Potassium (K+) starvation-induced oxidative stress triggers a general boost of antioxidant and NADPH-generating systems in the halophyte Cakile maritima

  Citation: Houmani, H.; Debez, A.; Freitas-Silva, L.d.; Abdelly, C.; Palma, J.M.; Corpas, F.J. Potassium (K+) Starvation-Induced Oxidative Stress Triggers a General Boost of Antioxidant and NADPH-Generating Systems in the Halophyte Cakile maritima.Antioxidants 2022,11, 401. https://doi.org/10.3390/ antiox11020401 Academic Editor: Sang Yeol Lee Received: 11 January 2022 Accepted: 14 February 2022 Published: 16 February 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). antioxidants Article Potassium (K+) Starvation-Induced Oxidative Stress Triggers a General Boost of Antioxidant and NADPH-Generating Systems in the Halophyte Cakile maritima Hayet Houmani 1,2, Ahmed Debez 2, Larisse de Freitas-Silva 1, Chedly Abdelly 2, JoséM. Palma 1 and Francisco J. Corpas 1,* 1Group of Antioxidants, Free Radicals and Nitric Oxide in Biotechnology, Food and Agriculture, Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, CSIC, Apartado 419, E-18080 Granada, Spain; [email protected] (H.H.); [email protected] (L.d.F.-S.); [email protected] (J.M.P.) 2 Laboratory of Extremophile Plants, Center of Biotechnology of Borj Cedria, P.O. Box 901, Hammam-Lif 2050, Tunisia; [email protected] (A.D.); chedly[email protected] (C.A.) *Correspondence: javier[email protected] Abstract: Potassium (K + ) is an essential macro-element for plant growth and development given its implication in major processes such as photosynthesis, osmoregulation, protein synthesis, and enzyme function. Using 30-day-old Cakile maritima plants as halophyte model grown under K + deprivation for 15 days, it was analyzed at the biochemical level to determine the metabolism of reactive oxygen species (ROS), key photorespiratory enzymes, and the main NADPH-generating systems. K + starvation-induced oxidative stress was noticed by high malondialdehyde (MDA) content associated with an increase of superoxide radical (O 2•− ) in leaves from K + -deficient plants. K + shortage led to an overall increase in the activity of hydroxypyruvate reductase (HPR) and glycolate oxidase (GOX), as well as of antioxidant enzymes catalase (CAT), those of the ascorbate-glutathione cycle, peroxidase (POX), and superoxide dismutase (SOD), and the main enzymes involved in the NADPH generation in both leaves and roots. Especially remarkable was the induction of up to seven CuZn-SOD isozymes in leaves due to K + deficiency. As a whole, data show that the K + starvation has associated oxidative stress that boosts a biochemical response leading to a general increase of the antioxidant and NADPH-generating systems that allow the survival of the halophyte Cakile maritima. Keywords: ascorbate peroxidase; Cakile maritima; catalase; CuZn-SOD isozymes; halophyte; NADPisocitrate dehydrogenase; pentose phosphate pathway; oxidative stress; potassium deficiency 1. Introduction Potassium is the fourth most abundant macro-element in the lithosphere (2.5%). In a soil solution K + concentration is about 0.01 to 20 mM [ 1 ], whereas this nutrient needs to be maintained within a range of 100–200 mM in the plant cell cytosol [ 2 ]. Multiple functions are ascribed to this macronutrient and have been recently reviewed [ 3 – 5 ]. K + availability for plants depends on the complex dynamics of the soil which are strongly influenced by root–soil interactions [ 6 ]. In soils, K + can be found as either soluble, exchangeable, fixed, or associated with minerals [ 7 ]. The release of the exchangeable form is slow and hence insufficient for plant growth and development [ 7 ]. Thus, imbalanced nutrition with K + is well known. Under K + limited conditions, impairment in photosynthetic apparatus occurs affecting notably the electron transfer chain and CO 2 fixation, thus enhancing the oxygen photoreduction in chloroplasts and leading to high production of reactive oxygen species (ROS). To cope with low K + availability, plants have evolved several strategies to increase K + uptake and maintain their ROS homeostasis. In the few reports addressing plant response to K + deficiency, the modulation of the antioxidant Antioxidants 2022,11, 401. https://doi.org/10.3390/antiox11020401 https://www.mdpi.com/journal/antioxidants Antioxidants 2022,11, 401 2 of 18 enzyme systems including superoxide dismutase (SOD), catalase (CAT), and all enzymatic components of the ascorbate-glutathione cycle was described [ 8 – 11 ]. However, the available information on the regulation of antioxidant defense upon K + starvation mostly concerns glycophytes and crops, whereas halophytes remain poorly addressed. These kinds of plants are particularly challenged with the nutrient