Effects of catalase on chloroplast arrangement in Opuntia streptacantha chlorenchyma cells under salt stress

In arid and semiarid regions, low precipitation rates lead to soil salinity problems, which may limit plant establishment, growth, and survival. Herein, we investigated the NaCl stress effect on chlorophyll fluorescence, photosynthetic-pigments, movement and chloroplasts ultrastructure in chlorenchyma cells of Opuntia streptacantha cladodes. Cladodes segments were exposed to salt stress at 0, 100, 200, and 300 mM NaCl for 8, 16, and 24 h. The results showed that salt stress reduced chlorophyll content, F v/F m, ΦPSII, and qP values. Under the highest salt stress treatments, the chloroplasts were densely clumped toward the cell center and thylakoid membranes were notably affected. We analyzed the effect of exogenous catalase in salt-stressed cladode segments during 8, 16, and 24 h. The catalase application to salt-stressed cladodes counteracted the NaCl adverse effects, increasing the chlorophyll fluorescence parameters, photosynthetic-pigments, and avoided chloroplast clustering. Our results indicate that salt stress triggered the chloroplast clumping and affected the photosynthesis in O. streptacantha chlorenchyma cells. The exogenous catalase reverted the H2O2 accumulation and clustering of chloroplast, which led to an improvement of the photosynthetic efficiency. These data suggest that H2O2 detoxification by catalase is important to protect the chloroplast, thus conserving the photosynthetic activity in O. streptacantha under stress.


Salt treatments effect on photosynthetic pigments content in O. streptacantha cladodes.
In order to determine the effect of salt stress on the photosynthetic pigments content in O. streptacantha, we quantified the chlorophyll a, b, total (a + b), and carotenoids (x + c) content in cladodes segments exposed to salt treatments at 0, 100, 200, and 300 mM NaCl during 8, 16, and 24 h. We observed that the chlorophyll a, and total (a + b) contents tends to decline along with the exposure time to NaCl concentrations ( Fig. 2). At 8 h of salt treatment, were observed a decrease in chlorophyll a, and total (a + b) levels at 100 and 200 mM NaCl concentrations compared to the control. However, no changes were observed in the Chl a, and total (a + b) levels at 16 h under the salt treatments evaluated. After 24 h of the salt treatments, we found a decrement of chlorophyll a, and total (a + b) levels in all the salt concentrations tested ( Fig. 2A and B). On the other hand, we did not observe a significant difference in the ratio of Chl a to Chl b (Chl a/b) at 8 h of salt treatments. Meanwhile, the Chl a/b ratio was significantly reduced at 16 and 24 h for all the salt treatments (Fig. 2C). The ratio of Chl a and Chl b to total carotenoids (a + b)/(x + c) was significantly reduced only at 24 h of salt treatments (Fig. 2D). Therefore, salt treatments negatively affected the chlorophyll and carotenoids content in O. streptacantha cladodes.

Salt treatments triggers chloroplasts clumping in O. streptacantha chlorenchyma cells.
In order to analyze the chloroplast arrangement in response to salt treatments, O. streptacantha cladode segments were incubated with 0, 100, 200, and 300 mM NaCl under continuous light conditions for 8, 16, and 24 h (Fig. 3). Under the control conditions, the chloroplasts were always dispersed in the cytosol of the cells. However, we observed chloroplast clumping in chlorenchyma cells when the salt concentration was increased. At 100 mM NaCl for 24 h the chloroplasts were redistributed. Furthermore, it was observed that in some cells chloroplasts began to cluster. Additionally, we detected aggregation of chloroplasts at 200 and 300 mM NaCl during the period analyzed. We also observed that NaCl-induced chloroplast clumping at 200 mM for 8 h was reversible when the salt stressed cladodes segments were washed and then incubated in distilled water ( Supplementary Fig. S2). These results evidence that salt treatment triggers chloroplasts clumping in O. streptacantha chlorenchyma cells.

Salt treatment induced ultrastructural changes in thylakoid membranes of O. streptacantha chlorenchyma cells.
