Kinetin mitigates Cd-induced damagesto growth, photosynthesis and PS II photochemistry of Trigonella seedlings by up-regulating ascorbate-glutathione cycle

About the Authors:

Gausiya Bashri

Roles Conceptualization, Data curation, Funding acquisition, Investigation, Writing – review & editing

* E-mail: [email protected] (GB); [email protected] (SMP); [email protected] (MJA)

Current address: Department of Botany, Aligarh Muslim University, Aligarh, India

Affiliation: Department of Botany, Ranjan Plant Physiology and Biochemistry Laboratory, University of Allahabad, Allahabad, India

Shikha Singh

Roles Formal analysis, Methodology, Software, Validation, Visualization, Writing – original draft

Affiliation: Department of Botany, Ranjan Plant Physiology and Biochemistry Laboratory, University of Allahabad, Allahabad, India

Sheo Mohan Prasad

Roles Conceptualization, Methodology, Project administration, Software, Supervision, Writing – review & editing

* E-mail: [email protected] (GB); [email protected] (SMP); [email protected] (MJA)

Affiliation: Department of Botany, Ranjan Plant Physiology and Biochemistry Laboratory, University of Allahabad, Allahabad, India

Mohammad Javed Ansari

Roles Conceptualization, Writing – review & editing

* E-mail: [email protected] (GB); [email protected] (SMP); [email protected] (MJA)

Affiliation: Department of Botany, Hindu College Moradabad, Mahatma Jyotiba Phule Rohilkhand University, Bareilly, India

ORCID logo https://orcid.org/0000-0002-8718-3078

Salma Usmani

Roles Formal analysis, Validation, Visualization, Writing – review & editing

Affiliation: Department of Biochemistry, D.K.M College for Women (Autonomous), Vellore, India

Saleh Alfarraj

Roles Conceptualization, Funding acquisition, Writing – review & editing

Affiliation: Zoology Department, College of Science, King Saud University, Riyadh, Saudi Arabia

Sulaiman Ali Alharbi

Roles Conceptualization, Funding acquisition, Writing – review & editing

Affiliation: Department of Botany & Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia

Marian Brestic

Roles Conceptualization, Funding acquisition, Writing – review & editing

Affiliation: Department of Plant Physiology, Slovak University of Agriculture, Nitra, Slovakia

Introduction

Contamination of air, soil and water due to various kinds of pollutants which are byproduct of industrial activity has created a worldwide problem [1–3]. In recent years, heavy metals are the most important contaminants causing serious threat to the life of every living being. Cadmium (Cd) is considered the most toxic of such pollutant metals because of its rapid uptake and translocation, combined with its ability to bind sulphahydryl group of enzymes/ proteins. Cadmium levels are increasing in agricultural soils due to the application of phosphate fertilizers and municipal Increased Cd inhibits plant growth by reducing photosynthesis by interfering with photosystem II (PS II) photochemistry, and by altering the leaf-ultrastructure, particularly that of chloroplasts [4]. A major mechanism of Cd- induced damage in plant is an increase in the generation of reactive oxygen species (ROS) due to disturbance of mitochondrion and chloroplast electron transport chain via the displacement of essential cations like Ca2+ and Zn2+ [5]. Increased generation of ROS, such as O2¯, H2O2, and HO¯ leads to oxidative stress by damaging the cell membranes through lipid peroxidation and proteins degradation, which results in disturbed cellular homeostasis [6, 7]. Plants protect themselves from ROS impairment through the mutual action of enzymatic and non-enzymatic antioxidants and the main pathway for scavenging of H2O2 is ascorbate (AsA)-glutathione (GSH) cycle [8, 9]. Here, the reduction of H2O2 into water is catalyzed by the enzyme ascorbate peroxidase (APX) with the help of AsA, which serve as an electronic donor. In addition, dehydroascorbic acid (DHA) is reduced by the enzyme dehydro ascorbate reductase (DHAR) by consuming the electrons provided by reduced glutathione (GSH) which oxidize into glutathione disulfide (GSSG). The reduction of GSSG into GSH is catalyzed by the enzyme glutathione reductase (GR). The AsA–GSH cycle is therefore crucial for maintaining the reductive environment in plant cells via the up-regulation of its enzymes under a variety of stress conditions [5, 9].

