Botany

Conocarpus lancifolius Engl. () photosynthetic apparatus suffers damage in heavy metal contaminated soil

Journal: Botany

Manuscript ID cjb-2018-0047.R5

Manuscript Type: Article

Date Submitted by the 04-Dec-2018 Author:

Complete List of Authors: Redha, Amina; Kuwait University, Biological Sciences Al-Hasan, Redha; Kuwait University, Biological Sciences Jose, Jacquilion; Kuwait University, Biological Sciences Saju, Divya; Kuwait University, Biological Sciences Afzal, Mohammad;Draft Kuwait University, Biological Sciences; Retired,

Conocarpus lancifolius, chlorophyll fluorescence, electron transport rate, Keyword: photosynthetic rate, photosystem II

Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? :

https://mc06.manuscriptcentral.com/botany-pubs Page 1 of 32 Botany

Revised Manuscript ID: cjb-2018-0047.R5

Conocarpus lancifolius Engl. (Combretaceae) photosynthetic

apparatus suffers damage in heavy metal contaminated soil

Amina Redha, Redah Al-Hasan, Jacquilion Jose, Divya Saju, Mohammad Afzal⌘

Department of Biological Studies, Faculty of Science, Kuwait University, Kuwait

Amina Redha: [email protected]

Redha Al-Hasan: [email protected]

Jacquilion Jose: [email protected]

Divya Saju: [email protected]

Running title: Conocarpus lancifolius responses to heavy metal stress

Corresponding author present address:

M. Afzal⌘,

2200-Traemoor Village Way,

Nashville, TN 37209,

USA.

Tel. +1 (352) 681 7347

email: [email protected]

1 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 2 of 32

Abstract

Conocarpus lancifolius Engl. (Combretaceae), a heat tolerant , could be used for phytoremediation of polluted soil. We aimed to analyze the physiological changes in C. lancifolius exposed to single and mixed heavy metals (HMs), cadmium (Cd2+), nickel

(Ni2+), and lead (Pb2+). Under controlled growth conditions, some groups of were exposed to a single HM at concentrations of 25 or 50 µM and other groups were exposed to 25 µM HM mixtures, for 30 days. Photosynthetic parameters such as electron transport rate, photosynthetic rate, chlorophyll fluorescence, chlorophyll content index, and photosynthetic pigments were measured. The chloroplast morphology was studied by transmission electron microscopy (TEM). In plants exposed to HM 25 µM, the photosynthetic parameters were unaffected,Draft whereas at HM 50 µM, all parameters significantly decreased until 20 days of exposure followed by an increase until 30 days, indicating a slow adaptability of plants under HM stress. Compared to single HMs, mixed

HMs were more toxic at the same concentration. All parameters indicated damage to the photosynthetic apparatus due to stress from mixed HMs at 25 µM and single HMs at 50

µM. TEM analyses showed a dispersion of grana in the chloroplast of the affected C. lancifolius plants.

Keywords: Conocarpus lancifolius, chlorophyll fluorescence; electron transport rate; photosynthetic rate; photosystem II

2 https://mc06.manuscriptcentral.com/botany-pubs Page 3 of 32 Botany

Introduction

Although heavy metals (HMs) are naturally present in the soil, their abundance increases

due to industrial activities and oil exploration, causing stress for the proximate animals

and plants. Because of the distinct toxicity of HMs, their elevated levels in ecosystems

affect the diversity, abundance, and composition of microbial communities, including the

micro-flora activities (Baath 1989; Friedlova 2010). Thus, vegetation growing in polluted

soil displays growth retardation due to changes in physiological and biochemical

parameters (Chibuike and Obiora 2014). The double-membrane-enclosed chloroplast is a

sensitive organelle that is most affected by environmental pollutants that enter the cytosol

and damage the chloroplast membrane and the photosynthetic apparatus. Factors such as

the moisture content of the leaves, theDraft water holding capacity of the soil, and the soil pH,

play major roles in determining the levels of HM uptake by the roots, thus affecting the

plant’s photosynthetic organelles.

Conocarpus lancifolius Engl. (Combretaceae) was introduced into Kuwait from

Djibouti where it has thrived in the arid environment and is now widespread. C.

lancifolius is also present throughout the Middle East, Southeast Asia, and East Asia,

where it is extensively planted along the main boulevards. The plant is seen as an

attractive shrub and is therefore purposely planted all over Kuwait and throughout its

geographic range. Its conservation, therefore, is important for the people in the region.

C. lancifolius is a significant plant with potential applications for health and

ecosystem remediation, and therefore, its conservation is of great importance. For

instance, Al-Taweel et al. (2016) reported a new ellagic acid derivative from C.

lancifolius that shows anti-inflammatory, cytotoxic, and peroxisome proliferator activated

3 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 4 of 32

receptor (PPAR) agonistic activities. Furthermore, Al-Musayeib et al. (2012) demonstrated antiplasmodial, antileishmanial, and antitrypanosomal activities in C. lancifolius, indicating their potential for human health benefits. In terms of ecosystem remediation, Yateem et al. (2008) reported the rhizo-remediation of hydrocarbon- contaminated soil by C. lancifolius and their results suggested that 85.7% of measurable total petroleum hydrocarbon could be degraded in rhizosphere soils associated with C. lancifolius.

We initiated a systematic study of C. lancifolius and found that this plant was not only resistant to drought and high salinity, but it could also withstand high desert temperatures and light intensities (Redha et al. 2011, 2012a,b,c, 2013). Currently, there are concerns about the long-term Draft survival of C. lancifolius in Kuwait environment, polluted with heavy crude oil. During the Gulf war of 1991, 6-8 million barrels of crude oil were spilled into the marine environment of Kuwait waters, in addition to oil well fires that released massive amounts of soot and toxic gasses into the environment, causing one of the largest man-made environmental disasters in human history. This disaster caused deleterious effects on the local ecosystem due to the persistent toxicity of several HMs and aromatic hydrocarbons, components of crude oil. Along the Wafra road

(an oil producing area) the oil-contaminated soil contains Ni at 120.96 mg/kg soil, Pb at

2.9 mg/kg, and Cd at 0.027 mg/kg soil (Kostecki and Behbehani 1995). This can be detrimental to the survival of C. lancifolius, causing public concern.

Heavy metal toxicity, caused by free radical generation (Dietz et al. 1999), induces increased synthesis of metal chelating proteins such as phytochelatin, offering a detoxification mechanism to plants for removal of the toxic metal ions and/or a metal

4 https://mc06.manuscriptcentral.com/botany-pubs Page 5 of 32 Botany

exchange (Tiecher et al. 2016). Metal stress, however, can damage the plasma membrane

(Janicka-Russak et al. 2008) through the formation of free radical and/or binding of metal

ions to sulfhydryl groups of membrane proteins and phospholipids, which may cause

cellular damage (Devi and Prasad 1999). In plants, this leads to a decline in membrane

integrity, undermining cellular transport, energy metabolism, growth, and development

(Surowy and Boyer 1991; Binzel 1995; Oufattole et al. 2000). Modification of the plasma

membrane, as induced by toxic metals also affects organelle membranes, and results in

alterations to the photosynthetic apparatus and consequently energy metabolism (Santos

et al. 2014).

