Influence of sub-lethal crude oil concentration on ...

Environ Sci Pollut Res DOI 10.1007/s11356-016-6976-7

RESEARCH ARTICLE

Influence of sub-lethal crude oil concentration on growth, water relations and photosynthetic capacity of maize (Zea mays L.) plants Habib-ur-Rehman Athar 1,2 & Sarah Ambreen 1,3 & Muhammad Javed 1 & Mehwish Hina 1 & Sumaira Rasul 3 & Zafar Ullah Zafar 1 & Hamid Manzoor 3 & Chukwuma C. Ogbaga 2 & Muhammad Afzal 4 & Fahad Al-Qurainy 5 & Muhammad Ashraf 5,6

Received: 25 May 2015 / Accepted: 24 May 2016 # Springer-Verlag Berlin Heidelberg 2016

Abstract Maize tolerance potential to oil pollution was assessed by growing Zea mays in soil contaminated with varying levels of crude oil (0, 2.5 and 5.0 % v/w basis). Crude oil contamination reduced soil microflora which may be beneficial to plant growth. It was observed that oil pollution caused a

Responsible editor: Philippe Garrigues Capsule abstract Oil pollution reduced the maize growth by affecting water relations and thylakoidal and stromal reactions, but it increased root length and thus helped in regulation of water and N uptake during acclimation. Highlights • Physiological and biochemical responses of Zea mays grown in soil contaminated with crude oil were assessed. • Crude oil pollution reduced the growth of maize plants, which is mainly associated with reduction in plant water status, reduction in N accumulation, and reduced photosynthetic rate. • Plants grown in soil contaminated with 2.5 % tried to acclimate by improving root traits. • Gas exchange measurements evidenced that reduction in photosynthetic rate due to crude oil was due to non-stomatal factors. • Fast chlorophyll a kinetic studies showed that reduction in quantum yield of PSII (Fv/Fm) was associated with PSII damage at both donor and acceptor side. • Quenching analysis suggested that PSII damage might have been due to lower turnover of D1 protein. Electronic supplementary material The online version of this article (doi:10.1007/s11356-016-6976-7) contains supplementary material, which is available to authorized users. * Habib-ur-Rehman Athar [email protected]

1

Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan 60800, Pakistan

2

Department of Plant Sciences, Faculty of Life Sciences, The University of Manchester, Manchester, UK

remarkable decrease in biomass, leaf water potential, turgor potential, photosynthetic pigments, quantum yield of photosystem II (PSII) (Fv/Fm), net CO2 assimilation rate, leaf nitrogen and total free amino acids. Gas exchange characteristics suggested that reduction in photosynthetic rate was mainly due to metabolic limitations. Fast chlorophyll a kinetic analysis suggested that crude oil damaged PSII donor and acceptor sides and downregulated electron transport as well as PSI end electron acceptors thereby resulting in lower PSII efficiency in converting harvested light energy into biochemical energy. However, maize plants tried to acclimate to moderate level of oil pollution by increasing root diameter and root length relative to its shoot biomass, to uptake more water and mineral nutrients. Keywords JIP test, nitrogen . Oil pollution . PIABS, photosynthetic rate, water potential

Introduction Crude oil pollution deriving from exploration and processing of petroleum is a widespread environmental problem (Pernar et al. 2006, Paulauskienė et al. 2014). Crude oil pollution is a major threat to the life near oil welling areas as well as survival of plants near ponds and canal banks and in agricultural fields (Agbogidi 2011). It causes acute and long-lasting impacts on agricultural land and crop plants by affecting soil characteristics (Anoliefo and Vwioko 1995, Lin et al. 2002, RodríguezBlanco et al. 2010). Plants growing in oil-polluted zones are reported to have poor growth resulting from the cumulative effect of toxic components of crude oil and insufficient aeration caused by air displacement from the pore spaces between the soil particles by the crude oil (Ellis and Adams 1961,

Environ Sci Pollut Res

Baker 1970, Pernar et al. 2006). Insufficient aeration also causes root stress and low water availability to the plant, which consequently reduces plant growth (Ashraf and Rehman 1999). Increasing level of oil in soil caused an exponential increase in growth retardation, which is mainly due to reduction in water and nutrient uptake (Anoliefo et al. 2003). This can be reasoned to the fact that absorption of toxic petroleum molecules by plants in contaminated soil can modify the permeability and the structure of the plasma membrane (Peña-Castroa et al. 2006). While working with alfalfa, (Marti et al. 2009) suggested that the extent of adverse effects of oil pollution on plant physiological status may directly affect the plant detoxification capacity. The adverse effects of crude oil on plants vary according to the concentration of petroleum, the time of exposure to the contaminant and the plant species (Adam and Duncan 2002, Baek et al. 2004, Merkl et al. 2005a, Naidoo et al. 2010). However, different plants have different abilities to respond or adapt to a variety of biotic stresses. Since plant species exhibited a degree of tolerance on soil contaminated with oil, it is important to understand the physiological aspects of tolerance to oil pollution. Of various physiological traits, photosynthetic capacity, maintenance of water balance and acquisition of nutrients are the most important characteristics (Agbogidi et al. 2007a, Agbogidi et al. 2007b, Njoku et al. 2009). However, detailed information about the effect of oil contamination photosynthetic activity is scarce. For example, recently it was reported that crude oil pollution reduces overall photosynthetic activity and chlorophyll contents of plants (Njoku et al. 2009). Moreover, most of studies were focused on effects of crude oil on plants growing in coastal and oil field areas. However, there is comparatively little information available on effects of oil pollution on crop plants. In view of all these reports, the aim of the present study was to assess up to what extent and how crude oil-contaminated soil affects the uptake of macronutrient, water relation and photosynthesis of maize (Zea mays), which is one of the most important cereal crops. In addition, one of the secondary objectives of the study is to draw the relationship between various biochemical and physiological processes with the growth in order to work out the mechanism of oil toxicity to maize plants. This information will be of great help in improving crop productivity on agricultural land polluted with oil. 3