shortage in their natural habitats, whether direct (due to soil poverty) or indirect (caused by the salt-induced restriction of nutrient uptake) [ 12 ]. Thus, understanding the responses to nutrient (particularly potassium) deficiency in halophytes with emphasis on the regulation of antioxidant defense upon K + starvation is pertinent, as it may enable the selection of species with high antioxidant capacity and tolerance to K+deficiency. The metabolism of ROS, which includes mechanisms of ROS generation and a set of antioxidant systems [ 13 ], keeps under control the potential ROS overproduction that can cause oxidative damages usually associated with many types of stresses, such as salinity [ 14 ], heavy metals [ 15 , 16 ], drought, and mechanical wounding [ 17 ]. Furthermore, NADPH is a basic indicator of cellular redox status required for cell growth, proliferation, and detoxification [ 18 – 22 ]. Thus, NADPH is strictly necessary by the antioxidant enzyme glutathione reductase (GR) in the ascorbate-glutathione pathway to regulate H 2 O 2 content in the different subcellular compartments including cytosol, chloroplasts, mitochondria, and peroxisomes. It is also needed by the NADPH-dependent thioredoxin reductases (NTRs) in the regulation of metabolic pathways through thiol group reduction. In addition to the ferredoxin-NADP reductase (FNR) in photosynthetic cells, NADPH is mainly generated by NADP-isocitrate dehydrogenase (NADP-ICDH), NADP-malic enzyme (NADP-ME) also named NADP-malate dehydrogenase, glucose-6-phosphate dehydrogenase (G6PDH), and 6-phosphogluconate dehydrogenase (6PGDH), the latter two belonging to the oxidative part of the pentose phosphate pathway. All these enzymes are also involved in diverse, basic metabolic pathways such as carbon and nitrogen metabolisms [23–27]. Cakile maritima L. (Brassicaceae) is an annual succulent halophyte with high tolerance to osmotic constraints [ 28 – 31 ] which has also great potential as a nutritious crop [ 32 ]. This species is also well known for its high seed oil content (up to 40% of seed DW) [ 33 ]. The natural ecosystems for C. maritima are sandy littoral dunes, which are known for their poor nutrient composition [ 34 ]. Given its high nutrient use and absorption efficiencies [ 35 ], its aptitude to substitute K + with Na + in many biological functions, and the performance of its antioxidant system [ 36 ], C. maritima is a useful candidate to investigate halophyte responses to nutrient deficiencies in its antioxidant response. Therefore, the present study aimed at better characterizing the response of C. maritima to K + deficiency with special emphasis on the plant growth, the water content, the antioxidative stress response, and the nutrient status. Overall, the data provide evidence that K + starvation provoked oxidative stress which triggers a general boost of the main antioxidant systems, and this was also accompanied by an increase in the NADPH-generating system in both roots and leaves, thus allowing the survival of C. maritima. 2. Materials and Methods 2.1. Plant Material and Growth Conditions C. maritima seeds were cleaned with commercial sodium hypochlorite (50%; w/v) for 4 min . Then they were washed five times with distilled water, and then sown in foilcovered Petri dishes containing two layers of filter paper imbibed with 15 mL H 2 O [ 28 ]. After seven days , plantlets were hydroponically grown in half-strength Hoagland’s nutrient solution (2.5 mM Ca(NO 3 ) 2· 4H 2 O, 2.5 mM KNO 3 , 0.5 mM KH 2 PO 4 , and 1 mM MgSO 4· 7H 2 O for the macronutrients; and 23.2 µ M H 3 BO 3 , 4.6 µ M MnCl 2· 4H 2 O, 1.2 µ M ZnSO 4· 7H 2 O, 0.185 µ M CuSO 4· 5H 2 O, and 0.06 µ M Na 2 MoO 4· 2H 2 O for the micronutrients) for an additional 7 days. Thereafter, plants were separated into two sets to study K + deficiency: control plants were kept in the Hoagland nutrient solution, and deficient plants were cultivated after transplanting to a medium without K+. The deficiency of NO3−and Antioxidants 2022,11, 401 3 of 18 PO 43− in the nutrient solution was corrected by the addition of appropriate amounts of NaNO 3 and (NH 4 )H 2 PO 4 [ 10 , 37 ]. The plant culture was carried out under greenhouse conditions (16 h photoperiod, 24/18 ◦ C light/dark temperature; 80% relative humidity). After another 15 days, plants were harvested and separated into roots and leaves and stored at − 80 ◦ C for biochemical assays. Figure 1shows the experimental design to study K+deficiency in C. maritima. Antioxidants 2022, 11, x FOR PEER REVIEW 3 of 19 MgSO4·7H2O for the macronutrients; and 23.2 μM H3BO3, 4.6 μM MnCl2·4H2O, 1.2 μM ZnSO4·7H2O, 0.185 μM CuSO4·5H2O, and 0.06 μM Na2MoO4·2H2O for the micronutrients) for an additional 7 days. Thereafter, plants were