Heatwave Effects on the Photosynthesis and Antioxidant Activity of the Seagrass Cymodocea nodosa under Contrasting Light Regimes

: Global climate change, speciﬁcally the intensiﬁcation of marine heatwaves, affect seagrasses. In the Ria Formosa, saturating light intensities may aggravate heatwave effects on seagrasses, particularly during low spring tides. However, the photophysiological and antioxidant responses of seagrasses to such extreme events are poorly known. Here, we evaluated the responses of Cymodocea nodosa exposed at 20 ◦ C and 40 ◦ C and 150 and 450 µ mol quanta m − 2 s − 1 . After four-days, we analyzed (a) photosynthetic responses to irradiance, maximum photochemical efﬁciency (Fv/Fm), the effective quantum yield of photosystem II ( φ PSII); (b) soluble sugars and starch; (c) photosynthetic pigments; (d) antioxidant responses (ascorbate peroxidase, APX; oxygen radical absorbance capacity, ORAC, and antioxidant capacity, TEAC); (d) oxidative damage (malondialdehyde, MDA). After four days at 40 ◦ C, C. nodosa showed relevant changes in photosynthetic pigments, independent of light intensity. Increased TEAC and APX indicated an “investment” in the control of reactive oxygen species levels. Dark respiration and starch concentration increased, but soluble sugar concentrations were not affected, suggesting higher CO 2 assimilation. Our results show that C. nodosa adjusts its photophysiological processes to successfully handle thermal stress, even under saturating light, and draws a promising perspective for C. nodosa resilience under climate change scenarios.


Introduction
Seagrass meadows are among the most productive ecosystems on earth and deliver significant ecosystem services in shallow marine habitats. Despite their relevance, seagrasses are under threat [1,2], with wide losses being attributed to disease [3], anthropogenic impacts [1], and to the increased frequency of extreme climatic events, such as marine heatwaves (MHW) [4][5][6][7]. MHW are generally defined as transient periods of extremely warm water, where the temperature exceeds the 90th percentile of a 30-year baseline database [8]. Marine heatwaves put seagrass growth and survival at risk and affect their distribution with subsequent impacts on the associated habitats [7].
Temperature is a key factor, affecting enzyme activity and metabolism of marine organisms [9], which, according to the species-specific tolerance, may be translated to an imbalance in photosynthesis and respiration. Reported effects of high temperatures (HT) on seagrasses include changes in photosynthesis [10] and respiration [11]. Inhibition or decrease of photosynthesis and increase in respiration due to HT or MHW was described in Z. marina [9], Thalassia hemprichii, Enhalus acoroides [11], and P. oceanica [12]. The negative impact of high temperature on chlorophyll fluorescence, indicating damage or inactivation of the photosynthetic apparatus, was observed in Z. marina and T. hemprichii [13]. In addition, high temperature can also induce changes in seagrass cell membranes [10], photosynthetic pigments (chlorophyll a and chlorophyll b), and strong oxidative damage [14].
In the Ria Formosa lagoon, southern Portugal (37 • N, 008 • W), Cymodocea nodosa is abundant in the shallow subtidal, where physiologically demanding conditions may occur, particularly in the summer during low spring tides, when the plants are confined to relatively small ponds where water temperature and light reach very high values. C. nodosa plants were collected in May and June 2011, when seawater temperature ranged between 18 • C and 30 • C (https://www.hidrografico.pt/boias, accessed on 30 May 2011), and light at the canopy level ranged between 230 and 400 µmol quanta m −2 s −1 (Li-192SA underwater quantum sensor connected to Li-1000 data logger; Li-Cor).

