Spatial Heterogeneity of Cadmium Effects on Salvia sclarea Leaves Revealed by Chlorophyll Fluorescence Imaging Analysis and Laser Ablation Inductively Coupled Plasma Mass Spectrometry

In this study, for a first time (according to our knowledge), we couple the methodologies of chlorophyll fluorescence imaging analysis (CF-IA) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), in order to investigate the effects of cadmium (Cd) accumulation on photosystem II (PSII) photochemistry. We used as plant material Salvia sclarea that grew hydroponically with or without (control) 100 μM Cd for five days. The spatial heterogeneity of a decreased effective quantum yield of electron transport (ΦPSΙΙ) that was observed after exposure to Cd was linked to the spatial pattern of high Cd accumulation. However, the high increase of non-photochemical quenching (NPQ), at the leaf part with the high Cd accumulation, resulted in the decrease of the quantum yield of non-regulated energy loss (ΦNO) even more than that of control leaves. Thus, S. sclarea leaves exposed to 100 μM Cd exhibited lower reactive oxygen species (ROS) production as singlet oxygen (1O2). In addition, the increased photoprotective heat dissipation (NPQ) in the whole leaf under Cd exposure was sufficient enough to retain the same fraction of open reaction centers (qp) with control leaves. Our results demonstrated that CF-IA and LA-ICP-MS could be successfully combined to monitor heavy metal effects and plant tolerance mechanisms.


Introduction
Cadmium (Cd), a non-essential element for plants, is considered to be as one of the most toxic elements for plants because it is not biodegradable in soil and it accumulates in the environment exhibiting toxic effects [1][2][3]. It can appear in the environment at high concentrations, due to several human activities (industrial and agricultural activities, such as mining and smelting of metalliferous ores, electroplating, wastewater irrigation, and abuse of chemical fertilizers) and subsequently becomes toxic to all living organisms [3][4][5][6]. However, some plants have established several mechanisms for Cd detoxification that result in acclimation and tolerance [2,6].
However, heavy metal accumulation does not influence its development, as well as the quality and quantity of the essential oils, which can be used in the perfumery and cosmetics [50][51][52]. To the best of our knowledge, the effects of Cd action on the photosynthetic apparatus and PSII photochemistry of Salvia leaves, and especially Cd distribution in the leaf area, have not been studied before.
In the present work we tested the hypothesis whether exposure of Salvia sclarea plants to Cd will result in spatial leaf heterogeneity of the effective quantum yield of electron transport (Φ PSII ), and if it does, whether the decreased Φ PSII values in the leaf area will correspond to the respective pattern of high Cd accumulation obtained by LA-ICP-MS analysis.

Plant Material and Growth Conditions
Seeds of Salvia sclarea L. collected from a field in the Rose Valley region of Bulgaria were kindly provided by Bio Cultures Ltd (Karlovo, Bulgaria), which is focused on growing several types of herbs for the production of essential oils.
Salvia seeds were germinated and grown on soil in a growth room for about a month. When one pair of true leaves fully expanded (height 4-5 cm), the seedlings were transferred to 1-L vessels (two seedlings per vessel) filled with a continuously aerated modified Hoagland nutrient solution composed of 1.5 mM KNO 3

Cd Treatment
About 2-month-old uniform plants were selected and subjected to treatment with 0 and 100 µM Cd (applied as 3CdSO 4 8H 2 O) for five days. The nutrient solution with or without Cd was renewed every three days.

Chlorophyll Fluorescence Imaging Analysis
An Imaging-PAM Fluorometer M-Series MINI-Version (Walz, Effeltrich, Germany) was used to measure in 15 min dark-adapted leaves of S. sclarea plants, grown with 0 (control) or 100 µM Cd for five days, the effects of Cd on PSII function. Five leaves were measured from five different plants with the actinic light intensity of 220 µmol photons m −2 s −1 . In each leaf, 14-16 areas of interest were selected from which chlorophyll fluorescence values were measured. The chlorophyll fluorescence parameters, measured as described in detail previously [53], were the minimum chlorophyll a fluorescence in the dark (F o ), the maximum chlorophyll a fluorescence in the dark (F m ), the maximum chlorophyll a fluorescence in the light (F m '), and the steady-state photosynthesis in the light (F s ). The minimum chlorophyll a fluorescence in the light was computed by the Imaging Win V2.41a software (Heinz Walz GmbH, Effeltrich, Germany) as . By using Win software, we calculated the allocation of absorbed light energy at PSII, that is the effective quantum yield of photochemistry (Φ PSII ), the quantum yield of regulated non-photochemical energy loss (Φ NPQ ), and the quantum yield of non-regulated energy loss (Φ NO ). The relative PSII electron transport rate (ETR), the fraction of open PSII reaction centers, the so-called photochemical quenching (q p ) and the non-photochemical quenching that reflects heat dissipation of excitation energy (NPQ) were also calculated.