separated into two sets to study K+ deficiency: control plants were kept in the Hoagland nutrient solution, and deficient plants were cultivated after transplanting to a medium without K+. The deficiency of NO3− and PO43− in the nutrient solution was corrected by the addition of appropriate amounts of NaNO3 and (NH4)H2PO4 [10,37]. The plant culture was carried out under greenhouse conditions (16 h photoperiod, 24/18 °C light/dark temperature; 80% relative humidity). After another 15 days, plants were harvested and separated into roots and leaves and stored at −80 °C for biochemical assays. Figure 1 shows the experimental design to study K+ deficiency in C. maritima. Figure 1. Experimental design to study potassium deficiency in the halophyte Cakile maritima L. Seeds were germinated in Petri dishes for 7 d. Then, the plantlets were hydroponically grown in half-strength Hoagland’s nutrient solution for an additional 7 days. Thereafter, plants were separated in two lots: control (C) plants kept in the Hoagland nutrient solution containing 3 mM K+ and deficient plants, cultivated after transplanting to a medium without K+. After 15 additional days, plants were harvested. 2.2. Plant Crude Extracts Plant organs (roots and leaves) were collected and frozen in liquid N2. Then, the samples were ground to a powder in a mortar with a pestle. Two grams of the powder were suspended in a ratio 1/2 (w/v) in 50 mM Tris-HCl buffer (pH 7.8) containing 0.1 mM EDTA, 0.02% (w/v) Triton X-100, 10% (v/v) glycerol, 1% (w/v) polyvinylpolypyrrolidone (PVPP), and 5 mM dithiothreitol (DTT). The crude extracts were then filtered through one layer of Miracloth and centrifuged at 27,000× g at 4 °C for 25 min. Finally, the supernatants were collected and used for assays. 2.3. Water Content Water content (WC) of each organ was calculated as the difference between the fresh (FW) and dry (DW) weights according to the following equation WC = (FW − DW)/DW. Plant material was dried for 5 days at 65 °C on a stove. 2.4. Potassium Content and Cellular Potassium Concentration To determine K content in leaves and roots, samples were oven-dried at 60 °C and then ground into fine powder. K was extracted from the obtained powder (approximately 30–60 mg) by digestion with nitric/perchloric acid, and K analysis was performed using atomic absorption spectrophotometry (Perkin Elmer 1100 B). Cellular K concentration, a crucial parameter for the evaluation of K+ status inside the plant cell, was estimated as the ratio of K+ and water contents. 2.5. Lipid Peroxidation and Histochemical Detection of Superoxide Radical (O2•−) in Leaves Lipid peroxidation products were estimated by measuring the malondialdehyde (MDA) content through the thiobarbituric acid reactive substances (TBARS) method [38]. For the histochemical detection of O2•−, leaves from control and K+-deficient plants were excised and vacuum-infiltrated for 5 min in a nitroblue tetrazolium (NBT) solution Figure 1. Experimental design to study potassium deficiency in the halophyte Cakile maritima L. Seeds were germinated in Petri dishes for 7 days. Then, the plantlets were hydroponically grown in half-strength Hoagland’s nutrient solution for an additional 7 days. Thereafter, plants were separated in two lots: control (C) plants kept in the Hoagland nutrient solution containing 3 mM K + and deficient plants, cultivated after transplanting to a medium without K + . After 15 additional days, plants were harvested. 2.2. Plant Crude Extracts Plant organs (roots and leaves) were collected and frozen in liquid N 2 . Then, the samples were ground to a powder in a mortar with a pestle. Two grams of the powder were suspended in a ratio 1/2 (w/v) in 50 mM Tris-HCl buffer (pH 7.8) containing 0.1 mM EDTA, 0.02% (w/v) Triton X-100, 10% (v/v) glycerol, 1% (w/v) polyvinylpolypyrrolidone (PVPP), and 5 mM dithiothreitol (DTT). The crude extracts were then filtered through one layer of Miracloth and centrifuged at 27,000 × gat 4 ◦ C for 25 min. Finally, the supernatants were collected and used for assays. 2.3. Water Content Water content (WC) of each organ was calculated as the difference between the fresh (FW) and dry (DW) weights according to the following equation WC = (FW − DW)/DW. Plant material was dried for 5 days at 65 ◦C on a stove. 2.4. Potassium Content and Cellular Potassium Concentration To determine K content in leaves and roots, samples were oven-dried at 60 ◦ C and then ground into fine powder. K was extracted from the obtained powder (approximately 30–60 mg) by digestion with nitric/perchloric acid, and K analysis was performed using atomic absorption spectrophotometry (Perkin Elmer 1100 B). Cellular K concentration, a crucial parameter for the evaluation of K + status inside the plant cell, was estimated as the ratio of K+and water contents. 