Heavy metals are highly mobile and are easily transported to the above-ground parts

of the plant, principally in plastidsDraft causing ultrastructural modification of the leaf

chloroplast. It is known that chloroplasts are the primary targets of metal stress (Solymosi

and Bertrand 2012). A damaged chloroplast can impair photosynthesis, transpiration and

electron transport rate, photochemical quenching, and photosystem II (PSII) quantum

yield. Changes in the thylakoid structures, may cause a reduction in the chlorophyll and

carotenoid content, triggering a diminished photosynthetic pigment biosynthesis, and

chloroplasts functions (Aggarwal et al. 2011; Cabrita et al. 2016). Changes in the

accumulation of polyamines, leading to free, thylakoid- and chromatin-bound polyamines

in Pb-exposed barley leaves, have been shown to reduce photosynthetic pigments and

photosynthetic parameters in barley leaves (Legocka et al. 2015).

As HMs present in crude oil have cumulative negative effects, we carried out this

study to assess the effects of HMs on the cultivation of C. lancifolius in soil contaminated

with HMs at different concentrations. From the data gathered through this study, we

5 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 6 of 32

report changes in thylakoid ultrastructure of the chloroplasts leading to alterations in

photosynthetic activity of C. lancifolius that may be detrimental to its survival in the

Kuwaiti environment.

Materials and methods

Plant materials and growth conditions

One hundred and twenty, one-month-old plants with a single shoot with

approximately ten leaves, cultivated in plastic pots using peat moss, were obtained from

the Public Authority for Agriculture and Fish Resources (PAAFR), a government

organization in Kuwait. The pots were transported to Kuwait University, Faculty of

Science greenhouse, where they were acclimatized for two weeks.

Seventy plants of uniform heightDraft with 13-15 leaves were randomly selected and

transferred to plastic pots (19.0 cm diameter and 16.3 cm depth) containing local sandy

soil and peat moss (3:1 v/v, 1.5 kg). Sand was mixed with peat moss to increase the soil

water potential, and C. lancifolius grows well in sandy soil. Plants were placed in a walk- in growth chamber (Ayios Dimitrios, Athens, Greece) maintained at 25°C, 45-55% relative humidity, and 150 mM quanta m-2s-1 white light intensity, for 30 days. The plants were arranged on a single shelf to ensure equal light intensity for all plants and irrigated with distilled water (50 mL) on alternate days.

The plants were divided into 11 groups (A-K) with six plants in each group. The remaining four plants were left untreated for comparative studies. Plants in Groups B-K were irrigated with 50 mL solution containing different concentrations of HM nitrate salts. Ni, Cd, and Pb nitrate salts, all prevalent in Kuwaiti crude oil were selected for plant exposure. Two stock solutions at concentrations of 25 µM and 50 µM were

6 https://mc06.manuscriptcentral.com/botany-pubs Page 7 of 32 Botany

prepared for each HM salt, and used for irrigation of plants. Treatments were as follows:

25 µM Cd (group B), 50 µM Cd (group C), 25 µM Ni (group D), 50 µM Ni (group E), 25

µM Pb (group F), 50 µM Pb (group G), 25 µM Cd+Pb (group H), 25 µM Cd+Ni (group

I), 25 µM Ni+Pb (group J), and 25 µM Cd+Ni+Pb (group K), for 30 days. The plants in

group-A were treated as control and were irrigated with distilled water (50 mL) on

alternate days for 30 days. In fresh mature leaves, physiological parameters were

measured every fifth day.

Determination of chlorophyll content index, chlorophyll fluorescence and

photosynthesis rate

The chlorophyll content index (CCI) of fresh mature leaves was determined using a

portable chlorophyll content meterDraft (CCM-200, Opti-Science, Tyngsboro, MA, USA).

Active fluorescence parameters, minimal (Fo), maximal (Fm), and variable fluorescence

(Fv = Fm − Fo) were determined to assess photosynthetic activity. Chlorophyll

fluorescence measurements were taken to determine the maximum quantum efficiency of

PSII, Fv/Fm = (FM – F0)/FM, the relative electron transport rate (ETR), which is the

effective photochemical yield of PSII, ϕP = ΔF/FM’ = (FM’ – F)/FM’, and the

photosynthetic photon flux density (PPFD), (Geel et al. 1997; Kromkamp et al. 1998).

The measurements were taken at midday on three dark-adapted (45 min) mature leaves

from each plant to assess the status or efficiency of PSII with a chlorophyll fluorometer

OS5-FL (Opti-Sciences, Hudson, NH, USA).

The rate of photosynthesis was measured using the LCi photosynthesis meter CI 340,

(ADC BioScientific Ltd., Hoddesdon, UK). The treatments were replicated four times

7 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 8 of 32

and measurements (±SEM, standard error mean) were taken from the mature leaves of

each plant.

Measurement of photosynthetic pigments content

For the measurement of chlorophyll and carotenoid contents, mature fresh leaves,

collected from the control and experimental plants, were taken on the first and then every

fifth day, during the experiment. A 0.5 g sample of leaf material was homogenized in

80% acetone (10 mL), photosynthetic pigments were measured according to Vimala and

Poonghuzali (2015) and Zhou et al. (2018), and the absorbance of the syringe filtered-

pigment extract was measured spectrophotometrically, and the pigment concentration

was calculated as follows:

Ca (μg/mL) = Draft12.7 × OD663 – 2.69 × OD645

Cb (μg/mL) = 22.9 × OD645 – 4.68 × OD663

Ca+b (μg/mL) = 20.2 × OD645 + 8.02 × OD663

Pheophytin = OD412/ε Pheophytin412

β-Carotene = OD480/ε β-Carotene480

Lycopene = OD503/ε Lycopene503

Ck = 4.7 × OD440 + 0.27 × Ca+b

Ck = OD480 + (0.114 × OD663) – (0.638 × OD645)

Ca = chlorophyll-a concentration, Cb = chlorophyll-b concentration, Ca+b = total chlorophyll concentration, Ck = carotenoid concentration, OD = absorbance at the specified wavelength.

8 https://mc06.manuscriptcentral.com/botany-pubs Page 9 of 32 Botany

Transmission electron microscopy

Plant leaf tissue sections (2×4 mm) were prepared for transmission electron microscopy

(TEM)-[JEM-2100 electron microscope (JEOL)] according to Hulskamp et al. (2010).

