Journal of Photochemistry and Photobiology B: Biology

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Spectroscopic studies of photosynthetic responses of tomato plants to the interaction of zinc and cadmium toxicity

Jaouhra Cherif

^a,b,^⇑^,1

, Najoua Derbel

^b,1

, Mohamed Nakkach

^b

, Hubertus von Bergmann

^c

, Fatma Jemal

^a

, Zohra Ben Lakhdar

^b

aUnité de Recherche «Nutrition et Métabolisme Azotés et Protéine de Stress», Département de Biologie, Faculté des Sciences de Tunis, Campus Universitaire, 2092 Tunis, Tunisia

bLaboratoire de Spectroscopie Atomique-Moléculaire et Applications (LSAMAs), Département de Physique, Faculté des Sciences de Tunis, Campus Universitaire, 2092 Tunis, Tunisia

cLaser Research Institute, Department of Physics, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa

a r t i c l e i n f o

Article history:

Received 27 October 2011

Received in revised form 25 February 2012 Accepted 5 March 2012

Available online 30 March 2012

Keywords:

Cadmium Chlorophyll content

Chlorophyll ﬂuorescence spectra Fluorescence intensity ratios Solanum lycopersicum Zinc

a b s t r a c t

Thein vivochlorophyll (Chl) fluorescence spectra ofSolanum lycopersicumleaves were recorded in the spectral region 650–800 nm using a spectroscopic method based on ultraviolet light emitting diode induced fluorescence spectroscopy (UV-LED IFS). These spectra have been used to analyze the interactive functions of cadmium (Cd²⁺) and zinc (Zn²⁺) on photosynthetic activities ofS. lycopersicumplants. The fluorescence intensity ratios (F690/F735) of the chlorophyll bands at 685 and 730 nm were calculated by evaluating curve fitted parameters using a Gaussian spectral function, for control as well as treated plants. The fluorescence induction kinetics (Kautsky effect) was also measured on dark adapted intact plant leaves at the chlorophyll bands for determining the variable chlorophyll fluorescence decrease ratio (RFdvalues) and the stress adaptation index (Ap). In addition, metal accumulation in plants, plant growth and photosynthetic pigments content were estimated. It was found that theRFd(690),RFd(730) and Ap values decreased whereas theF690/F735ratio increased in the case of 10lM Cd²⁺treated plants, indicating an impairment of the photosynthetic efficiency. Zn²⁺supplementation, at low concentration (10 and 50lM), in combination with Cd²⁺protect the photochemical functions. However, the high Zn²⁺concentration exacerbated the negative effects of Cd²⁺and showed a severe decrease ofRFd(690),RFd(730) and Ap values compared to Cd²⁺alone. It is seen thatF690/F735ratios are strongly correlated with chlorophyll contents. The results demonstrate the usefulness ofF690/F735, Ap andRFdvalues in determining the potential photosynthetic activity of an intact attached leaf in a non-destructive way.

1. Introduction

In many areas of plant biology, there is an increasing require- ment for rapid and non-destructive screening techniques to identify changes in the metabolism and growth of plants. Chl ﬂuo- rescence spectroscopy is widely used as an indicator of the functional state and damage of the photosynthetic apparatus under environmental stresses. Heavy metals, which naturally occur at low levels in the environment, tend to accumulate to toxic concentrations as a consequence of mining, smelting as well as excessive use of phosphate fertilizers and sewage sludge in agriculture. It is well known that Cd²⁺is one of the most toxic heavy metals without any metabolic signiﬁcance. It can be transferred to the food chain by plant uptake. Studies carried out in different plant species have

revealed that Cd²⁺can interfere with a number of metabolic processes. It diminishes water and nutrient uptake[1]results in visible symptoms of injury in plants such as chlorosis and necrosis of leaves and reduced length and browning of roots. The photosynthetic apparatus is one of the target sites of Cd²⁺action in plants.

In fact, Cd²⁺can directly or indirectly interact with different components of the photosynthetic apparatus and can decrease electron transport efﬁciency, inhibit chlorophyll biosynthesis and reduce the photosynthetic carbon assimilation[2]. Cd²⁺ions directly affect the structure of the thylakoid membrane through peroxidation and oxidative stress and lead to disorganization and changes in the lipid composition of the thylakoid membranes[3]. On the other hand, zinc is an important component of a large number of enzymes, it is associated with the carbohydrate metabolism, proteins synthesis, and gene expression and regulation[4]. Moreover, zinc plays critical roles in the defence system of cells against oxidative stress, and thus represents an excellent protective agent against the oxidation of several vital cell components such as membrane lipid and chlorophyll[5], but is toxic in higher concentrations[6,7].

http://dx.doi.org/10.1016/j.jphotobiol.2012.03.002

⇑ Corresponding author at: Unité de Recherche «Nutrition et Métabolisme Azotés et Protéine de Stress», Département de Biologie, Faculté des Sciences de Tunis, Campus Universitaire, 2092 Tunis, Tunisia.