2.5. Lipid Peroxidation and Histochemical Detection of Superoxide Radical (O2•−) in Leaves Lipid peroxidation products were estimated by measuring the malondialdehyde (MDA) content through the thiobarbituric acid reactive substances (TBARS) method [38]. For the histochemical detection of O 2•− , leaves from control and K + -deficient plants were excised and vacuum-infiltrated for 5 min in a nitroblue tetrazolium (NBT) solution (0.5 mg mL −1 in 100 mM phosphate buffer, pH 6.8). After infiltration, the samples were incubated for 1 h at 25 ◦ C in darkness. Then, leaves were illuminated until the appearance of dark blue spots, characteristic of blue formazan precipitates [39]. Antioxidants 2022,11, 401 4 of 18 2.6. Anthocyanins Anthocyanin content was determined according to the Gould et al. (2000) [ 40 ] method. Samples were preserved in 2 mL of a solution containing (HCl/H 2 O/methanol) (v/v/v; 1/3/6) and stored at 4 ◦ C in the dark until the subsequent pigment extraction. The absorbance was read at 530 and 653 nm. The following formula was used to determine the anthocyanin content: Anthocyanins concentration (µg mL−1) = OD530 −0.24·OD653 2.7. In-Gel Isozyme Profile Analyses of Superoxide Dismutase (SOD), Peroxidase (POX), and Ascorbate Peroxidase (APX) The SOD isozymes were separated by non-denaturing polyacrylamide gel electrophoresis (PAGE) on 8% acrylamide gels and visualized by a photochemical NBT reduction method [ 41 ]. The type of SOD isozymes was identified according to its sensitivity to different inhibitors, 5 mM KCN or 5 mM H 2 O 2 . CuZn-SOD is inhibited by KCN and H 2 O 2 ; Fe-SOD is only inhibited by H 2 O 2 while Mn-SOD is unaffected by either KCN or H 2 O 2 [ 28 ]. The POX isozymes were separated by non-denaturing PAGE on 6% acrylamide gels and detected as previously described by [ 42 ]. Briefly, gels were incubated for 20 min in sodium acetate buffer 0.1 M, pH 5.5 containing 3,3-diaminobenzidine 1 mM, and H 2 O 2 (0.03%), and brown bands appeared at the end of the reaction. APX isozymes were separated as described by [ 43 ]. Briefly, gels were prepared at 10% acrylamide and run for 30 min at 120 V before samples were loaded. Electrophoresis was conducted for 3 h (120 V, 4 ◦ C). Then, gels were incubated three times in 50 mM potassium phosphate buffer, pH 7.0, containing 2 mM ascorbic acid for 10 min each, and once in 50 mM potassium phosphate buffer, pH 7.0, containing 4 mM ascorbic acid and 0.5 µ M H 2 O 2 for 10 min. After the last incubation, gels were rinsed twice with distilled water and equilibrated for 1–2 min in 50 mM potassium phosphate buffer pH 7.8. The staining reaction was started by adding 50 mM potassium phosphate buffer pH 7.8 containing 14 mM TEMED and 2.45 mM NBT. The reaction was stopped when the first blue bands became visible by decanting the staining solution and rinsing the gels with distilled water. 2.8. Determination of Enzyme Activities Glycolate oxidase (GOX; EC 1.1.3.1) was assayed by determining the formation of the complex glyoxylate-phenylhydrazone as described previously by [ 44 ]. NADH-dependent hydroxypyruvate reductase (HPR; EC 1.1.1.2 9) was assayed according to Schwitzguébel and Siegenthaler (1984). Catalase activity (CAT; EC 1.11.1.6) was determined using the [ 45 ] method which consists of measuring the disappearance of H 2 O 2 at 240 nm. Ascorbate peroxidase (APX; EC 1.11.1.11) was determined by monitoring the initial ascorbate oxidation by H 2 O 2 at 290 nm [ 46 ]. Monodehydroascorbate reductase (MDAR; EC 1.6.5.4) was assayed by measuring the monodehydroascorbate-dependent NADH oxidation, with monodehydroascorbate being generated by the ascorbate/ascorbate oxidase system [47]. The rate of monodehydroascorbate-independent NADH oxidation (without ascorbate and ascorbate oxidase) was subtracted from the monodehydroascorbate-dependent reaction. Glutathione reductase (GR; EC 1.6.4.2) was measured following the Edwards et al. (1990) [ 48 ] method by monitoring the NADPH oxidation at 340 nm coupled to the reduction of GSH (the reaction rate was corrected for the small, non-enzymatic oxidation of NADPH by glutathione disulfide, GSSG). Dehydroascorbate reductase (DHAR; EC 1.8.5.1) was determined by following the increase of ascorbate formation at 265 nm using N 2 -saturated buffer [ 49 ]. The reaction rate was corrected by the non-enzymatic reduction of dehydroascorbate by glutathione (GSH). A factor of 0.98, to account for the small contribution to the absorbance by GSSG, was also considered. NADP-dependent dehydrogenase (NADP-DH) activities were determined spectrophotometrically by recording the reduction of NADP + at 340 nm. The assays were performed at 25 ◦ C in a reaction medium (1 mL) containing 50 mM HEPES, pH 7.6, 2 mM MgCl 2 , Antioxidants 2022,11, 401 5 of 18 and 0.8 mM NADP. The reaction was initiated by the addition of a specific substrate for each enzyme. Thus, NADP-ICDH (EC 1.1.1.42) activity was started by the addition of 10 mM 2R,3S-isocitrate; G6PDH (EC 1.1.1.49) activity was initiated by the addition of 5 mM glucose-6-phosphate; to determine 6PGDH (EC 1.1.1.44) activity, the substrate was 5 mM 6-phosphogluconate was initiated; and, in the case of NADP-ME (EC 1.1.1.40) activity, the reaction was started by the addition of 1 mM L-malate [ 50 – 52 ]. Protein concentration was determined using the Bio-Rad protein assay with bovine serum albumin as standard. 