Photorespiration in eelgrass (Zostera marina L.): A photoprotection mechanism for survival in a CO2-limited world

Photorespiration, commonly viewed as a loss in photosynthetic productivity of C3 plants, is expected to decline with increasing atmospheric CO₂, even though photorespiration plays an important role in the oxidative stress responses. This study aimed to quantify the role of photorespiration and alternative photoprotection mechanisms in Zostera marina L. (eelgrass), a carbon-limited marine C3 plant, in response to ocean acidification. Plants were grown in controlled outdoor aquaria at different [CO₂]_aq ranging from ~55 (ambient) to ~2121 μM for 13 months and compared for differences in leaf photochemistry by simultaneous measurements of O₂ flux and variable fluorescence. At ambient [CO₂], photosynthesis was carbon limited and the excess photon absorption was diverted both to photorespiration and non-photochemical quenching (NPQ). The dynamic range of NPQ regulation in ambient grown plants, in response to instantaneous changes in [CO₂]_aq, suggested considerable tolerance for fluctuating environmental conditions. However, 60 to 80% of maximum photosynthetic capacity of ambient plants was diverted to photorespiration resulting in limited carbon fixation. The photosynthesis to respiration ratio (P_E : R_D) of ambient grown plants increased 6-fold when measured under high CO₂ because photorespiration was virtually suppressed. Plants acclimated to high CO₂ maintained 4-fold higher P_E : R_D than ambient grown plants as a result of a 60% reduction in photorespiration. The O₂ production efficiency per unit chlorophyll was not affected by the CO₂ environment in which the plants were grown. Yet, CO₂ enrichment decreased the light level to initiate NPQ activity and downregulated the biomass specific pigment content by 50% and area specific pigment content by 30%. Thus, phenotypic acclimation to ocean carbonation in eelgrass, indicating the coupling between the regulation of photosynthetic structure and metabolic carbon demands, involved the downregulation of light harvesting by the photosynthetic apparatus, a reduction in the role of photorespiration and an increase in the role of NPQ in photoprotection. The quasi-mechanistic model developed in this study permits integration of photosynthetic and morphological acclimation to ocean carbonation into seagrass productivity models, by adjusting the limits of the photosynthetic parameters based on substrate availability and physiological capacity.

Introduction

Photosynthesis and photorespiration are competing processes due to the bi-functionality of ribulose 1,5-biphosphate carboxylase/oxygenase (Rubisco) (Spreitzer and Salvucci, 2002). Since the oxygenation reaction of Rubisco decreases photosynthetic carbon gain, it has been viewed as an inefficient legacy of evolution that might be engineered out of terrestrial plants in a quest for increased productivity (Andrews and Lorimer, 1978; Somerville, 2001; Xin et al., 2015). Recent work, however, suggests that Rubisco’s CO₂/O₂ specificity in different species may approach optimal acclimation to their gaseous environment in which the plants are grown (Tcherkez et al., 2006; Bathellier et al., 2018). More importantly, especially for carbon-limited seagrasses, photorespiration may serve as an important metabolic “clutch” to protect the photochemical pathway at high irradiance (Heber and Krause, 1980; Osmond, 1981; Osmond et al., 1997; Igamberdiev et al., 2001). When the Calvin Benson cycle is limited by the availability of CO₂, continuation of light reactions over-reduces the thylakoid electron transport chain and generates O₂ and reactive oxygen species (ROS) that potentiates oxidative stress (Voss et al., 2013). Photorespiration helps to balance the redox state and minimize the accumulation of ROS by dissipating the excess reducing equivalents (NADPH) as well as energy (ATP) (Foyer et al., 2009). By recycling the photorespired CO₂, photorespiration may also facilitate carbon assimilation in CO₂ limited environments, thereby minimizing photosynthetic inefficiencies resulting from C-limitation (Busch et al., 2013; Xin et al., 2015).

Photorespiration is often considered to be of minor importance in aquatic systems as a result of carbon concentrating mechanisms (CCMs) that facilitate the transport of HCO₃^- and its dehydration by algal pyrenoids that effectively deliver CO₂ to Rubisco (Frost-Christensen and Sand-Jensen, 1992; Madsen et al., 1993; Meyer et al., 2017). In today’s oceanic water (pH ~8.2), 89% of the DIC is in form of HCO₃^- and only 0.5% exists as dissolved CO₂ (Zeebe, 2012). However, not all aquatic C₃ plants have similar efficiencies to use both forms of DIC for photosynthesis (Raven and Beardall, 2003; Raven et al., 2011; Raven and Beardall, 2014). Additionally, CO₂ acquisition by simple diffusion through the leaf surface is more difficult for submerged plants due to the 10,000-fold lower diffusion rates of gases in a liquid environment relative to air (Borum et al., 2006). Consequently, for aquatic C₃ plants such as seagrasses that do not use CCMs effectively, carbon limitation likely increases the photorespiratory function of Rubisco (Tolbert and Osmond, 1976; Touchette and Burkholder, 2000).

Seagrasses are flowering marine plants that evolved from terrestrial monocots in the middle Cretaceous (Larkum et al., 2006b) when higher atmospheric and oceanic CO₂ concentrations likely supported photosynthesis and minimized photorespiration (Kuypers et al., 1999; Zeebe, 2012). In colonizing the aquatic habitat, seagrass evolved adaptations to a submerged environment that produced important anatomical differences from their terrestrial ancestors (Zimmerman et al., 1997; Larkum et al., 2006a). Seagrass leaves have no stomatal openings as gas exchange occurs across both leaf surfaces by diffusion, which uncouples carbon uptake from water relations. Seagrasses also have a lacunal system with aerenchyma extending from the roots to the leaves that facilitates the transport of O₂ to the roots buried in permanently flooded anoxic sediments, and allows transport of CO₂ from the roots to leaves, providing an alternative carbon source (Madsen and Sand-Jensen, 1991). Like their terrestrial ancestors, however, seagrass chloroplasts lack pyrenoids that serve as an important CCM in most aquatic algae (Meyer et al., 2017) and seagrasses are typically less efficient in utilizing HCO₃^- than macroalgae (Beer et al., 1991). Although Rubisco activity in seagrasses is lower than the typical activities in freshwater emergent angiosperms and marine red algae, it is comparable to that observed in marine green and brown macroalgae (Beer et al., 1991). Simulations of nearshore seawater DIC distribution during the Cretaceous period have predicted that photosynthetic rates of seagrasses would have been similar to macroalgae (Beer and Koch, 1996). However, in today’s oceans, seagrass photosynthesis is generally considered to be carbon limited (Durako, 1993; Beer and Koch, 1996; Zimmerman et al., 1997; Invers et al., 2001).