Statistical analyses

All experiments were run in triplicate for a period of 30 days. The triplicate samples were

collected on day 1, 5, 15, 20, 25, and 30. For comparison of data, Kruskal-Wallis

nonparametric, one-way ANOVA, and two-tailed student’s t-test were performed. Graph

Pad Prism software (Version 5.0) was used for statistical analyses. For all graphics and

results, p < 0.05 was considered statistically significant (n = 3), and for all graphs, the

level of significance only applies to the final data point taken on 30th. Day. Draft Results

The initial fluorescence Fo and the maximum fluorescence Fm in C. lancifolius were

determined after exposure of the plants to HMs at different concentrations, and the results

are shown in Figure 1a-f. In experimental plants exposed to single HMs Cd, Pb, or Ni at

25 μM (Fo and Fm; Figure 1a and 1d) did not significantly differ (p > 0.05) from the

control plants, however, at 50 μM, single HM exposure, the Fo (Figure 1b) and Fm

(Figure 1e) decreased significantly (p < 0.05), compared with the control plants. Different

mixtures of HMs resulted in significant deleterious effects (p < 0.05) of different

intensities on Fo and Fm (Figure 1c and 1f) as compared to single HMs. A highly

significant decrease (p < 0.04) in Fm was observed when a mixture of all three HMs Ni,

Cd, Pb, was present in the soil at 25 µM (Figure 1f). Plants did not survive after exposure

to mixed HMs at the 50 µM concentration. In plants exposed to 50 μM of the single

HMs, a highly significant % decrease (p < 0.04) in Fo and Fm was observed for Pb and

9 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 10 of 32

Cd while exposure to Ni at 50 µM, showed a significant % decrease (p < 0.05) in Fo and

Fm (Figure 2a, 2c). The plants exposed to 25 μM single HMs, however, had a non- significant % decrease (p > 0.05) in Fo and Fm values (Figure 2a, 2c), compared with the control plants. After 30 days of exposure, mixed HMs at 25 µM had more toxic effect on

Fo and Fm. Thus, exposure to a mixture of HMs at 25 μM (Figure 2b, 2d), showed a highly significant % difference (p < 0.04) in Fo, and Fm (Figure 2b, 2d).

The above results were confirmed by the fluorescence efficiency Fv/Fm ratio, shown in Figure 3a-c. The influence of single HMs at 25 μM, on the quantum yield of photosynthesis, was non-significant (Figure 3a). The quantum yield of photosynthesis, however, underwent a significant (p < 0.05) decrease after exposure to single HMs at 50

μM (Figure 3b) and mixed HMs atDraft 25 μM (Figure 3c). The HM Ni showed a highly significant decrease in Fv/Fm (Figure 3b). Plants exposed to a mixture of all three HMs showed a highly significant decrease (p < 0.03) in Fv/Fm (Figure 3c). The highly significant decrease in Fv/Fm (Figure 3c) indicates that mixed HMs at lower concentration (25 μM), exert higher toxicity than single HMs at higher concentration (50

μM).

After quantifying the fluorescence efficiency, alterations in ETR in PSII and chlorophyll content index (CCI) were evaluated. The plants exposed to 25 µM single HM solutions showed a non-significant drop (p > 0.05) in ETR, compared with the control plants, over a period of 10-30 days (Figure 5a) but with the 50 µM single HMs, and mixed HMs at 25 μM, a highly significant drop in ETR (p < 0.03) was observed (Figure

5b, 5c). Exposure of the plants to 25 µM single metal solution had a non-significant (p >

0.05) influence on CCI (Figure 5d) however, the CCI significantly dropped (p < 0.05)

10 https://mc06.manuscriptcentral.com/botany-pubs Page 11 of 32 Botany

when plants were exposed to single HMs 50 µM (Figure 5e). An exposure to mixed HMs

at 25 µM solution showed a highly significant (p < 0.03) drop in CCI (Figure 5f). This

again suggests that mixed HMs at a lower concentration (25 µM) were more toxic,

compared with single HMs at higher concentration (50 µM).

Measurements of photosynthetic pigments by spectrophotometry (Table 1)

demonstrated that chlorophyll-a and -b significantly decreased (p < 0.05) in plants

exposed to 50 µM of all single HMs, but the decline was highly significant (p < 0.03)

when the plants were exposed to mixed HMs at 25 µM.

Chlorophyll-a and -b decreased by 16.5% and 23.3%, respectively, in plants exposed

to a mixture of Ni/Cd/Pb (25 µM), on the 30th day, compared to those of the control

plants. A corresponding increase Draft in pheophytin was observed. The lycopene and

carotenoid contents suffered the highest decrease in plants exposed to HMs stress.

Therefore, β-carotene, lycopene and total carotenoids were significantly lower (p < 0.05)

in response to either single HM or mixed HMs. Lycopene declined by 41.2% after

exposure of the plants to mixed HMs (Cd/Ni/Pb), at 25 µM. Ni at 50 µM provoked a

large decline in chlorophyll-b (18.8% decline) compared to Pb (15.4%; Table 1). With

mixed HMs Ni/Cd/Pb at 25 µM, a 23.3% decline in chlorophyll-b was observed

compared with those of the control plants on the 30th. day (Table 1).

Discussion

In plants and algae, chlorophyll fluorescence is widely used as a parameter to measure

photosynthetic performance and it is used as a marker for HM stress affecting the

photosynthetic apparatus (Cabrita et al. 2016). Depending on its concentration, the

fluorescence spectrum of chlorophyll-a (Chl-a) mainly includes two maxima, one in the

11 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 12 of 32

red (685-690 nm) and the other in the far-red, NIR (710-740 nm) reflecting a spectral

measurement range (Buschmann 2007). The fluorescence is influenced by Chl-a-protein

complex in photosystem II (PSII) (Govindjee 1995). Some of the fluorescence is also

caused by photosystem I (PSI), with an emission at 730 nm, which is highest at low

temperature, and is largely reduced at room temperature. PSI fluorescence is useful since

it can be measured at physiological temperature using fluorescence techniques. The total

fluorescence refers to the fluorescence from PSII-associated chlorophyll-a and PSI

associated chlorophyll-a and is used to measure Fv/Fm and ETR. PSII operating

efficiency, electron flux and CO2 assimilation are correlated and over a range of light

intensities, a linear relationship between PSII and PSI is observed. Thus, a change in

fluorescence indicates damage to theDraft photosynthetic apparatus.