2.9. Statistical Analysis Statistical analysis was performed using the Statgraphics program and data were analyzed by one-way ANOVA. Asterisk denotes that means are significantly different at p< 0.05. 3. Results 3.1. Effect of Potassium Starvation on C. maritima Growth Parameters To corroborate the potassium deficiency in the experimental design, the potassium content in roots and shoots was evaluated. Table 1shows that in C. maritima plants grown in nutrient solutions deficient in potassium, the K + content and concentration were significantly lower in both organs (on average 86%) in comparison to plants grown under optimal conditions. Table 1. K + content and K + concentration (mg mL −1 ) in shoots and roots of C. maritima under optimal or limiting K + conditions. Results are the mean of at least three different experiments ± SEM. Asterisk indicates that values in the same row are significantly different at p< 0.05. Organs 3 mM K+0 mM K+ Shoot K content (mg g−1dry weight) 4.05 ±0.14 0.46 ±0.04 * Root K content (mg g−1dry weight) 3.89 ±0.57 0.56 ±0.09 * Shoot K concentration (mg mL−1)0.43 ±0.02 0.065 ±0.003 * Root K concentration (mg mL−1)0.29 ±0.02 0.063 ±0.010 * Figure 2a shows a representative picture of 30-day-old C. maritima plants grown under potassium deficiency (0 mM K + ) which showed a 33% decrease in plant biomass production (Figure 2b) as compared to plants cultivated on optimal conditions (3 mM K + ). A 53% reduction in leaf number was also observed under the same conditions (Figure 2c). The lack of K + in the culture medium negatively affected both leaf and root water content (17% and 30%, respectively), suggesting the induction of water stress by K+deficiency (Figure 2d). Antioxidants 2022,11, 401 6 of 18 Antioxidants 2022, 11, x FOR PEER REVIEW 6 of 19 Figure 2. Phenotype and growth attributes of 30-day-old C. maritima plants grown in the presence or absence of K+ in the culture medium for 15 days. (a) Phenotype of C. maritima growth in the hydroponic cultivation and leaf detail. (b) Plant fresh weight (FW). (c) Leaf number per plant. (d) Shoot and root water contents (WC). Results are the mean of at least three different experiments ± SEM. Asterisks indicate that differences between values were statistically significant at p < 0.05. 3.2. Metabolism of ROS and Photorespiration under K+ Deficiency in C. maritima Lipid peroxidation, as a marker of membrane oxidative damages, and production of O2•− were used to evaluate the impact of ROS metabolism under K+ deficiency. The in vivo production of O2•− was assayed as the reduction and further precipitation of NBT leading to the appearance of dark spots of blue formazan in leaves from C. maritima plants subjected to K+ deficiency, but not in leaves from control plants (Figure 3A). As a potential result of the accumulation of O2•− and dismutation to H2O2, oxidative damage to membrane lipids was investigated by evaluating the MDA content, the final product of lipid peroxidation. There was a significant increase in MDA content (49%) in leaves from plants grown under K+ deficiency; in roots, the MDA values remained close to the control (Figure 3B). On the other hand, the content of anthocyanins increased about 53% in leaves of C. maritima plants grown under K+ deficiency (Figure 3C). Figure 2. Phenotype and growth attributes of 30-day-old C. maritima plants grown in the presence or absence of K + in the culture medium for 15 days. ( a ) Phenotype of C. maritima growth in the hydroponic cultivation and leaf detail. ( b ) Plant fresh weight (FW). ( c ) Leaf number per plant. ( d ) Shoot and root water contents (WC). Results are the mean of at least three different experiments ±SEM . Asterisks indicate that differences between values were statistically significant at p< 0.05. 