Carbon limited photosynthesis also restricts seagrasses to shallow, high light environments, where low daytime CO₂:O₂ ratios in the water column may increase seagrass vulnerability to photorespiration (Buapet et al., 2013b). The photorespiratory pathway was confirmed in marine plants and macrophytes by showing that photosynthesis could be inhibited by increasing the O₂ concentration, resulting in higher concentrations of glycolate pathway intermediates (Hough, 1974; Black et al., 1976; Burris et al., 1976; Downton et al., 1976; Hough and Wetzel, 1977; Andrews and Abel, 1979). The decreasing O₂ evolution rates relative to electron transfer rates measured by PAM fluorometry at high irradiances in Zostera marina and Halophila stipulacea also suggested a role for photorespiration in these seagrass species (Beer et al., 1998). More recent studies demonstrated the influence of oxygen concentrations and temperature on photorespiration in seagrass that fluctuate in natural environment because of eutrophication, high community productivity and elevated ocean temperatures; and therefore, will play a role in predicting the health status of these plants in warmer climate scenarios (Buapet and Björk, 2016; Rasmusson et al., 2020). The plastochron interval, which defines leaf life span, leaf turnover and elongation rates, plays an important role in photoacclimation strategies that differ among species at the chloroplast, leaf and shoot levels (Schubert et al., 2018). However, we still do not understand how long-term acclimation to climate warming and ocean acidification/carbonation will affect photorespiration and photoprotection in seagrasses (Koch et al., 2013).

Several experiments simulating ocean acidification/carbonation impacts on time scales of hours to >1 year have quantified the positive impacts of CO₂ availability on carbon balance, growth, survival and reproductive output in seagrasses (Zimmerman, 2021). During the most recent of these studies, the down-regulation of pigment content with increasing CO₂ resembled the photoacclimation response to high light environment that pointed to the importance of metabolic acclimation regulating the redox state of the chloroplast in eelgrass (Zimmerman et al., 2017; Celebi-Ergin et al., 2021). Therefore, the objectives of this study were to estimate the importance of photorespiration in the marine angiosperm Zostera marina L. (eelgrass) under today’s oceanic carbon concentrations and explore the potential acclimation response to prolonged ocean acidification/carbonation by 1) quantifying the photochemical rates under different light and CO₂ availability by using eelgrass grown in a high light low CO₂ environment, i.e., representing the baseline photosynthetic capacity under today’s oceanic conditions; and 2) comparing the relative contribution of different photochemical pathways in eelgrass after 13 months of acclimation to different CO₂ environments superimposed open daily and seasonal patterns of solar radiation, temperature and salinity.

Materials and methods

Table 1 provides a complete list of all abbreviations, acronyms, and symbols along with their units used throughout this paper.

TABLE 1

Table 1 List of symbols, their definitions, and dimensions.

The experimental facility and sampling from pH treatments

Eelgrass shoots used in this study were grown in an outdoor aquatic climate research facility at the Virginia Aquarium and Marine Science Center, VA, USA. The experimental design and control of manipulations for this-long term project were detailed in Zimmerman et al. (2017). Briefly, eelgrass plants, harvested in May 2013 from a subtidal population growing in South Bay, a coastal lagoon on the Virginia portion of the DelMarVa Peninsula, USA, were transplanted into 20 fiberglass open top aquaria (3 m³ each) plumbed with running seawater from Owl’s Creek, VA and exposed to natural sunlight. Temperature, pH, salinity, and irradiance were monitored continuously in all aquaria. Beverage-grade CO₂ gas was used to enrich the experimental aquaria from June 2013 to October 2014 using a system of pH-controlled solenoid valves. pH treatment levels ranged from pH 6 ([CO_2(aq)] ≅ 2121 µM) to ambient (no CO₂ addition, pH ≅ 7.7, [CO_2(aq)] ≅ 55 µM), with 0.5 pH intervals between the treatments (4 aquaria at each pH). The experimental CO₂ manipulation produced consistently different levels of [CO_2(aq)] and pH among the treatments day and night throughout the duration of the 13-month experiment. Plant performance was monitored monthly while environmental parameters, which varied daily and seasonally, were recorded at 10-minute intervals.

During July 2014, after 13 months of cultivation in the experimental aquaria, freshly collected 2^nd youngest leaves from pH 6.1 (2121 µM CO_2(aq)), pH 6.9 (371 µM CO_2(aq)) and ambient pH 7.7 (55 µM CO_2(aq)) treatments were harvested for laboratory measurements of photochemistry under fully controlled incubation conditions. Hereafter, the three treatments will be referred to as G_pH6, G_pH7 and G_pH8, for simplicity. During these measurements, the daily seawater temperature in aquaria ranged from 25 to 28°C; allowing all the incubation measurements described here to be conducted at the optimal temperature of 25°C without inducing heat stress. The daily total surface irradiance ranged from 10 to 29 mol photons m^-2 d^-1; corresponding to more than 6 h of photosynthetically saturating irradiance (>200 µmol photons m^-2 s^-1) per day, under which conditions the leaves of all plants should have been acclimated to a high light environment (Cummings and Zimmerman, 2003).

Incubation measurements of leaf photochemistry

Photosynthesis and respiration rates were measured using polarographic O₂ electrodes in temperature controlled, water-jacketed glass metabolic incubation chambers (Rank Bros., Cambridge, UK). Variable fluorescence was measured simultaneously on each leaf using a Pulse Amplitude Modulated (PAM) fluorometer (Mini PAM, Walz, Germany). Incubation seawater pH (a proxy for dissolved inorganic carbon (DIC) concentration) was measured using an epoxy mini-electrode and pH meter (Cole-Parmer) calibrated with NBS buffers. The lid of the incubation chamber was modified to hold the pH electrode in the incubation water and the miniature fiberoptic probe of the PAM device in close proximity to the leaf surface. The chamber was continuously mixed by a magnetic stirrer which homogenized the incubation medium and provided turbulent flow to reduce boundary layer limitation of gas exchange across the leaf surface. Continuous analog signals from the three sensors were recorded digitally using custom software written with LabView (2009 edition, National Instruments). Voltage data were post processed into metabolic rates using MATLAB R2014 (The MathWorks Inc.). A Kodak slide projector fitted with a halogen (ELH) bulb provided photosynthetically active radiation (PAR). The intensity of PAR was adjusted with neutral density filters and calibrated daily with a QSL 2100 scalar radiometer (Biospherical Instruments Inc.).