E-mail address:cherif_jaouhra@yahoo.fr(J. Cherif).

1 Both these authors contributed equally to this work.

Contents lists available atSciVerse ScienceDirect

Journal of Photochemistry and Photobiology B: Biology

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / j p h o t o b i o l

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Cadmium is often associated with Zn²⁺as a contaminant, up to 5% in the processed Zn²⁺-ores of Zn²⁺mines and smelters[8]. Since both Cd²⁺and Zn²⁺belong to group II transition elements with sim- ilar electronic configuration and valency, they have many physical and chemical similarities. Biologically, however, these two elements have different properties. The fact that Cd²⁺is a toxic heavy metal and Zn²⁺ is an essential element makes this association interesting as it raises the possibilities that the toxic effect of Cd²⁺may be preventable by Zn²⁺. Interactions between Cd²⁺and Zn²⁺and their transfer in soil–crop systems under field conditions, in solution culture experiments have been reported[9]. However, the influence of Zn²⁺–Cd²⁺interaction on the photosynthetic processes has not been investigated.

The study of thein vivo Chl ﬂuorescence of leaves of green plants provided basic information on the functioning of the photosynthetic apparatus and on the capacity and performance of the photosynthesis. Under optimum physiological conditions the major part of light absorbed by the photosynthetic pigments, chlorophylls and carotenoids is used for photosynthetic quantum conversion. This phenomenon consists of a complex reduction–

oxidation chain along which electrons are transported through the photosynthetic apparatus from water to NADP via photosystem II (PSII) and photosystem I (PSI)[10]. Some amount of light absorption is de-excited by heat dissipation and a small proportion via Chl fluorescence, whose spectrum exhibits two maxima in the red (near 685–690 nm), and far-red region (near 730–740 nm) [11]. However, under many stress conditions plants use less radi- ant energy for photosynthesis and have evolved numerous mecha- nisms that safely dissipate excess light energy to avoid photoinhibition and photooxidation with a concomitant increase in red and far-red Chl fluorescence[12]. The inverse relationship betweenin vivoChl fluorescence and photosynthetic activity can be used to study the potential photosynthetic activity of leaves and to detect stress effects on the green plants[13].

The red Chl fluorescence centered near 690 nm, when emitted deeper inside the leaf tissue, is partially reabsorbed by the absorption bands of the in vivo chlorophyll, whereas, the far-red Chl fluorescence centered near 730–740 nm is little affected by re-absorption [11], whereby the ratio between the red and the far-red Chl fluorescence maxima (near 690 and 730–740 nm, respectively), usually expressed asF690/F730orF690/F735, decreases with increasing chlorophyll content [14]. Lichtenthaler [15], showed that the ratioF690/F730is equivalent to the ratioF690/F735. The latter is determined by measuring two broad fluorescence bands and the 730 nm band (±18 nm) including the 735 nm fluorescence peak. The ratio F690/F730(=F690/F735) increases not only due to a lower chlorophyll content, but also when the process of photosynthesis declines due to a fast response to stress which does not yet change the chlorophyll content[11,14,16].

The measurement ofF690andF730during the induction kinetics with the resultingRFdvalues should provide quick information on the active photosynthetic performance of the leaf, and leads to the study of the consecutive loss of photosynthetic activity of leaves under stress conditions. The comparison ofF690andF730provides more information than the measurement at just one wavelength region alone[17]. Moreover, from theRFd(690) andRFd(730) values one can determine the stress adaptation index (Ap)[17]. This index is a measurement of how a leaf can reorganize the structure of the photosynthetic apparatus for best adaptation to the applied stress conditions.

Chl ﬂuorescence parameters are not limited to the detection of changes in photosynthetic performance during the induction of stress and damage, but allow also to check a possible regeneration of the photosynthetic activity of plants when the stressors factors are alleviated. No information is available on the role of nutrients against toxic metal on the photochemical processes, especially in

Solanum lycopersicumplant. Hence, the purpose of this study was to investigate the inﬂuence of Cd²⁺–Zn²⁺interaction on photosynthetic response in this plant using ultraviolet light emitting diode induced ﬂuorescence spectroscopy (UV-LED IFS).