3.2. Metabolism of ROS and Photorespiration under K+Deficiency in C. maritima Lipid peroxidation, as a marker of membrane oxidative damages, and production of O 2•− were used to evaluate the impact of ROS metabolism under K + deficiency. The in vivo production of O 2•− was assayed as the reduction and further precipitation of NBT leading to the appearance of dark spots of blue formazan in leaves from C. maritima plants subjected to K + deficiency, but not in leaves from control plants (Figure 3A). As a potential result of the accumulation of O 2•− and dismutation to H 2 O 2 , oxidative damage to membrane lipids was investigated by evaluating the MDA content, the final product of lipid peroxidation. There was a significant increase in MDA content (49%) in leaves from plants grown under K + deficiency; in roots, the MDA values remained close to the control (Figure 3B). On the other hand, the content of anthocyanins increased about 53% in leaves of C. maritima plants grown under K+deficiency (Figure 3C). Two peroxisomal photorespiratory enzymes were also analyzed. Thus, whereas hydroxypyruvate reductase (HPR) grew about 20% (Figure 4a), glycolate oxidase (GOX), which is considered one of the main sources of H 2 O 2 in green tissues, increased 2.8-fold (Figure 4b) under K + starvation. On the other hand, catalase, the main H 2 O 2 -removing enzyme also located in peroxisomes, increased 1.8-fold in leaves and 1.3-fold in roots of plants grown under K+deficiency (Figure 4c). Antioxidants 2022,11, 401 7 of 18 Antioxidants 2022, 11, x FOR PEER REVIEW 7 of 19 Figure 3. ROS parameters and anthocyanin content in 30-day-old C. maritima plants grown in the presence or absence of K+ in the culture medium for 15 days. (A) Histochemical detection of superoxide radical with NBT staining in leaves. Arrow indicates the precipitated blue formazan product. (B) Lipid peroxidation (MDA) in leaves and roots. (C) Leaf anthocyanin content. Results are the mean of at least three different experiments ± SEM. Asterisks indicate that differences between values were statistically significant at p < 0.05. Two peroxisomal photorespiratory enzymes were also analyzed. Thus, whereas hydroxypyruvate reductase (HPR) grew about 20% (Figure 4a), glycolate oxidase (GOX), which is considered one of the main sources of H2O2 in green tissues, increased 2.8-fold (Figure 4b) under K+ starvation. On the other hand, catalase, the main H2O2-removing enzyme also located in peroxisomes, increased 1.8-fold in leaves and 1.3-fold in roots of plants grown under K+ deficiency (Figure 4c). Figure 3. ROS parameters and anthocyanin content in 30-day-old C. maritima plants grown in the presence or absence of K + in the culture medium for 15 days. ( A ) Histochemical detection of superoxide radical with NBT staining in leaves. Arrow indicates the precipitated blue formazan product. ( B ) Lipid peroxidation (MDA) in leaves and roots. ( C ) Leaf anthocyanin content. Results are the mean of at least three different experiments ± SEM. Asterisks indicate that differences between values were statistically significant at p< 0.05. Antioxidants 2022, 11, x FOR PEER REVIEW 7 of 19 Figure 3. ROS parameters and anthocyanin content in 30-day-old C. maritima plants grown in the presence or absence of K+ in the culture medium for 15 days. (A) Histochemical detection of superoxide radical with NBT staining in leaves. Arrow indicates the precipitated blue formazan product. (B) Lipid peroxidation (MDA) in leaves and roots. (C) Leaf anthocyanin content. Results are the mean of at least three different experiments ± SEM. Asterisks indicate that differences between values were statistically significant at p < 0.05. Two peroxisomal photorespiratory enzymes were also analyzed. Thus, whereas hydroxypyruvate reductase (HPR) grew about 20% (Figure 4a), glycolate oxidase (GOX), which is considered one of the main sources of H2O2 in green tissues, increased 2.8-fold (Figure 4b) under K+ starvation. On the other hand, catalase, the main H2O2-removing enzyme also located in peroxisomes, increased 1.8-fold in leaves and 1.3-fold in roots of plants grown under K+ deficiency (Figure 4c). Figure 4. Activities of photorespiratoy enzymes and catalase in C. maritima plants grown in the presence or absence of K + in the culture medium for 15 days. ( a ) Glycolate oxidase activity. ( b ) Hydroxypruvate reductase activity. ( c ) Catalase activity. Results are the mean of three different experiments ±SEM . Asterisks indicate that differences between values were statistically significant at p< 0.05. Given the importance of the ascorbate-glutathione cycle to control the cellular H 2 O 2 content in coordination with catalase, the activity of its enzymatic components (APX, MDAR, DHAR, and GR) was spectrophotometrically assessed (Figure 5a–d). In general, Antioxidants 2022,11, 401 8 of 18 the activity of these enzymes was higher in roots than in leaves, and under K + deficiency a significant increase of all these activities in both organs was found, except for MDAR in