Laser Ablation Inductively Coupled Plasma Mass Spectrometry
Leaf tissues were analysed in vivo using an ICP-QMS spectrometer (Elan DRC II, Perkin-Elmer Sciex, Guelph, ON, Canada) equipped with a laser ablation system (LA; model LSX-500, CETAC Technologies, Omaha, NE, USA) operating at a wavelength of 266 nm. The instrumentation was optimized on a daily basis by ablating the standard reference glass material NIST SRM 610 and adjusting the nebulizer gas flow, RF generator power and ion lens voltage in order to obtain the maximum signal intensity for 24 Mg + , 115 In + , 238 U + . Plasma robustness was monitored via the 232 Th 16 O + / 232 Th, doubly charged ions and the 238 U/ 232 Th intensity ratios. ThO + /Th + intensity ratios were always below 0.2%, doubly charged ions 42 Ca 2+ / 42 Ca + < 0.5% and 238 U + / 232 Th + intensity ratio was less than 1.2. For leaf sample analysis optimization of the parameters, such as energy of laser beam, spot size, shot frequency and scanning speed, was performed. The laser ablation conditions were chosen so that the ablation of the sample was completed. In the experiment, the volume of standard ablation chamber was reduced to~10 mL, which shortened the washout time of the aerosol and improved the LA images. The instrumental and analytical conditions of LA-ICP-MS are summarized in Table 1. For bioimage generation, LA-iMageS software was used [54].

Statistical Analysis
Chlorophyll fluorescence analysis data are presented as the mean ± SD. Statistical analysis of means from five leaves from different plants was performed using the Student's t-test. Differences were considered statistically significant at p < 0.05.

Photosynthetic Heterogeneity Revealed by Chlorophyll Fluorescence Imaging Analysis in Salvia sclarea Leaves under Cd Exposure
The imaging area in the MINI-Version of the Imaging-PAM Fluorometer M-Series that was used is 24 × 32 mm. Thus, we studied such an area from the distal leaf area of S. sclarea. CF-IA revealed a photosynthetic heterogeneity in the studied leaf area of S. sclarea leaves under Cd exposure. The spatial heterogeneity was observed mainly in the effective quantum yield of photochemistry (Φ PSII ), the quantum yield of regulated non-photochemical energy loss (Φ NPQ ) and the quantum yield of non-regulated energy loss (Φ NO ) after five days exposure of S. sclarea to 100 µM Cd. We observed three clearly distinguishable leaf areas in the chlorophyll fluorescence images of Φ PSII , Φ NPQ and Φ NO . More specifically we marked in the chlorophyll fluorescence images of Φ PSII , a leaf area at the leaf edge, than a top leaf area with lower Φ PSII values than those of the leaf edge, and a second leaf area with Φ PSII values higher than the top leaf area (Figure 1a). The lower Φ PSII values, of the top leaf area, were found near the midvein (Figure 1a). The same three areas appeared at the chlorophyll fluorescence images of Φ NPQ with the top leaf area having higher Φ NPQ values compared to the other two areas (leaf edge and second leaf area) (Figure 1b). The higher Φ NPQ values were found near the midvein of the top leaf area (Figure 1b). In the chlorophyll fluorescence images of Φ NO , the higher values were found in the second leaf area (Figure 2). Representative chlorophyll fluorescence images of Φ PSII , Φ NPQ , and Φ NO of Salvia sclarea leaves from plants grown under control conditions (0 µM Cd) are shown in Figure S1. At control growth conditions, a photosynthetic homogeneity was observed in S. sclarea leaves. ΦNPQ and ΦNO. More specifically we marked in the chlorophyll fluorescence images of ΦPSΙΙ, a leaf area at the leaf edge, than a top leaf area with lower ΦPSII values than those of the leaf edge, and a second leaf area with ΦPSII values higher than the top leaf area (Figure 1a). The lower ΦPSII values, of the top leaf area, were found near the midvein (Figure 1a). The same three areas appeared at the chlorophyll fluorescence images of ΦNPQ with the top leaf area having higher ΦNPQ values compared to the other two areas (leaf edge and second leaf area) ( Figure 1b). The higher ΦNPQ values were found near the midvein of the top leaf area (Figure 1b). In the chlorophyll fluorescence images of ΦNO, the higher values were found in the second leaf area ( Figure 2). Representative chlorophyll fluorescence images of ΦPSΙΙ, ΦNPQ, and ΦNO of Salvia sclarea leaves from plants grown under control conditions (0 μM Cd) are shown in Figure S1. At control growth conditions, a photosynthetic homogeneity was observed in S. sclarea leaves.
(a) (b) Figure 1. Representative chlorophyll fluorescence images of ΦPSΙΙ (a) and ΦNPQ (b) of Salvia sclarea leaves exposed to 100 μM Cd for five days. The different leaf areas: Leaf edge, top leaf part, and second leaf part, are marked. The color code depicted at the right of the images ranges from 0 to 1.