Concentrations of CO_2(aq) in the aquaria and metabolic incubation chambers were determined from measured values of pH, alkalinity, salinity and temperature using CO2SYS (Ver. 2.1; Lewis and Wallace, 1998). Leaves, harvested from plants grown at pH/CO₂ treatments G_pH6 ([CO_2(aq)] = 2121 µM), G_pH7 ([CO_2(aq)] = 371 µM) and G_pH8 ([CO_2(aq)] =55 µM), were used to measure the photosynthetic response at pH/CO_2(aq) levels of 6 (M_pH6), 7 (M_pH7) and 8 (M_pH8). Seawater [DIC] and pH in the incubation chambers were adjusted by bubbling with a gas mixture of CO_2, O₂ and N₂ that maintained [O₂] at air saturation (~215 µM). Seawater temperature was maintained at 25°C by a circulating water bath. Leaves were cleaned of epiphytes by gently scraping with a razor blade and kept in the dark for 20 minutes before the incubation measurements. A separate three cm long pieces of leaf tissue, cut approximately one cm above the meristem, was used during each ten min dark (i.e., dark respiration) and ten min light (i.e., net photosynthesis) measurement. The pigment content and optical properties of the leaf tissues (Table 1) were measured after each incubation as described by Celebi-Ergin et al. (2021).

The seawater used during all incubations was collected in April 2014 from Owl’s Creek, the tidal estuary used as source water for the experimental facility (Zimmerman et al., 2017). This seawater stock, with salinity of 24 (PSS-78, (Lewis, 1980) was filtered through 0.2 µm Nucleopore membrane filters and stored under refrigeration in dark bottles until used in these experiments. After incubations, alkalinity was determined on aliquots of seawater taken from the chamber using an automated potentiometric titrator (Metroohm). Table 2 summarizes measured parameters of seawater used in metabolic incubation chamber.

TABLE 2

Table 2 Distribution of dissolved inorganic carbon and dissolved oxygen concentrations in seawater during the incubation measurements of net photosynthesis at different light levels, including dark respiration measurements.

Determination of photochemical rates

Oxygen evolution rates of each tissue were separately normalized to fresh weight, leaf area and total pigment concentration to explore the effects of phenotypic differences resulting from acclimation to different growth conditions. Parameters of photosynthesis (P) vs Irradiance (E) curves were estimated by fitting the data to a cumulative one-hit Poisson model pioneered for photosynthesis by Webb et al. (1974):

$\begin{array}{ll}{P}_{\text{net}}{\text{ = }P}_{\text{g}}{-R}_{\text{D}}& (1)\end{array}$

$\begin{array}{ll}{P}_{\text{net}}{\text{ = [ }P}_{\text{E}}·\left({1\text{- e}}^{{-E\text{/ }E}_{\text{k}}}\right){\text{]-}R}_{\text{D}}& (2)\end{array}$

where P_net was the measured rate of net photosynthesis and R_D was the measured rate of dark respiration, from which the gross photosynthesis (P_g) was calculated according to Equation (1). P_g was defined as a function of light, where P_E represented the light-saturated rate of gross photosynthesis that varied with [CO₂] and [HCO₃^-] (sensu McPherson et al. (2015)). E_k was the irradiance threshold for photosynthetic saturation. E was separately defined as photosynthetically available radiation ($\text{PAR}={\displaystyle {\sum }_{400}^{700}E[\lambda }]$) and as photosynthetically utilized radiation $\left(\text{PUR}={\displaystyle {\sum }_{400}^{700}\left[E\left(\lambda \right)·A\left(\lambda \right)\right]}\right)$, where A(λ) was the spectral leaf absorptance that integrated the variability of light capture efficiency due to changes in leaf optical properties and pigment content/composition. The quantum yield of oxygen evolution (Φ_O2) at different irradiances (in units of mol O₂ mol^-1 absorbed photon) was calculated by Φ_O2 = P_g/PUR. Maximum quantum yield was calculated as Φ_max = P_E/E_{k (PUR)}.

Although Equation (1) represents the typical method for determining gross photosynthesis from measured values of P_net and R_D, the model does not separately account for O₂ consumed by photorespiration in the chloroplast. It also assumes that the Mehler Ascorbate Peroxidase pathway does not affect net O₂ exchange even though it may facilitate ATP generation and electron flow, which might be detected by fluorescence measurements (Larkum et al., 2006a). Following the principle explained by Raghavendra (2000) gross photosynthesis (P_g) can be detailed as the difference between true photosynthesis (P_T) and photorespiration (P_R):

$\begin{array}{ll}{P}_{\text{g}}{\text{ = }P}_{\text{T}} {\text{– }P}_{\text{R}}& (3)\end{array}$

Under CO₂-saturation (i.e., at low pH that increases CO₂:O₂ ratio in seawater, Table 2), P_R should approach a minimum (~ 0), so that P_g will be an approximate estimate of true photosynthetic O₂ production (P_T). In this study, O₂ production rates measured at pH 6 were assumed to approximate the true photosynthesis (P_T) for each growth condition. Therefore, photorespiration was calculated by subtracting the carbon limited P_g measured at pH > 6 from P_g measured at pH 6:

$\begin{array}{ll}{P}_{\text{R}}{}_{\mathrm{[pH>6]}}=P_{\text{g}}{}_{\mathrm{[pH6]}}-P_{\text{g}}{}_{\mathrm{[pH>6]}}& (4)\end{array}$

$\begin{array}{ll}{P}_{\text{g}}{}_{\mathrm{[pH>6]}}=\text{ [ }{P}_{\text{E}}·\left({1-\text{ e}}^{{-E\text{/ }E}_{\text{k}}}\right){\text{] }}_{\mathrm{[pH>6]}}& (5)\end{array}$

$\begin{array}{ll}{P}_{\text{g}}{}_{\mathrm{[pH6]}}=\text{ [ }{P}_{\text{m}}·\left({1-\text{ e}}^{{-E\text{/ }E}_{\text{k}}}\right){]}_{\mathrm{[pH6]}}& (6)\end{array}$

Thus, P_g approached P_E when saturated by light and flow, and it approached the true physiological capacity (P_m) when saturated by CO₂, light and flow. In this formulation, the limit of P_m is set by availability of cellular components such as enzyme and pigment concentrations that may change under different growth conditions.