roots and DHAR in leaves that were unaffected. The data on APX activity correlates with the isozyme pattern obtained after native PAGE and specific in-gel activity staining. Two APX isozymes were detected in leaves and roots, being the most prominent isozymes in roots. Under K + deficiency, there was a slight increase of APX isozymes in both organs (Figure 6a). Antioxidants 2022, 11, x FOR PEER REVIEW 8 of 19 Figure 4. Activities of photorespiratoy enzymes and catalase in C. maritima plants grown in the presence or absence of K+ in the culture medium for 15 days. (a) Glycolate oxidase activity. (b) Hydroxypruvate reductase activity. (c) Catalase activity. Results are the mean of three different experiments ± SEM. Asterisks indicate that differences between values were statistically significant at p < 0.05. Given the importance of the ascorbate-glutathione cycle to control the cellular H2O2 content in coordination with catalase, the activity of its enzymatic components (APX, MDAR, DHAR, and GR) was spectrophotometrically assessed (Figure 5a-d). In general, the activity of these enzymes was higher in roots than in leaves, and under K+ deficiency a significant increase of all these activities in both organs was found, except for MDAR in roots and DHAR in leaves that were unaffected. The data on APX activity correlates with the isozyme pattern obtained after native PAGE and specific in-gel activity staining. Two APX isozymes were detected in leaves and roots, being the most prominent isozymes in roots. Under K+ deficiency, there was a slight increase of APX isozymes in both organs (Figure 6a). Figure 5. Ascorbate-glutathione cycle activities in leaves and roots of 30-day-old C. maritima plants grown in the presence or absence of K+ in the culture medium for 15 days. (a) Ascorbate peroxidase (APX) activity. (b) Monodehydroascrobate reductase (MDAR) activity. (c) Dehydroascorbate reductase (DHAR) activity. (d) Glutathione reductase (GR) activity. Results are the mean of three different experiments ± SEM. Asterisks indicate that differences between values were statistically significant at p < 0.05. The analysis of POX isozyme patterns in C. maritima revealed essentially the same profile in leaves and roots, with a total of five isozymes designated I to V according to their increased mobility in the gel, with the intensity of the bands considerably higher in roots than in leaves (Figure 6b). Under K+ starvation, in leaves POX I, III, and IV increased. However, in roots there were no apparent changes due to K+ deficiency, except for a slight decrease of POX V, an isozyme that was not detected in leaves. Figure 6c shows the analysis of SOD isozyme activity patterns obtained after native PAGE and NBT staining of gels in both leaves and roots from C. maritima grown under optimal and low K+ supply. In leaves from control plants, only a single Fe-SOD was present in crude extracts. However, under K+ deficiency this isozyme was undetectable, Figure 5. Ascorbate-glutathione cycle activities in leaves and roots of 30-day-old C. maritima plants grown in the presence or absence of K + in the culture medium for 15 days. ( a ) Ascorbate peroxidase (APX) activity. ( b ) Monodehydroascrobate reductase (MDAR) activity. ( c ) Dehydroascorbate reductase (DHAR) activity. ( d ) Glutathione reductase (GR) activity. Results are the mean of three different experiments ± SEM. Asterisks indicate that differences between values were statistically significant at p< 0.05. The analysis of POX isozyme patterns in C. maritima revealed essentially the same profile in leaves and roots, with a total of five isozymes designated I to V according to their increased mobility in the gel, with the intensity of the bands considerably higher in roots than in leaves (Figure 6b). Under K + starvation, in leaves POX I, III, and IV increased. However, in roots there were no apparent changes due to K + deficiency, except for a slight decrease of POX V, an isozyme that was not detected in leaves. Figure 6c shows the analysis of SOD isozyme activity patterns obtained after native PAGE and NBT staining of gels in both leaves and roots from C. maritima grown under optimal and low K + supply. In leaves from control plants, only a single Fe-SOD was present in crude extracts. However, under K + deficiency this isozyme was undetectable, whereas until seven CuZn-SODs isozymes (designated as I to VII) were induced, CuZn-SODs I to III were the most prominent. In roots, two Mn-SOD and four CuZn-SOD (I to IV) isozymes were identified, with a light activity increase under K deficiency (Figure 6c). Antioxidants 2022,11, 401 9 of 18 Antioxidants 2022, 11, x FOR PEER REVIEW 9 of 19 whereas until seven CuZn-SODs isozymes (designated as I to VII) were induced, CuZn-SODs I to III were the most prominent. In roots, two Mn-SOD and four CuZn-SOD (I to IV) isozymes were identified, with a light activity increase under K deficiency (Figure 6c). Figure 6. Analysis of APX, POX, and SOD isozymes in C. maritima plants grown in the presence or absence of K+ in the culture medium for 15 days. (a) Ascorbate peroxidase (APX) isozymes (40 μg of proteins per lane). (b) Peroxidase (POX) isozymes (80 μg of proteins per lane). (c) Superoxide dismutase (SOD) isozymes (100 and 40 μg of proteins per lane were used for leaves and roots, respectively). The different isozymes were separated by native PAGE (8% for SOD, 10% for APX, and 6% for POX). 