2. Material and methods

2.1. Plant material and growth conditions

Seeds of tomato (S. lycopersicum cv. Chebli) were sterilized in 10% hydrogen peroxide for 20 min. The seeds were then thor- oughly washed with distilled water and germinated on moistened ﬁlter paper at 25°C in the dark. The uniform seedlings were then transferred to continuously aerated nutrient solutions containing KH2PO4, 0.50 mM ; Ca(NO3)2, 1.25 mM ; KNO3, 1.00 mM; MgSO4, 0.50 mM; Fe–K–EDTA, 50

l

M; MnSO44H2O, 5

l

M; ZnSO47H2O, 1

l

M; CuSO45H2O, 1

l

M; H3BO3, 30

l

M; (NH4)6 MO7O244H2O, 1

l

M. The seedlings were cultivated in pots of 5 l (six seedlings per pot). After an initial growth period of 10 days, 10

l

M CdCl2

was added to the medium. Cd²⁺concentration of 10

l

M was se- lected for the analysis based on its intermediate level of growth inhibition. Zn²⁺supplementations (10, 50, 100, and 150

l

M) were given to the plant as ZnCl₂ along with the Cd²⁺ concentration.

Plants were grown and treated in a growth chamber (26°C/70%

relative humidity during the day, 20°C/90% during the night). A photoperiod of 16 h was used with a light irradiance of 150

l

mol m²s²at the canopy level, using ﬂuorescent lamps (OS- RAM HQL 250 W). The experimental conditions that we used are those which promote the optimal growth of tomato plants under regular agricultural conditions. However, in a growth chamber the experimental conditions are controlled, which allows repeti- tion under identical conditions. After 7 days of treatments, Chl ﬂuorescence was measured by the UV-LED IFS technique. Leaves from six plants were harvested and the fresh weight was immedi- ately determined, then leaves were quickly frozen in liquid nitro- gen and stored at 80°C until analysis of photosynthetic pigments. To determine biomass production, other plants were harvested and divided into roots and shoots. Fresh weights were measured and the dry matter determined after drying for 48 h at 60°C.

2.2. Determination of metal contents

Cadmium and zinc contents in various plant tissues was ana- lyzed by digestion of dried samples with an acid mixture (HNO3/ HClO4, 4/1,V/V). Metal concentrations were determined by atomic absorption spectrophotometry (AAnalyst 300, ﬂame spectrometer, Perkin–Elmer, USA).

2.3. Estimation of photosynthetic pigments contents

Photosynthetic pigments were extracted in 80% acetone for 24 h in darkness, at 4°C. The resulting suspension was centrifuged for 5 min at 3000 g, then the absorbance of the supernatant was measured at 460, 645 and 663 nm with an UV/Vis spectrometer (Lamb- da 25, Perkin–Elmer, USA). The pigment concentrations were calculated by equations allowing a simultaneous determination of chlorophylla(Chla) andb(Chlb) and carotenoids (Car) in the same supernatant, as in the work of Lichtenthaler[18].

2.4. Measurement of chlorophyll ﬂuorescence and induction kinetics

Spectroscopic measurements of Chl fluorescence of stressed and non-stressed tomato plants were performed by using a portable fiber-probe fluorescence detection system comprising a continu-

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ous-wave high-power ultraviolet (UV) light emitting diode (LED), which has a peak wavelength of 365 nm with 8 nm spectral bandwidth [19]. Prior to measurements, tomato plants were kept in darkness for twenty minutes to ensure deactivation of photosynthetic electron transport. Fluorescence emitted by the irradiated region was collected by a bifurcated multimode optical fiber probe with 1 m length. The fiber consists of one center fiber with a diam- eter of 600

l

m surrounded by six 600

l

m fibers. The UV light trav- els along the surrounding fibers and then the fluorescence is picked up by the single center fiber and fed back into a high resolution Ocean Optics Spectrometer (USB 4000 spectrometer (Oceanoptics, USA)). The spectrometer displayed the fluorescence spectrum in the range from 400 to 1100 nm. A computer with dedicated software (by Ocean Optics) was connected to the spectrometer to record the fluorescence spectra from 600 to 875 nm. The measured spectra were fitted with two Gaussian curves, which correspond to the red and the far-red bands, using IGOR Pro 6.1, (WaveMetrics, USA) software. The Gaussian spectral function was used because it provides an acceptable matching fit of spectral data with good standard error for the peak amplitude, peak center and full width at half maximum (bandwidth)[20]. The fluorescence intensity ratios (FIR)F690/F735of peak height and band area were calculated from the fitting curve.