Institute of Molecular Biology and Biotechnology, Bahauddin Zakariya University, Multan, Pakistan

4

National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan

5

Department of Botany and Microbiology, King Saud University, Riyadh, Saudi Arabia

6

Pakistan Science Foundation, Islamabad, Pakistan

Materials and methods In view of the information that tolerance in plants to contaminated soil with oil pollution varies with concentration of the oil in soil and duration of exposure of plants to soil contaminated with oil, the present study was conducted in two experiments. The duration of oil pollution stress was 6 weeks. Both experiments were conducted in Botanic Gardens of Bahauddin Zakariya University, Multan, Pakistan (30° 11 N and 71° 28 E). The first experiment was conducted in July to August in normal sunlight with average day and night temperatures of 39 + 6 and 28 + 4 °C, respectively. The relative humidity ranged from 34.5 to 56.5 % and day length from 11 to 12 h. Crude oil used to contaminate soil was sourced from Oil and Gas Development Corporation (OGDC), Pakistan. Varying amounts of crude oil (0, 2.5 and 5 % on v/ w basis) were added to the soil and mixed thoroughly using mechanical skills and then filled in 15 plastic pots (30 cm diameter) with (7 kg) soil in each. The maize seeds (Zea mays, var. Pak-F) were obtained from Punjab Seed Corporation, Khanewal, Pakistan. Seeds of maize were surface-sterilized in 5 % sodium hypochlorite for 5 min and rinsed with distilled water two times before further experimentation. Twenty-five maize seeds were allowed to germinate in each pot. After 1 week, plants were thinned to equidistantly placed four plants of uniform size per pot. After 2 weeks of germination, Hoagland nutrient solution was applied to each pot (1 l/pot). The experiment was arranged in a completely randomized design. Pots were maintained in well-watered state throughout the experiments. After 6 weeks of germination, plants were harvested, washed with distilled water, blotted dry and separated into shoots and roots, and data for fresh biomass was recorded. These plants were then oven-dried at 65 °C for 72 h and dry biomass was recorded. At the time of harvest, data for shoot or root length were also recorded. However, 2 days before harvest, chlorophyll contents, chlorophyll fluorescence and gas exchange parameters were measured. Total chlorophyll content of plant samples was determined with the help of a chlorophyll meter (SPAD-502, Konica Minolta, Sensing, Inc., Japan). Chlorophyll fluorescence The polyphasic rise of fluorescence transients was measured by a handheld chlorophyll fluorescence meter (PAR-FluorPen FP 1 00-MAX-LM, Photon Sy ste ms Instruments, Czech Republic) according to (Strasser et al. 1995). The transients were induced in a 4-mm diameter leaf by red light at 3000 μmol m−2 s−1 provided by an array of six light emitting diodes (peak 650 nm), which focused on the sample surface to give homogenous illumination over the exposed area of the sample surface. All the samples were dark-adapted for 30 min prior to fluorescence measurements. Data collected were used


to calculate the OJIP parameters as described by Strasser et al. (1995). For the quantification and performance of photosystem II (PSII) response of maize cultivar grown in crude oilcontaminated soil, biophysical and phenomenological parameters derived from OJIP analysis were assessed. After the fluorescence induction, the following energy fluxes with their ratios were measured: (a) the specific energy fluxes per reaction centres for absorption (ABS/RC), trapping (TRo/RC), dissipation (DIo/RC) at antenna chlorophyll level and electron transport (ETo/RC) and (b) maximum quantum yield of primary photochemistry ΦPo, with Ψo the efficiency of trapped exciton that can move an electron into electron transport chain further than QA–, the quantum yield of electron transport (ETo/ ABS or φPo o). The performance index (PI) is the ratio of two structures and function indexes and calculated as h . i . PIABS ¼ ChlRC Chlantenna ΦPo ð1− ΦPo Þ h . i Ψ o ð1 − Ψ o Þ