3.3. Metabolism of NADP-Dehydrogenases under K+ Deficiency in C. maritima Figure 7 illustrates the activity of the four main NADPH-generating enzymes, NADP-ICDH (Figure 7a), NADP-ME (Figure 7b), and the two enzymes of the oxidative pentose phosphate pathway, G6PDH and 6PGDH (Figure 7c,d, respectively). In general, the activity of these NADP-dehydrogenases was higher in roots than in leaves, and under K+ deficiency all activities underwent a significant increase in both organs. Figure 6. Analysis of APX, POX, and SOD isozymes in C. maritima plants grown in the presence or absence of K + in the culture medium for 15 days. ( a ) Ascorbate peroxidase (APX) isozymes (40 µ g of proteins per lane). ( b ) Peroxidase (POX) isozymes (80 µ g of proteins per lane). ( c ) Superoxide dismutase (SOD) isozymes (100 and 40 µ g of proteins per lane were used for leaves and roots, respectively). The different isozymes were separated by native PAGE (8% for SOD, 10% for APX, and 6% for POX). 3.3. Metabolism of NADP-Dehydrogenases under K+Deficiency in C. maritima Figure 7illustrates the activity of the four main NADPH-generating enzymes, NADPICDH (Figure 7a), NADP-ME (Figure 7b), and the two enzymes of the oxidative pentose phosphate pathway, G6PDH and 6PGDH (Figure 7c,d, respectively). In general, the activity of these NADP-dehydrogenases was higher in roots than in leaves, and under K + deficiency all activities underwent a significant increase in both organs. Antioxidants 2022, 11, x FOR PEER REVIEW 10 of 19 Figure 7. NADP-dehydrogenase activities in leaves and roots of 30-day-old C. maritima plants grown in the presence or absence of K+ in the culture medium for 15 days. (a) NADP-isocitrate dehydrogenase (ICDH) activity. (b) NADP-malic enzyme (ME) activity. (c) Glucose-6-phosphate dehydrogenase (G6PDH) activity. (d) 6-phosphogluconate dehydrogenase activity (6PGDH). Data represent the mean ± SEM of at least three different experiments. Asterisks indicate that differences between values were statistically significant at p < 0.05. 4. Discussion 4.1. K+ Deficiency Alters C. maritima Growth and Induces Oxidative Stress K+ deficiency disturbs water status in leaves and roots of C. maritima, suggesting osmotic stress caused by the lack of this element in the medium. The root is the first organ that senses K+ deficiency and consequently, a series of responses occur at physiological, biochemical, and molecular levels. According to [53], at an earlier phase of K+ deficiency exposure, cytosolic K+ content in plant tissue is regarded as one of the “master switches” which is responsible for plant transition from the ordinary metabolism to a “hibernated state”. In C. maritima roots, water stress could be explained by the fact that plant K+ status affects greatly the activity of aquaporins, as revealed by [54] who demonstrated that the activity of aquaporins in tomatoes was suppressed under low K+ supply and was associated with a decrease of water transport to shoots. Given its implication in osmotic adjustments, K+ deficiency is known to disturb transpiration causing a severe inhibition of leaf expansion [55], supporting our findings on leaves of C. maritima subjected to K+ starvation. K+ is implicated in the regulation of guard cells during stomatal movements and its deficiency leads to stomatal closure. Such behavior was documented in sunflowers and olives, in which the K+ status greatly affected stomata closure level [56]. This condition leads to an inhibition of photosynthesis rate, transpiration, stomatal conductance, and disturbing plant water relations [57] and, as a consequence, an imbalance between photosynthetic CO2 fixation and excessive accumulation of non-scavenged electrons may provoke the generation of reactive oxygen species (ROS) [58,59]. In Arabidopsis thaliana it was found that the loss of function of the chloroplast K+ Efflux Antiporters KEA1 and KEA2, located in the inner envelope membrane, provoked inefficient photosynthesis [60] and altered ROS homeostasis in leaves and roots [61]. 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