In order to record the kinetics at 690 nm and 735 nm, plants were placed in front of the bifurcated optical fiber. Leaves were illuminated by the UV light, for 3.5 min and simultaneously the fluorescence at 690 nm and 735 nm was transmitted to the Spectrometer determining the decay rate. The induction kinetics consists of a fast fluorescence rise to a maximum intensity level (Fm) followed by a slow fluorescence decay (Fd) and a steady-state level (Fs). Chl fluorescence decrease ratios,RFd(690) andRFd(735), were calculated from these kinetics.RFdis especially defined as ratio of fluorescence decrease (F_d) to the steady state Chl fluorescence (Fs) with the following equation[21,22]:

RFd¼ ðFmFsÞ=Fs¼Fd=Fs ð1Þ

FromRFd(690) andRFd(735) the stress–adaptation index Ap[17,23]

was determined as following equation:

Ap¼1 ð1þRFdð735ÞÞ=ð1þRFdð690ÞÞ ð2Þ From Eqs. (1) and (2), Ap can also be calculated as following equation:

Ap¼1 ðFmð735Þ=Fsð735ÞÞ=ðFmð690Þ=Fsð690ÞÞ ð3Þ

2.5. Statistics

The data presented in this work are the average of at least six replicates per treatment and each experiment was conducted in duplicate. The mean values ±SE are reported in the ﬁgures and table. The signiﬁcance of differences between control and treatment was determined at the 0.05 level of probability.

3. Results and discussion

3.1. Plant growth and metals accumulation

GrowingS. lycopersicumfor 7 days on nutrient solution supplemented with 10

l

M Cd²⁺, resulted in the inhibition of shoot growth (Table 1). Chlorosis and necrosis of leaves are the main visual toxicity symptoms. However, plant growth was promoted at the low concentrations of Zn²⁺(10 and 50

l

mol/l) added to Cd²⁺(Table 1) and did not show any of the above symptoms. Thus, it can be con- cluded that Zn²⁺, at low concentration, had a signiﬁcant effect on the alleviation of Cd²⁺toxicity on plant growth. This effect was re-

ﬂected directly by analysis of Cd²⁺and Zn²⁺accumulation. Plants exposed to Cd²⁺ alone showed an accumulation of Cd²⁺ and a reduction in Zn²⁺content in their leaves (Table 1). Zn²⁺addition induced a decrease in Cd²⁺uptake and simultaneously an increase in Zn²⁺accumulation, indicating a strong competition between these two metals in the plant system. Since Cd²⁺and Zn²⁺, both taken as divalent cations belong to the group II transition metals with eight electrons in their outer orbital, Cd²⁺can readily inhibit most of the Zn²⁺-dependent processes[24]and hence increased Zn²⁺concentration is able to replace a non-physiological metal like Cd²⁺, which may bind to the crucial and functional membrane and enzyme active sites and inactivate their functions. The antagonistic effect between Cd²⁺and lower Zn²⁺concentrations on plant growth is attributable largely to reduced Cd²⁺ uptake. Conversely, when Zn²⁺ was added at higher doseP100

l

M in combination with Cd²⁺, a signiﬁcant reduction in biomass production was observed (Table 1). This toxic effect is due to the important accumulation of Zn²⁺in leaves (Table 1). Excessive Zn²⁺in plants can profoundly affect normal ionic homeostatic systems by interfering with the uptake, transport, and regulation of essential ions[6]and results in the disruption of metabolic processes such as photosynthesis responsible for growth reduction[25].

3.2. Levels of photosynthetic pigments

The levels of photosynthetic pigments also indicated the toxic nature of Cd²⁺ to the plant system. The content of Chla, band Car was decreased by 50%, 28% and 45% when the plants were grown under 10

l

M Cd²⁺in comparison to the control (Table 1).