Figure 2.
A representative chlorophyll fluorescence image of ΦNO of Salvia sclarea leaves exposed to 100 μM Cd for five days. The different leaf areas: Leaf edge, top leaf part, and second leaf part, are marked. The color code depicted at the right of the image ranges from 0 to 1. ΦNPQ and ΦNO. More specifically we marked in the chlorophyll fluorescence images of ΦPSΙΙ, a leaf area at the leaf edge, than a top leaf area with lower ΦPSII values than those of the leaf edge, and a second leaf area with ΦPSII values higher than the top leaf area (Figure 1a). The lower ΦPSII values, of the top leaf area, were found near the midvein (Figure 1a). The same three areas appeared at the chlorophyll fluorescence images of ΦNPQ with the top leaf area having higher ΦNPQ values compared to the other two areas (leaf edge and second leaf area) ( Figure 1b). The higher ΦNPQ values were found near the midvein of the top leaf area (Figure 1b). In the chlorophyll fluorescence images of ΦNO, the higher values were found in the second leaf area ( Figure 2). Representative chlorophyll fluorescence images of ΦPSΙΙ, ΦNPQ, and ΦNO of Salvia sclarea leaves from plants grown under control conditions (0 μM Cd) are shown in Figure S1. At control growth conditions, a photosynthetic homogeneity was observed in S. sclarea leaves.
(a) (b) Figure 1. Representative chlorophyll fluorescence images of ΦPSΙΙ (a) and ΦNPQ (b) of Salvia sclarea leaves exposed to 100 μM Cd for five days. The different leaf areas: Leaf edge, top leaf part, and second leaf part, are marked. The color code depicted at the right of the images ranges from 0 to 1.

Figure 2.
A representative chlorophyll fluorescence image of ΦNO of Salvia sclarea leaves exposed to 100 μM Cd for five days. The different leaf areas: Leaf edge, top leaf part, and second leaf part, are marked. The color code depicted at the right of the image ranges from 0 to 1.

Figure 2.
A representative chlorophyll fluorescence image of Φ NO of Salvia sclarea leaves exposed to 100 µM Cd for five days. The different leaf areas: Leaf edge, top leaf part, and second leaf part, are marked. The color code depicted at the right of the image ranges from 0 to 1.

Cadmium Imaging in Salvia sclarea Leaves by Laser Ablation Inductively Coupled Plasma Mass Spectrometry
Three leaves from three different plants were studied by LA-ICP-MS. The area that was selected for analysis corresponds to the two areas that were marked in CF-IA, a top leaf part and a second leaf part. Thus, from each leaf, the corresponding area of 20 × 18 mm was cut and placed on the polyethylene terephthalate slide. In order to normalize the signal, compensating plasma variations and ablations process, two candidates for internal standards, such as 13 C and 34 S were evaluated [33]. Finally, isotope of carbon 13 C was selected as the internal standard as its distribution in the S. sclarea leaves was homogeneous. The leaves from S. sclarea plants grown under control conditions (0 µM Cd) were analyzed by the whole area ( Figure S2), while the leaves from plants exposed to Cd were analyzed in two parts that corresponded to the two leaf parts studied by CF-IA ( Figure 3).