Gas exchange parameters Gas exchange characteristics were measured using an opensystem LCA-4 ADC portable infrared gas analyzer (Analytical Development Company, Hoddesdon, England). These measurements were carried out from 10:00 to 14:00 h with the following specifications/adjustments: leaf surface area 11.25 cm 2 , ambient CO 2 concentration (C r e f ) 371 μmol mol−1, temperature of leaf chamber varying from 25 to 28 °C, leaf chamber volume gas flow rate (v) 296 mL min −1 , leaf chamber molar gas flow rate (U) 400 μmols−1, ambient pressure (P) 97.95 kPa and PAR (Qleaf) at leaf surface maximum up to 779 μmol m−2 s−1. Measurements of net CO2 assimilation rate (A), transpiration rate (E), ambient carbon dioxide and sub-stomatal CO2 concentration (Ci) were made on the youngest fully emerged leaf (usually third leaf from top) of each plant. The second experiment was conducted in the same way. However, the duration of oil stress is 8 weeks. Before the harvest, various physiological and biochemical attributes of maize plants measured are given below. Measurement of water potential, osmotic potential and turgor potential The third leaf (fully expanded, youngest) from the top of each plant was excised at 7.00 a.m., and the leaf water potential measurements were made with a Scholander-type pressure chamber (Chas W. Cook and Sons, Birmingham, UK). A part of the lamina of the same leaf (used in measuring leaf water potential) was frozen in 2-cm3 polypropylene microfuge tubes

in an ultra-low temperature freezer at −18 °C for 2 days. Then, the frozen leaf samples were thawed and the sap was extracted by pressing the material with a glass rod. The sap was used directly for osmotic potential determination in a vapor pressure osmometer (Wescor 5500). Leaf osmotic potential values were corrected for the dilution of the symplastic sap by apoplastic water which occurs when the sap is extracted. Apoplastic water was considered 10 % following Wilson et al. (1980). Leaf turgor potential was calculated as the difference of water potential and osmotic potential, i.e. Turgor Potential ¼ Water Potential – Osmotic Potential

Measurement of total soluble proteins, total free amino acids and nitrogen Total soluble proteins were determined as described by Bradford (1976). Fresh leaf material from healthy and diseased plants (0.2 g) was homogenized in 4 mL of sodium phosphate buffer solution (pH = 7.0) and centrifuged. The extract was used for the estimation of soluble proteins and free amino acids. For estimation of total soluble proteins, 0.1 mL of each sample extracts was reacted with 5 mL of Bradford’s reagent and the optical densities read at 620 nm using a spectrophotometer (Hitachi U-2000). For estimation of total free amino acids, 1 mL of each sample as extracted for soluble protein determinations was treated with 1 mL of 10 % pyridine and 1 mL of 2 % ninhydrin solution. The optical densities of the solutions were read at 570 nm using a spectrophotometer (Hitachi U-2000). For the analysis of N, a fully expanded youngest leaf from each plant was sampled. One hundred milligrams of ground dry leaf and root samples were digested in 2 mL of sulphuric-peroxide digestion mixture until a clear and almost colorless solution was obtained. After digestion, the volume of the sample was made 100 mL with distilled deionized water. Nitrogen contents were measured by titration method following (Allen et al. 1986). At the end of the experiment, total hydrocarbon contents of the soil samples were determined employing infrared spectroscopy as described earlier (Yousaf et al. 2010). Moist soil (5 g) was dehumidified with Na2SO4 and then extracted with 30 mL of 1,1,2-trichloro-trifluoro-ethane (C2Cl3F3). The soil was allowed to settle, and 3 mL of supernatant was further purified with aluminium oxide columns. The filtered extract was measured with FT-IR spectroscopy.

Statistical analysis The data obtained from the two experiments were subjected to analysis of variance using COSTAT computer package (Cohort Software, Berkeley, California). The mean values


were compared with least significance difference (LSD) test following Snecdecor and Cochran (1989).

Results A significant increase in hydrocarbon in soil was observed with an increase in crude oil concentration in soil (Table 1). In addition, total bacterial colony count decreased significantly with an increase in crude oil concentration in soil (Table 1). A significant reduction in the fresh and dry biomass of shoots and roots was observed in plants of Zea mays grown in the soils contaminated with crude oil (Fig 1). Lengths of shoots and roots also exhibited a significant decrease with the addition of crude oil (Fig 2). Root diameter was also affected by crude oil contamination. The root diameter of maize plants significantly increased in plants growing in 2.5 % oilcontaminated soils while it is similar to that in control plants at 5 % contamination level (Fig 3). Total photosynthetic pigments in the leaves of maize plants decreased consistently with consistent increase in oil concentration in the growth medium (Fig 4a). Exposure of maize plants to crude oil caused a significant decline in Fv/Fm, quantum yield of PSII photochemistry and quantum yield of electron transport, and density of active reaction centre was observed, indicating the possible occurrence of damage in PSII centres (Fig 4b; Table 2). Performance index, activities of PSII and PSI, efficiency of light reaction and efficiency of biochemical reaction all are greatly reduced in maize due to soil contamination with crude oil (Table 2; Fig 5a, b, e). Efficiency to transfer energy to reaction centre, non-photochemical reduction of QA and stability of OEC (ΔVOJ) were also reduced due to increasing oil concentration in soil as is obvious from the semi-quantitative analysis of OJIP curves (Fig 5b; Supplementary Fig. S1). Relative variable fluorescence between I and P points (indicator of electron flow from plastocyanin to PSI, ferredoxin (fd) and ferredoxin-dependent NADP reductase) as VOI >1 increased in maize plants due to oil-contaminated soil, which indicates a decrease in rate of electron flow from the intersystem to PSI (Fig 5c). However, the I-P phase assessed as VIP by plotting the transient normalized data over a time interval of 30–200 μs exhibited a similar hyperbolic pattern with no increase in fluorescence amplitude Table 1 Hydrocarbon analysis and colony counting in soil contaminated with varying concentration of crude oil Crude oil (% age)