Fluorescence is used to monitor changes in photosystem II (PSII, P680), electron flux

from PSII to the primary quinone acceptor (QA) and the quantum yield of CO2 assimilation øCO2 (Baker 2008). A linear relationship between CO2 fixation and PSII quantum yield measured by chlorophyll fluorescence has been reported (Krause and Weis

1991). Photochemical quenching is measured from the minimal level of fluorescence Fo and the maximal fluorescence in the dark (Fm). A non-significant (p > 0.05) increase in

Fo (Figure 1a) and Fm (Figure 1d) compared to the control, indicated minimal damage to the photosynthetic apparatus in the plants after exposure to single HMs at 25 μM. A significant decrease (p < 0.05) in Fo and Fm values at higher concentration (50 μM) of single HMs (Figure 1b, 1e), however, indicated severe damage to the photosynthetic apparatus of the plants. The adverse impact of the mixed HMs at 25 μM, on Fo and Fm was highly significant (p < 0.03) (Figure 1c and 1f). These results were supported by the

12 https://mc06.manuscriptcentral.com/botany-pubs Page 13 of 32 Botany

% differences in Fo and Fm for single HMs at 25 and 50 μM and also the mixed HMs at

25 μM, (Figure 2a-2d). Cojocaru et al. (2016) and Wang et al. (2016), have reported a

higher toxicity of mixed HMs and increased effects on the plant’s morphometric

parameters. Our results revealed that the plants exposed to mixed HMs displayed a highly

significant decline in Fo and Fm, suggesting a severe impediment to the synthesis of

chlorophyll-a and consequently to the photosynthetic apparatus and/or to the extracellular

structure of the leaf. Under these conditions, reduced transpiration with a reduced ϕp/ϕO2

ratio, due to thickening of the leaf structure, could reduce the Fo and the photosynthetic

rate in plants (Masojidek et al. 2001).

It is known that the fluorescence ratio F690/F736, (red/far-red), is an effective

bioindicator of HM stress (SchuergerDraft et al. 2003; Buschmann 2007). Once HMs are

inside the cell, they can trigger numerous alterations in the cell physiology, for example,

membrane permeability, enzyme inhibition, photosynthetic electron transport, and other

cellular functions (Kupper et al. 2002). The red to far-red fluorescence ratio decreases

with an increase in chlorophyll-a concentration in a curvilinear fashion and it is

negatively correlated with the chlorophyll content of the leaf (Buschmann 2007). The

maximum quenching efficiency of PSII is measured from the Fv/Fm ratio, which

indicates the maximum efficiency of light absorbed by PSII, used for the reduction of QA.

A non-significant decrease in fluorescence ratio (Fv/Fm), was observed for single HM at

25 μM concentration, compared with the control plants (Figure 3a). This indicated that

the plants were not adversely affected by single HMs at this concentration (Figure 3a).

The experimental plants exposed to 50 µM HMs, however, showed a significant decrease

in Fv/Fm, ratio compared with a minor increase in that of the control plants (Figure 3b).

13 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 14 of 32

This implied that the plants were sensitive to this concentration of HMs, resulting in a significant drop in PSII activity. It was also observed that after 30 days of exposure under the 50 μM HM treatment, a highly significant decrease in Fv/Fm, was observed in the plants exposed to Ni (Figure 3b) These data indicated that C. lancifolius was sensitive to

HM stress at 50 µM, and that Ni was the most toxic HM for this plant. Ni-stress is known to alter cell wall structure, nucleus, mitochondria, and stomata structure, and causes chloroplast abnormalities (Mosa et al. 2016). Figure 3c shows the mixed HM stress at 25

µM, with a highly significant drop in Fv/Fm confirming that the mixed HMs at a lower concentration exert higher toxicity than the single HMs at a higher concentration

(Cojocaru et al. 2016; Wang et al. 2016).

Photosynthetic efficiency on theDraft 20th day of exposure to 25 µM mixed HMs (Cd/Pb,

Cd/Ni, Ni/Pb, and Cd/Pb/Ni) and 50 µM single HMs is shown in Figure 3c and 3b, respectively. The Fv/Fm ratio significantly dropped due to the plant’s exposure to mixed

HMs at 25 µM, indicating a highly significant inhibition of photosystem II impeding the photosynthetic electron transport ETRmax (μM e-1 m-2 s-1) with a disruption of the chloroplast envelop and the thylakoid membranes (Figure 4h, i) (Park and Jung 2017).

C. lancifolius is a C3 plant and the plants in this category have high rates of photorespiration, lower CO2 assimilation rates and respond to all types of biotic and abiotic stresses (Noctor et al. 2002). The primary effect of stress is on the photosynthetic apparatus with a reduction of the CO2 uptake, resulting in a loss of biomass. Under HM stress, the maximal photochemical efficiency (Fv/Fm) of PSII is adversely affected with increasing concentration of the HM (Zhou et al. 2018) and the protein-photosynthetic complexes involved in energy transport may also be affected. At low photon flux

14 https://mc06.manuscriptcentral.com/botany-pubs Page 15 of 32 Botany

densities <1000 µM m-2 s-1, the thylakoid membrane is not affected. At higher photon

flux densities > 1000 µM m-2 s-1, however, the plant is under severe stress, resulting in

lower rates of open-chain electron transport and photophosphorylation (Haubner et al.

2014).

Our data suggest that the conocarpus plants experienced a maximum toxicity on the

20th day of the HM exposure (Figure 3b, 3c), and thereafter, the Fv/Fm ratio continues to

return to near normal values. This signifies that the plants switch to an

adaptation/resistance mode after the 20th day of exposure to single or mixed HM stress

(Figure 3b, 3c). The data was supported by examining the leaf tissue structure under a

Transmission Electron Microscope (TEM). The micrographs showed the presence of

irregular thylakoids at 20 days of Draft exposure; whereas at 30 days, the thylakoids were

relatively better symmetrically arranged in the chloroplast (Figure 4a-4d).

Individual and combined trace metals including Cd, Pb and Ni, have been recently

reported in the diatom model species Phaeodactylum tricornutum (Cabrita et al. 2016).

This study showed that the trace metals associated with the treated cells were at a

significantly higher concentration than in the control cells, resulting in a significantly

reduced fluorescence and growth. The authors further concluded that the F685/F735 ratio

could be used to monitor chlorophyll content, and chlorophyll fluorescence and as a

biomarker for plant stress.

A decline in the photosynthetic efficiency was confirmed from the observed

alterations in electron transfer rates (ETR) in PS II and CCI, in plants, after exposure to

HMs. The plants exposed to 25 µM of single HMs (Figure 5a) showed a non-significant

drop in ETR but at 50 µM single HMs, or at 25 µM mixed HMs, the ETR decline was

15 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 16 of 32

highly significant (Figure 5b, 5c). The decline in ETR may be associated with CO2 absorption and a related decline in the activity of the photosynthetic enzyme Rubisco (Fu et al. 2016). Costa et al. (2016) reported a decline in ETR drop in the brown seaweed

Sargassum cymosum when exposed to a combination of HMs (Cu+Pb). In our study, a decline in the CCI (Figure 5d-5f) was comparable to the decline in ETR values which confirmed that mixed HMs at lower concentrations (25 μM) were as toxic as single HMs at higher concentration (50 µM).

