, 2010;Liao, Huang, Yu, & Zhang, 2012;Małolepsza & Różalska, 2005). Since the positive roles of reactive oxygen species have been recognized in normal plant growth and metabolism, importance of H 2 O 2 emerged in different plant developmental aspects. As the dose-dependent duality of H 2 O 2 was accepted, species and genotypic responses of it were tested under abiotic stress and normal plant growth conditions. The external application of hydrogen peroxide has been proved promising in improving the growth responses of several plants including legumes, modulating the physiological and biochemical responses (Ashfaque, Khan, & Khan, 2014;Deng et al., 2012;Ishibashi et al., 2011). Since a threshold level of reactive oxygen species is important to trigger the plant growth (Dat et al., 2000), hydrogen peroxide is recognized as a versatile molecule in this network (İşeri, Körpe, Sahin, & Haberal, 2013;Quan, Zhang, Shi, & Li, 2008).
Plants never grow in optimal growth conditions and face a range of diurnal changing environments of water, heat, radiations, and edaphic alterations to manifest suboptimal growth of their genetic potential. An optimal concentration of plant growth regulator, based on age and mode of treatment may unleash this genetic potential to provide better outcome. The role of H 2 O 2 as a secondary messenger is well established in the literature (Neill, Desikan, Clarke, Hurst, & Hancock, 2002;Orozco-Cardenas et al., 2001;Quan et al., 2008;Veal, Day, & Morgan, 2007) and it has been reported to have roles in physiological regulation under abiotic and biotic stress conditions (Małolepsza & Różalska, 2005;Orozco-Cardenas et al., 2001). Application of H 2 O 2 has also been shown to improve the plant growth metabolism and physiological performance in various crop plants (Ishibashi et al., 2011;Kao, 2014) in concentration-dependent manner (Razem, 2008).
The present study was carried out to evaluate the potential of plant growth regulators, hydrogen peroxide, against physiological responses in cowpea emphasizing on growth, photosynthesis, and antioxidant system activity. Cowpea (Vigna unguiculata L.) has been tested against their different concentrations sprayed on leaves at 30 days of sowing (DAS). The aim was to test the comparative growth responses in correlation with photosynthetic attributes and activity of antioxidant system in V. unguiculata treated with H 2 O 2 .

Preparation of plant growth regulators
Hydrogen peroxide (H 2 O 2 ) was obtained from Sigma Chemicals, USA. Molar stock solution of H 2 O 2 was prepared by dissolving required quantity of double distilled water (DDW) in 100 cm 3 volumetric flasks. The desired concentrations of hydrogen peroxide (0, 0.1, 0.5, 1.0, and 1.5 mM H 2 O 2 solutions) were prepared by the dilution of stock solution. Five cubic centimeter surfactant "Tween-20" was added to the solution using DDW at the time of spray.

Plant material and experimental setup
Healthy, uniform seeds of Vigna unguiculata L. Walp were obtained from authentic source. Surfacesterilized seeds (with 0.01% HgCl 2 solution for 2 min) were washed repeatedly with DDW to remove adhering particles of HgCl 2 . The experiment was arranged in a completely randomized block design in the natural environment. The experiment was set up in late February of 2014-2015, under ambient environmental conditions with optimum temperature varied from 10 to 30°C.

Soil characteristics
Seeds were sown in earthen pots (25 × 25 cm) filled with garden soil and farmyard manure in ratio 6:1 v/v. The soil samples were analyzed in the Soil Testing Laboratory, Government Agriculture Farm, Quarsi, Aligarh. The physical-chemical properties of soil included; sandy loam in texture, pH 7.94, E.C. (1:2) 0.48 m mhos cm −1 , organic carbon 1.22%, available N; 123.36-134.2 kg/ha (low), P; 31.67-35.46 kg/ha (low) and K; 118.64-122.52 kg/ha (medium), respectively. The farmyard manure was prepared using cow dung and vegetable domestic waste mixture when completely decomposed. The soil was amended with uniform recommended basal dose of N, P, and K from urea, single superphosphate and muriate of potash added at 40, 138, and 26 mg kg −1 of soil, respectively. The seeds of cowpea were sown at the rate of eight seeds per pot. After one week, plants were thinned to three plants per pot. The foliage of plants were sprayed with hydrogen peroxide (0, 0.1, 0.5, 1.0, or 1.5 mM H 2 O 2 ) at 29 DAS and sampled at 30 and 45 DAS of vegetative stage for various parameters.