Hydrocarbon Total bacterial count (CFU/g soil)

0 (Control)

2.5

0.0 6.4 × 108

0.28 %

5.0 0.66 % 7

1.2 × 10

5.6 × 106

as in VOI >1, but fluorescence kinetics or rate of reduction of PSI end electron acceptors (fd, ferredoxin-dependent NADP reductase) significantly lowered in maize plants due to crude oil contamination in soil as indicated by increased Km points (Fig 5d). From quenching analysis, it is clear that recovery of quantum yield in the dark is delayed in plants grown in oilcontaminated soil than in plants grown in control conditions (Fig 6). Moreover, at the highest crude oil concentration, partial recovery of quantum yield occurred indicating damages to PSII. Similarly, a lower increase in non-photochemical quenching (NPQ) was observed in non-stressed plants of maize during the induction phase, while in relaxation phase a considerable delay was observed in stressed plants (Fig 6). Moreover, net CO2 assimilation rate declined significantly (P < 0.001) with increase in crude oil contents of the soils. Although least assimilation rates were recorded for plants grown in 5 % oil pollution, the reducing effect of oil pollution on net assimilation rate was the same at both oil pollution levels (Fig 7). Transpiration rate of plants grown in oilcontaminated soils was also reduced, particularly at 5 % oil contamination level (Fig 7). Water use efficiency calculated as A/E reduced in maize plants due to the addition of crude oil in soil indicating more water transpired per molecule of CO2 fixation. Higher relative rate of transpiration in maize plants grown in oil-contaminated soil could be due to changes in stomatal conductance. Although sub-stomatal CO2 or internal CO2 (Ci) slightly increased (data not shown) in maize plants grown in oil-contaminated soil, the ratio of Ci/Can reduced significantly in maize plants grown at 2.5 and 5 % oil concentrations (Fig 7). A positive linear correlation was observed between net CO2 assimilation rate and ratio of Ci/Can, photosynthetic pigments and with Fv/Fm. Moreover, there was no relationship between net CO2 assimilation rate and Ci. The results recorded for the second experiment exhibited a similar reducing trend for the fresh weights, dry weights and lengths of shoots and roots of maize plants, grown in crude oil-treated soils for 8 weeks (data not shown). Leaf water potential significantly decreased (negative term) in maize plants due to crude oil stress (Fig 8). However, leaf osmotic potential did not change at 2.5 % oil pollution but it slightly increased (negative term) at 5 % crude oil pollution (Fig 8). Negative effects of crude oil contamination were also prominent on leaf turgor potential of maize plants with an increase in oil contamination percentage (Fig 8). Total soluble proteins in the leaves of maize plants remained unaffected due to the increasing concentration of oil in soil (Fig 9), whereas total free amino acids decreased significantly due to crude oil pollution. Nitrogen in the leaves and roots of maize plants also decreased with the increase in crude oil pollution. A positive relationship was found between leaf N and total free amino acids, photosynthetic pigments and net CO2 assimilation rate. Similarly, a slight relationship was found between total free amino acids and leaf osmotic

Fig. 1 Fresh and dry weights of shoots and roots of maize plants grown for 6 weeks in soil contaminated with varying concentrations of crude oil (v/w)

3

Shoot dry weight. (g/plant)


a

2 1.5 1

b c

0.5

a Control

b

b

2.5

5

Root d.wt (g/plant)

1

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0.3 0.25 0.2 0.15

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Root f.wt (g/plant)

Shoot f. wt (g/plant)

2.5

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a Control

b

b 2.5

5

Crude oil percentage in soil (v/w basis)

potential and leaf turgor potential indicating that the extent of accumulation of total free amino acids played a role in maintaining leaf turgor potential. This may be attributed to the fact that decreased nitrogen uptake primarily affects root nitrogen, whereby shoot nitrogen and overall nitrogen deficiency prevail with passage of time.