The reductions of Chlaand ChlbinS. lycopersicumwere probably caused by a deﬁciency of chlorophyll biosynthesis. In fact, Cd²⁺

interferes with enzymes of chlorophyll biosynthesis such as d-aminolevulinic acid dehydratase (ALA dehydratase)[26], as well as the protochlorophyllide reductase[27]. The reductions of chlorophyll content can be also due to the oxidation coming from an overproduction of reactive oxygen species (ROS), which are gener- ated by Cd²⁺ toxicity [28]. In Cd²⁺-treated plants with supplemented Zn²⁺ (especially 10

l

M), we observed that there is full protection and restoration of the chlorophyll contents. Zn²⁺ at low level probably maintains chlorophyll synthesis through sul- phydryl group protection, a function primarily associated with Zn²⁺[5]. Zn²⁺plays also a role in activating ALA dehydratase, and hence protochlorophyllide to chlorophyllide conversion facilitating the formation of complete chlorophyll moiety[29]. Not only Zn²⁺

can protect chlorophyll content against toxicity of cadmium, but also other divalent ions like Mg²⁺, Hermans et al.[30]reported that Mg pretreatment of 7 days alleviated the bleaching of young leaves caused by Cd²⁺. The protective effect of Mg²⁺against Cd²⁺toxicity could be attributable partly to the maintenance of Fe status but also to the increase in antioxidative capacity, detoxiﬁcation and/

or protection of the photosynthetic apparatus. The highest Zn²⁺

concentration (150

l

M), when added in combination with Cd²⁺, induced a severe reduction in the Chlaand Chlblevels compared to Cd²⁺-alone-treated plants. We have earlier reported that excess Zn²⁺, when added alone in the nutrient medium, induces chlorosis and loss of total chlorophyll. We found that variations in Chlaand bcontent caused a decrease in the Chla/Chlbratio, especially at 100 and 150

l

M[7].

3.3. Chlorophyll ﬂuorescence spectra excited by 365 nm of UV-LED

The curve-fitted Chl fluorescence spectra of control as well as treatedS. lycopersicumplants exhibited maxima in the red (F690 band) and far-red (F735 band) regions (Fig. 1). The curve-fitted fluorescence parameters such as peak-position, height and band area are given inTable 2. These spectra indicated that the intensity

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of red Chl fluorescence was strongly affected by the Cd²⁺ treatment, in fact, there is an important increase of this band compared to the control, which could be due to inhibition in electron flow at the reducing site of photosystems. This result coincides with the occurrence of visible symptoms of chlorosis caused by the degradation of the photosynthetic pigments. As seen in Table 2, the red band showed minimal shifting in the peak position while the far-red band showed quite significant shift under Cd²⁺treatment.

Indeed, the peak centers of the red and far-red chlorophyll bands were observed at 687.10 and 729.30 nm, respectively for control plants, and at 684.74 and 716.05 nm for plants treated by 10

l

M Cd²⁺. This shift in the peak position towards shorter wavelengths may be due to the decrease of chlorophyll content. However, in the case of Cd²⁺-treated plants with supplemented Zn²⁺ (10–100

l

M), the intensity of the ﬂuorescence emission at 690 nm peak was depressed (especially at 10

l

M Zn²⁺). This result is probably

due to higher re-absorption of 690 nm by increased photosynthetic pigments content (Fig. 1andTable 1). In fact, the chlorophyll re- absorbs mainly the red Chl ﬂuorescence band[11]. On the contrary, when Zn²⁺was added to Cd²⁺at high level (150

l

M) the fluorescence emitted at 690 nm was significantly higher than that observed in S. lycopersicum plants treated with Cd²⁺ alone. The fluorescence emission at 735 nm peak remained unchanged with treatments.

Fig. 2shows the Chl FIRF690/F735determined from the curve-ﬁt- ted Chl ﬂuorescence spectra for control as well as treated plants.

Results showed that the FIRF690/F735peak height and band area increased in the case of 10

l

M Cd²⁺treatedS. lycopersicumby about 271 and 46%, respectively, compared to the control. Supplementa- tion of Zn²⁺at low concentration650

l

M to Cd²⁺treatment caused a signiﬁcant decrease in the FIRF690/F735peak height and band area compared to Cd²⁺-alone.

The variations in the Chl FIRF₆₉₀/F₇₃₅of the control as well as the Cd²⁺and Zn²⁺treatedS. lycopersicumare due to the different Table 1

Inﬂuence of Cd²⁺and Zn²⁺on the metal accumulation, shoots growth and photosynthetic pigment contents inSolanum lycopersicumplants. Each value represents the mean ± SE of six individual replicates.