Growth analysis of plants
The plants were gently dig from the pots along with adhered soil and dipped in a bucket filled with tap water. The soil particles were gently removed from plant roots. The length and fresh mass of shoot and root separately were measured using a meter scale and squares covered by the three fully developed upper leaves were counted on graph paper for average leaf area. The plant parts were then placed in an oven at 80°C for 72 h. The dried shoot and root were then weighed to record dry mass.

Leaf pigment content and photosynthetic parameters
The leaf chlorophyll and carotenoid content was measured following the method of Mackinney (1941). Photosynthetic parameters [net photosynthetic rate (P N ), water use efficiency (WUE), and maximum quantum yield of PSII (Fv/Fm)] were measured in a well-expanded upper third leaf while attached to the plant using an infrared gas analyzer portable photosynthetic system (Li-COR 6400, Li-COR, Lincoln, NE, USA), between 11:00 and 12:00 h under clear sunlight. The atmospheric conditions during measurement were photosynthetically active radiation, 1,016 ± 6 μmol m −2 s −1 , relative humidity 60 ± 3%, atmospheric temperature 22 ± 1°C, and atmospheric CO 2 360 μmol mol −1 . The duration of the measurement of each sample was 10 min after the establishment of steady-state conditions inside the measurement chamber.

Carbonic anhydrase (CA) activity
The activity of carbonic anhydrase was determined by adopting the procedure described earlier by Dwivedi and Randhawa (1974). Fresh leaf samples (0.2 g) were cut into small pieces and suspended in 10 ml of 0.2 M cysteine hydrochloride solution. The samples were incubated at 4°C for 20 min. The leaf pieces were blotted dry and transferred to test tubes containing 4 ml of phosphate buffer (pH 6.8) followed by the addition of 4 ml of 0.2 M alkaline bicarbonate solution and 0.2 ml of 0.002% bromothymol blue indicator. The test tubes were incubated at 4°C for 20 min. The reaction mixture was titrated against 0.05 N HCl, after the addition of 0.2 ml of methyl red indicator. The results are expressed as mol (CO 2 ) kg −1 (leaf FM) s −1 .

Nitrate reductase (NR) activity
Nitrate reductase activity was measured by the method of Jaworski (1971) in fresh leaf samples that were cut into small pieces. The fresh leaf samples were cut into small pieces and transferred to plastic vials, containing phosphate buffer (pH 7.5), KNO 3 , and isopropanol that were incubated at 30°C for 2 h. After incubation, sulfanilamide and N-1-naphthylethylenediamine hydrochloride solutions were added. The absorbance was read at 540 nm and the activity of NR [n mole NO 2 g −1 (FM) s −1 ] was calculated.

Antioxidant system activity
The activity of peroxidase (POX) and catalase (CAT) was assayed following the procedure described by Chance and Maehly (1955). The activity of superoxide dismutase (SOD) was assayed by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) using the method of Beauchamp and Fridovich (1971). The amount of enzyme which causes 50% inhibition in photochemical reduction of NBT was considered as one enzyme unit. The proline content in fresh leaf samples was determined by the method of Bates, Waldren, and Teare (1973). The absorbance of the toluene layer was read at 528 nm, on a spectrophotometer (Milton & Roy, USA).

Statistical analysis
The experiment was conducted according to simple randomized block design. Each treatment was replicated five times and three plants per pot were maintained where each pot was considered as a replicate. Treatment means were compared by the analysis of variance using R ver. 3.1.0 for Windows (http://www.r-project.org/). Least Significant Difference between treatment means was calculated at 5% probability level (p < 0.05).

