ФИЗИОЛОГИЯ РАСТЕНИЙ, 2015, том 62, № 3, с. 349-354


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© 2015 R. Bansal, J. P. Srivastava

Department of Plant Physiology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, India

Received July 30, 2014

We studied the effect of waterlogging stress on photosynthetic and biochemical parameters in pigeonpea [Cajanus cajan (L.) Millsp.] genotypes viz. ICPL-84023 (waterlogging resistant) and MAL-18 (waterlogging susceptible). Plants at early growth stage (20 days) were subjected to stress by keeping the pots in water filled containers. Stress was imposed for six days. Waterlogging reduced carbon exchange rate, stomatal conductance, intercellular CO2 concentration and transpiration rate. Photosynthesis was limited by stomatal as well as non stomatal components under waterlogging. Reduction in chlorophyll, starch and increase in alcohol dehydrogenase activity along with the membrane injury was reported under waterlogging. High carboxylation efficiency, more chlorophyll content, starch availability, alcohol dehydrogenase activity and membrane stability were associated with better survival of ICPL-84023 under waterlogging.

Keywords: Cajanus cajan - photosynthesis - waterlogging - alcohol dehydrogenase - chlorophyll

DOI: 10.7868/S0015330315030033


Waterlogging is one of the major abiotic stresses adversely affecting crop productivity. It may develop due to several direct (improper irrigation practices) and indirect (global warming) anthropogenic factors and natural consequences (meteorological). Intergovernmental panel on climate change reported that man induced climate change may increase the frequency of waterlogging events in future.

Waterlogging leads to development of rapid hy-poxia and anoxia in the soil because of slow diffusion of oxygen in water as compared to air. Oxygen is the terminal acceptor in mitochondrial electron transport. In the absence of oxygen, plant shifts its metabolism to anaerobic mode [1]. ATP production is hampered and all the metabolic activities are adversely affected. Many proteins are induced in response to waterlogging. Most of the induced proteins are glycolytic and fermentation enzymes [2]. Alcohol dehydrogenase (ADH) is the key enzyme involved in anaerobic fermentation. ADH converts acetaldehyde into ethanol with the oxidation of NADH into NAD+. Though, in anaerobic fermentation, efficacy of ATP production is

1 This text was submitted by the authors in English.

Abbreviations: ADH - alcohol dehydrogenase; DTT - dithio-threitol; EU - enzyme units; IRGA - infra red gas analyzer; MDA - malondialdehyde; ROS - reactive oxygen species. Corresponding author: J. P. Srivastava. Department of Plant Physiology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, India; fax: 0915422368993, e-mail: jpsbhu25@yahoo.co.in

much lower than aerobic respiration, but the induction of fermentative metabolism is an adaptive response to waterlogging. Waterlogging leds to increase in ADH activity in mustard [3], maize [4, 5], and mu-ng bean [6].

Waterlogging prevents the aerial influx of CO2, which leads to reduction in photosynthesis. Under waterlogging, ABA-mediated stomatal closure was observed in tomato [7]. Effect of waterlogging on photosynthesis has been extensively studied. Reduction in photosynthetic parameters was observed in maize [8], barley [9], mung bean [10], and tomato [7].

Chlorophyll is the important constituent of light harvesting complex in photosynthetic apparatus of plants. Therefore, chlorophyll content is directly associated with the photosynthesis. Under waterlogging, reduction in the chlorophyll content was evident in maize [8], barley [9], mung bean [7], and soybean [11]. Reduction in photosynthesis leads to restrictions in carbohydrate metabolism in plants, which adversely affects the availability of sugars [12]. Reduced in sugar content under waterlogged condition was observed in maize [4], while accumulation of starch was observed in leaves of luffa [13].

Low energy supply and altered redox state of the cells are responsible for reactive oxygen species (ROS) production under hypoxic and anoxic conditions. Increase in ROS concentration may cause lipid peroxi-dation, enzyme inactivation and oxidative damage to DNA. Oxidative injury due to waterlogging leads to membrane disintegrity due to lipid peroxidation. Re-

duction in membrane stability under waterlogging was reported in corn [8], winter rape [14], and barley [15].

Pigeonpea is an important legume crop of rain-fed agriculture in tropics and sub-tropics. The crop is generally sown during June-July. At early growth stages, the crop frequently faces the problem of waterlogging. This crop is very sensitive to waterlogging stress and wilting, senescence, chlorosis, and abscission of lower leaves may occur within two days after imposing stress [16]. A few studies have been conducted in the crop regarding physiological, biochemical and molecular responses to waterlogging. In pigeonpea, study of carbohydrate metabolism under waterlogging revealed enhanced expression of sucrose synthase gene [17]. Role of root antioxidant enzyme system in waterlogging resistance has also been studied in the crop [18].