Experimental Design
C. nodosa plants, including rhizomes and roots, with the apical meristem, and three or more shoots with 3-4 leaves each, were harvested from the middle of a large meadow and immediately replanted in glass aquaria (9.5 × 15 × 7.5 cm, 23 shoots each), using sterilized fine sand (pore size < 2 mm) as substrate to cover the rhizomes (ca 2-4 cm depth).
Three independent aquaria were used as replicates for each treatment (n = 3). Aquaria were filled with natural seawater that was continuously aerated and renewed every 48 h. Prior to renewal, seawater temperature was brought to the experimental level for 24 h. The experiment was run in a walk-in growth chamber, with incorporated temperature control (Aralab 1000 Thin), in a factorial four-day set-up of water temperature (20 • C and 40 • C) and light (150 and 450 µmol quanta m −2 s −1 ), provided by OSRAM Powerstar HOI-BT 400W/D Daylight lamps, in combination with 100W Halolux Ceram lamps) with a photoperiod of 12 h:12 h. The lower light level was obtained by covering the aquaria with neutral greenhouse shading nets. The temperature was monitored continuously with a HOBO temperature logger, and light intensity inside the leaf canopy was checked daily with a Li-192SA underwater quantum sensor connected to a Li-1000 data logger (Li-Cor). The choice of an extreme heatwave temperature of 40 • C was selected based on temperatures previously measured by the authors in the water ponds formed during low spring tides in the Ria Formosa; 40 • C is 5 • C higher than the highest temperatures measured. The higher light intensity was selected to saturate for C. nodosa (minimum saturation irradiance: 397.68 ± 40.08 µmol quanta m −2 s −1 , [28]).

Photosynthesis and Dark Respiration
Photosynthesis-irradiance (P-I) curves (n = 3) were determined at the experimental temperatures with an oxygen electrode (DW3/CB1, Hansatech, Norfolk, UK) according to Silva et al. [29]. Briefly, the middle part of the second or third leaf (mature leaves) of each replicate was mounted vertically inside the measuring chamber, for an even exposure to the different incident light intensities. Dark respiration was measured using the same procedure in the dark. Data points that deviated from the upper and lower quartiles more than 1.5-fold the interquartile range were considered outliers and, thus, were not included in the analysis [30]. P-I curves were fitted with the mathematical model equation by Jassby and Plat [31].
Both the maximum quantum efficiency of photosystem II photochemistry (Fv/Fm) and the effective quantum yield of photochemical energy conversion in PSII (φPSII) were measured at the end of the experiment (four days), using a pulse amplitude modulated fluorometer (Diving-PAM, Heinz Walz, Effeltrich, Germany). For Fv/Fm measurements, plants were dark-adapted for 30 min prior to fluorescence measurements.

Biochemical Analysis
To avoid the influence of tissue age on the responses to temperature and light [29,32], only the middle parts of the second and third leaf of each shoot of C. nodosa (excluding the apex and the meristem) were used for biochemical analysis. C. nodosa leaf samples were collected and prepared as described in [33]. Briefly, the samples were cleaned of epiphytes, rinsed with distilled water, blotted dry and immediately frozen in liquid nitrogen, and stored at −80 • C until analysis. The whole procedure took about 5 min per sample. Leaf sampling was completely performed inside the walk-in growth chamber at the experimental light and temperature conditions.