Growth Parameters
All the growth values (leaf area per plant, length, fresh and dry mass of shoot and root) increased with the treatment, as compared to the water-treated (Control) plants at 45 DAS (Figures 1(a)-(g)). All the concentrations of H 2 O 2 increased the above-mentioned growth parameters. However, the best concentration was recorded to be 0.5 mM H 2 O 2 foliar spray which induced the growth more effectively than the other concentrations. This concentration registered percent increase in root and shoot lengths by 25 and 35%, for fresh masses by 39, 33%, and for dry masses by 20, 29%, respectively, compared to controls. The increase in leaf area was by 25% as compared to water-sprayed control plants.

Leaf pigment contents and photosynthetic parameters
The foliar treatment (H 2 O 2 ; 0, 0.1, 0.5, 1.0, or 1.5 mM) significantly improved the pigment (total chlorophyll and carotenoid) content at later growth age (45 DAS) as compared to early growth stage (30 DAS), where 0.5 mM foliar spray of H 2 O 2 induced the level of these pigments irrespective of growth stage (Figures 2(b) and (c)). The net photosynthetic rate (Pn) along with their attributes (WUE and Fv/ Fm; maximum quantum yield of PSII) also increased as the growth progressed from 30 to 45 DAS, as compared to the control plants (Figures 2(a), (d) and (e)). Leaves of V. unguiculata sprayed with 0.5 mM H 2 O 2 showed best improvement in chlorophyll and carotenoid level 28 and 39% (30 DAS) and by 34% (45 DAS), while P N , WUE, and Fv/Fm increased by 38, 36, and 26 and 45%, 32, and 20%, at 30 and 45 DAS, respectively, as compared to their controls. However, the order of improvement of pigments level and photosynthetic attributes was 0.5 mM > 1.5 mM > 1 mM > 0.1 mM > 0.0 mM (control).

Activity of carbonic anhydrase (CA) and nitrate reductase (NR) enzyme
The activity of CA and NR both increased significantly with respect to foliar application of different molar solutions (0, 0.1, 0.5, 1.0, or 1.5 mM) H 2 O 2 (Figures 2(f) and (g)). Out of the various concentrations of H 2 O 2 , the maximum activity was recorded in plants treated with 0.5 mM solution, whereas lowest activity was recorded in plants received 1.5 mM solution as compared to control. Both the enzymes reflected increasing values of enzymes activity as the growth progressed from 30 to 45 DAS. The percent increase in CA activity by different molar solutions of H 2 O 2 was 27, 52, 36, and 23%, whereas in NR activity was 38, 52, 36, and 32%, at 45 DAS, respectively, compared to control plants. The increase in CA and NR activity was more at 30 DAS as compared to later growth stage (45 DAS).

Antioxidant system activity
The data depicted in Figures 3(a)-(d) clearly indicated an increase in the activity of all the antioxidant enzymes as growth progressed and also in response to different concentrations (H 2 O 2 ; 0, 0.1, 0.5, 1.0, or 1.5 mM) of treatments. Among the four tested concentrations, 0.5 mM H 2 O 2 maximum enhanced the activity of POX, CAT, and SOD. Higher activity was recorded at growth stage sampled after foliar treatment than latter growth stage. Percent increase of CAT, POX, and SOD was by 57, 27, and 38% at 30 DAS, while it increased by 29, 42, and 19% at 45 DAS, respectively, than the control plants. Also a significant increase in the leaf proline content in response to different concentrations H 2 O 2 was recorded as compared to control. As the growth progressed, the proline content decreased similar to that of antioxidant enzymes.