Pulsed Amplitude Modulation (PAM) fluorescence measurements were analyzed following the calculations outlined in Baker (2008). The maximum (F_m) and minimum (F₀) fluorescence emissions were measured in the dark after at least 10 min of acclimation while simultaneously measuring respiration. The maximum variable fluorescence yield (F_v = F_m - F₀) was used to quantify the maximum quantum yield of fluorescence (F_v/F_m), which is a measure of maximum efficiency at which absorbed light by photosystem II (PSII) can be used for photochemistry. The maximum (F’_m) and minimum fluorescence (F_t) emissions induced by the short saturating pulse of PAM were measured again in the light while simultaneously measuring P_net. Based on these emissions under the presence of the actinic background light, the effective quantum yield of PSII (Φ_PSII), also known as photochemical quenching, was determined as:

$\begin{array}{ll}{\Phi }_{\text{PSII}}{=(F\text{'}}_{\text{m}}{-F}_{\text{t}}{)/F\text{'}}_{\text{m}}& (7)\end{array}$

Φ_PSII provides an estimate of the quantum yield of linear electron flow through PSII at a given irradiance. The other non-radiative energy loss that quenches fluorescence, called Non-Photochemical Quenching (NPQ), results from the dissipation of excess excitation energy as heat via the Xanthophyll cycle. NPQ was estimated as:

$\begin{array}{ll}{\text{NPQ = ( }F}_{\text{m}}{\text{- }F\text{ '}}_{\text{m}}{\text{) / }F\text{ '}}_{\text{m}}& (8)\end{array}$

For comparisons among the treatments and incubations, NPQ and Φ_PSII at different light levels were fitted to a four-parameter sigmoid curve, which is commonly used for dose response analysis (Motulsky and Christopoulos, 2004), with the following formula:

$\begin{array}{ll}{\text{NPQ = NPQ}}_{\text{min }}\text{+ }\frac{{\text{(NPQ}}_{\text{max}}{\text{+ NPQ}}_{\text{min}})}{1+{\left(\text{PUR/}EC\mathit{5}0\right)}^{-H}}& (9)\end{array}$

where the exponent H was Hill slope that controlled the steepness of the dose-response curve. EC50 was the PUR level required to provoke a response halfway between the baseline and maximum responses. The threshold for NPQ_max was constrained to 10 based on literature values (Kalaji et al., 2014).

The electron transport rate (ETR) was estimated from Φ_PSII as:

$\begin{array}{ll}{\text{ETR (}\mu \text{mol electrons m}}^{-2}{\text{s}}^{-1}\text{) = PUR }·{\text{ F}}_{\text{II}}·\text{Φ}{}_{\text{PSII}}& (10)\end{array}$

where F_II was the fraction of PUR captured by PSII and its light harvesting complexes (LHC). The typical value of F_II for Chlorophyta and seagrasses is about 0.5 (Figueroa et al., 2003; Larkum et al., 2006a). Photosynthetic parameters of ETR curves (i.e., ETR_max, α_ETR and E_{k -ETR}) were calculated by modifying the model of O₂ based P vs E. curves (Equation 2):

$\begin{array}{ll}{\text{ETR = ETR}}_{\text{max}}·\left({1\text{- e}}^{{-E\text{/ }E}_{\text{k}}}\right)& (11)\end{array}$

Linear electron flow through PSII is directly related to photosynthetic oxygen production, therefore the gross photosynthesis based on fluorescence measurements (P_g-ETR) were estimated from ETR as:

$\begin{array}{ll}{P}_{\text{g-ETR}}\text{ (}\mu \text{mol\hspace{0.17em}}{\text{O}}_2{\text{ m}}^{-2}{\text{s}}^{-1})=\text{ETR }·\tau & (12)\end{array}$

where τ was the ratio of oxygen evolved per electron generated at PSII. Since four stable charge separations are necessary to generate one mole of O₂ at PSII, τ is equal to 0.25.

Statistical analysis

Effects of growth [CO₂] on pigment content and optical properties of leaves were analyzed by one-way Analysis of Variance (ANOVA) followed by the Tukey multiple comparison method when significant overall effects were identified. Effects of growth [CO₂] and measurement [CO₂] on dark respiration rates, measured with the O₂ evolution method, were analyzed by Analysis of Covariance (ANCOVA).

O₂ evolution and fluorescence models were implemented by using the non-linear curve fitting tools in SigmaPlot (Systat Software Inc., Version 13.0). This tool provided the mean estimates of the model parameters with their error estimates and significances using computational procedures described by Draper and Smith (1981) and Zimmerman et al. (1987). Additionally, analysis of variance for the regression models were presented to account for the goodness of fit of the P vs E curves for each experimental condition (Supplementary Tables 1–3). Significant effects of measurement [CO₂] and growth [CO₂] on model parameters obtained by non-linear regression were analyzed by ANCOVA, with growth pH as the primary (categorial) factor and measurement pH as the continuous covariate.

Results

Photoacclimation to growth CO₂

Pigment content and optical properties varied significantly among the leaves grown in different [CO₂] treatments (Table 3). Both total chlorophyll and carotenoid content decreased with increasing growth [CO₂], while the molar ratio of Total Car : Total Chl remained constant across CO₂ treatments at about 0.27. The decrease in total chlorophyll resulted in an increased optical cross section (a_L*(λ)) with growth [CO₂], thereby reducing the package effect that results in Chlorophyll self-shading. Growth [CO₂] increased the thickness of the unpigmented mesophyll, thereby increasing the leaf biomass per unit of surface area. These phenotypic responses, consistent with the long term acclimation responses described by Celebi-Ergin et al. (2021), had important consequences for the comparison of photosynthetic efficiencies when metabolic rates were normalized to different leaf properties.

TABLE 3

Table 3 Pigment content and optical properties of leaves used in photosynthesis measurements.

Light response curves of oxygen flux

Rates of dark respiration, whether normalized to biomass (R_{D (FW)}) or leaf area (R_{D (LA)}), were not affected by growth [CO₂] or instantaneous variations of [CO₂] within the metabolic incubation chambers (Table 4). Therefore, the average rate of dark respiration for all samples combined was 5.96 ± 0.31 µmol O₂ hr^-1 g^-1 FW or 0.50 ± 0.03 µmol O₂ m^-2 s^-1. Dark respiration rates were also independent of pH within the range examined here, indicating no negative impact of changing ionic composition on respiration.