Metal treatment (lM) Shoot Cd²⁺content (lg g¹DW)

Shoot Zn²⁺content (lg g¹DW)

Shoots growth (g DW)

Photosynthetic pigment contents (mg g¹FW)

Chla Chlb Carotenoids

Control 0 ± 0 10.44 ± 0.42 0.120 ± 0.004 1.34 ± 0.045 0.58 ± 0.064 0.52 ± 0.048

10 Cd²⁺ 31.63 ± 2.41 03.58 ± 0.30 0.058 ± 0.001 0.65 ± 0.054 0.35 ± 0.062 0.26 ± 0.021

10 Cd²⁺+10 Zn²⁺ 24.92 ± 2.39 11.78 ± 1.08 0.128 ± 0.008 1.40 ± 0.040 0.55 ± 0.036 0.59 ± 0.045

10 Cd²⁺+50 Zn²⁺ 17.46 ± 0.67 28.03 ± 3.27 0.099 ± 0.010 1.25 ± 0.048 0.46 ± 0.011 0.47 ± 0.017

10 Cd²⁺+100 Zn²⁺ 13.95 ± 0.80 30.91 ± 2.83 0.063 ± 0.002 0.90 ± 0.086 0.38 ± 0.023 0.42 ± 0.031

10 Cd²⁺+150 Zn²⁺ 11.84 ± 0.26 43.57 ± 7.19 0.041 ± 0.006 0.52 ± 0.051 0.31 ± 0.016 0.31 ± 0.019

Fig. 1.Gaussian curve-fitted chlorophyll fluorescence spectra of the control and metal treatedSolanum lycopersicumleaves. a.u, arbitrary unit. Each spectrum in the figure is the average of 12 spectra.

Table 2

Fluorescence parameters of the curve-ﬁtted spectra of the control and treatedS. lycopersicumleaves, excited by 365 nm of ultraviolet light emitting diode (UV-LED).

Metal treatment (lM) Curve-ﬁtted chlorophyll ﬂuorescence parameters

F690 F735

Peak position (nm) Peak height (a.u.) Band area (a.u.) Peak position (nm) Peak height (a.u.) Band area (a.u.)

Control 687.10 1558 53,974 729.30 2099 131,459

10 Cd²⁺ 684.74 6057 120,835 716.05 2196 201,139

10 Cd²⁺+10 Zn²⁺ 685.60 2113 59,272 726.68 2081 132,774

10 Cd²⁺+50 Zn²⁺ 684.48 2780 64,787 725.60 2105 136,963

10 Cd²⁺+100 Zn²⁺ 683.79 4870 92,617 722.19 2060 161,317

10 Cd²⁺+150 Zn²⁺ 683.93 6661 119,940 720.49 2106 178,911

Fig. 2.Fluorescence intensity ratios (FIR)F690/F735of the curve-ﬁtted parameters of Chl ﬂuorescence spectra of control and metal treatedS. lycopersicumleaves. Data are means ±SE (n= 12).

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concentrations of chlorophyll contents in the plants’ leaves. The FIR F690/F735peak height was inversely correlated with the Chlacon- tent (r²= 0.968) and Chlbcontent (r²= 0.970) (Fig. 3A and B). There

also exists a signiﬁcant inverse correlation forF690/F735band area with the Chlacontent (r²= 0.960) and Chlbcontent (r²= 0.950) (Fig. 3C and D).

Chl FIR has been extensively used as a method of early stress detection and it is inversely correlated to the photosynthetic activity[31]. The increase of the red ﬂuorescence (F690) intensity and the FIRF₆₉₀/F₇₃₅of tomato plants under Cd²⁺stress could be correlated to the interaction of Cd²⁺with the reaction center of PSII. The Cd²⁺ion has multiple effects on both the donor side and the acceptor side of PSII. According to Faller et al.[32]and Pagliano et al.

[33], a possible mechanism for Cd²⁺toxicity on the donor side of PSII may be due to the substitution of the essential Ca²⁺with a non-functional Cd²⁺ cation, resulting in inhibition of photosynthetic oxygen evolution. Cd²⁺ also inhibits the electron transfer from the amino acid, tyrosineYZto the Chlacation (P680⁺)[34].