Plant growth regulators (PGRs) play crucial roles in the growth and development of the plants, by altering various important physiological and biochemical processes required by plants when exposed to heavy metal toxicity [10]. In recent years, many PGRs have been shown to ameliorate the negative impacts of heavy metal toxicity [5, 11]. Cytokinins (CKs) for example, take part in numerous physiological activities such as cell division and morphogenesis, flower and seed development and chloroplast development [12–16]. CKs can also regulate the abiotic stresses by the up-regulation of nitrogen metabolism and antioxidant defense system of plants [12, 17–21]; the importance of CKs in the regulation of heavy metal stress tolerance in plants is well known [11, 17, 18, 22], but the mechanism of CKs induced regulation of heavy metal stress tolerance needs further investigation. The work reported here aimed to determine the effect of exogenous application of different level of KN (KNL, KNM and KNH) on Trigonella seedlings (in order to counteract Cd phyto-toxicity) by examining their effects on growth and photochemistry of the PS II and AsA–GSH cycles.

Material and methods

Plant material and growth conditions

Seeds of Trigonella foenum-graecum L. var. Antara were procured from the Suttind seeds Pvt. Ltd, Delhi. The growth conditions used are the same as described by Bashri and Prasad [5]. The selected doses i.e. 3 mg Cd kg-1 soil (Cd1) and 9 mg Cd kg-1 soil (Cd2) of Cd were applied in the soil before seed-sowing. After 15 days of growth the seedlings were treated with three doses of KN, i.e., 10 μM (low, KNL), 50 μM (medium, KNM) and 100 μM (high, KNH) exogenously. The tested doses (KNL, KNM and KNH) of KN were selected on the basis of screening experiment with 1, 5, 10, 25, 50, 75 and 100 μM of KN. The experimental scheme contains twelve combinations i.e. control, Cd1, Cd2, KNL, Cd1+KNL, Cd2+KNL, KNM, Cd1+KNM, Cd2+KNM, KNH, Cd1+KNH, Cd2+KNH. All experiments were performed following 15 days of KN application.

Determination of growth, and pigments: Chlorophylls and carotenoids

Plant growth was analyzed by determining the fresh and dry weight, height and leaf area. Seedlings were harvested and their fresh weight was determined immediately; dry weight determination was achieved by drying the plant samples at 80°C for 48 h in hot air oven. The height of the plants was recorded by the meter scale. The leaf area of seedlings was analyzed by leaf area meter (Model—211, Systronics, India). For the estimation of pigments (chlorophylls and carotenoids), 20 mg fresh leaves were extracted with 80% (v/v) acetone and optical density of the supernatant was measured at 663.2, 646.5 and 470 nm spectrophotometrically [23].

Determination of carbonic anhydrase (CA) activity and rates photosynthesis and respiration

The activity of CA in leaves was measured by the method of Wilbur and Anderson [24]. The rate of photosynthesis and respiration was estimated in leaf discs using a Clark type oxygen electrode in terms of O2 evolution / consumption in the presence and absence of light, respectively and expressed as μmol oxygen evolved / consumed g-1 FW h-1 [25].

Chlorophyll a fluorescence (PS II photochemistry) measurements

Different JIP (JIP is a dark adapted chlorophyll fluorescence technique that is used for plant stress measurement)-parameters i.e. φP0 or Phi_P0, Ψ0 or Psi_0, φE0 or Phi_E0, PIABS, ABS/RC, TR0/RC, ET0/RC and DI0/RC of PS II photochemistry of control and treated seedlings were measured as chlorophyll a fluorescence through leaf fluorometer (FluorPen FP 100, Photon System Instrument, Czech Republic) in 30 minutes dark adapted leaves [26]. The values presented in the form of radar chart by normalizing all the data with their respective controls and control value for all parameters in radar chart is one.

Determination of hydrogen peroxide (H2O2): In vitro and in vivo analysis

Hydrogen peroxide determination was performed using 40 mg fresh leaves, and the supernatant was obtained by homogenization of these leaves in 3 ml of 0.1% (w/v) trichloro acetic acid after centrifugation (10,000 g for 15 min). The method described by Velikova et al. [27] was used; the amount of H2O2 in each sample is expressed as nmol g—1 FW. The localization of H2O2 in leaves was determined using a 1% solution of 3, 3’-diaminobenzidine, and bleaching with boiling ethanol, then photographed by digital camera [28].