Discussion
Most of the work on H 2 O 2 application at low concentration treatments, studied in seed germination or seedling growth has given positive response. The experimental results suggested that H 2 O 2 treatment increased the plant growth parameters (length, fresh and dry weight of shoot and root, leaf area) at 45 DAS when sprayed at 29 DAS on foliage. However, higher and lower concentration of H 2 O 2 than the optimum level (here 0.5 mM) has not induced growth parameters optimally. Lower concentrations of H 2 O 2 alone or with nitric oxide positively influence adventitious root growth of marigold and sweet potato seedlings (Deng et al., 2012;Liao, Xiao, & Zhang, 2009;Liao et al., 2012). A different set of plant hormones have been recorded to interact with H 2 O 2 during germination and early plant growth (Barba-Espin et al., 2010) and under different set of growth conditions. H 2 O 2 -mediated auxin-induced gravitropic responses have also been worked out in maize roots (Joo, Bae, & Lee,  (Ivanchenko et al., 2013). The increased shoot length was also calculated in this experiment (Figures 1(b) and (c)) which might be due to auxin-directed H 2 O 2 signaling (Joo et al., 2001). H 2 O 2 induced the germination and early seedling growth of barley and wheat (Çavuşoğlu & Kabar, 2010;Hameed, Farooq, Iqbal, & Arshad, 2004), probably counteracting the ABA action. The growth of plant and plant organs depends upon the induction of water and mineral uptake through root hair plasma membranes, regulated by the hormonal network to reset the growth redox in meristematic tissues. Qiu, Li, Bi, and Yue (2011) suggested that H 2 O 2 metabolism in wheat seedling was involved as a signal in the processes of laser-induced water acclimation, the osmo-protective effect they related with NADPH oxidasedependent H 2 O 2 production. The fresh and dry weight of the plants in our experiment (Figures 1(d) and (f)) also increased with H 2 O 2 treatment. Increased adventitious root growth and absorption of mineral (especially P) ions favor root nodule formation (D'Haeze et al., 2003). A positive role of SODs is tested by Rubio et al. (2004) as a source of H 2 O 2 in indeterminate alfalfa (Medicago sativa) and pea (Pisum sativum) nodules where abundant H 2 O 2 reflected the oxidative stress inducing nodule senescence which was associated with the degrading bacteroids in the senescent zone. The leaves of soybean foliar pre-treated sprayed with H 2 O 2 under drought stress had higher relative water content than non-treated soybean leaves (Ishibashi et al., 2011). Treatment with H 2 O 2 or salicylic acid caused significant increase in growth parameters in tomato cultivars, which was due to marked increase in endogenous growth regulators as GA 3 , IAA, and ABA (Orabi, Dawood, & Salman, 2015). Exogenous H 2 O 2 application to legumes also increased the dry matter production (Figures 1(e) and (g)). Increased plant dry matter was also recorded in the leaves of wax apple (Khandaker, Boyce, Osman, & Hossain, 2012), maize plants with induced mineral content and level of osmotic solutes (Guzel & Terzi, 2013;Terzi, Kadioglu, Kalaycioglu, & Saglam, 2015) by exogenous H 2 O 2 treatment. Generation of H 2 O 2 in the cell wall apoplastic space is reported and has been shown to be required for the formation of cell cross-linking of wall polymers (Elstner & Heupel, 1976;Mader, Ungemach, & Schloss, 1980). The increased leaf area in tested plants (Figure1(a)) appears due to induction of cell proliferation. In multicellular organisms, H 2 O 2 can also activate signaling pathways to stimulate cell proliferation (Foreman et al., 2003;Geiszt & Leto, 2004), differentiation (Konieczny, Banaś, Surówka, Michalec, & Miszalski, 2014;Potikha, Collins, Johnson, Delmer, & Levine, 1999), and seedlings elongations ( Barba-Espin et al., 2010). Similar result in mung bean was also obtained by Fariduddin, Khan, and Yusuf (2014).