A significant decrease in photosynthetic and auxiliary pigments (Table 1) after exposure to 50 µM single HMs was observed. As discussed above, mixed HMs proved to be more toxic for the plant’s photosynthetic apparatus with a significant decline in the pigment concentration. The ultrastructuralDraft damage to the chloroplast is shown in Figure 4

Our results shown in Table 1, demonstrate an abnormal amount of pheophytin as compared to chlorophyll in experimental plants exposed to 50 µM single or mixed HMs.

This may be due to HM induced oxidative damage to the chloroplast, degrading chlorophyll to pheophytin. In addition, chlorophyll concentration may influence the functioning of the photosynthetic apparatus thus affecting the metabolism of the whole plant (Solymosi and Bertrand 2012). The carotenoids and lycopene with their extended conjugation system offer resistance to overproduction of reactive oxygen species generated in plants in response to HMs. A significant decrease in the auxiliary pigments in plants under HM stress indicates a complete breakdown of the plant antioxidant defenses as well as a reduction in energy metabolism causing severe damage to the chloroplast (Schaller and Diez 1991) as shown in Figure 4.

16 https://mc06.manuscriptcentral.com/botany-pubs Page 17 of 32 Botany

Transmission electron microscopy (TEM) of the experimental plant tissues showed

extensive damage to the plastids (Figure 4). Distorted and distended chloroplasts,

deformed thylakoid, retreating lamellae nucleus, and cell wall due to metalloproteins

exchange, were apparent abnormalities in the stressed plants (Figure 4b and 4c). The cell

wall and chloroplast envelop ruptured when plants were exposed to 50 μM single or 25

μM mixed HMs (Figure 4b and 4c).

Photosynthetic pigment analyses of the chlorophyll, implied that Ni was the most

toxic element for the photosynthetic apparatus in C. lancifolius. Our results suggest that

mixed HMs present in heavy crude oil, may have a clear detrimental effect on the PSII in

C. lancifolius, threatening its survival in the oil-polluted soil of Kuwait.

Our results further indicate thatDraft C. lancifolius, due to tolerance/adaptation and

detoxification mechanisms, can adapt to lower concentrations of HMs, but if the

concentration of the HM increases or if the HMs are mixed together in the soil, it may

suppress plant growth to various degrees. These results are consistent with reports in the

literature (Markina and Aizdaicher 2006; Horvatic and Persic 2007; Cabrita et al. 2014).

Our results show that C. lancifolius experiences a concentration dependent toxicity in the

order of Ni > Pb > Cd, and that toxicity increases with the mixed HMs. This study shows

that the distribution of HMs in the polluted soil affect the growth and sustainability of C.

lancifolius in Kuwait and East Asian countries.

Conclusions

Our results suggest that heavy metal contaminated soil adversely affects photosynthetic

ability causing physiological damage to Conocarpus lancifolius and threatening its

17 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 18 of 32

survival in the Kuwaiti environment. The plant’s photosynthetic efficiency is dependent upon the concentration of metals in the soil and also whether those metals are present in isolation or are mixed. For crude oil contaminated soils, plant growth is adversely affected since the crude oil contains a combination of heavy metals. The mechanism of action of these HMs may be through the generation of free radicals, which are responsible for disrupting the envelop and plasma membrane of the chloroplasts, consequently disrupting the thylakoid membranes and negatively affecting photosynthetic efficiency.

Conflicts of interest: The authors declare no conflict of interest.

Acknowledgements Draft

The authors thankfully acknowledge research grant # KFAS P214-42SL-05 to carry out this work. Instrumental facilities from the Science Analytical Facility Lab. # GS-0210,

GS-0103, and SRUL-01/13 are also thankfully acknowledged. The authors would like to thank Editage for English language editing.

References

Aggarwal, A., Sharma, I., Tripathi, B.N., and Sharma, V. 2011. Metal toxicity and

photosynthesis: Overviews on recent progress & future perspective, First Edition. IK

International Publishing house, New Delhi, pp. 229-236.

Al-Musayeib, N.M., Mothana, R.A., Al-Massarani, S., Matheeussen, A., Cos, P., and

Maes, L. 2012. Study of the in vitro antiplasmodial, antileishmanial and

antitrypanosomal activities of medicinal plants from Saudi Arabia. Molecules, 17(10):

11379-11390.

18 https://mc06.manuscriptcentral.com/botany-pubs Page 19 of 32 Botany

Al-Taweel, A.M., Perveen, S., Fawzy, G.A., Mehmood, R., Khan, A., and Khan, S.I.

2016. New ellagic acid derivative from the fruits of heat-tolerant plant Conocarpus

lancifolius Engl. and their anti-inflammatory, cytotoxic, PPAR agonistic activities.

Pak. J. Pharm. Sci. 29(5): 1833-1837.

Baath, E. 1989. Effect of heavy metal in soil on microbial processes and populations (A

Review). Water Air Soil Pollut. 47(3-4): 335-379.

Baker, N.R. 2008. Chlorophyll fluorescence: A probe of photosynthesis in vivo. Annu.

Rev. Plant Biol. 59: 89-113.

Binzel, S.W. 1995. NaCl induces accumulation of tonoplast and plasma membrane H+-

ATPase message in tomato. Physiol. Plant. 94: 722-728.

Buschmann, C. 2007. VariabilityDraft and application of the chlorophyll fluorescence

emission ratio red/far-red of leaves. Photosyn. Res. 92: 261-271.

Cabrita, M.T., Raimundo, J., Pereira, P., and Vale, C. 2014. Immobilized Phaeodactylum

tricornutum as bioindicator of trace element availability in water column during

dredging. Environ. Sci. Pollut. Res. 21(5): 3572-3581.

Cabrita, M.T., Gameiro, C., Utkin, A.B., Duarte, B., Cacador, I., and Cartaxana, P. 2016.

Photosynthetic pigment laser-induced fluorescence indicators for the detection of

changes associated with trace element stress in the diatom model species

Phaeodactylum tricornutum. Environ. Monit. Assess. 188, 285. doi: 10.1007/s10661-

016-5293-4.

Chibuike, G.U., and Obiora, S.C. 2014. Heavy metal polluted soils: Effect on plants and

bioremediation methods. Appl. Environ. Soil Sci. vol. 2014, Article ID 752708, 12

pages. doi: 10.1155/2014/752708.

19 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 20 of 32

Cojocaru, P., Gusiatin, P., and Cretescu, I. 2016. Phytoextraction of Cd and Zn as single

or mixed pollutants from soil by rape (Brassica napus). Environ. Sci. Pollut. Res. Int.

23(11), 10693-10701.

Costa, G.B., de Felix, M.R., Simioni, C., Ramlov, F., Oliveira, E.R., Pereira, D.T.,

Maraschin, M., Chow, F., Horta, P.A., Lalau, C.M., da Costa, C.H., Matias, W.G.,

Bouzom, Z.L., and Schmidt, E.C. 2016. Effects of copper and lead exposure on the

ecophysiology of the brown seaweed Sargassum cymosum. Protoplasma, 253: 111-

125.