Determination of activities of enzymes of ASC and GSH cycle

The activity of APX was determined by the method described by Nakano and Asada [18], with one unit of enzyme activity being defined as 1 nmol AsA oxidized min-1. Activity of DHAR was analyzed by extinction coefficient of 7.0 mM−1 cm−1; one unit of DHAR activity is defined as 1 nmol DHA reduced min-1 [29]. GR activity was estimated by extinction coefficient of 6.2 mM-1 cm-1; one unit of GR activity is defined as 1 nmol NADPH oxidized min -1 [30].

Determination of ascorbate and glutathione

Ascorbate content was determined as described by Gossett et al. [31]. The absorbance for ascorbate content was recorded at 525 nm and calculated using a standard curve prepared with L-ascorbic acid. Glutathione content was determined using the method described by Brehe and Burch [32] in a leaf homogenate, absorbance was recorded at 412 nm. Glutathione amount was then determined using a standard curve prepared with GSH.

Statistical analysis

Data presented is the means of triplicate (n = 3). One-way ANOVA test was performed to test the significance of data which was carried out at P<0.05 significance level using Duncan’s multiple range test (DMRT).

Results

Effect of KN on growth and photosynthetic pigments under Cd stress

Cadmium treatments significantly (P<0.05) reduced plant fresh and dry weight with increasing Cd concentration (Fig 1). Trigonella seedlings grown under Cd1 and Cd2 stress showed decrease in both fresh weight (by 13 and 20%) and in dry weight (by 8 and 14%);the reduction in leaf area was 6 and 12%, respectively. Supplementation of KNL (10 μM KN) significantly improved the repressing effects of Cd on growth, the effect being more pronounced under Cd1 stress.

[Figure omitted. See PDF.]

Fig 1. Impact of foliar application of kinetin on growth attributes of Trigonella seedlings exposed to Cd stress.

Data represent the mean value ± standard error from three replicates (n = 3). Values followed by the different letters at each bar differ at P<0.05 among treatments by the DMRT.

https://doi.org/10.1371/journal.pone.0249230.g001

Similarly, KNM (50 μM KN) marginally alleviated the Cd toxicity in Trigonella seedlings. High dose of KN (KNH,100 μM KN), in contrast, increased the Cd induced toxicity showing the decline of 17 and 26% in fresh weight and 18 and 34% in dry weight exposed to Cd stress (Cd1 and Cd2, respectively). Plant height also declined by 7 and 17% under Cd stress (Cd1 and Cd2, respectively). Exogenous KNL under the similar conditions, mitigated the Cd induced toxicity, the reduction being only 3 and 8% under Cd stress (Cd1 and Cd2, respectively). In contrast, KNH aggravate the Cd induced effect and the decline was 15 and 27%, respectively (Fig 1). Cadmium application at both doses significantly (P<0.05) decreased the Chl a, Chl b and Car contents in Trigonella seedlings. The exogenous application of kinetin at KNL and KNM doses in Cd treated seedlings resulted in a considerable reduction in all the pigment contents. On the other hand, KNH application lead to an additional declined in Chl a, Chl b and Car contents of Trigonella seedlings exposed to Cd stress (Table 1).

[Figure omitted. See PDF.]

Table 1. Effect of KN on photosynthetic pigment contents of Trigonella seedlings exposed to Cd stress.

https://doi.org/10.1371/journal.pone.0249230.t001

Effect of KN on CA activity, rate of photosynthesis and respiration under Cd stress

Cadmium toxicity decreased CA activity by 35 and 47% exposed to Cd stress (Cd1 and Cd2, respectively). Exogenous supplementation of KNL dose completely reinstated the CA activity at Cd1 dose while at Cd2 dose enhancement was recorded, showing only 5% decrease in CA activity. The application of KNH further decreased the CA activity under Cd stress (Fig 2B). As with CA activity, the rate of photosynthesis was significantly (P<0.05) inhibited by Cd in Trigonella seedlings which showed a 16 and 24% decrease, respectively under Cd1 and Cd2 stress. Exogenous kinetin at KNL and KNM doses significantly ameliorated Cd-induced inhibition while KNH treatment further inhibited the photosynthetic activity showing 24 and 29% reduction, respectively under Cd1 and Cd2 stress (Fig 2A). The respiratory activity was enhanced significantly under both the doses of Cd in leaves Trigonella seedlings, showing increases of 7 and 16%, respectively under Cd1 and Cd2 stress. The exogenous application of KNL and KNM doses considerably decreased the O2 uptake under Cd stress. On the other hand, KNH dose supplementation resulted in an enhancement of 11 and 17% in the respiratory activity under Cd1 and Cd2 dose, respectively (Fig 2C).