TABLE 4

Table 4 Dark respiration (R_D) rates measured with O₂ evolution method and estimated by non-linear model fit to P vs E curves.

In contrast, net O₂ production rates increased with light and incubation [CO₂] for all plants, regardless of the CO₂ environment in which they were grown (Figure 1). The biomass specific rate of light-saturated photosynthesis (P_{E (FW)}) averaged 14.1 µmol O₂ hr^-1 g^-1 FW at low incubation [CO₂] for all plants and increased as a function of incubation [CO₂] (Figure 1). However, P_{E (FW)} of the plants grown under ambient conditions (G_pH8) was twice as sensitive to increasing incubation [CO₂] as plants grown under the highest CO₂ enrichment (G_pH6) (Table 5, 86.8 vs 33.5 µmol O₂ hr^-1 g^-1 FW at M_pH 6 respectively). This difference was associated with 2-fold higher biomass specific pigment content of the plants grown under ambient [CO₂] (Table 3). Thus, low rates of oxygen evolution by ambient plants in their natural low CO₂ environment resulted mainly from photorespiration and not the lack of photosynthetic capacity characterized by light harvesting, electron transport and carbon fixation.

FIGURE 1

Figure 1 Net photosynthesis of eelgrass leaves (biomass normalized) as a function of irradiance. O₂ production rates were measured at different pH levels (red: pH6, black: pH7 and blue: pH8) using leaves grown at pH6 (2121 µM CO_2(aq)) (A), pH7 (371 µM CO_2(aq)) (B) and ambient pH8 (55 µM CO_2(aq)) (C). Curves were fit using Equation (2).

TABLE 5

Table 5 Model estimates (mean ± 1 SE) of photosynthesis parameters generated by non-linear regression fit to the experimental data using Equation (2) (N.S. stands for non-significant parameter estimate).

For all plants, increased incubation [CO₂] also increased the irradiance required to saturate photosynthetic oxygen production (E_{k (PAR)} and E_{k (PUR)}); rather than changing the photosynthetic efficiency (α) within the light limited region of P versus E response curves (Table 5 and Supplementary Table 1). Overall, photoacclimation of eelgrass leaves to ocean carbonation increased E_{k (PUR)} values from 17 to 44 and 48 µmol absorbed photon s^-1 m^-2 for pH 8 (55 µM CO_2(aq)), pH 7 (371 µM CO_2(aq)) and pH 6 (2121 µM CO_2(aq)) respectively.

Chlorophyll specific rates of light-saturated photosynthesis (P_{E (Chl)}) were the same for all plants grown at different CO₂ environments and produced an identical response to incubation [CO₂] (Figure 2). Consequently, the O₂ production efficiency per unit chlorophyll was not affected by the CO₂ environment in which the plants were grown (Table 5 and Supplementary Table 2) and the stimulatory effect of [CO₂] on O₂ evolution was instantaneous (Figure 3A). The most likely explanation for this instantaneous response would be a reversible and light dependent O₂ consuming process involving the chloroplast, such as photorespiration (P_R), that is competitively inhibited by increasing [CO₂]. Therefore, for all plants grown under all treatments, P_{E (Chl)} rates at high incubation [CO₂] (i.e., at M_pH6) were assumed to be the true physiological photosynthetic capacity (P_m) under light, carbon and flow saturation. Based on this assumption, photorespiration rates were quantified by solving the Equation 4 with the chlorophyll specific gross photosynthesis models (Figure 3B). Normalizing the models to pigment, rather than biomass or area, eliminated the effect of morphological differences among the plants on net oxygen metabolism.

FIGURE 2

Figure 2 Net photosynthesis of eelgrass leaves (Chlorophyll normalized) as a function of absorbed irradiance. O₂ production rates were measured at different pH levels (red: pH6, black: pH7 and blue: pH8) using leaves grown at pH6 (2121 µM CO_2(aq)) (A), pH7 (371 µM CO_2(aq)) (B) and ambient pH8 (55 µM CO_2(aq)) (C). Curves were fit using Equation (2).

FIGURE 3

Figure 3 Modeled gross photosynthesis (A) and photorespiration (B, C) of eelgrass leaves as a function of absorbed irradiance. Colors represent different pH/CO₂ levels at which the measurements (M_pH) were performed; line styles represent the different pH/CO₂ levels at which the plants were grown (G_pH). Photorespiration at M_pH6 were zero.

Like photosynthesis, photorespiration increased with light under constant [CO₂], but decreased with increasing incubation [CO₂], as carboxylation became increasingly favored over oxygenation (Figure 3B). Predicted P_R rates increased rapidly with light to a maximum of 60 to 80% of P_m at low [CO₂] (i.e., M_pH8) (Figure 3C). When aqueous [CO₂] was equal to aqueous [O₂] (at M_pH7, Table 2), maximum P_R rates were only 20% of P_m, which is equivalent to the inherent carboxylation: oxygenation ratio of Rubisco.

All plants reached the lowest gross photosynthesis to dark respiration ratio (P_g : R_D) of 2 at low incubation [CO₂] when light saturated (Figure 4A). This ratio increased instantaneously when saturated with CO₂ in the incubation medium, maximally up to 12 for ambient plants (G_pH8). However, the P_E : R_D ratio of high CO₂ grown plants peaked at 8 when saturated with CO₂ in the incubation medium, illustrating the consequence of pigment acclimation on metabolic balance of plants grown in a high CO₂ environment (Figure 4B, grey arrows). Having excess pigment content in a CO₂-limited environment (as observed in ambient plants) did not improve the P_E : R_D under normal growth conditions even though it allowed the instantaneous 6-fold increase of P_E : R_D when incubation [CO₂] increased. High CO₂ acclimated plants, on the other hand, maintained a 4-fold higher P_E : R_D above ambient plants at their respective growth [CO₂] even though pigment content of the high CO₂ plants decreased by half.