Devi, S.R., and Prasad, M.N.V. 1999. Membrane lipid alterations in heavy metal exposed

plants. In Heavy metal stress in plants. From molecules to ecosystems. Edited by

M.N.V. Prasad and J. Hagemeyer.Draft Springer. pp. 99-116.

Dietz, K.J., Kramer, U., and Baier, M. 1999. Free radicals and reactive oxygen species as

mediators of heavy metal toxicity. In Heavy metal stress in plants. From molecules to

Ecosystems. Edited by M.N.V. Prasad and J. Hagemeyer. Springer. pp. 73-97.

Friedlova, M. 2010. The influence of heavy metals on soil biological and chemical

properties. Soil Water Res. 5(1): 21-27.

Fu, J., Gates, R.N., Xu, Y., and Hu, T. 2016. Diffusion limitations and metabolic factors

associated with inhibition and recovery of photosynthesis following cold stress in

Elymus nutans Griseb. J. Phytochem. Photobiol. B 163: 30-39.

Geel, C., Versluis, W., and Snel, J.F.H. 1997. Estimation of oxygen evolution by marine

phytoplankton from measurement of the efficiency of photosystem II electron

flow. Photosyn. Res. 51: 61–70.

20 https://mc06.manuscriptcentral.com/botany-pubs Page 21 of 32 Botany

Govindjee, N. 1995. Sixty three years since Kautsky: chlorophyll fluorescence. Aust. J.

Plant Physiol. 22: 131-160.

Haubner, N., Sylvander, P., Vuori, K., and Snoeijs, P. 2014. Abiotic stress modifies the

synthesis of alpha-tocopherol and beta-carotene in phytoplankton species. J. Phycol.

50(4): 753-759.

Horvatic, J., and Persic, V. 2007. The effect of Ni2+, Co2+, Zn2+, Cd2+, Hg2+ on the

growth rate of marine diatom Phaeodactylum tricornutum Bohlin:microplate growth

inhibition test. Bull. Environ. Contam. Toxicol. 79: 494-498.

Hulskamp, M., Schwab, B., Grini, P., and Schwarz, H. 2010. Transmission electron

microscopy of plant tissues, Cold Spring Harbor Protocols. 2010 Jul

1;2010(7):pdb.prot4958. doi:10.1101/pdb.prot4958;Draft Available from http://intl-

cshprotocols.cshlp.org

Janicka-Russak, M., Kabala, K., Burznski, M., and Kobus, G. 2008. Response of plasma

membrane H+-ATPase to heavy metal stress in Cucumis staiva roots. J. Exp. Bot.

59(13): 3721-3728.

Kostecki, P., and Behbehani, M. 1995. Assessment and remediation of oil contaminated

soils. Proceedings of workshop, Kuwait, March 18-27, Arab School of science and

technology, Kuwait, New age International Limited Publisher, New Delhi, India. pp.

70. ISBN:81-224-1204-1.

Krause, G.H., and Weis, E. 1991. Chlorophyll fluorescence andphotosynthesis: The

basics. Annu. Rev. Plant Physiol. Plant Mol. Biol. 42: 313–349.

21 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 22 of 32

Kromkamp, J., Barranguet, C., and Peene, J. 1998. Determination of microphytobenthos

PS II quantum efficiency and photosynthetic activity by means of variable chlorophyll

fluorescence. Mar. Ecol. Prog. Ser. 162: 45–55.

Kupper, H., Setlik, I., Spiller, M., Kupper, F.C., and Prasil, O. 2002. Heavy-metal-

induced inhibition of photosthnthesis:targets of in vivo heavy metal chlorophyll

formation. J. Phycol. 38: 429-441.

Legocka, J., Sobiesczzuk-Nowicka, E., Wojtyla, L., and Samardakiewicz, S. 2015. Lead-

stress induced changes in the content of free, thylakoid- and chromatin-bound

polyamines, photosynthetic parameters and ultrastructural in greening barley leaves. J.

Plant Physiol. 186-187: 15-24.

Markina, Z.V., and Aizaicher, N.A.Draft 2006. Content of photosynthetic pigments, growth

and cell size of microalgae Phaeodactylum tricornutum in the copper-polluted

environment. Russ. J. Plant Physiol. 53(3): 305-309.

Masojidek, J., Grobbelaar, J.U., Pechar, L., and Kobizek, M. 2001. Photosystem II

electron transport rates and oxygen production in natural water blooms of freshwater

cyanobacteria during diel cycle. J. Plankton Res. 23(1): 57-66.

doi:10.1093/plamkt/23.1.57.

Mosa, A., El-Banna, M.F., and Gao, B. 2016. Biochar filters reduce the toxic effects of

nickel on tomato (Lycopersicon esculentum L.) grown in nutrient film technique

hydroponic system. Chemosphere, 149: 254-262.

Noctor G., Veljovic-Jovanovic, S., Driscoll, S., Novitsk Y, L., and Foyer, C.H. 2002.

Drought oxidative load in the leaves of C3 plants: a predominant role of

photorespiration? Ann. Bot. 89(7): 841-850.

22 https://mc06.manuscriptcentral.com/botany-pubs Page 23 of 32 Botany

Oufattole, M., Arango, M., and Boutry, M. 2000. Identification and expression of three

new Nicotiana plumbaginfolia genes which encode isoforms of a plasma membrane

H+-ATPase and one which is induced by mechanical stress. Planta, 210: 715-722.

Park, J.H., and Jung, S. 2017. Perturbations of carotenoids and tetrapyrrole biosynthesis

pathways result in differential alterations in chloroplast function and plastid signaling.

Biochem. Biophys. Res. Commun. 482(4): 672-677.

Redha, A., Al-Mansour, N., Suleman, P., Afzal, M., and Al-Hasan, R. 2011. Leaf Traits

and Histochemistry of Trichomes of Conocarpus lancifolius a Combretaceae in Semi-

arid Conditions. Am. J. Plant Sci. 2(2): 165-174.

Redha, A., Al-Mansour, N., Suleman, P., Al-Hasan, R., and Afzal, M. 2012a. Modulation

of antioxidant defenses in ConocarpusDraft lancifolius under variable abiotic stress.

Biochem. Sys. Ecol. 43: 80-86.

Redha, A., Al-Mansour, N., Suleman, P., Al-Hasan, R., and Afzal, M. 2012b. Drought,

salinity and temperature responses to photosynthesis in Conocarpus lancifolius. J.

Agric. Food Environ. 10 (2): 1320-1325.

Redha, A., Suleman, P., Al-Hasan, R., and Afzal, M. 2012c. Responses of Conocarpus

lancifolius to environmental stress: a case study in the semi-arid land of Kuwait.

Phyton, 81: 181-190.

Redha, A., Patrice, S., Al-Hasan, R., and Afzal, M. 2013. Conocarpus lancifolius

biochemical responses to variable UV-B radiation. Biochem. Syst. Ecol. 48: 157-162.