Cadmium Imaging in Salvia sclarea Leaves by Laser Ablation Inductively Coupled Plasma Mass Spectrometry
Three leaves from three different plants were studied by LA-ICP-MS. The area that was selected for analysis corresponds to the two areas that were marked in CF-IA, a top leaf part and a second leaf part. Thus, from each leaf, the corresponding area of 20 × 18 mm was cut and placed on the polyethylene terephthalate slide. In order to normalize the signal, compensating plasma variations and ablations process, two candidates for internal standards, such as 13 C and 34 S were evaluated [33]. Finally, isotope of carbon 13 C was selected as the internal standard as its distribution in the S. sclarea leaves was homogeneous. The leaves from S. sclarea plants grown under control conditions (0 μM Cd) were analyzed by the whole area ( Figure S2), while the leaves from plants exposed to Cd were analyzed in two parts that corresponded to the two leaf parts studied by CF-IA ( Figure 3). The area with high Cd signal intensity was the top leaf area, and, more specifically, the area in the midvein of the top leaf area (Figure 3). In contrast, the presence of Cd was not revealed in leaves of control plants ( Figure S2).

Changes in the Light Energy Use at PSII Under Cd Exposure
We calculated for all chlorophyll fluorescence parameters whole leaf values, top leaf part area values and second leaf part area values (leaf part areas are marked in Figures 1 and 2 and Figure S1). We estimated the allocation of absorbed light energy at PSII, that is the effective quantum yield of photochemistry (ΦPSII), the quantum yield of regulated non-photochemical energy loss (ΦNPQ), and the quantum yield of non-regulated energy loss (ΦNO) of S. sclarea leaves from plants exposure to 0 and 100 μM Cd. ΦPSΙΙ whole leaf values, after five days exposure to Cd, decreased significantly The area with high Cd signal intensity was the top leaf area, and, more specifically, the area in the midvein of the top leaf area (Figure 3). In contrast, the presence of Cd was not revealed in leaves of control plants ( Figure S2).

Changes in the Light Energy Use at PSII Under Cd Exposure
We calculated for all chlorophyll fluorescence parameters whole leaf values, top leaf part area values and second leaf part area values (leaf part areas are marked in Figures 1 and 2 and Figure S1). We estimated the allocation of absorbed light energy at PSII, that is the effective quantum yield of photochemistry (Φ PSII ), the quantum yield of regulated non-photochemical energy loss (Φ NPQ ), and the quantum yield of non-regulated energy loss (Φ NO ) of S. sclarea leaves from plants exposure to 0 and 100 µM Cd. Φ PSII whole leaf values, after five days exposure to Cd, decreased significantly compared to controls as did also top leaf part area values compared to their corresponding controls (Figure 4a). Φ PSII values of the second leaf part area did not differ compared to the corresponding control values (Figure 4a). The second leaf part values after five days exposure to Cd were significantly higher than the top leaf part area Φ PSII values (Figure 4a). Φ NPQ values after five days exposure to Cd, increased significantly in the whole leaf compared to control, and also in the other two parts compared to their corresponding controls (Figure 4b). Top leaf part Φ NPQ values after Cd exposure were significantly higher than second leaf part area values (Figure 4b).
Materials 2019, 12, x FOR PEER REVIEW 7 of 14 compared to controls as did also top leaf part area values compared to their corresponding controls (Figure 4a). ΦPSΙΙ values of the second leaf part area did not differ compared to the corresponding control values (Figure 4a). The second leaf part values after five days exposure to Cd were significantly higher than the top leaf part area ΦPSΙΙ values (Figure 4a). ΦNPQ values after five days exposure to Cd, increased significantly in the whole leaf compared to control, and also in the other two parts compared to their corresponding controls (Figure 4b). Top leaf part ΦNPQ values after Cd exposure were significantly higher than second leaf part area values (Figure 4b).
(a) (b) ΦNO values after five days exposure to Cd, decreased significantly in the whole leaf compared to control, and also in the other two parts compared to their corresponding controls ( Figure 5). Top leaf part ΦNO values after five days exposure to Cd were significantly lower than second leaf part area values ( Figure 5). Φ NO values after five days exposure to Cd, decreased significantly in the whole leaf compared to control, and also in the other two parts compared to their corresponding controls ( Figure 5). Top leaf part Φ NO values after five days exposure to Cd were significantly lower than second leaf part area values ( Figure 5).