2001). Auxin-induced H 2 O 2 concentration in tomato root tips inhibits root growth
Present study indicated that the treatment of H 2 O 2 significantly increased the pigment levels and photosynthetic attributes i.e. net photosynthetic rate, WSE, and maximum quantum yield of PS II, in the cowpeas (Figures 2(a)-(e)). H 2 O 2 treatment with BRs caused significant increases in chlorophyll level in Vigna radiata (Fariduddin et al., 2014) and in sand-cultured tomato cultivars (Orabi et al., 2015), which was suggested due to marked increase in endogenous growth regulators. Formation of H 2 O 2 can also take place through thermal dissipation from antenna inducing excess electron leakage from photosynthetic electron transport chain, to molecular oxygen (Mehler reaction). It was indicated that H 2 O 2 can diffuse through the chloroplast envelope aquaporins, where CA presumably remains attached (Borisova et al., 2012). Activity of CA with H 2 O 2 -mediated increased influx of stomatal CO 2 can cooperatively induce the net photosynthetic rate as was reported in Vicia faba stomatal guard cells (Jannat et al., 2011;Zhang et al., 2001) and maize plants (Gondim et al.;. A significantly increased quantum efficiency of PSII (Fv/Fm value) was obtained with the lower concentration (0.1, 0.5, and 1.0 mM) application of H 2 O 2 , as observed in the experiment. The effect of H 2 O 2 on Fv/Fm might be due to interaction with ABA and NO mediated which was also shown by (Neill, 2007;Neill et al., 2002;Yang, Yun, Zhang, & Zhao, 2006). Hydrogen peroxide works as a secondary messenger for Brassinosteroids induced CO 2 assimilation and carbohydrate metabolism (Jiang et al., 2012). It was also recorded that exogenous H 2 O 2 increased photosynthetic rates in the leaves of wax apple under field conditions (Khandaker et al., 2012).
Often elevated level of H 2 O 2 is correlated with oxidative stress which resulted into increased level of antioxidant molecules including carotenoids such as beta carotene and xanthophyll (Upadhyaya, Khan, & Panda, 2007). The carotenoid endoperoxide produced from a reaction between β-carotene and reactive oxygen species Ramel, Mialoundama, and Havaux (2012). Carotenoids besides working as antenna are important antioxidants in photosynthetic systems (Larson, 1988) absorbing short wavelength energy. Increased carotenoid level concomitant with carotenoid level is often correlated with increasing age or stress (Prochazkova, Sairam, Srivastava, & Singh, 2001). Cowpea Plants sprayed with chitosan reflected increased accumulation of H 2 O 2 concomitantly increased the carotenoid level in leaves (Farouk, Ramadan, & Showler, 2013).
As it is clear from the results, application of H 2 O 2 significantly induced the activity of two enzymes i.e. carbonic anhydrase and nitrate reductase (Figures 1(f)-(g)). Among the different concentrations of H 2 O 2 solutions (0, 0.1, 0.5, 1.0, 1.5 mM) used as foliar spray, 0.5 mM best optimized the enzyme's activity which could be due multiple factors viz. increased substrate availability, redox regulation of enzymes transcripts and their translation to increase their cellular pool, and availability of co-factors. Hydrogen peroxide (H 2 O 2 ) regulated increased adventitious rooting suggesting increased surface area of absorption for important critical mineral ions including N, P, and K. Phosphorus is known to be required for the formation of root nodules (Tang, Hinsinger, Drevon, & Illard, 2001) which further increase the N assimilation from root hairs (Fujikake et al., 2003). Increased substrate (nitrate) availability could possibly upregulate the activity of NR in legume leaves. Carbonic anhydrase activity is regulated by the availability of cellular availability of CO 2 taken up through stomatal activity. H 2 O 2 regulates the stomatal conductance and hence the CO 2 exchange (Gondim, Miranda, Gomes-Filho, & Prisco, 2013;Jannat et al., 2011;Zhang et al., 2001). Here, again increased availability could positively regulate the activity of CA in photosynthesizing leaves. The enzyme CA has also been detected near the thylakoid aquaporins, and was also suggested earlier to have role in the regulation of photosynthetic electron transport chain (Borisova et al., 2012;Stemler, 1997). Root absorption of mineral ions (e.g. Zn, Co, Fe, etc.) to work as co-factors helping to catalyze the activity of CA and NR may favorably be suggested for the increased activity of these enzymes by H 2 O 2 foliar treatment.
The level of proline increased in the plants exogenously sprayed with H 2 O 2 on legumes foliage (Figure 1(a)). The increased relative water content due to H 2 O 2 application (as discussed above) was positively correlated with the induced level of osmolites such as sugars, polyamines, and proline has been recorded in plant tissues which creates a negative potential for the absorption of water. Increased proline level was also recorded in several plants against H 2 O 2 signaling (Fariduddin et al., 2014;Guzel & Terzi, 2013;Jiang et al., 2012;Moskova et al., 2014).

Conclusion
The positive role of H 2 O 2 on plant growth and development is concentration and age dependent to promote its root system, photosynthesis and redox status for better growth and physiology. In cowpeas, 0.5 mM concentration optimally produced most of the growth and physiological features of plant favorably at 45 days after sowing (DAS) of plants when sprayed with H 2 O 2 at 29 DAS on leaves.