Antioxidant Responses
The methods used to evaluate the antioxidant scavenging capacity can be divided in two groups [34]: assays that quantify the protection capacity against peroxyl radicals (ROO•) through hydrogen atom transfer (HAT) and assays that quantify the total antioxidant capacity based on a single electron transfer (ET). Here we assessed the global antioxidant capacity of the seagrass C. nodosa using both types of assay, respectively oxygen reactive absorbance capacity (ORAC) and trolox equivalent antioxidant capacity (TEAC). ORAC quantifies the antioxidant capacity of phenolic compounds and tocopherols. It has been extensively used by pharmaceutical, nutritional and food industries to determine total antioxidant capacity [35]. TEAC quantifies the antioxidant activity related with carotenoids, phenolic compounds, superoxide dismutase, ascorbate peroxidase, and reducing agents such as ascorbate, glutathione, and NADPH. It has been widely used to determine the antioxidant capacity of terrestrial plants [36,37]. The extraction for ORAC and TEAC quantification was performed simultaneously. Frozen leaf samples (300 mg, n = 3), were powdered in liquid nitrogen, suspended in 0.1 N hydrochloric acid (HCl), and kept overnight under constant agitation at 4 • C followed by centrifugation at 4700× g for 30 min. Quantifications were performed on the supernatant.
To assess the ascorbate peroxidase (APX) activity, frozen leaf tissue (800 mg, n = 3) was powdered in liquid nitrogen with polyvinylpolypyrrolidone (PVPP) and sodium ascorbate and then extracted in 5 mL of 100 mM potassium phosphate buffer (pH 7.8) with 2% triton-x and 10 mM ascorbate. Extracts were centrifuged at 4 • C, 3500× g for 30 min. Supernatants were purified by filtration with Sephadex PD-10 G-25 columns (GE Healthcare) [39], previously equilibrated with 20 mL of 100 mM potassium phosphate buffer (pH 7.0) with 1 mM ascorbate. APX activity was measured at 25 • C, by following for 3 min the decrease in absorbance at 290 nm of a mixture containing 50 mM potassium phosphate buffer (pH 7.0), 8 mM ascorbate, and 20 mM hydrogen peroxide (adapted from [40]). APX activity was calculated after subtraction of the control rates, where the enzyme extract was replaced by the potassium phosphate buffer, using ε = 2.8 mM −1 cm −1 . One unit (U) of APX is equivalent to the protein necessary to oxidize one µmol of ascorbate per minute. Enzyme activity was expressed in U mg −1 of leaf dry weight (DW).
To evaluate oxidative damage, malondialdehyde (MDA) was quantified. Lipids are a major component of cell membranes, and the breakdown products of membrane fatty acid peroxides, such as MDA, are commonly used as reliable markers of oxidative stress [41].
MDA quantification was performed according to [42]. Frozen leaf tissue (300 mg, n = 3) was powdered in liquid nitrogen, suspended in 80% aqueous ethanol and then centrifuged at 3000× g for 10 min. The supernatant was added to a solution of 20% trichloroacetic acid (TCA) with 0.65% thiobarbituric acid (TBA) and 0.015% butylated hydroxytoluene (BHT). Two blanks were performed, either without TBA or with 80% ethanol instead of sample extract. All samples and blank reaction mixtures were incubated at 90 • C for 25 min., then cooled for 15 min and again centrifuged. Absorbance of the supernatants were read at 440, 532, and 600 nm in a Beckman Coulter DU-650 spectrophotometer, and MDA equivalents were calculated as in [42].

Photosynthetic Pigments
Frozen leaf tissue (200 mg, n = 3) was powdered in liquid nitrogen and sodium ascorbate, and photosynthetic pigments were extracted under dim light in 5 mL of acetone 100% and NaHCO 3 [43]. Extracts were filtered sequentially with 5.0 µm LS membrane and PTFE 0.2 µm hydrophobic filters. Chlorophylls a and b were quantified spectrophotometrically, using the equations of [44], while carotenoids were analyzed by isocratic high performance liquid chromatography (HPLC) as in [45] after [46]. HPLC analysis of extracts and standards (20 µL) were carried out in an Alliance Waters 2695 separation module, with a Waters 2996 photodiode array detector and a Waters Novapak C18 radial 8 × 100 mm compression column (4 µm particle size). During injection, extracts were maintained at 5 • C. The mobile phase was pumped at a 1.7 mL flow. The mobile phase A, acetonitrile:methanol:triethylamine (7.5:1:0.7), was fluxed through the column, in an isocratic 3.5 min step, followed by mobile phase B, acetonitrile:methanol:miliq water:ethyl acetate (7:0.96:0.04:8) in a 6.5 min isocratic step. Between injections, the column was equilibrated with mobile phase A for 5 min. All eluents were prepared with HPLC grade solvents (VWR HiPerSolv Chromanorm), filtered, and sonicated prior to use. During all chromatographic analysis, the column was kept at a steady temperature (24 • C). Calibration was done with commercially available pigments (CaroteNature, Lupsingen, Switzerland). Peak areas were monitored at 450 nm. Pigment concentrations were calculated based on peak areas from standards in known concentrations. For calibration curves, all standard dilutions were injected eight times for each pigment. The de-epoxidation state (DES) of the xanthophyll cycle pigments was calculated as (A+Z)/(V+A+Z) [47,48], where A stands for antheraxanthin, Z for zeaxanthin, and V for violaxanthin.