Changes in Non-Photochemical Quenching and the Redox State of PSII under Cd Exposure
Non-photochemical quenching (NPQ) that reflects heat dissipation of excitation energy, increased significantly after five days exposure to Cd in the whole leaf compared to control, and also in the other two parts compared to their corresponding controls (Figure 6a). Top leaf part area NPQ values after five days exposure to Cd were significantly higher than second leaf part area values

Changes in Non-Photochemical Quenching and the Redox State of PSII under Cd Exposure
Non-photochemical quenching (NPQ) that reflects heat dissipation of excitation energy, increased significantly after five days exposure to Cd in the whole leaf compared to control, and also in the other two parts compared to their corresponding controls (Figure 6a). Top leaf part area NPQ values after five days exposure to Cd were significantly higher than second leaf part area values (Figure 6a). The redox state of PQ pool (q p ), decreased significantly in the top leaf part area compared to the corresponding control, but remained the same with control at the whole leaf level and at the second leaf part area (Figure 6b). Top leaf part q p values after five days exposure to Cd were significantly lower than second leaf part values (Figure 6b). Figure 5. Changes in the quantum yield of non-regulated energy loss (ΦNO) in Salvia sclarea plants grown at 0 (control), or 100 μM Cd for five days. Error bars on columns are standard deviations based on five leaves from different plants. Error bars on columns are standard deviations based on five leaves from different plants. Columns with a different letter (lower case for controls and capitals for 100 μM Cd) are statistically different between different leaf areas (p < 0.05). An asterisk represents a significantly different mean between controls and 100 μM Cd of the same leaf area (p < 0.05).

Changes in Non-Photochemical Quenching and the Redox State of PSII under Cd Exposure
Non-photochemical quenching (NPQ) that reflects heat dissipation of excitation energy, increased significantly after five days exposure to Cd in the whole leaf compared to control, and also in the other two parts compared to their corresponding controls (Figure 6a). Top leaf part area NPQ values after five days exposure to Cd were significantly higher than second leaf part area values (Figure 6a). The redox state of PQ pool (qP), decreased significantly in the top leaf part area compared to the corresponding control, but remained the same with control at the whole leaf level and at the second leaf part area (Figure 6b). Top leaf part qP values after five days exposure to Cd were significantly lower than second leaf part values (Figure 6b).

Changes in the Electron Transport Rate in Response to Cd Exposure
The relative electron transport rate at PSII (ETR) decreased significantly at the whole leaf level and at the top leaf part, after five days exposure to Cd, compared to their corresponding controls, while retained the same ETR values with controls at the second leaf part (Figure 7). Top leaf part ETR values after five days exposure to Cd were significantly lower than second leaf part values (Figure 7).

Changes in the Electron Transport Rate in Response to Cd Exposure
The relative electron transport rate at PSII (ETR) decreased significantly at the whole leaf level and at the top leaf part, after five days exposure to Cd, compared to their corresponding controls, while retained the same ETR values with controls at the second leaf part (Figure 7). Top leaf part ETR values after five days exposure to Cd were significantly lower than second leaf part values ( Figure  7).

Discussion
Among the different techniques that have been developed for elemental imaging, including secondary ion mass spectrometry, X-ray fluorescence, scanning electron microscopy with energy-dispersive X-ray analysis, and LA-ICP-MS, the latter one has emerged as the prevailing, with high sensitivity and more comprehensible tool for bioimaging of mineral elements in plant tissues