FIGURE 4

Figure 4 Modeled ratio of gross photosynthesis to dark respiration as a function of absorbed irradiance (A) and as a function of Chlorophyll content at saturating irradiances (B). Colors represent different pH/CO₂ levels at which the measurements (M_pH) were performed; line styles and symbols (▲, at pH6 ◼ at pH7, ● at ambient pH) represent the different pH/CO₂ levels at which the plants were grown (G_pH). (B) Ellipses highlight when plants from different treatments were incubated at their corresponding growth pH/CO₂. Gray arrows show the trajectory of P_E : R_D as a result of phenotypic acclimation to the increasing CO₂ environment.

Light response curves of variable fluorescence

Maximum quantum yields of fluorescence by dark-adapted leaves were above 0.7 regardless of incubation [CO₂], indicating leaves from all growth treatments were healthy during the experiments (Φ_PSII at PUR 0 µmol absorbed photon s^-1 m^-2, Figure 5). For all plants, effective quantum yields of fluorescence (Φ_PSII) decreased faster with increasing light when the incubation [CO₂] was low (M_pH8). The decreased photochemical yield resulted from rapid induction of non-photochemical quenching (NPQ) when [CO₂] was limited under light saturation (Figure 6). Increasing the growth [CO₂], however, caused the light-dependent onset of NPQ to increase, as the NPQ pathway saturated more quickly due the decreased carotenoid content of leaves grown under high [CO₂] (Table 3).

FIGURE 5

Figure 5 PAM fluorescence parameters of eelgrass leaves as a function of absorbed irradiance. PAM fluorescence measurements were performed at different pH/CO₂ levels (red: pH6, black: pH7, blue: pH8) using leaves grown at pH6 (2121 µM CO_2(aq)) (A), pH7 (371 µM CO_2(aq)) (B) and ambient pH8 (55 µM CO_2(aq)) (C). Curves were fit using Equation (9).

FIGURE 6

Figure 6 PAM fluorescence parameters of eelgrass leaves as a function of absorbed irradiance. PAM fluorescence measurements were performed at different pH/CO₂ levels (red: pH6, black: pH7, blue: pH8) using leaves grown at pH6 (2121 µM CO_2(aq)) (A), pH7 (371 µM CO_2(aq)) (B) and ambient pH8 (55 µM CO_2(aq)) (C). Curves were fit using Equation (9).

Under saturating irradiance (350 µmol absorbed photon s^-1 m^-2, Figure 6), NPQ values of ambient plants increased 5-fold as incubation [CO₂] became increasingly limiting. In contrast, the plants grown under high [CO₂] (G_pH6) yielded the same light-saturated NPQ of 2.5 regardless of incubation [CO₂]. The dynamic range of NPQ regulation in ambient grown plants in response to instantaneous changes in [CO₂] suggests considerable tolerance for fluctuating environmental conditions (Figure 6C).

The relation between quantum yield of fluorescence (Φ_PSII) and quantum yield of oxygen evolution (Φ_O2) was nonlinear, and their ratios were closest to the theoretical value of 8 only at low light and high [CO₂] conditions (Figure 7). For this ratio to be higher than 8, either less than half of the photons are directed to PSII (i.e., F_II<0.5, Equation 10), and/or more than four electrons are processed to evolve one mole of oxygen (i.e., τ<0.25, Equation 12). Both outcomes highlight deviation from linear electron flow. For ambient plants, Φ_O2 decreased faster than Φ_PSII with increasing light resulting in a drastic increase in Φ_PSII:Φ_O2, especially at their growth CO₂ (G_pH8), suggesting that these plants were using an alternative pathway to maintain electron flow without the production or consumption of O₂.

FIGURE 7

Figure 7 Ratio of the quantum yield of fluorescence (Ф_PSII) to the quantum yield of oxygen (Ф_O2) as a function of light and incubation pH/CO₂. O₂ production and fluorescence were measured simultaneously at different pH levels using eelgrass leaves grown at different CO₂ treatments. Yields were calculated using PUR. Growth specific 3D relationships, at pH6 (2121 µM CO_2(aq)) (A) and ambient pH8 (55 µM CO_2(aq)) (B), were generated by the combination of non-linear and linear regression fits. First, the Ф_PSII/Ф_O2 ratio as a function of PUR were described by the exponential rise to maximum models separately for each incubation pH/CO₂. Parameter estimates of these non-linear regression models were fitted as a function of incubation pH using linear regression.

Similar to net photosynthesis rates, electron transport rates (ETR) of all plants increased with light and were lowest at low incubation [CO₂] (i.e., M_pH8) (Figure 8 and Table 5). However, the increase of ETR_max with incubation CO₂ was not consistent among the plants due to the non-monotonic response of Φ_PSII with incubation [CO₂] (Figure 5), in agreement with the findings by Celebi (2016). Only ETR_max of plants grown at G_pH7 increased consistently with increasing incubation [CO₂]. For all incubation experiments, PUR levels required to saturate ETR (E_k-ETR) were consistently higher than the E_k values required to saturate O₂ production (Table 5 and Supplementary Table 3). For all plants, estimated gross photosynthesis based on ETR were also higher than the gross photosynthesis measured by the O₂ evolution method (Figure 9). However, this overestimation was not consistent among plants grown at different CO₂ environments. The P_{E (LA)} to ETR_max ratio was around 0.1 for pH 6 and pH 8 plants when incubated at pH 6 and pH 8, instead of the theoretical value (τ) of 0.25 (Table 5).

FIGURE 8

Figure 8 Electron transport rates of eelgrass leaves as a function of absorbed irradiance. PAM measurements were performed at different pH/CO₂ levels (red: pH6, black: pH7 and blue: pH8) using leaves grown at pH6 (2121 µM CO_2(aq)) (A), pH7 (371 µM CO_2(aq)) (B) and ambient pH8 (55 µM CO_2(aq)) (C). Curves were fit using Equation (11).

FIGURE 9

Figure 9 Modeled gross photosynthesis of eelgrass leaves as a function of absorbed irradiance for plants growing at pH6 (2121 μM CO_2(aq)) (A), pH7 (371 μM CO_2(aq)) (B) and ambient pH8 (55 μM CO_2(aq)) (C). Solid lines are calculated from leaf area normalized O₂ production rates (Equation 1) and dashed lines are estimated from ETR measurements (Equation 12). Colors represent incubation pH/CO₂ levels. Green dot-dashed lines represent the theoretical O₂ production per absorbed photon under non-limiting environmental conditions.