[Figure omitted. See PDF.]

Fig 2. Impact of foliar application of KN on photosynthetic O2 yield, carbonic anhydrase activity and respiration rate of Trigonella seedlings exposed to Cd stress.

Data represent the mean value ± standard error from three replicates (n = 3). Values followed by the different letters at each bar differ at P<0.05 among treatments by the DMRT.

https://doi.org/10.1371/journal.pone.0249230.g002

Effect of KN on PS II photochemistry under Cd stress

The Photochemistry of PS II were analyzed in Trigonella seedlings exposed to Cd stress showed a marginal decrease in φP0 or Phi_P0, Ψ0 or Psi_0 φE0 at both the doses of Cd whereas PIABS declined significantly. Foliar application of KNL and KNM doses under Cd stress showed a significant alleviation in φP0, Ψ0, φE0 and PIABS however, with KNH dose (Cd+KNH) the values of φP0, Ψ0, φE0 and PIABS were showing further reduction. Moreover, Cd stress enhanced the energy flux parameters i.e. ABS, TR0, ET0 and DI0 per RC (Fig 3). Kinetin application at KNL and KNM doses significantly reduced the energy flux parameters in Trigonella seedlings exposed to Cd stress. On the other hand, KNH application caused a further increase in energy flux parameters (Fig 3).

[Figure omitted. See PDF.]

Fig 3. Impact of foliar application of KN on JIP—parameters in Trigonella seedlings exposed to Cd stress.

https://doi.org/10.1371/journal.pone.0249230.g003

Impact of KN on hydrogen peroxide level under Cd stress

Cadmium at Cd1 and Cd2 doses led to an increase in H2O2, by 28 and 67%, respectively. The exogenous application of KN at low and middle doses led to a significant (P<0.05) lowering in H2O2 content. In contrast, higher doses of KN (KNH) caused a further rise in H2O2 content over the values recorded in Cd1 and Cd2 treated seedlings (Fig 4A). The in vivo localization of H2O2 was performed with DAB. Cadmium treated seedlings showed a significant accumulation of brown precipitate as compared to the control which depends upon H2O2 content. Brown precipitates were less intense in the low and middle doses of KN treated Trigonella seedlings under Cd stress, indicating that H2O2 accumulation was lower in exogenously treated KNL and KNM seedlings. In contrast, higher dose (KNH) alone and together with Cd stressed seedlings exhibited a more intense brown color in leaf than that seen in the respective control (Fig 4B).

[Figure omitted. See PDF.]

Fig 4. Impact of foliar application of KN on in—vivo localization of hydrogen peroxide in Trigonella seedlings exposed to Cd stress.

Trigonella leaves stained with DAB, and brown area shows hydrogen peroxide content.

https://doi.org/10.1371/journal.pone.0249230.g004

Impact of KN on AsA-GSH cycle under Cd stress

Cadmium treatments at both the doses significantly (p<0.05) increased the activities of APX, DHAR (except at Cd2) and GR. The exogenous application of KN at low and middle doses further increased the rate of activity of these enzymes exposed to Cd stress; and these effects being most pronounced following KNL treatments. The application of KNH under Cd treatment showed variable responses (Fig 5). Cadmium stress caused a significant decrease in contents of AsA and GSH while the contents of DHA and GSSG were increased that result in sharp decrease in the ratios of AsA/DHA and GSH/GSSG. The exogenous application of KN at low (KNL) and middle (KNM) doses increased AsA and GSH and decreased DHA and GSSH as a result of this ratios of AsA/DHA and GSH/GSSG significantly increased in Cd-exposed seedlings; KNH application resulted in opposite trends in the contents of AsA, DHA, GSH, GSSG and the ratios of AsA/DHA and GSH/GSSG (Table 2).

[Figure omitted. See PDF.]