Several results indicate that Cd²⁺ binds at the acceptor side of the PSII. According to Sigfridsson et al.[34]the Cd²⁺slows down the kinetics in forward electron transfer from the primary quinone electron acceptor,Q_A, either by perturbing the function of the secondary quinone electron acceptor,Q_B, or by modifying the electron transfer at the level of the reduction ofQB. Zn²⁺, at low concen- tration650

l

M, on the other hand induced a decrease in the red ﬂuorescence (F690) intensity and the FIRF₆₉₀/F₇₃₅(Fig. 2) indicating a restoration of the lost photosynthetic activity and proving the action of Zn²⁺against the toxic nature of Cd²⁺. Zn²⁺by controlling the levels of Cd²⁺ entering into the system not only controls its intracellular levels, but also replaces the toxic metal, Cd²⁺, thereby maintaining the integrity at the oxygen-evolving centers and preventing oxidative burst at antenna Chl molecules and probably also preventing binding of Cd²⁺to the major thylakoid proteins like D1 of the PS II[35]. However, the high Zn²⁺concentration (150

l

M) exacerbated the negative effects of Cd²⁺and showed an increase of FIRF₆₉₀/F₇₃₅peak height and band area by about 14% and 11%, respectively, compared to Cd²⁺alone. Excess Zn²⁺can inhibit the electron transfer at the reducing side of the PSII reaction center, inducing an inhibition of the PSII activity[36]. Moreover, Zn²⁺induced a decline in the energy transfer from PSII-Chlaantenna to the PSII reaction center[37]. Jin et al.[38]reported that the exposure of Sedum alfrediito higher external Zn²⁺resulted in chloroplasts structural degradation, disorganization of grana, increased number of plastoglobuli, greater size starch grains and breakdown of the chloroplast membrane.

3.4. Effects on induction kinetics

The fluorescence induction kinetics measured on pre-darkened leaves of S. lycopersicumconsists of a fast fluorescence rise to a maximum intensity level (Fm) followed by a slow fluorescence decay (F_d) and a steady-state level (F_s) (Fig. 4). The variable chlorophyll fluorescence decrease ratio (RFd) values were calculated from the fluorescence induction kinetics parametersFm,FsandFd, measured from fluorescence induction kinetics curves recorded at 690 nm and 735 nm. Our results illustrated that theRFdvalues of the leaves of control plants showed the values of 3.05 for RFd(690) and 2.13 for RFd(735) (Fig. 5A) usually found in plants grown at normal physiological conditions[16]. When exposed to Cd²⁺treatment, theRFdvalues were found to be decreased compared to those of the control plants and were always higher at 690 nm than at 735 nm (Fig. 5A). BothRFd(690) andRFd(735) are good criteria for the potential photosynthetic activity of a leaf, andRFd(690) values higher than 2.5 indicate a very good photosynthetic activity[39]. This is further demonstrated by the fact that theRFd(690) values are linearly correlated with the photosynthetic CO2 fixation rate [14,40]. The decrease of RFd(690) (1.64) and RFd(735) (1.43) in the case of Cd²⁺treatment suggests that this metal reduced CO2fixation. Raziuddin et al.[41]have observed that Fig. 3.An inverse relationship between the FIRF690/F735peak height and Chl a

content(A)and Chl b content(B), and between the FIR F690/F735band area and Chl a content(C)and Chl b content(D)ofS. lycopersicum.

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during Cd²⁺stress the reduction of photosynthetic rates can also be attributed to a certain limitation of carbon dioxide (CO2) supply due to a decreased stomata conductance, associated with a partial closure of stomata. This can also be responsible for the rate of oxygen evolution, which probably decreases parallel to theRFdvalues.

It was reported that other divalent ion like Ca²⁺antagonized the toxicity effects of Cd²⁺and increased the electron transport rate of PSII, effective quantum yield of photochemical energy

conversion in PSII, photosynthetic active radiation, coefﬁcient of photochemical quenching, and chlorophyll ﬂuorescence decrease ratioRFd[42]. In addition, Mn²⁺content in the medium, can protect the photosynthetic apparatus of low-CO₂cells against toxicity of cadmium[43].

In tomato plants Cd²⁺treatment induced an increase in theFm, F_sandF_d, (data not shown), nevertheless, the increase ofF_sis greater than that ofFd, which is responsible for decreasedRFdvalues.

During the Chl ﬂuorescence induction kinetics, the decline from F_mtoF_sparallels the oxygen evolution[17]. Moreover, among the different reactions of PSII, the O2-evolving process is particularly sensitive to Cd²⁺stress[33]. The decreased photochemical activity in this condition was correlated with a declined value of the stress adaptation index Ap (Fig. 5B). The stress adaptation index Ap has also successfully been applied to quantify stress adaptation and damage of plants to petrol engine exhaust pollutants and drought [44] and permitted to differentiate more stress sensitive plants from more stress tolerant ones.