Soluble Sugars and Starch
Freeze-dried leaf tissue (10 mg, n = 3) was ground to powder, in a ball mil (RETSCH, MM 300, extracted with ethanol 80% for 10 min at 80 • C and centrifuged at 2000× g 5 min (adapted from [49]). The supernatant was collected, and the pellet resuspended in ethanol for an additional extraction. This procedure was repeated three consecutive times. The supernatants from the three-step extraction were combined and used for soluble sugars quantification by phenol-sulfuric method [50]. Calibration was performed using glucose as standard.
For starch quantification, the pellet from soluble sugars extraction was washed 3 times with distilled water, resuspended in 1 mL of distilled water, and incubated for 10 min at 100 • C. Starch was hydrolyzed by a 24 h incubation in an enzymatic solution with 1000 U/mg α-amylase and 14U/mL amyloglucosidase. After hydrolysis, starch was quantified in the extracts as glucose equivalents as described for soluble sugars.

Data Analysis
All results are presented as mean ± standard error. The effects of temperature (20 • C, 40 • C) and light (150 and 450 µmol quanta m −2 s −1 ) on the variables measured were tested using Two-way analyses of variance (ANOVA) after checking parametric assumptions. The Student-Newman-Keuls post-hoc test was used for significant differences between factor levels. All data treatment and statistical analysis were performed using Sigma Stat/SigmaPlot (SPSS Inc., v. 11) software package.

Photosynthesis and Dark Respiration
Both the maximum quantum efficiency (Fv/Fm) and the effective quantum yield of photochemical energy conversion in PSII (φPSII) decreased significantly with light (Figure 1a,b). As opposed to Fv/Fm, φPSII increased with temperature (Figure 1a,b).
Maximum photosynthesis (Pmax) (Figure 1c) was higher at 20 • C than at 40 • C, independently of light. Although, Pmax was not affected by light, at 40 • C and 450 µmol quanta m −2 s −1 , P-I results were to disperse and did not allow de mathematical adjustment of the curve.
Dark respiration increased 3.95-fold with temperature (Figure 1d), and although it was not significantly affected by light, at 20 • C and 450 µmol quanta m −2 s −1 , dark respiration was lower that at 150 µmol quanta m −2 s −1 .
Maximum photosynthesis (Pmax) (Figure 1c) was higher at 20 °C than at 40 °C, independently of light. Although, Pmax was not affected by light, at 40 °C and 450 μmol quanta m −2 s −1 , P-I results were to disperse and did not allow de mathematical adjustment of the curve.
Dark respiration increased 3.95-fold with temperature (Figure 1d), and although it was not significantly affected by light, at 20 °C and 450 μmol quanta m −2 s −1 , dark respiration was lower that at 150 μmol quanta m −2 s −1 .

Antioxidant Responses
The experimental heatwave of 40 • C increased both the antioxidant activity quantified by TEAC (Figure 2a) and of the APX activity (Figure 2b). APX activity was 1.4-fold higher in leaves at 40 • C than at 20 • C. ORAC was not affected by temperature (Figure 2c) and none of the antioxidant responses tested were affected by light (Figure 2).

Antioxidant Responses
The experimental heatwave of 40 °C increased both the antioxidant activity quantified by TEAC (Figure 2a) and of the APX activity (Figure 2b). APX activity was 1.4-fold higher in leaves at 40 °C than at 20 °C. ORAC was not affected by temperature (Figure 2c) and none of the antioxidant responses tested were affected by light (Figure 2). The increase in light or temperature did not cause oxidative damage, as measured by malondialdehyde MDA (Figure 2d).

Photosynthetic Pigments
Significant changes occurred in the balance between carotenoids and chlorophylls in response to the higher temperature (40 °C) where, in general, carotenoids content decreased relatively to chlorophyll due to the significant increase in leaf total chlorophyll content (Supplementary Materials Table S1). The increase in light or temperature did not cause oxidative damage, as measured by malondialdehyde MDA (Figure 2d).