Fig 5. Impact of foliar application of KN on ascorbate peroxidase, dehydroascorbate reductase and glutathione reductase activities of Trigonella seedlings exposed to Cd stress.

Data represent the mean value ± standard error from three replicates (n = 3). Values followed by the different letters at each bar differ at P<0.05 among treatments by the DMRT.

https://doi.org/10.1371/journal.pone.0249230.g005

[Figure omitted. See PDF.]

Table 2. Impact of KN on the contents of reduced asorbate (AsA), dehydroascorbate (DHA), reduced glutathione (GSH), and oxidized glutathione (GSSG), and ratios of AsA/DHA and GSH/GSSG in Trigonella seedlings exposed to Cd stress.

https://doi.org/10.1371/journal.pone.0249230.t002

Discussion

In present study, cadmium (Cd1 and Cd2) inhibited the growth of Trigonella seedlings, as shown by decreased plant height, leaf area and fresh and dry weight (Fig 1). The exogenous application of KN at KNL (10 μM) and KNM (50 μM) improved growth under Cd stress and the effect of KNL (10 μM) being more pronounced. In consistent with our results, Zhou et al. [33] also reported better growth on exogenous application of cytokinin trans-zeatine riboside (10 μM) in Kosteletzkyapenta carpos under Zn stress. The observed KN-induced abatement of Cd toxicity on growth may be due to a decrease in Cd content in the plant as reported in earlier studies of eggplant [17]. In contrast to KNL and KNM, KNH application aggravates Cd-induced toxicity. This may be explained on the basis of excess accumulation of ROS and less efficient antioxidant system.

Since the growth of autotroph depends on photosynthesis; hence the negative impact on growth of Cd stress can be correlated with an alteration in photosynthesis. Cadmium at both the doses decreased photosynthetic pigments (Chl a, Chl b and Car), a result which agrees with the findings of Ahanger et al. [18], in which Cd was shown to repress chlorophyll biosynthesis. The ameliorating effect of KN on photosynthetic pigment content could result from the regulatory effects of cytokinins on the biogenesis of chloroplast, as observedn wheat plants exposed to drought stress [34]. Further, cytokinins have been shown to stimulate the tetrapyrrole synthesis, hence supported the functioning of chloroplast in barley seedlings [35]. Another possibility could be due to decrease in reactive oxygen species like H2O2 (Fig 4) which is known to induce damaging effects on these pigments following Cd exposure under KN treatment. In contrast, the further degradation in pigment content seen in test seedlings treated with high doses of (KNH) of KN may be explained on the basis of excess accumulation of ROS (Fig 4) which probably triggered the degradation process at a greater rate under Cd stress. Photosynthetic oxygen yield during light reaction showed a direct link with photo-fixation of CO2 during Calvin cycle, hence Cd induced decrease in O2 yield in Trigonella seedlings, which leads to a decline in plant biomass accumulation, as observed in the present study (Fig 1). Popova et al. [36] reported that the exposure of pea plants to Cd at early stages of their establishment caused a decrease in the rate of CO2 fixation and the activity of Rubisco in the pea leaves. The significant improvement in photosynthetic activity seen in KN treated seedlings could be explained on the basis of (i) rise in light harvesting pigments i.e. Chl a, Chl b and Car contents (Table 1) and (ii) improved PS II activity as indicated by chlorophyll fluorescence activity (Fig 3). Aldesuquy et al. [34] reported that KN pre-treated wheat plant showed significant increase in the PS II activity and under salt stress; KN also ameliorated the PS II activity appreciably. Cadmium declined the activity of CA in test seedlings which might have resulted following the down regulation of genes for CA, as has been reported in Populus tremula under Cd stress [37]. It is also possible that Cd treatment may reduce Zn transport across the cell membrane, as Cd enters the cell via a Zn transporter protein [38]. Thus, decrease in Zn concentration in the leaf tissue may be one of the causes of decreased CA activity seen in the present study. Trigonella seedlings treated with low and middle doses of KN showed significant (P<0.05) improvement in CA activity under Cd stress. Fariduddin et al. [39] have similarly reported that KN and brassinosteroid treatment increased CA activity in Vigna radiata. The rate of O2 uptake was significantly increased by exposure to both doses of Cd, which can be explained on the basis of supply of ATP needed to carry out basic metabolism of plants, following a possible increase in rate of respiratory electron transport [40]. Cadmium treated seedlings on KN exposure exhibited a lower increase in rate of O2 uptake (Fig 2C). Al-Hakimi [41] also observed KN-induced decrease in respiration rate during Cd-stress in pea seedlings. To further validate the action of Cd on PS II, the photochemistry of PS II was analyzed in Trigonella seedlings exposed to Cd stress. Decrease in the values of φP0, Ψ0, φE0 and PIABS on treatment with the Cd (Fig 3) showing the inactivation of PS II and these results are in consistent with the earlier report on Pontederia cordata [4]. Further, Cd-induced increase in ABS/RC, TR0/RC, ET0/RC and DI0/RC could be explained on the basis of inactivation of active RC might be due to greater dissipation of flux [42]. KN induced improvements in JIP parameters under Cd exposure could be correlated with (i) anti senescing effect of KN, (ii) restoration in the status of D1 protein by increasing the antioxidants (Fig 5 and Table 2), as reported in cytokinin enriched Arabidopsis seedlings exposed to light stress [43], (iii) increased number of active RC (Fig 3). Tarakhovskaya et al. [44] also reported that KN increases the structural elements of Chl a including the PS II RC.