To analyze if the ultrastructure of chloroplasts gets affected by exposure to salt treatment, cladode segments of O. streptacantha were incubated with 200 mM NaCl during 8, 16, and 24 h. Subsequently, the chloroplast membranes were analyzed by a transmission electron microscope (TEM). At the ultra-structural level, chloroplast distortion was observed in the cladode segments exposed to salt treatment for 8, 16, and 24 h compared to the control (Fig. 4A). Initially, we observed a stacking in the thylakoid membranes at 8 and 16 while at 24 h, a distortion of chloroplasts was highly visible where the thylakoid membranes were fragmented. These results show that the thylakoid membranes were notably affected by the salt treatment. In order to confirm the stacking of thylakoid membranes, measurements of lumen thickness were made on grana lamellae (GL) in separate micrographs (Fig. 4B). Electron microscopy data showed that the width of the thylakoid lumen was significantly diminished by the exposure to 200 mM NaCl during 8 and 16 h compared to the control. The lumen thickness at 24 h under salinity was not examined because the thylakoid membranes were severely distorted. These results indicate that salt treatment induced thylakoid membranes stacking in O. streptacantha.

Catalase treatment avoid the clustering of chloroplasts under salt treatment.
To determine if the exogenous catalase (CAT) application may counteract the aggregative effect of chloroplasts under salt stress, we incubated cladode segments in solutions with 200 mM NaCl supplemented with 100, 200, and 300 UmL −1 CAT during 8 h (Fig. 5). Under the application of 100 UmL −1 CAT, we observed that chloroplast grouping was diminished in comparison with the treatment of 200 mM NaCl without CAT. Conversely, we detected that the chloroplasts were completely dispersed throughout the cell when the highest concentrations of CAT were applied (200 and 300 UmL −1 ). In this regard, our data indicate that the application of CAT may inhibit the clustering of chloroplasts during salt stress in O. streptacantha chlorenchyma cells. Catalase prevented photosynthetic pigments degradation. To assess whether the exogenous application of CAT prevents the pigments degradation, we quantified the content of chlorophylls and carotenoids in O. streptacantha cladode segments exposed to 200 mM NaCl supplemented with 300 UmL −1 CAT for 8, 16, and 24 h.
As we previously described in Fig. 2, the treatment of 200 mM NaCl showed a decrement of chlorophyll content. However, CAT exogenous application reversed the negative effect of 200 mM NaCl on chlorophyll levels, reaching values similar to the control without salt stress (Fig. 8). These results support the notion that exogenous CAT prevents photosynthetic pigments degradation induced by salt stress in O. streptacantha cladodes.

Discussion
Abiotic stress affects the development, establishment and survival of wild-plants and crops. Photosynthesis is one of the most important physiological processes through which plants produce the essential energy for their growth and development 16 . Chloroplasts are specialized organs that capture sunlight required to perform photosynthesis 1 . However, when plants are exposed to various environmental stresses, such as salinity 10, 17 , drought 18 , or high light conditions 31 , plant's chloroplasts can get damaged, leading to photosynthesis inhibition.
In this study, we analyzed the effect of salt stress on the movement of chloroplasts in the chlorenchyma cells of O. streptacantha. Notably, we observed that the chloroplasts were densely clumped towards the center of the cell in response to NaCl treatments. Similar results were reported by Yamada et al. 10 , who showed that salinity Values are means and bars indicate ± SD, (n = 9). Different letters indicate significant difference between treatments and time (hours) according to Duncan's multiple range tests at P < 0.05. stress induced aggregative movement of chloroplasts in Eleusine coracana cells under normal intensity light. The increase of salt causes adverse effects on the functions and structure of the photosynthetic apparatus, leading to a decline in the F v /F m , ΦPSII, and qP parameters. These changes indicate that the reaction centers (RCs) got photochemically inactive, which reduced electron transport capacity in PSII and that decreased the photosynthetic capacity of O. streptacantha cladodes under salt stress.