Photosynthetic Pigments
Significant changes occurred in the balance between carotenoids and chlorophylls in response to the higher temperature (40 • C) where, in general, carotenoids content decreased relatively to chlorophyll due to the significant increase in leaf total chlorophyll content (Supplementary Materials Table S1).
Foliar chlorophyll b concentration was higher under 40 • C no matter the light intensity, as opposed to chlorophyll a that was not affected by temperature and light intensity (Supplementary Materials Table S1). In addition, higher chlorophyll b at 40 • C resulted in significantly higher total chlorophyll (Supplementary Materials Table S1).
There were no changes in total carotenoid foliar content with temperature or light (Supplementary Materials Table S1). Additionally, the sum of the pigments of the xanthophyll cycle, violaxanthin (V), antheraxanthin (A) zeaxanthin (Z) did not change with light or temperature (Supplementary Materials Table S1). However, the de-epoxidation index (DES) decreased under high temperature, but this decrease was only significant under low light (150 µmol quanta m −2 s −1 ) (Figure 3a). As expected, DES was higher under the higher irradiance at both the temperatures tested.  Table S1). In addition, higher chlorophyll b at 40 °C resulted in significantly higher total chlorophyll (Supplementary Materials Table S1).
There were no changes in total carotenoid foliar content with temperature or light (Supplementary Materials Table S1). Additionally, the sum of the pigments of the xanthophyll cycle, violaxanthin (V), antheraxanthin (A) zeaxanthin (Z) did not change with light or temperature (Supplementary Materials Table S1). However, the de-epoxidation index (DES) decreased under high temperature, but this decrease was only significant under low light (150 μmol quanta m −2 s −1 ) (Figure 3a). As expected, DES was higher under the higher irradiance at both the temperatures tested. Values represent mean ± SE (n = 3). Different letters indicate significant differences among light levels under the same temperature (p < 0.05, SNK test); * indicate significant differences among temperatures under the same light level (p < 0.05, SNK test); capital letters indicate differences among temperature independently of light level (no interaction among factors; p < 0.05, SNK test).

Soluble Sugars and Starch
There were no significant differences among soluble sugars concentrations in C. nodosa leaves submitted to irradiance and temperature treatments (Figure 4a). However, starch concentration in those leaves was almost twice higher at 40 °C than at 20 °C ( Figure  4b). Light treatments did not influence starch concentrations. Values represent mean ± SE (n = 3). Different letters indicate significant differences among light levels under the same temperature (p < 0.05, SNK test); * indicate significant differences among temperatures under the same light level (p < 0.05, SNK test); capital letters indicate differences among temperature independently of light level (no interaction among factors; p < 0.05, SNK test).

Soluble Sugars and Starch
There were no significant differences among soluble sugars concentrations in C. nodosa leaves submitted to irradiance and temperature treatments (Figure 4a). However, starch concentration in those leaves was almost twice higher at 40 • C than at 20 • C (Figure 4b). Light treatments did not influence starch concentrations.

Discussion
This work revealed that Cymodocea nodosa might show signs of stress under a heatwave of four days at 40 °C, as indicated by the increase in antioxidant response (TEAC and APX concentrations). These signs of stress were not aggravated by high light. There were also important changes in photosynthetic pigments as well as in photosynthetic and dark respiration processes. However, the species was able to tolerate well this experimental heatwave since no oxidative damage, measured by MDA, was detected.
Relevant changes of the relative concentrations of chlorophyll a, b, and carotenoids/chlorophyll ratio occurred at 40 °C. The decrease of the C. nodosa chlorophyll a/b