Discussion Contamination of soil and water with petroleum hydrocarbons is posing serious threads to the environment and the health of the ecosystem (Zhang et al. 2010, Kaczyńska et al. 2015). Crude oil pollution caused a reduction in growth of maize 30

Shoot length (cm)

25

a

20 15

plants. These results match earlier reports in which reduced shoot development in plants growing in soil contaminated with petroleum and/or its by-products has been documented (Adam and Duncan 2002, Chupakhina and Maslennikov 2004, Merkl et al. 2004, Tomar and Jajoo 2013, Tomar et al. 2015). Similarly, lengths of shoots and roots of maize plants were shorter in petroleum-contaminated soil. The observed inhibition of plant height in maize plants grown in soils treated with oil corresponds with the report of Asuquo et al. (2002) on okra and fluted pumpkin. The reduced growth of maize plants due to oil contaminated to soil may be attributable to reduction in leaf growth and/or delay in cell expansion as has been observed earlier (Agbogidi et al. 2007a). However, toxic effects of soil contaminated with crude oil on growth of maize plants were less at 2.5 % as compared to higher levels (5 %). The degree of toxicity of the soil contaminated with petroleum on shoot development depends on type of species, the concentration of oil and the time of exposure (Dorn and Salanitro 2000, Pezeshki et al. 2000, Adam and Duncan 2002). For example, Baek et al. (2004) observed that Zea mays was more sensitive to soil contamination by crude oil than was

b 1.6

c

10

5 c 10 b 15 20

Root diameter (mm)

0 0 Root length (cm)

b

1.4

5

1.2

a

1

a

0.8 0.6 0.4 0.2

a control

2.5

5


Fig. 2 Lengths of shoots and roots of maize plants grown for 6 weeks in soil contaminated with varying concentrations of crude oil (v/w)

0

Control

2.5

5


Fig. 3 Diameter of roots of maize plants grown for 6 weeks in soil contaminated with varying concentrations of crude oil (v/w)


Chlorophyll (SPAD Units)

45 40

a

a

35 30 b

25 20

c

15 10 5 0 0.90 0.80

b

a

0.70

Fv/Fm

0.60

b

0.50 0.40

c

0.30 0.20 0.10 0.00

Control

2.5

5


Fig. 4 Chlorophyll contents and efficiency of PSII in the leaves of maize plants grown for 6 weeks in soil contaminated with varying concentrations of crude oil (v/w)

Table 2 Quantum efficiencies and specific energy fluxes derived from JIP test of maize grown in soil contaminated with varying concentrations of crude oil

Phaseolus nipponensis. Moreover, while assessing the comparative effect of crude oil on growth of grasses and legumes, Merkl et al. (2004) suggested that grassy species seem to be more sensitive to petroleum-contaminated soil than do leguminous plants. Contaminating substances such as hydrocarbons generally inhibit plant growth. The primary inhibiting factors are considered to be toxicity of low molecular weight compounds and hydrophobic properties that limit the ability of plants to absorb water and nutrients (Kirk et al. 2005). In the present study, total hydrocarbon in soil increased and total microbial count reduced to a great extent due to soil contamination with crude oil (Table 1). Reduction in microbial count in soil due to oil contamination may have been due to the toxic impact of hydrocarbons. This microbial community may include bacteria which colonize the endosphere of plants without exhibiting pathogenicity to maize plants which may either degrade organic contaminants after plant uptake or improve plant stress tolerance (Yousaf et al. 2010). Crude oil contamination caused reduction in root biomass and root length. Reduction in root growth due to soil contamination with crude oil has previously been found in a number of studies (Reilley et al. 1996, Davies and Bacon 2003, Merkl et al. 2004, 2005b). However, an interesting finding was the increase in root diameter at 2.5 % crude oil contamination, whereas it decreased at 5 % crude oil contamination. This increase in root diameter due to crude oil contamination may probably be due to higher crude oil concentration in soil which Crude oil (% age)

Quantum efficiencies

0 (Control)

2.5

5.0

ΦPo Maximum quantum yield of primary photochemistry Ψo Efficiency/probability with which a PSII trapped electron is transferred from QA to QB ΦEo The quantum yield of electron transport ΦDo The quantum yield of heat dissipation Specific energy fluxes ABS/RC Absorbed photon flux per PSII reaction centre, antenna size of an active PSII TRo/RC Maximum trapped exciton flux per PSII ETo/RC Electron transport flux from QA to QB per PSII reaction centre DIo/RC Heat dissipation per reaction centre Performance indices PIABS Performance index Density of reaction centre = (RC/ABS) Density of active reaction centre per cross section of leaf at minimal fluorescence = (RC/Cso) Density of active reaction centre per cross section of leaf at maximal fluorescence = (RC/Csm) Efficiency of light reaction = ΦPo / (1 − ΦPo) Efficiency of biochemical reaction = Ψo / (1 − Ψo)