In addition, the effect of NaCl treatments on the photosynthetic pigment content in chlorenchyma cells of O. streptacantha cladodes was analyzed. Our data showed that the decrease in the content of chlorophylls at 8 and 24 h was more pronounced at a low than at a higher salt concentration. One possible explanation could be that O. streptacantha cells, when sensing high salt concentrations activate several mechanisms to induce de novo chlorophyll synthesis, and thus counteract osmotic and ionic shock. This behavior has been previously described in cell lines of Medicago sativa and Nicotiana tabacum, which showed a greater accumulation of chlorophylls under salt stress 32,33 . These authors postulate that these changes may be an indicative of physiological processes activation in chloroplasts against saline stress. Therefore, O. streptacantha cells could activate mechanisms such as chloroplast biogenesis, osmoprotectants synthesis pathways, detoxification, heat shock protein and late embryogenesis abundant proteins involved in chloroplast protection. Moreover, no significant changes in the chlorophyll content at We showed that salt stress causes photosynthesis damage, which in turn would provoke the clustering of chloroplasts like a mechanism of protection to prevent light to penetrate to deeper layers and then reduce the photo-oxidation. However, plants have additional mechanisms to protect themselves against damage by an excess of energy 34 . The reduction in the chlorophyll a/b ratio in cladode segments under the salt treatments showed in O. streptacantha can be interpreted as an enlargement in the light-harvesting complex II (LHCII) antenna of PSII. Thus, we suggest that the increase in antenna size could reduce the excitation energy from the antenna to RCs of PSII, or as a direct response of photosynthetic apparatus to salt stress. Similar results were presented by  Moreover, it has been demonstrated that a re-arrangement on antenna leads to the stacking of thylakoid membranes 36 . The ultrastructural analysis in O. streptacantha chloroplast exhibited that salt treatment induced thylakoid membranes stacking at 8 and 16 h. Dynamics in the stacking of the thylakoid membranes are essential for regulatory processes of the photosynthesis under different abiotic stress conditions 34 . The ability to control the lateral separation of PSI from PSII is considered a functional consequence of grana stacking to minimize the formation of ROS in the PSI through of the decrease of electron transport between photosystems 37, 38 . Thus, our results suggest that changes of thylakoid membranes permit a balance in the excitation energy between the two photosystems 37 . These features can therefore be considered as a response of O. streptacantha cladodes to salt stress.
Instead, the ratio of chlorophyll to carotenoids at 24 h for the NaCl treatments was low, which indicate damage to the photosynthetic machinery. This result was consistent with the breakdown of chlorophyll caused by chloroplast damage with 200 mM NaCl at 24 h. Several studies have reported that salt stress induce the degradation of photosynthetic pigments in plants by the accumulation of toxic ions and ROS, changing the ultrastructure of the photosynthetic apparatus 16,[39][40][41][42][43] .
Although the chloroplasts could experience damage under salt stress, our results suggest that the clumping of chloroplasts plays an important role to minimize the excitation pressure (1-qP) on the photosynthetic machinery in O. streptacantha cladodes. The aggregative arrangement of chloroplasts in O. streptacantha seedlings also occurs under drought and high sunlight conditions 9 . Our research group has proposed that chloroplasts move towards the vacuole facilitating malate transportation; thus maintaining the photosynthetic activity under water deficit. The chloroplast-clumping phenomenon is a typical mechanism that has been observed in plants as a response to high solar radiation 11 , salinity 10, 39 , drought 7,9,44 , and low temperatures 8 . It has been proposed that the aggregation of chloroplasts may provide protection against photodamage and help to maintain the photosynthetic activity under stressful conditions 10,44,45 . Therefore, this phenomenon could be a common adaptive strategy used by plants for their survival under abiotic stress.
We also examined the effect of exogenous catalase (CAT) on chloroplasts clumping of O. streptacantha cladodes under NaCl treatments. Our data showed that CAT avoided the chloroplast clumping in chlorenchyma cells. Therefore, CAT generated an increase in the F v /F m , ΦPSII, qP values, and photosynthetic pigment levels in cladodes under salt treatments. Also, the cladode segments treated with CAT experienced less photo-inhibition under salt treatment than those without CAT. These results showed that the CAT application improved the photosynthesis under salt stress, counteracting for the clustering of chloroplasts. Similar photosynthesis protective mechanisms in plants under salt stress have been showed with exogenous application of non-enzymic antioxidants such as ascorbate (AsA) 46, 47 , glutathione (GSH) 48,49 , and ∝-tocopherols 50 . It is known that enzymatic antioxidants such . A high level of endogenous CAT is essential to maintain the antioxidant system that protects plants from oxidative damage due to various environmental stresses 52 . Thus, the exogenous CAT applied to cladode segments of O. streptacantha produced a protection against oxidative damage by H 2 O 2 scavenging activity.