Discussion

Long-term growth under high [CO₂] produced a remarkable combination of morphological and metabolic changes in eelgrass. Although pigment content decreased in plants grown at high CO₂, leaf biomass increased as a direct result of the CO₂-stimulated increase in photosynthetic carbon gain. The equivalent responses of chlorophyll normalized O₂ production rates to increased incubation [CO₂], independent of the growth CO₂, allowed us to quantify the impact of [CO₂] on photorespiration in eelgrass because the instantaneous difference in O₂ production rates in CO₂-saturated vs. CO₂-limited incubation media corresponded to the amount of O₂ consumed in the photorespiratory pathway. Thus, photosynthesis and photorespiration as a function of light for each growth condition were precisely predictable using the P versus E curves, although the responses to incubation CO₂ differed between biomass and pigment normalization due to changes in leaf morphology. Presently, models of eelgrass performance do not consider these long-term morphological and metabolic acclimation responses (Zimmerman, 2003; Zimmerman, 2006; Zimmerman et al., 2015). Thus, the quasi-mechanistic model developed in this study permits integration of the photosynthetic and morphological acclimation due to ocean carbonation into seagrass productivity models, by adjusting the limits of the photosynthetic parameters based on substrate availability and physiological capacity.

Morphological acclimation and regulation of pigment content, Rubisco activity, light capture and carbon fixation as a function of CO₂ availability have been previously observed in freshwater angiosperms (Madsen et al., 1996). Increasing P_g : R_D due to the enhancing impact of [CO₂] on P_E was detected even in short term (2-6 weeks) studies using temperate and tropical seagrass species without any CO₂ effect on pigment content (Zimmerman et al., 1997; Ow et al., 2015). Long term studies, moreover, reported significant increases in total shoot biomass, carbon allocation to roots and rhizomes (blue carbon), shoot survival and reproductive output by eelgrass in response to CO₂ availability (Palacios and Zimmerman, 2007; Zimmerman et al., 2017). Despite the decreases in pigment content and leaf absorptance observed here, plants grown at high CO₂ were able to maintain higher P_g : R_D ratios than plants grown under ambient CO₂; indicating a strong coupling between the regulation of photosynthetic structure and metabolic carbon demands. This coupling between photosynthetic regulation and growth might be poor for organisms that undergo photodamage because photosynthesis might accommodate the biochemical costs associated with protection and recovery rather than fueling the energy towards growth (Barra et al., 2014). On the other hand, the eelgrass used in these experiments show no sign of photodamage, either in the growth aquaria or in laboratory incubations even when photosynthesis was carbon limited but light saturated.

When measured at low [CO₂], plants grown under ambient CO₂ had the same photosynthetic O₂ production as the plants grown at high [CO₂]. These same photosynthetic rates highlighted the apparent lack of carbon concentrating mechanisms inducible by low CO₂ availability in eelgrass, in contrast with marine algae and cyanobacteria that are capable of upregulating their carbon concentrating mechanisms via e.g., generation of pyrenoids, carboxysomes and periplasmic carbonic anhydrases when CO₂ availability becomes limiting (Björk et al., 1993; Raghavendra, 2000; Falkowski and Raven, 2007; Meyer et al., 2017). This was also consistent with the limited sensitivity of eelgrass photosynthesis to the aqueous presence of acetazolamide, an inhibitor of periplasmic carbonic anhydrase (McPherson et al. (2015) and Celebi-Ergin - unpublished data). Seagrasses living in shallow estuarine environments, like the Chesapeake Bay eelgrass used in this study, are subject to highly variable CO₂/pH levels daily and seasonally, which might explain the unresponsiveness of CCMs for ambient plants (Buapet et al., 2013a; Duarte et al., 2013; Ruesink et al., 2015; Zimmerman et al., 2017; Cyronak et al., 2018). Similarly, all plants had the same P_{E (Chl)} when measured at saturating [CO₂] due to minimized P_R, indicating all plants approached the same physiological oxygen production capacity per available photosynthetic apparatus (i.e., P_{m (Chl)} was constant across all treatments). Therefore, the difference in P_E : R_D among growth [CO₂] treatments when all were incubated at high [CO₂] resulted from the downregulation of light harvesting components by plants grown in the high CO₂ environment.

Despite phenotypic acclimation across the CO₂ gradient, the maximum photosynthetic efficiency (Φ_max) remained constant for all plants (~0.08 mol O₂ mol^-1 absorbed photon) but photosynthesis-saturating light levels (E_k) increased, as was predicted by the model of McPherson et al. (2015). Photosynthetic efficiency within and among seagrass species vary with efficiency of light absorption and the subsequent conversion of that energy into carbon assimilation (Ralph et al., 2007). Although the observed values of α in this study were in agreement with previous estimates for eelgrass (Frost-Christensen and Sand-Jensen, 1992), constant α across different CO₂ regimes represents an interesting contrast to that observed in terrestrial C3 plants in which α increases with [CO₂] availability (Raghavendra, 2000). This difference between responses of aquatic and terrestrial plants may result because CO₂ responses are coupled to water stress in terrestrial plants but not in aquatic plants. The increased E_k and P_E values for high CO₂ acclimated plants will decrease the estimates of H_sat (i.e., average daily period of P_E) required to maintain positive carbon balance for the whole plant. The H_sat requirement is a useful modeling tool in predicting the depth distribution of eelgrass in variable light environments (Dennison, 1987; Zimmerman et al., 1991; Zimmerman et al., 1994; Zimmerman et al., 1995).

A strong correlation between diurnal NPQ cycle (i.e., xanthophyll cycle) and high light exposure has been confirmed for eelgrass to avoid photodamage under fluctuating light environments (Ralph et al., 2002). High light acclimated eelgrass leaves have higher NPQ activity, and higher photosynthetic capacity, than low light acclimated leaves (Ralph and Gademann, 2005). Here, we demonstrated a similar effect of [CO₂] availability on NPQ activity. Under ambient CO₂ concentrations, the onset of photosynthesis CO₂ limitation (E_k) occurred at lower irradiance, accelerating the diversion of excess photon absorption to NPQ, likely using the xanthophyll cycle as a photoprotective mechanism to prevent photoinhibition. The high CO₂ incubations reduced this carbon limitation and increased the E_k, consequently reducing NPQ. Due to increased E_k, the same light environment became less damaging at high CO₂, which may explain the reduction in both photosynthetic and photoprotective pigments observed in response to growth CO₂. Thus, by reducing CO₂ limitation of Rubisco, ocean carbonation should also reduce the vulnerability of eelgrass to excess reactive oxygen species (ROS) and therefore the need for photoprotection.