0.805a 0.553a

0.777ab 0.498b

0.761b 0.482b

0.445a 0.194a

0.387b 0.222b

0.369c 0.238b

2.388a

2.619b

2.633b

1.924a 1.064a 0.464a

2.035b 1.014a 0.583b

1.992b 0.956b 0.641c

2.156a 0.419a 2873a

1.380b 0.384b 2689b

1.254c 0.385b 2476c

14799a

12248b

10636c

4.145a 1.239a

3.521b 1.003b

3.270c 0.949c

Environ Sci Pollut Res 2.5 % Crude oil 5% Crude oil

0.085

0.03

2.5 % Crude oil 5% Crude oil

0.065 0.02

0.025

Δ VOJ

Δ VOP

0.045

0.01

a

0.005 -0.015 1

100 10000 Time (μS)

-0.035 1.25

1000000

0

500

1.15 Control 2.5 % Crude oil 5% Crude oil

1.1 1.05

0

100000 Time (μS)

0.8 0.6

Control 2.5 % Crude oil 5% Crude oil

0.4 0.2 Km

0

200000

DIo/RC 40 ETo/RC 30 TRo/RC 20 10 ABS/RC 0 -10 Pi_Abs -20 -30 Phi_Pav -40 -50 Phi_Do

Fo

100000 Time (μS)

200000

2.5 % Crude oil 5% Crude oil Control

Fj Fi Fm Fv Vj Vi Fm/Fo

Phi_Eo

Fv/Fo

Psi_o

Fv/Fm

Phi_Po

Mo N Ss

increased the soil compaction and mechanical impedance to plant roots which in turn resulted in root thickening (Davies and Bacon 2003, Merkl et al. 2005b, Cubera et al. 2009). However, at higher concentration of crude oil contamination, a reduction in root diameter was observed. Reduction in root diameter has previously been found for roots suffering from water stress and nutrient deficiency (Sharp et al. 1988, Reilley et al. 1996, Hinsinger et al. 2009). The results for root growth together with shoot growth are likely to indicate petroleum contamination which may cause water and nutrient deficiency, inducing changes in root growth and root diameter to reduce blockage of nutrient entry to improve the exploration of the soil volume at low expense of carbohydrates. It could be a result of combined effect of cell maceration resulting from hydrocarbon penetration in cell walls and loss of turgor of cells of plants growing in soils with crude oil (Fitter et al. 2002, Ebere et al. 2011). Growth modifications in shoots and roots were observed in maize plants grown in petroleum-contaminated soil, and it is

2000

1

0

1

1500

d

1.2

c

1000 Time (μS)

-0.01

1.2

e

b

0

VIP= (Ft-FI)/(FM-FI)

VOI= (Ft-FO)/(FI-FO)

Fig. 5 Difference kinetics of chlorophyll a transients between O and P (a), O and J points (b), OI (c) and IP phases (d), and radar plot from JIP test in the leaves of maize plants grown for 3 weeks in soil contaminated with varying concentrations of crude oil (v/w). Fo, minimal fluorescence; Fj, fluorescence at J step; Fi, fluorescence at I step; Fm, maximal fluorescence; Fv, variable fluorescence; Vj and Vi, relative variable fluorescence at J and I steps; Fm/Fo, Fv/Fo and Fv/ Fm, ratios of minimal and maximal fluorescence; Mo, rate of accumulation of reduced reaction centre; Sm, pool size of electron carriers; ΦPo, Ψo, ΦEo and ΦDo, quantum efficiencies; PIABS, performance index; ABS/RC, TRo/RC, ETo/RC and DIo/RC, specific energy

Sm

Area Fix Area

most likely accompanied by modifications in plant physiological and biochemical processes such as plant water status, mineral nutrient status, photosynthetic capacity, etc. (Baker 1970, Njoku et al. 2009). Of all these, photosynthetic capacity is central to growth of plants. Photosynthetic capacity of plants is mainly determined by the amount of photosynthetic pigments, amount of light absorbed and quenched by photosynthetic apparatus and rate of CO2 assimilation. From the results of the present studies, a substantial reduction in total chlorophyll content of maize plants was observed due to crude oil contamination. This decrease in total chlorophyll content is in agreement with the findings of previous studies in which it was observed that crude oil contamination caused chlorosis in leaves or reduced photosynthetic pigments (Amakiri and Onofeghara 1983, Fitter et al. 2002, Adenipekun et al. 2008). In the present study, a positive correlation was found between shoot N and photosynthetic pigments. Moreover, leaf water potential and leaf turgor potential were greatly reduced in petroleum-stressed plants of maize, and this reduction is


0.9

Fig. 6 Quantum yield and nonphotochemical quenching from NPQ induction and relaxation analysis in the leaves of maize plants grown for 3 weeks in soil contaminated with varying concentrations of crude oil (v/w)

0.8 0.7

QY

0.6 0.5

Control

0.4

2.50%

0.3

5%

0.2 0.1 0 0

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200

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600

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NPQ

2 Control 1.5

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1 0.5 0 0

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a

7

b

6

300

b

5 4 3 2 1

Transpiration rate ( mol m-2 s-1)

Net CO2 assimilation rate ( mol m-2s-1)