The apoplast is an important site for H 2 O 2 production in acclimation response of plants to salinity 19,53,54 . It has been proposed that H 2 O 2 accumulation in the apoplast could activate a signal for the chloroplast due to their location close to the plasma membrane. Then, the chloroplast may transmit the ROS signaling to the nucleus for the photosynthesis acclimation through nuclear gene expression 20 . Moreover, Wen and Zhang 55 reported that high blue light exposure can induce H 2 O 2 generation in the plasma membrane, and that H 2 O 2 is involved in chloroplast movements in Arabidopsis thaliana. Thus, we proposed that H 2 O 2 generated by salt stress could be acting as a signaling molecule which promotes clumping of chloroplasts, particularly as an acclimation mechanism for mitigation of photo-inhibition in salt-stressed cladodes of O. streptacantha. However, the role of H 2 O 2 in chloroplast movement is still poorly understood.
In higher plants, ROS and cytosolic Ca 2+ ([Ca 2+ ] cyt ), are largely recognized to be important signaling messengers of many biological responses 56 . It is known that high NaCl concentrations, particularly under excess of chloride (Cl − ) ions in the cytoplasm, leads to an increase of [Ca 2+ ] cyt concentrations, which initiates the stress signal transduction pathways in plants under salt stress [57][58][59] . Furthermore, studies in Arabidopsis leaves reported that calcium is involved in the signal transduction for the movement of chloroplast in response to blue light 5, 60-62 . The authors work shows that the stress by high blue light can increase [Ca 2+ ] cyt , which may trigger the activity of nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) to generate H 2 O 2 . In turn; the H 2 O 2 generated may promote the chloroplast movements. Thus, it is possible that the clustering of chloroplast in cladodes segments can be regulated by internal Ca 2+ stores, produced by salt stress. However, the cross-talk between Ca 2+ and H 2 O 2 in the regulation of chloroplast movements in O. streptacantha cells is an open question arising from this work that demands future research efforts. Finally, the physiological responses of CAM cacti to salinity are complex and the mechanisms underlying this phenomenon have not been completely elucidated, opening a variety of exciting new questions.

Conclusions
The chloroplasts are usually more sensitive to salinity than other organelles. Our findings show that salt stress causes photosynthesis damage and accumulation of H 2 O 2 in the O. streptacantha cells. We suggest that H 2 O 2 acts as a messenger molecule for the clustering of chloroplasts. The exogenous application of CAT alleviates salt-induced oxidative stress in O. streptacantha cladodes most likely through H 2 O 2 scavenging activity. CAT activity avoids chloroplasts clustering and protects photosynthetic machinery function in salt-stressed O. streptacantha. In this regard, further studies about the effect of H 2 O 2 in chloroplast clustering will help to understand the role of this phenomenon during stress.  replicates) and we take three segments of cladode per plant (technical replicates) for each treatment, under continuous white light (300 μmol m −2 s −1 ) at 25 °C.

Materials and Methods
For CAT mixed with NaCl solution treatments, the CAT stock solution was prepared by pre-dissolving 1 mg of CAT (Sigma-Aldrich Ref: C1345) in 1 mL 50 mM potassium phosphate buffer pH 7.0. The CAT solutions at final concentrations of 100, 200, and 300 UmL −1 respectively, were added to 200 mM NaCl solution. Subsequently, the cladode segments were completely immersed in these mixtures (NaCl + CAT) for 8, 16, and 24 h. As control, we used distilled water without NaCl or CAT. The CAT concentration was selected according to Aroca 65 . The assays were performed using three different cladodes of O. streptacantha plants (biological replicates) and we take three segments of cladode per plant (technical replicates) for each treatment, under continuous white light (300 μmol m −2 s −1 ) at 25 °C.