The simultaneous measurements of variable fluorescence, and O₂ flux performed here yielded quantitative estimates of changes in photoprotective pathways of eelgrass acclimated to different CO₂ environments. The difference between the theoretical O₂ evolution (i.e., the linear increase of O₂ with light) and the ETR estimates of gross photosynthesis (P_g-ETR) was most pronounced for plants grown at high CO₂, accounting for the absorbed photons that did not contribute to the electron transport pathway (not exciting electrons at PSII), but explained by quenching pathways, such as fluorescence and NPQ. This trend was consistent with their lower area-specific O₂ production rates at high CO₂ incubations when compared to pH7 and ambient pH grown plants. These plants downregulated their pigment content but increased the light-dependent NPQ at lower irradiances even at high incubation [CO₂]. This may indicate that phenotypic acclimation to ocean carbonation by downregulating the photosynthetic apparatus reduces the role of photorespiration but increases the role of NPQ in photoprotection.

On the other hand, as observed in all treatments, the difference between the ETR estimated gross photosynthesis (P_g-ETR) and the gross photosynthesis measured by oxygen production (P_g) may result from inaccurate assumptions of F_ii and/or τ (Equation 12). In theory, 8 photons absorbed equivalently both by PSI and PSII (F_ii= 0.5) excites a total of 4 electrons producing 1 mole of O₂ (τ =0.25). This equilibrium of linear electron flow is valid when there is no limitation of resources such as CO₂ and/or accumulation of byproducts such as reducing equivalents and ROS (Scheibe et al., 2005; Dietz and Pfannschmidt, 2011; Pfannschmidt and Yang, 2012). Under limiting conditions, this balance shifts towards pathways that ensure the optimal redox state of the chloroplast resulting in altered photon: electron: O₂ ratios (Foyer et al., 2012).

Following the linear assumption that 4 electrons produce 1 O₂ (τ = 0.25) resulted in overestimation of the PG_ETR in all treatments. Since the molecular chemistry of water splitting at PSII is well-known, τ can only be reduced in an apparent sense. This apparent ratio can result from the excitation of four electrons (as detected with PAM) either without producing O₂, indicating cyclic electron flow around PSII, or consumption of O₂ in the chloroplast that would remain undetected by the gas exchange method (Foyer et al., 2009; Ananyev et al., 2017). Two possible pathways to explain a reduction in τ due to O₂ consumption are (i) the Mehler reaction and (ii) photorespiration. The Mehler reaction increases the pH gradient that may induce NPQ (Demmig-Adams and Adams, 1996; Kanazawa and Kramer, 2002). However, in this study NPQ induction did not happen until Φ_PSII values fell below 0.6 while O₂ yield continuously decreased. Therefore, the observed nonlinearity between quantum yield of fluorescence and quantum yield of oxygen most likely resulted from O₂ consumption via photorespiration, which probably represents the primary pathway to remove excess O₂ buildup and use the ATP energy from light reactions for this purpose. NPQ was then triggered when photorespiration is incapable of consuming enough ATP to lower the pH gradient forming across lumen at very high irradiances.

Other pathways that keep the electron flow continuous without contributing to CO₂ assimilation are the malate valve and the cyclic electron flow around PSI, which triggers NPQ by generating a pH gradient (Munekage et al., 2004; Johnson, 2005; Miyake, 2010). If PSI cyclic electron flow plays an important role, then the assumption of half of the absorbed photons going to PSII (e.g., F_ii=0.5) would be inaccurate. Although PAM measurements are easily made under field conditions (including underwater) and provide non-intrusive information about the photoprotection of eelgrass through NPQ, the fluorescence measurements with PAM overestimate P_g-ETR and therefore are not equivalent to true carbon assimilation. Fluorescence measurements may account for the number of absorbed photons used in electron excitation but not necessarily towards the rates of oxygen production/consumption or carbon assimilation, especially when alternative electron sinks are available (Beer et al., 1998). Still, by having quantified the ratio of Φ_PSII to Φ_O2 as a function of light and carbon availability in response to acclimation to ocean carbonation, the alternative electron pathways can be accounted for in the future estimation of photosynthesis in eelgrass.

To conclude, photorespiration likely provides an important metabolic clutch to protect the photochemical pathway in CO₂-limited eelgrass by maintaining electron flow to prevent the inhibitory damage to photosystems due to light saturation when carbon assimilation is limited by CO₂ supply. In addition to providing a photoprotective role, photorespiration could serve multiple purposes by connecting different metabolic pathways that allow instantaneous energy and reductant modulation under fluctuating environmental conditions. Further, photorespiration may provide a carbon concentrating mechanism via recycling of photorespired CO₂ and removing excess intracellular O₂. Therefore, even though carbon limitation causes eelgrass photosynthesis to saturate at relatively low light levels in the modern ocean, longer daily periods of saturating irradiances might be required to keep the photosynthetic apparatus running to produce ATP to support photorespiration. Consequently, understanding photoprotection mechanisms employed by these remarkable plants that are permanently rooted in highly variable shallow-water environments, becomes important when high water column productivity causes [O₂] to rise and [CO₂] to fall just as daily irradiances begin to peak. More importantly, this study demonstrated that acclimation of photoprotective mechanisms in response to CO₂ availability accounted for the previously reported physiological acclimations of enhanced growth and survival of this species under ocean acidification scenarios.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Author contributions

BC-E, RZ and VH conceived the project. BC-E, RZ and VH performed the research. BC-E and RZ analyzed the data. BC-E, RZ and VH wrote the article. All authors contributed to the article and approved the submitted version.

Funding

Financial support for this research was provided by the National Science Foundation (Award OCE-1061823 to RZ and VH), Virginia Sea Grant/NOAA (Award NA14OAR4170093 to RZ and BC-E) and the Department of Ocean, Earth & Atmospheric Sciences, Old Dominion University (to BC-E). This research was performed in partial completion of the requirements for the Ph.D. degree (Oceanography) at Old Dominion University.

Acknowledgments

Thanks to W. M. Swingle and the staff of the Virginia Aquarium & Marine Science Center for assistance with maintenance of the experimental facility, and to D. Ruble, M. Jinuntuya, C. Zayas-Santiago, T. Cedeno and M. Smith for assistance with experimental procedures and maintenance of the experimental plants.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2022.1025416/full#supplementary-material

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