9

3.5

600

b bc

2 1.5 1 0.5 0 9

a

1

b

b

0.8 0.6 0.4

8

a

7

b

6

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5 4 3 2

0.2 0

500

a

2.5

WUE (A/E) ( molCO2/mmolH2O)

1.2

400

3

0

Ci/Ca ratio

Fig. 7 Gas exchange characteristics of maize plants grown for 6 weeks in soil contaminated with varying concentrations of crude oil (v/w)

100

1 0 Control

2.5

5

Control


2.5

5

Environ Sci Pollut Res 1.2 Water potential (-MPa)

1 0.8

b

b a

0.6 0.4 0.2

Osmotic potential (MPa)

0 1.2 1.15

a

a

1.1 b

1.05 1

Turgor potential (Mpa)

0.95 0.39 0.34

a

0.29 0.24 0.19 0.14

b

0.09 0.04

c

-0.01

Control 2.5 5 -0.06 Crude oil percentage in soil (v/w basis)

Fig. 8 Leaf water potential, osmotic potential and turgor potential of maize plants grown for 6 weeks in soil contaminated with varying concentrations of crude oil (v/w)

positively correlated with chlorophyll contents. These results suggested that this decrease in chlorophyll may have been due to degradation of chlorophyll by crude oil contamination which induced water stress, or reduced biosynthesis of chlorophyll contents by crude oil contamination which induced nutrient deficiency such as N and Mg, or the consequence of biodegradation of essential metabolites due to prevailed toxic effects of crude oil, or the combination of these effects (Ibemesim 2010). Generally, plants suffering from abiotic stresses show reduced growth which is mostly attributed to reduced photosynthetic rate. However, reduction in photosynthetic rate is either due to stomatal factors or metabolic factors. In the present study, decrease in net CO2 assimilation rate and transpiration rate was accompanied with decrease in internal CO2 and with higher ambient CO2 (Ci/Can ratio) indicating that stomatal closure was less responsible for inhibiting photosynthesis (Iwegbue et al. 2007) and growth of the plants (Gill et al. 1992, Okonwu et al. 2010). Moreover, increase in Ci in maize plants due to crude oil contamination pointing that CO2 is sufficiently available for its fixation and reduction in photosynthesis at the highest level of crude oil contamination might

have been due to factors other than stomatal limitation such as reduced amount and activity of rubisco, amount of photosynthetic pigments and energy capture efficiency of PSII. Chlorophyll fluorescence analysis has proven to be a sensitive method for the detection and quantification of stress-induced changes in PSII, and both light- and dark-adapted measurements can be used to determine whether photodamage has occurred in leaves or not (Maxwell and Johnson 2000). Crude oil contamination caused a decline in Fv/Fm of maize plants suggesting that damages to the PSII reaction centre along with non-stomatal limitations are responsible for the inhibition of photosynthesis. A number of evidences from different kinetics of stressed and non-stressed plants at J, I and P phases (Fig 6) suggested that crude oil contamination damages PSII at both donor and acceptor ends, and such damages were more prominent at higher oil concentration. For example, more positive L-bands in oil-stressed plants indicated that crude oil contamination decreased the energetic connectivity among PSII units. In other words, crude oil contamination reduced the utilization efficiency of excitation energy trapped by chlorophyll molecules in antennae of PSII (Chen et al. 2015) as has been observed in salt stress (Athar et al. 2015, Khalid et al. 2015). Similarly, positive O–J or K band values in oil-stressed plants of maize suggested that crude oil contamination reduced the oxygen evolving complex or increased the antennae size of PSII. These results can be related with those of Guha et al. (2013) who found that drought stress induces positive L and K bands in mulberry and confirmed through protein expression analysis that such changes in fluorescence is associated with PSII damages. Similarly, Khalid et al. (2015) found that salt stress caused a positive increase in L and K bands in canola which is associated with PSII photoinhibition. Likewise, while assessing detrimental effects of environmental pollutant polycyclic aromatic hydrocarbon on the photosynthetic efficiency of crop plants, different researchers reported that changes in fluorescence level between O and J phases are associated with an increase in the number of inactive reaction centre or lower energy transfer from LHCII to PSII reaction centre such as in wheat (Tomar and Jajoo 2013) and soybean (Tomar et al. 2015). A similar impact of other environmental pollutants such as heavy metals has also been observed in wheat (Mathur et al. 2016). From the results of the present study, the number of active PSII centres (RC/Cso, RC/Csm) decreased in maize plants due to increase in crude oil concentration in soil (Table 2). These results suggested that crude oil converted the active PSII centres into heat sinks and became inactive which cannot reduce QA, resulting in lower rates of photochemistry and damage to the photosynthetic apparatus. From J–I and I–P phases of O–J–I–P, transients are associated with electron flow from reduced PQ (PQH2) to PSI end electron acceptors (Tsimilli-Michael and Strasser 2013). Crude oil contamination decreased energy absorption (фPo),