Keeping in view the high sensitivity of the crop to waterlogging, present study was undertaken to investigate the effect of waterlogging stress on photosynthesis and biochemical parameters in pigeonpea.


A pot experiment was conducted with pigeonpea [Cajanus cajan (L.) Millsp.] genotypes ICPL-84023 (waterlogging resistant) and MAL-18 (waterlogging susceptible). Seeds were procured from Department of Genetics and Plant Breeding, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, India. Plants were grown in plastic pots (diameter 9.5 cm) filled with 750 g sandy soil. The plants were supplemented with half strength Hoagland solution on each alternate day [19]. Waterlogging stress was imposed at 20 days after sowing by keeping the pots in water filled containers. Water level was maintained at 4-5 cm above the soil surface in the pots. Control plants were maintained at normal supply of moisture. All the observations were recorded at 0, 2, 4, and 6 days of imposing stress.

Chlorophyll content and all the photosynthetic parameters were studied in first fully expanded leaf from the top of the plant. All the observations were recorded between 9:00 to 10:30. For biochemical studies (starch, ADH and membrane injury), root samples were collected by uprooting the plants such a way that roots were least damaged and were immediately stored at —20°C. All the analyses were done as soon as possible.

Chlorophyll content was recoded with the help of SPAD meter (Minolta) and photosynthetic parameters (Carbon exchange rate, intercellular CO2 concentration, transpiration and stomatal conductance) were measured by Infrared Gas Analyzer (IRGA, "ADC BioScientific Ltd.", United Kingdom). Photosynthet-ic parameters were calculated by using the empirical equations as mentioned in LCi Portable Photosynthesis System Instruction Manual [20]. All the photosyn-thetic parameters were calculated as per the formulae given below.

Carbon exchange rate, A (^mol/(m2 s)) = usAc,

where us - mass flow of air per m2 of leaf area, mol/(m2 s); Ac - difference in CO2 concentration through chamber, ^mol/m.

Intercellular CO2 concentration, Ci (^mol/mol) = = [{fec - E/2)C;n} - A]/(gc + E/2),

where gc - 1/(1.6rs + 1.37rb); can - CO2 flowing out from leaf chamber, ^mol/mol; E - transpiration rate, mol/(m2 s); A - carbon exchange rate, ^mol/(m2 s); rb - boundary layer resistance to water vapour, m2/(s mol); rs - stomatal resistance to water vapour, m2/(s mol).

Transpiration rate, E (mol/(m2 s)) = Aeujp,

where Ae - differential water vapour concentration, mbar; us - mass flow of air into leaf chamber per m2 of leaf area, mol/(m2 s); p - atmospheric pressure, mbar; gs - stomatal conductance, mol/(m2 s); gs = 1/rs, where rs - stomatal resistance to water vapour, m2/(s mol).

Starch content was determined by anthrone method [21]. To analyze the ADH activity, 100 mg root tissue was homogenized in 0.15 M Tris buffer, pH 8.0, with 10 mM dithiothreitol (DTT). After centrifuga-tion at 5000 g for 10 min at 4°C, the supernatant was collected. Reaction mixture consisted of 2.3 mL Tris buffer, pH 9.0, 0.1 mL ethanol, 0.1 mL NAD+ (5 mg/mL) and 0.5 mL enzyme extract. Change in ab-sorbance, due to conversion of NAD+ to NADH + H+, was measured at 340 nm with a UV visible spectrophotometer (SL196, "Elico Ltd.", India) [22]. Protein in the crude extract was measured by Coomasie Brilliant Blue G-250 dye binding method [23].

To determine root membrane injury, in one set, 100 mg root tissue was taken in 10 mL double distilled water and was heated at 40°C for 30 min in a water bath. Electrical conductivity of the solution was recorded with the conductivity meter (C1). Second set was boiled at 100°C on a boiling water bath for 10 min, and its conductivity was also measured (C2) [24]. Per cent membrane injury was calculated as:

Per cent membrane injury = [1 - (1 - C1/C2)] x 100.

All the experimental data recorded are mean values for three independent observations with three replicates each. The data were subjected to ANOVA for completely randomized design factorial. Differences atp < 0.01 were considered significant [25].


Photosynthetic parameters

Waterlogging led to reduction in all the photosyn-thetic parameters (carbon exchange rate, intercellular CO2 concentration, transpiration rate and stomatal conductance) in both the genotypes (fig. 1). Under



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