Discussion
Among the different techniques that have been developed for elemental imaging, including secondary ion mass spectrometry, X-ray fluorescence, scanning electron microscopy with energy-dispersive X-ray analysis, and LA-ICP-MS, the latter one has emerged as the prevailing, with high sensitivity and more comprehensible tool for bioimaging of mineral elements in plant tissues [55,56].
Bioimaging of mineral elements in plant tissues has revealed that the distribution of trace elements in leaves is highly heterogeneous [57][58][59]. The accumulation, distribution and localization of Cd in plant leaves, reported by numerous studies [33,47,60], proposed that the accumulation and distribution of Cd and also of other elements depends on the element, the plant species, the organ and the age of the organ [61][62][63]. In Salvia leaves information regarding the distribution of any element is lacking. In our experiment, the distribution of Cd in Salvia leaves, under 100 µM Cd, shows that high Cd signal intensity was detected in the midvein of the top leaf part (Figure 3). In contrast, no Cd could be detected in S. sclarea grown under control conditions ( Figure S2).
Photosynthetic perturbations to heavy metal exposure do not develop homogeneously over the whole leaf area, thus, making chlorophyll fluorescence measurements at a specific point on the leaf surface non-reliable [43,44]. CF-IA detects spatial and temporal heterogeneity of photochemical efficiency under heavy metal stress and can provide further information on the particular leaf area that is most sensitive to heavy metal stress [6,45].
No significant photosynthetic heterogeneity was detected in leaves of control grown clary sage plants, but we were able to identify a spatial photosynthetic heterogeneity in the leaves of clary sage exposed to Cd. This spatial heterogeneity was observed in Φ PSII , Φ NPQ and Φ NO after exposure to Cd. The lower Φ PSII values, of the top leaf area, that were found near the midvein (Figure 1a) are linked to the high Cd signal intensity in the midvein of the top leaf area (Figure 3). These observations confirm the previous suggestion [6] that spatial leaf heterogeneity of the effective quantum yield of electron transport (Φ PSII ) arise from differences in the distribution of Cd across the leaf.
The high increase of Φ NPQ values after five days exposure to Cd, in the whole leaf area compared to control values, and especially at the top leaf part (Figure 4b), resulted in lower Φ NO values, compared to control, with the lower values to be observed at the top leaf part ( Figure 5). Φ NO consists of chlorophyll fluorescence internal conversions and intersystem crossing, which leads to the formation of singlet oxygen ( 1 O 2 ) via the triplet state of chlorophyll ( 3 chl * ) [39,[64][65][66]. Consequently, after five days exposure to Cd, 1 O 2 decreased in the whole leaf area, compared to control values, and especially at the top leaf part, where the higher Cd signal intensity was scored. This can be explained by the photoprotective mechanism of non-photochemical quenching, that allows for the sequestration of reactive oxygen species (ROS) below critical levels [61]. Otherwise, an increase in ROS triggers remarkable damage to the metabolic machinery, inducing photoinhibition and a generalized damage response [67][68][69].
Non-photochemical chlorophyll fluorescence quenching (NPQ) is a process that takes place in the photosynthetic membranes of plants, algae, and cyanobacteria in which surplus absorbed light energy is dissipated as heat [70][71][72]. This is a molecular adaptation process that represents the fastest response of the photosynthetic membrane to the surplus light energy [70,71]. Thus, the excess light causes rapid saturation of the photosynthetic reaction centers and their eventual closure [69,70]. The excess light energy that cannot be used for photochemistry can damage the most delicate part of the photosynthetic apparatus, the PSII reaction center, which drives the oxidation of water and liberation of molecular oxygen [18]. In order this photodamage to be avoided, the excess excitation energy has to be safely removed by the photoprotective mechanism of NPQ [71,73,74].
The presence of Cd ions increased the heat dissipation of energy as NPQ [75], but this is a photoprotective response mechanism to avoid ROS generation and damage to PSII [71,76,77]. In general, the influence of Cd on photosystems is more serious in PSII than in PSI [78,79]. The photoprotective dissipation of excess light energy (NPQ) under stress conditions can be regarded as efficient only if it is adjusted in such a way to retain the same fraction of open reaction centers as in control conditions [80]. The photoprotective mechanism of NPQ was sufficient in the leaves of clary sage exposed to Cd, since the fraction of open reaction centers at the whole leaf area remained the same to controls (Figure 6b).
Although excess Cd accumulation is harmful to plants [1,2,7], detoxification mechanisms to Cd toxicity that are involved in Cd tolerance and accumulation exist in the hyperaccumulators [6,45]. Plants have developed complicated mechanisms to control concentrations of essential nutrient elements and to diminish the injury from exposure to non-essential metals, but the mechanisms regarding the regulatory network of metal uptake, chelation, transport, sequestration and detoxification which contributes to the alleviation of heavy metal toxicity and photosynthetic tolerance remain to be further elucidated [81][82][83][84].

Conclusions
Exposure of S. sclarea plants to Cd resulted in spatial leaf heterogeneity of Φ PSII , with the decreased Φ PSII values in the leaf area to correspond to the respective pattern of high Cd accumulation obtained by LA-ICP-MS analysis. We propose that combining the methodologies of chlorophyll fluorescence imaging analysis (CF-IA) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) can identify the effects of heavy metals on plants and provide information on tolerance mechanisms. We suggest that S. sclarea could be characterized as a heavy metal accumulator, as it is tolerant to Cd, and could also potentially be used for phytoremediation.