Environ Sci Pollut Res 12.00

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energy entrapment (ψo) and electron transport (фEo) in maize plants, and this decreasing effect was greater at 5 % crude oil contamination in soil. This might have been due to decrease in over-excitation of PSII as a protective effect or re-oxidation capacity of QA– by intersystem electron carriers (Cyt-b6f, PSI) as has been earlier observed in soybean (Tomar et al. 2015). From the results of the normalised I–P part of the transients measured as VOI >1, it is clear that crude oil phytotoxicity caused an increase in this part of transient (Fig 5c) and suggested that pool of PSI end electron acceptors (ferredoxin and NADPH) increased. However, normalization between FI and Fm, the kinetic rate of electron flow to PSI end electron acceptors, is greatly reduced. From all these results, it can be concluded that electron transport rate is greatly reduced due to damages to intersystem electron carriers including PSI end electron acceptors (ferredoxin and NADPH) (TsimilliMichael and Strasser 2013). Another evidence comes from the results of performance indices which represent structural and functional statuses of both PSII and PSI, and PIABS reduced in maize plants grown in oil-contaminated soil (Table 2). Components of PIABS (density of active PSII centres, light reaction efficiency and efficiency of biochemical dark redox reaction, NADPH production and utilization) are also reduced with an increase in concentration of crude oil in the soil (Table 2). These results can be explained in view of

some earlier analogous studies with environmental phytotoxicants (Tomar and Jajoo 2013, Tomar et al. 2015, Mathur et al. 2016) as the electron generated by PSII photochemistry in maize plants grown in crude oil-contaminated soil transported to PSI, but all electrons are not utilized in CO2 fixation and electrons are diverted to other electron acceptors from the donor side of PSI. This is the probable justification for reduction in photosynthetic rate of maize plants subjected to crude oil toxicity (Fig 7). Quenching analysis (Fig 6) suggested that crude oil stress reduced PSII efficiency by decreasing lower induction of NPQ which might have been due to its association with repairable damages to PSII (loss of D1 protein) as indicated by a delay in relaxation in the dark phase. Accumulation of N in leaves and roots of maize plants greatly reduced due to crude oil contamination. The reduction in nutrient uptake may have been due to the presence of hydrocarbons in crude oil that changes soil physical properties leading to reduced mineral release from soil and thus nutrient availability (Agbogidi et al. 2007b). Moreover, crude oil in the soil caused an increase in soil organic matter, improving soil binding properties while reducing water drainage capacity and root penetration in the soil. These results are in accordance with other studies which state a net decrease in total nitrogen of plants with the crude oil application (Ogbo et al. 2007, Onyeneke and Aguebor-Ogie 2007). Reduced uptake, transport


and accumulation of N from roots to leaves caused the adverse effects on N metabolism such as biosynthesis of amino acids and proteins in different crops such as cotton and okra biotic and abiotic stresses (Zafar et al. 2010, Yasmeen et al. 2013, Zafar and Athar 2013, Razaq et al. 2014). In the present study, total free amino acids in leaves of maize plants decreased due to oil stress and have a positive relationship with leaf N. These results suggested that lower accumulation of N in maize plants due to oil stress reduces the biosynthesis of amino acids. However, in the present study, total soluble proteins remained unchanged in leaves of maize plants due to oil stress. The relationship between total free amino acids and total soluble proteins can be explained due to the fact that stress-inducible proteins are biosynthesized at the expense of degraded proteins by stress (Ashraf and Rehman H-u- 1999, Athar et al. 2011).

Conclusions Present studies concluded that soil contaminated with crude oil had an effect on plant performance by altering in aeration and biological properties of the soil, thereby affecting uptake of water and mineral nutrients in maize plants. It finally results in inhibition of physiological processes like photosynthesis and transpiration and metabolic processes like chlorophyll biosynthesis, thus affecting overall growth of plants. Crude oil toxicity reduced the efficiency of light reaction by increasing in inactive reaction centres, rate of electron transport and biochemical reactions of photosynthesis by diversion of electrons at PSI acceptor end and reducing stomatal conductance. However, at lower toxic level, maize plants tried to adjust by the downregulation of electron transport to protect PSII. Since nutrient deficiency is one of the main factors reducing growth of maize plants grown on oil-contaminated soil, further experimentation is suggested to elucidate whether the adverse effect of crude oil on maize performance can be ameliorated by nutrient supplementation. Compliance with ethical standards Authors’ contribution Habib-ur-Rehman Athar and Zafar Ullah Zafar designed the experiment; Sarah Ambreen, Muhammad Javed, Mehwish Hina and Sumaira Rasul conducted the experiment; Muhammad Afzal did the soil analysis and microbial count; Habib-ur-Rehman Athar, Chukwuma C Ogbaga, Hamid Manzoor and Zafar Ullah Zafar did the physiological analysis; and Habib-ur-Rehman Athar, Fahad-Al-Qurainy and Muhammad Ashraf wrote and edited the manuscript. All authors agreed to submit this MS to Environmental Science and Pollution Research.

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