Santos, D., Durate, B., and Cacador, I. 2014. Unveiling Zn hyperaccumulation in Juncus

acutus:implications on the electronic energy fluxes and on oxidative stress with

23 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 24 of 32

emphasis on non-functional Zn-chlorophylls. J. Photochem. Photobiol. B. 140: 228-

239.

Schaller, A., and Diez, T. 1991. Plant specific aspects of heavy metal uptake and

comparison with quality standards for food and forage crops, In Der Einfluß

von festen Abfällen auf Böden. Edited by P.D. Sauerbeck and S. Lübben. KFA,

Jülich, Germany, pp.92-125.

Schuerger, A.C., Capelle, G.A., Di Denedetto, J.A., Mao, C., Chi, N., Mark, T., Evans,

D., Richard, J.T., Blank, T.A., and Stryjewski, E.C. 2003. Comparison of hyperpectral

imaging and two laser-induced fluorescence instruments for the detection of zinc stress and chlorophyll concentration inDraft bahiagrass (Paspalum notatum Flugge). Remote Sens. Environ. 84: 572-588.

Solymosi, K., and Bertrand, M. 2012. Soil metals, chloroplasts, and secure crop

production: A review. Agron. Sust. Dev. 32(1): 245-272.

Surowy, T.K., and Boyer, S.J. 1991. Low water potential affect expression of genes

encoding vegetative storage proteins and plasma membrane proton ATPase in

soybean. Plant Mol. Biol. 16: 252-262.

Tiecher, T.L., Tiecher, T., Ceretta, C.A., Ferreira, P.A., Nicoloso, F.T., Soriani, H.H.,

Tassinari, A., Paranhos, J.T., De Conti, L., and Brunetto, G. 2016. Physiological and

nutritional status of black oat (Avena strigosa Schreb) grown in soil with interaction of

high doses of copper and zinc. Plant Physiol. Biochem. 106: 253-263.

Vimala, T., and Poonghuzhali, T.V. 2015. Estimation of pigments from seaweeds by

using acetone and DMSO. Int. J. Sci. Res. 4(10): 1850-1854. ISSN, 2319-7064

24 https://mc06.manuscriptcentral.com/botany-pubs Page 25 of 32 Botany

Wang, W., Wu, Y., Akbar, S., Jia, X., He, Z., and Tian, X. 2016. Effect of heavy metals

combined stress on growth and metal accumulation of three Salix species with

different cutting position. Int. J. Phytoremediation, 18(8): 761-767.

Yateem, A., Al-Sharrah, T., and Bin-Haji, A. 2008. Investigation of microbes in the

rhizosphere of selected for the rhizoremediation of hydrocarbon-contaminated

soils. Int. J. Phytoremediation, 10: 311-324.

Zhou, J., Zhang, Z., Zhang, Y., Wei, Y., and Jiang, Z. 2018. Effects of lead stress on the

growth, physiology, and cellular structure of privet seedlings. PLoS One,

13(3):e0191139. doi: 10.1371/journal.pone.0191139. Draft

25 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 26 of 32

Figure legends:

Fig. 1 Measurement of initial (Fo) and maximum (Fm) fluorescence in response to different heavy metal concentrations against exposure time. (a): Fo after plant exposure to single heavy metal at 25 µM (b): Fo after exposure to mixed heavy metals at 50 µM; (c): Fo after plant exposure to mixed heavy metals at 25 µM; (d): Fm after plant exposure to single heavy metals at 25 µM; (e): Fm after plant exposure to single heavy metals at 50 µM; (f): Fm after plant exposure to mixed heavy metals at 25 µM. The level of significance only applies to the final data points. * NS; ** Significant; *** highly significant

Fig. 2 Percentage variation in Fo & Fm after exposure of plants with heavy metal (a): Fo after plant exposure to single heavy metal at 25 and 50 μM; (b): Fo after plant exposure to mixed heavy metals at 25 µM; (c): Fm after plant exposure to single heavy metal at 25 and 50 μM; (d): Fm after plant exposure to mixed metal treatment at 25 µM. * NS; ** Significant; *** highly significant

Fig. 3 a-b (a): Percentage variation in Fo after exposure of plants with single heavy metals at 25 and 50 µM; (b): after exposure of the plants to mixed heavy metals at 25μM. Plant did not survive after exposure to mixed heavy metals at 50 µM. Fig. 3 c-e show quantum yield of photosynthesis, Fv/Fm in plants exposedDraft to individual metals: (c) 25 μM; (d) 50 μM; (e) 25 μM mixed metals. The level of significance only applies to the final data points. * NS; ** Significant; *** highly significant

Fig. 4 (a): control plants: mature chloroplasts with organized thylakoid membranes; (b): 50 μM single and 25 μM mixed metal, detached chloroplast, disrupted cell wall; (c): 50 μM, single and 25 μM mixed metal, swollen chloroplasts, lipid globules and disrupted chloroplast membrane; (d): 25 μM, single metal, chloroplast with thylakoid stacks and lipid globule; (e): 50 μM, single metal, contracted thylakoids and no defined chloroplasts; (f): 25 μM, two mixed metals, large lipid globule with unorganized grana; (g): 25 μM, three mixed metals, agitated grana with lipid globules, chloroplast detached from membrane; (h): 25 μM, three mixed metals, impaired grana and no defined chloroplast membrane, no visible organelles; (i): 25 μM, two mixed metals, impaired grana with no defined chloroplast membrane.

Fig. 5 Measurement of electron transport rate (ETR; 5a-c) and chlorophyll content (5d-f) index (CCI) in response to different concentrations of heavy metal stress and exposure time. The level of significance only applies to the final data points. * NS; ** Significant; *** highly significant

Table 1 Changes in photosynthetic pigments concentration mg/g FW, after 30 days of plant exposure to individual and mixed heavy metals at 25 μM and 50 μM. a = NS; b = significant; c = highly significant.

https://mc06.manuscriptcentral.com/botany-pubs Page 27 of 32 Botany Influence of single Cd, Pb, Ni 25 µM Influence of single Cd, Pb, Ni 50 µM Influence of mixed Cd, Pb, Ni metals 25 µM on initial fluorescence Fo 120 on initial fluorescence Fo on initial fluorence Fo 120 120 Control Ni/Cd Control Pb Control Pb Cd/Pb Ni/Cd/Pb Cd Ni * Cd Ni Ni/Pb 110 110 110 ** ** Fluorescence Fo Fluorescence