Chlorophyll fluorescence measurements. The chlorophyll fluorescence analysis is a powerful technique that allows to obtain detailed information about of the process of photosynthesis in plants 66 and can also be applied to know how plants respond to abiotic stress factors 67 . Cladode segments of 10 mm diameter were removed from O. streptacantha plants using forceps and scalp. Subsequently, these were subjected to the aforementioned treatments. Chlorophyll fluorescence was measured using a MINI-PAM II fluorometer (H. Walz, Effeltrich, Germany) following the manufacturer's instructions. The chlorophyll fluorescence measurements were realized using a pulse the actinic light with an intensity of 820 μmol m −2 s −1 . The maximum quantum yield of photosystem II (F v /F m ) was determined after dark adaptation of cladodes segments for 30 min. The F v /F m values were calculated as described by Kitajima and Butler 68 . On the other hand, light-adapted cladodes segments were used to measure the fluorescence parameters as follows: the effective photochemical quantum yield of PSII was calculated using the equation: ΦPSII = (F m ′ − F)/F m ′ = ΔF/F m ′ presented by Genty et al. 69 . The photochemical quenching was calculated as qP = (F m ′ − F)/(F m ′ − F O ′) and used to determine the fraction of closed (reduced) PSII reaction centers, also known as excitation pressure, and calculated as 1-qP. The non-photochemical fluorescence quenching, NPQ = (F m − F m ′)/F m ′ was determined according to Bilger and Björkman 70 . The photosynthetic electron transport rate (ETR) was estimated with the following equation ETR = ΦPSII × PPFD × 0.5 × 0.84, where PPFD is photosynthetic photon flux density, the factor 0.5 assumes that photosystems II and I are similarly excited by the irradiance. The factor 0.84 considers that only 84% incident irradiance will be absorbed by the two photosystems 71 . The assays were performed using three different cladodes of O. streptacantha plants (biological replicates) and we take three segments of cladode per plant (technical replicates) for each treatment (n = 9).
Determination of chlorophylls and carotenoids content. The chlorophyll (a and b) and carotenoids extraction was performed according to the methodology reported by Lichtenthaler 72,73 . The cladode chlorenchyma fresh segments were homogenized in 80% acetone and incubated in dark at 4 °C for 5 min. Subsequently, it was centrifuged at 13,000 rpm at 4 °C for 5 min. The chlorophyll and carotenoids contents were measured in 200 μL supernatant using a microplate reader (Epoch 2, Biotek, Winooski, VT, United States of America) at 663 (chlorophyll a), 646 (chlorophyll b), and 470 (carotenoids) nm wavelengths. The assays were performed using three different cladodes of O. streptacantha plants (biological replicates) and we take three segments of cladode per plant (technical replicates) for each treatment (n = 9). The pigments content, the ratio of chlorophyll a (Chl a) to chlorophyll b (Chl b) (Chl a/b), and Chl a and Chl b to total carotenoids (a + b)/(x + c) ratio were estimated using the equations proposed by Lichtenthaler 73 74 . Ultrathin sections were contrasted using aqueous uranyl acetate (2% w/v) and aqueous lead citrate (2% w/v). Samples were examined with TEM JEOL 200CX (JEOL, Welwyn Garden City, UK) using a 100 kV acceleration voltage. The ultrastructural analysis of chloroplasts to determine the thickness of the thylakoid lumen was realized using the methodology reported by Kirchhoff et al. 38 . The distances of stacking repeat unit (R), which includes the two thylakoid membrane bilayers (M) and the widths of one partition gap (P) were realized with Image J software 75 . Subsequent, the thickness of the thylakoid lumen (L) were estimated using the equations proposed by Kirchhoff et al. 38 : L = R − M − P. The measurements of lumen thickness were made on grana lamellae (GL) using three separate micrographs and we take three grana lamellae per micrographs for each treatment (n = 9). KCl (pH 6.1) for 30 min, images were visualized using an Epi-fluorescence microscope (Axio Imager M2; Carl Zeiss Microscopy, LLC, USA). The assays were performed using three different cladodes of O. streptacantha plants (biological replicates) and we take three segments of cladode per plant (technical replicates) for each treatment (n = 9). Data analysis. All data obtained from chlorophyll fluorescence parameters and pigments content were statistically analyzed with STATISTICA version 7 software 76 , using Duncan's multiple range test at the P < 0.05 level of significance between treatments and time (hours). The results are expressed as mean values ± SD (standard deviation) (n = 9).