Discussion
This work revealed that Cymodocea nodosa might show signs of stress under a heatwave of four days at 40 • C, as indicated by the increase in antioxidant response (TEAC and APX concentrations). These signs of stress were not aggravated by high light. There were also important changes in photosynthetic pigments as well as in photosynthetic and dark respiration processes. However, the species was able to tolerate well this experimental heatwave since no oxidative damage, measured by MDA, was detected.
Relevant changes of the relative concentrations of chlorophyll a, b, and carotenoids/ chlorophyll ratio occurred at 40 • C. The decrease of the C. nodosa chlorophyll a/b ratio, also detected by other authors in other seagrass species submitted to heatwaves [51], suggests an unbalance between light harvesting and electron release into the electron transport chain, favoring the former and thus potentially leading to the overexcitation of chlorophyll a and the formation of chlorophyll triplets ( 3 Chl*). Furthermore, the generalized decrease of carotenoids relative to chlorophyll unveils a waning capacity to dissipate excess energy, both in the reaction centers and light harvesting complexes. Both conditions favor the energy transfer from overexcited chlorophyll a to ground state O 2 generating singlet oxygen ( 1 O 2 ) and other reactive oxygen species (ROS) [52].
The de-epoxidation state of the pigments of the xanthophyll cycle (DES) typically increases with increasing light, especially when light intensity approaches photosynthetic saturation [53]. Zeaxanthin accumulation protects the photosynthetic antennae complexes by promoting the dissipation of excess excitation energy as heat and by quenching the chlorophyll triplet ( 3 Chl*) and oxygen singlet ( 1 O 2 ) [54][55][56]. As expected, we also observed higher DES under high light (450 µmol quanta m −2 s −1 ), driven by higher zeaxanthin concentration (Supplementary Materials Table S1), which indicates higher photoprotection capacity. The increase of DES under high light was followed by the decrease in the maximum quantum efficiency of PSII (Fv/Fm) and in the maximum photosynthetic rate (Pmax), as expected, reflecting the increase of energy dissipation away from the PSII reaction centers [48].
The antioxidant response of C. nodosa, quantified by TEAC, was higher under the experimental heatwave, revealing processes of maintaining ROS concentrations below the level that causes cell damage [57], such as tocopherols, zeaxanthin, and carotenes, among others [58,59]. Tocopherols act together with VAZ-cycle to scavenge species as oxygen singlet under stressful situations [60,61]. The increased activity of C. nodosa APX under the heatwave is probably a response to the accumulation of H 2 O 2 [62], which is an important signaling ROS molecule for stress acclimation [57,63,64]. APX removes excess H 2 O 2 , preventing the formation of the highly destructive hydroxyl radical [65,66], thus decreasing or eliminating oxidative stress and the associated membrane lipid peroxidation. No signs of lipid peroxidation (measured with MDA) were observed in C. nodosa.
Despite the higher dark respiration rates induced by higher temperature, the foliar soluble sugar concentration was not affected, and the starch concentration increased, suggesting higher CO 2 assimilation. The increase of the effective quantum yield of photochemical energy conversion in PSII (φPSII) at 40 • C indicates higher electron transport [67] that may feed the Calvin cycle and potential sinks, such as photorespiration, the Mehler reaction (reduction of O 2 on the acceptor side of photosystem I), nitrogen and sulfur metabolism, and the export of reducing equivalents to mitochondria or peroxisomes [68]. Both photorespiration and the Mehler reaction are known producers of H 2 O 2 and, although H 2 O 2 produced in the peroxisomes by photorespiration is eliminated by catalase, the H 2 O 2 that results from the Mehler reaction is scavenged by APX in the chloroplast [69]. The increase on APX activity we reported here may indicate an increase in the leaf production and scavenging of H 2 O 2 and thus the redirection of excess electrons to the Mehler reaction. Additionally, the export of reducing equivalents to mitochondria, besides alleviating the reducing pressure next to photosystem I, would have resulted in higher O 2 consumption in the mitochondria, and may help to explain the increased dark respiration rates without affecting soluble sugar concentration.
No signs of lipid peroxidation were observed in C. nodosa under the experimental heatwave, as evidenced by the leaf MDA concentrations [42]. In fact, MDA was always within the values previously determined for non-stressed C. nodosa plants from the Ria Formosa (between ca 60 nmol g DW −1 [28] and 140 nmol g DW −1 (unpublished data)), and other non-stressed plants [70,71].
In conclusion, we showed here that C. nodosa could adjust its photophysiological processes and antioxidant defense mechanisms to successfully handle a short period of thermal stress. Light did not exert a synergistic effect with temperature and no oxidative damage was caused by the experimental heatwave. The positive response of C. nodosa to thermal stress draws a good perspective for its resilience under climate change scenarios, particularly when heatwaves are increasing in frequency and intensity, but further investigation, including successive heatwave events and recovery periods, is needed.