Fluorescence Fo Fluorescence 100 Fluorescence Fo Fluorescence 100 100

a b 90 c 90 90 0 10 20 30 0 10 20 30 0 10 20 30 Day DraftDay Day Influence of single Cd, Pb, Ni metals at 25 µM Influence of single Cd, Pb, Ni metals at 50 µM Influence of mixed Cd, Pb, Ni metals at 25 µM on maximum fluorescence Fmax on maximum fluorescence Fmax on maximum fluorescence Fmax 700 700 700 * Control Ni/Cd Control Cd Cd/Pb Ni/Cd/Pb Control Pb

max Pb Ni Ni/Pb max ** max 650 Cd Ni ** 650

650 600 *** Fluorescence F Fluorescence Fluorescence F Fluorescence 600 F Fluorescence 550

600 d e f 550 500 0 10 20 30 0 10 20 30 0 10 20 30 Day Day Day https://mc06.manuscriptcentral.com/botany-pubs Fig. 1 Botany Page 28 of 32 % initial Fluorescence difference F % initial Fluorescence difference Fo o 20 at 25 and 50µM after 30 days for mixed metals 25µM after 30 days 20

o o

10 10 *** *** }

0 0 } * * -10 * ** -10 % difference in F fluorescence % difference in F fluorescence -20 -20

Ni/Pb Ni/Cd Ni-25 Ni-50 Cd/Pb Cd-25Pb-25 Pb-50Cd-50 Control Control Cd/Pb/Ni Drafta b

% Max fluorescence difference Fm % Max fluorescence difference Fm at 25µM mixed metals after 30 days 20 at 25 and 50 M after 30 days µ 20 m

m

10 10 *** ***

0 } 0 } * * -10 * ** -10 % difference in F fluorescence

-20 % difference in F fluorescence -20

Pb-25Cd-25 Ni-25 Pb-50Cd-50 Ni-50 Control Cd/Pb Ni/Pb Ni/Cd c Control Ni/Cd/Pb d

https://mc06.manuscriptcentral.com/botany-pubs Fig. 2 Page 29 of 32 Botany Quantum yield of photosynthesis in Response of Cd, Pb, Ni at 50 µM conc. response to 25 µM Cd, Pb, Ni on quantum yield of photosynthesis 0.90 0.85 0.88 Control Pb Control Pb Cd Ni Cd Ni 0.86 ** 0.80 0.84 } 0.82 * 0.80 0.75 * * * (Yield) Fv/Fm 0.78 0.76 0.70

Photosynthetic Efficiency Fv/Fm Efficiency Photosynthetic 0 10 20 30 Day 0 10 20 30 Day Drafta b

Response of mixed Cd, Pb, Ni metals at 25 µM conc. on quantum yield of photosynthesis 0.9 Cd/Ni Cd/Pb Cd/Pb/Ni Ni/Pb

0.8 ***

0.7 (Yield) Fv/Fm

0 10 20 30 Day c https://mc06.manuscriptcentral.com/botany-pubs Fig. 3 Botany Page 30 of 32

Draft

58x44mm (300 x 300 DPI)

https://mc06.manuscriptcentral.com/botany-pubs Page 31 of 32 Botany 170 Influence of single Cd, Pb, Ni * Influence of single Cd, Pb, Ni Influence of mixed Cd, Pb, Ni 25 µM on ETR 50 µM on ETR 25 µM on ETR -1 -1 160 -1 160 s s s -2 -2 -2 160 Control Pb m m m -1 -1 Cd Ni -1 Control Ni Control Ni/Pb/Cd Cd Pb Ni/Pb Cd/Pb mol e μ mol mol e μ mol mol e μ mol

* * * * * * 140 Ni/Cd 140 ETR ETR 150 ETR

a b c 120 0 10 20 30 0 10 20 30 0 10 20 30 Day DraftDay Day 75 Influence of single Cd, Pb, Ni Influence of single Cd, Pb, Ni Iinfluence of mixed Cd, Pb, Ni 25 µM on CCI 50 µM on CCI 25 µM on CCI

70 70 70 Control Cd/Ni Control Pb Control Pb Cd/Pb Cd/Ni/Pb * Ni/Pb 65 Cd Ni Cd Ni ** * 60 C C I C C C C I C C *

C C I C C 60 60 ** *

55 50 d 50 e f 50 0 10 20 30 0 5 10 15 20 25 30 0 10 20 30 Day Day Day

https://mc06.manuscriptcentral.com/botany-pubs Fig. 5 Botany Page 32 of 32

Table 1 Changes in photosynthetic pigments after exposure of the plants to individual and mixed heavy metals at 25 μM and 50 μM Metals Concentration Chlorophyll a Chlorophyll b β carotene Pheophytin Lycopene Total Carotenoids Metal µM mg/gm Fw mg/gm Fw mg/gm Fw mg/gm Fw mg/gm Fw mg/gm Fw Control Control 53.01 ± 0.0113 44.61 ± 0.0062 38.61± 0.0035 69.82± 0.0054 0.015± 0.0063 0.9866 ± 0.0073

25 51.41 ± 0.0060a 40.42± 0.0071a 35.53± 0.0062a 65.18± 0.0058a 0.013± 0.0053a 0.8966 ± 0.0073b Cd 50 49.42 ± 0.0116b 36.28 ± 0.0050bDraft33.23± 0.0057b 63.25± 0.0062b 0.012 ± 0.006b 0.8033 ± 0.0058b

25 49.64 ± 0.0055a 39.63± 0.0060a 35.02± 0.0057a 64.83± 0.0059a 0.009± 0.0063a 0.8656 ± 0.0056a Pb 50 48.98 ± 0.0063b 36.22± 0.0060b 32.45± 0.0054b 63.01± 0.0057b 0.009± 0.0051b 0.8016 ± 0.0047b

25 48.97 ± 0.0032b 40.15± 0.0052b 33.95± 0.0054b 63.85± 0.0051b 0.010 ± 0.006b 0.7915 ± 0.0045b Ni 50 48.55± 0.0107b 37.75± 0.0063b 32.42± 0.0048b 61.58± 0.0062c 0.010± 0.006b 0.7816 ± 0.0066b

Cd+Pb 25 50.12 ± 0.0044b 35.04 ± 0.0060b 33.75± 0.0132b 59.28± 0.0064c 0.009± 0.006b 0.6981 ± 0.0060c

Ni+Pb 25 48.61 ± 0.0164b 34.62± 0.0048b 32.79± 0.0058b 56.61± 0.0069c 0.009± 0.006b 0.6133 ± 0.0072c

Ni+Cd 25 49.92± 0.0069b 34.81 ± 0.0059b 33.32± 0.0066b 56.73± 0.0057c 0.009 ± 0.006b 0.6757 ± 0.0065c

Ni+Cd+Pb 25 44.22± 0.0113c 34.20± 0.0053c 31.58± 0.0126c 55.83± 0.0049c 0.008 ± 0.005c 0.6008 ± 0.0056c Table 1 Changes in photosynthetic pigments concentration mg/g FW, after 30 days of plant exposure to individual and mixed heavy metals at 25 μM and 50 μM. a = NS; b = significant; c = highly significant.

https://mc06.manuscriptcentral.com/botany-pubs