The inhibition of the photosynthetic activity can be also due to the Zn²⁺deﬁciency. Results of our study showed that Cd²⁺stress led to a decrease in Zn²⁺concentration of the tomato plants. This impairment was effectively controlled and alleviated by Zn²⁺ in Cd²⁺-treated plants with supplements of Zn²⁺at low concentration (10 and 50

l

M) (Fig. 5). The higher capacity for photosynthetic quantum conversion of tomato leaves was also reflected by the higher values of the Chl fluorescence ratiosRFdand the stress adaptation index Ap compared to those detected in plants treated with only Cd²⁺. Zinc at low level can also enhance carbonic anhydrase (CA) activity, which is very beneficial for plants in order to facili- tate the supply of CO2from the stomatal cavity to the site of CO2

ﬁxation, and thus enhances the general growth of the plants[45].

However, the highest Zn²⁺concentration (150

l

M), when added in combination with Cd²⁺, strongly decreased theR_Fd(690) (0.84) andRFd(730) (0.80) (Fig. 5A) and leaves exhibited more chlorosis.

Such stress-induced decline ofRFd-values to less than 1.0 indicates an irreversible damage to the photosynthetic apparatus[2,17]and plants died off[16]. This result was conﬁrmed by the strong decline of the Ap index at this treatment which Ap was reduced by 70%

compared to Cd²⁺alone (Fig. 5B). This decline in Ap index indicates that older senescing leaves are more stress sensitive than fully functional green leaves and can no longer recover the photosynthetic activity. This result shows that at high concentrations of Zn²⁺, the negative effects, on photosynthetic efﬁciency, were greater than with Cd²⁺alone.

4. Conclusion

The present study has demonstrated that the remote sensing of the UV-LED IFS in the 690 and 735 nm regions from plant leaves, with subsequent determination of the ratioF690/F735andRFdvalues as stress indicators, appears to be a suitable instrument (method) for remote sensing of the development and health of plants as well as to detect stress events. The results of this investigation show that the intensity of the Chl fluorescence spectra is greatly dependent on the Chl content of leaves. A lower chlorophyll content due to Cd²⁺stress increased fluorescence intensity in the 690 nm region and the values of the FIRF690/F735ratio compared to normal green leaves. An increased FIRF690/F735is not only indicative of lower chlorophyll content, but the values also increase when the process of photosynthesis quantum conversion is affected. The R_Fdvalues calculated from the fluorescence induction kinetics were decreased under Cd²⁺stress, indicating a reduction in the photosynthetic CO2fixation rate. The Chl fluorescence intensity together with the FIRF690/F735and the RFd values revealed that in Cd²⁺- treated plants with supplemented Zn²⁺, at low level, there is hence Fig. 5.(A)RFdvalues for control and metal treatedS. lycopersicumplants. These

values were calculated from the fluorescence induction kinetics curve, recorded at both red (690 nm) and far-red (735 nm) of Chl fluorescence bands. (B) Influence of Cd²⁺and Zn²⁺on the adaptation index Ap.RFd, variable chlorophyll fluorescence decrease ratio. Data are means ± SE (n= 12).

Fig. 4.Fluorescence induction kinetics curve of leaves excited by 365 nm of ultraviolet light emitting diode (UV-LED). This curve is the average of twelve curves.

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restoration of photosynthetic activity. Zn²⁺also enhanced the biosynthesis of chlorophyll ultimately proving beneﬁcial for the photosynthetic machinery of the plant system. The results demonstrate the usefulness ofF₆₉₀/F₇₃₅, Ap andR_Fdvalues in determining the potential photosynthetic activity of an intact attached leaf and its application in understanding the regeneration of photosynthetic functioning on alleviation of Cd²⁺toxicity by adequate Zn²⁺level. All these ﬂuorescence signals and ratios can be applied for non-destructive monitoring of terrestrial vegetation in basic photosynthesis research.

5. Abbreviations

Ap stress–adaptation index

Car carotenoids

Chl chlorophyll

Fd ﬂuorescence decrease fromFmto

Fs

Fm maximum chlorophyll

ﬂuorescence

Fs steady state Chl ﬂuorescence

FIR ﬂuorescence intensity ratio

FW fresh weight

F690 the red Chl ﬂuorescence centered

near 690 nm

F735 the far-red Chl ﬂuorescence

centered near 735 nm

PSI photosystem I

PSII photosystem II

RFd variable chlorophyll ﬂuorescence

decrease ratio

ROS reactive oxygen species

UV-LED IFS ultraviolet light emitting diode induced ﬂuorescence

spectroscopy

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