In the present study, an over accumulation of H2O2 was observed following cadmium treatment (Fig 4) which points towards the oxidative stress induced by Cd [17, 18]. However, kinetin application, at low and medium concentration declined the level of H2O2, under Cd stress. In another study, kinetin treatment has been shown to improve the growth of root and shoot of maize seedlings, while it decreased the electrolyte leakage from leaf discs under Zn and Ni stress [45]. To remove the excess level of H2O2, one of the major mechanisms is AsA-GSH cycle. Although, Cd treatments increased the activities of APX, DHAR (except at Cd2) and GR but this increase was not sufficient for curtailing oxidative damage as evident with decreased growth (Fig 1). The exogenous application of KN at low and middle doses further enhanced the activity of APX, DHAR and GR in Trigonella seedling under Cd stress (Fig 5), suggesting the detoxification of H2O2 via AsA-metabolizing pathways by enzyme up-regulation. Cadmium stress caused significant decline in the contents of AsA and GSH and ratios of AsA/DHA and GSH/GSSG. The decrease in the level of AsA and GSH contents might be resulted due to their utilization in AsA-GSH cycle as electron donors and in synthesis of phytochelatins (particularly GSH) for metal detoxification. The exogenous application of KN at low (KNL) and middle (KNM) doses improved the AsA and GSH contents and declined the DHA and GSSG contents, resulting in increased ratios of AsA/DHA and GSH/GSSG in Cd exposed seedlings this may help Trigonella seedlings to preserve redox status under Cd stress. Wu et al. [46] also observed a BAP-induced increase of AsA and GSH contents under Zn stress. The application of KNH under Cd treatment showed a variable response (Fig 5). In addition, glutathione synthetase and GR maintained the level of GSH, which ise involved in the biosynthesis and recycling pathways of GSH, respectively [8]. In the present work, a KN-induced increase of GSH content within the Cd-treated plants suggests that KN alters the regeneration of the GSH pool by stimulating GR enzyme. Our findings agree with those of Piotrowska-Niczyporuk et al. [11] who showed accelerated sulfur assimilation pathway under auxins and cytokinins treatment which led to increases in the synthesis of GSH in A. obliquus resulting in enhanced tolerance under Pb stress.

Conclusions

We conclude that Cd has caused oxidative stress in Trigonella seedlings, and AsA-GSH cycle was not able to control the Cd induced damage which disturbed the redox status of the cell as manifested by decreased AsA/DHA and GSH/GSSG ratios. Supplementation of KN at low and medium doses up-regulated the enzymes of AsA-GSH cycle that maintains the redox status of cells, as evidenced by the increased content of AsA and GSH and ratios of AsA/DHA and GSH/GSSG which enhanced the tolerance of Trigonella seedlings under Cd stress.

Citation: Bashri G, Singh S, Prasad SM, Ansari MJ, Usmani S, Alfarraj S, et al. (2021) Kinetin mitigates Cd-induced damagesto growth, photosynthesis and PS II photochemistry of Trigonella seedlings by up-regulating ascorbate-glutathione cycle. PLoS ONE 16(6): e0249230. https://doi.org/10.1371/journal.pone.0249230

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