Significance of HCO3 alkalinity in calcification and utilization of dissolved inorganic carbon in Chara vulgaris

To investigate the influence of HCO3 alkalinity on calcification in thalli of the genus Chara, we studied the effects of increased light level and additional HCO3 on calcification rate in C. vulgaris at various hours (30 min, 8 h, 12 h, 12.5 h, 20 h and 24 h after treatment) in a 24 h experiment (12 h light:12 h dark). We identified a significant Pearson’s correlation between exogenous dissolved inorganic carbon (DIC) concentrations and the utilization of DIC (μmol C g−1 fresh weight). Plotting the daily rhythm of DIC utilization produced negative quadratic curves. Furthermore, calcification rate (μmol Ca h−1 g−1) was linearly related to DIC utilization rate (DICuptake; μmol C h−1 g −1), indicating that the calcification rate is dependent on DICuptake. However, ratios of calcification to the utilization of DIC were decreased at high light intensity and increased with HCO3 addition, which was mainly ascribed to changes in the ratio of calcification to photosynthesis. Chlorophyll fluorescence results provided direct evidence for the promotion of photosynthesis in Chara thalli by both high light and DIC addition and their positive influence on maximum relative electron transport rate. These results suggest that calcification in calcareous C. vulgaris is mainly restrained by HCO3 alkalinity, which could explain the correlation between calcification of Chara thalli and alkalinity of water bodies in the field.


INTRODUCTION
CaCO 3 encrustations are common on the stems, branchlets, and surfaces of oogonia in Chara spp.(hereafter 'Chara') (Wood & Imahori 1965).However, intra-and inter-specific variations in the external encrustations on the cell wall are present within this genus (Ana dón et al. 2002), as well as between ecorticate species and corticated species (Kawahata et al. 2013).Furthermore the average percentage content of encrustation in dry charophyte mass varied from 15% (Siong & Asaeda 2009a) to 60% (Hutchinson 1975), 61.5% (Kufel et al. 2013), 70% (Blindow 1992), and 77% (Urbaniak 2010), depending on the studied species and the water environment where those species were collected.However, carbonate deposited by dense charophyte vegetation accounts for a significant portion of calcium carbonate in the sediments of so-called Chara lakes and represents an important record of environmental conditions (Apolinarska et al. 2011, Pelechaty et al. 2013, Kufel & Strzałek 2016).
Light and dissolved inorganic carbon (DIC) are 2 of the most important environmental variables that affect the morphology and distribution of Chara plants (McConnaughey 1998) because of their fast at ten uation and limited solubility in the water column, respectively.In natural habitats, most calcareous species of Chara inhabit waters of relatively high alkalinity and calcium content (Kufel & Kufel 2002).Obvious encrustation has been observed under high light intensity (Wang 2008).However, there is much debate on the influence of light and HCO 3 − alkalinity on calcification.According to the cis-calcification model, calcification on alkaline surfaces of Chara is a by-product of bicarbonate assimilation from alkaline water, and light therefore promotes calcification (McConnaughey & Whelan 1997).However, in the trans-calcification model, Ca 2+ −2H + exchange catalysed by Ca 2+ ATPase is responsible for calcification in Chara (McConnaughey & Falk 1991).Therefore, in the trans-calcification model, calcification does not require light, and calcification functions as a proton generator.Since the 1990s, many studies have demonstrated the beneficial effects for macrophytes from proton generation by calcification and how this is related to bicarbonate utilization (McConnaughey 1991, McConnaughey & Falk 1991), nutrient assimilation (McConnaughey & Whelan 1997, Siong & Asaeda 2006, 2009b, Gomes & Asaeda 2010, Wang et al. 2013), and hyper-accumulation of heavy metals (McConnaughey 1991, Siong & Asaeda 2009a).Furthermore, Wang et al. (2013) demonstrated that calcification could mitigate the toxic effects of high NH 4 -N on calcareous Chara plants.
In mildly alkaline water, DIC is typically decreased in 2 ways, either by uptake through the photosynthesis of algae or by becoming incorporated into the cell walls of calcareous Chara by calcification (Ray et al. 2003).Owing to the sound understanding of the relationships between fluorescence parameters and photosynthetic electron transport in vivo (Maxwell & Johnson 2000, Baker 2008), fluorescence parameters such as photosynthetic efficiency estimates (F v /F m ) have been widely used to assess the photosynthetic characteristics of submerged plants or macroalgae (Schwarz et al. 2000 andDurako &Kunzelman 2002 for seagrass, Beer & Axelsson 2004 for macroalgae, Asaeda et al. 2014 for Chara spp.).In addition, the rapid light curve (RLC) is a powerful tool for the assessment of photosynthetic performance and for providing detailed information on the saturation characteristics of electron transport (Ralph & Gademann 2005).
C. vulgaris L. is a polymorphic charophyte with a worldwide distribution between 70°N and 50°S.C. vulgaris is prone to accumulate lime and form apparently non-banded encrustation on its stems (Anadón et al. 2002), accounting for more than 60% of ash mass under 50% of sunlight (Wang et al. 2008).To clarify the influence of light and HCO 3 − -alkalinity on calcification, we investigated the effects of high light intensity and HCO 3 − addition on the calcification rate of C. vulgaris by using an open experimental system.Furthermore, RLC was used to assess the effects of light level increase and HCO 3 − addition on photosynthetic performance.The aims of this study were to: (1) investigate the occurrence of dark calcification by monitoring the daily rhythm of the calcification rate of Chara thalli under constant light and (2) identify the rate-limiting factor for calcification of calcareous plants.

Plant culture
On 5 April 2010, 24 clusters of shoots (10−15 cm in length) of Chara vulgaris were collected from a depth of 1−1.5 m in Liangzi Lake (30°05' to 30°18' N, 114°21' E to 114°39' E), a shallow lake located in the middle reach of the Yangtze River in China.These specimens were then transplanted into 3 cylindrical aquaria (1 m in radius) located at Donghu Experimental Station of Lake Ecosystems, Institute of Hydrobiology, Chinese Academy of Sciences.These aquaria were filled with tap water and sediment from the Tanglinhu area, as described by Feng et al. (2006).After 1 mo, the shoots of the plants had developed towards the water surface, with a branched morphology and visible calcified encrustation on the internodes.On 15 May 2010, more than 120 apical tips with 3 or 4 internodes (about 4−5 cm in length) were cut using scissors and pre-incubated in 1 l glass beakers (n = 4) filled with plant-containing water (PCW).To avoid the effect of Ca 2+ and Mg 2+ contents on calcification, PCW was obtained by filtering water from the previously used circle aquaria through GF/C membranes (Whatman), with DIC content of 0.98 ± 0.04 mM C, Ca 2+ content above 60 mg l −1 and Mg 2+ content below 30 mg l −1 (McCon naughey 1998, Kufel & Kufel 2002, Gomes & Asaeda 2010, Asaeda et al. 2014).All beakers were placed in the plant growth incubator under controlled conditions: temperature, 25 ± 2°C; illumination on the water surface, 90 µmol m 2 s −1 ; and a photo period of 12:12 h (light:dark).After 2 wk of pre-culture, inorganic carbon accumulated within the plants was removed by transferring all apical tips to 1 l beakers containing ultrapure water for 6 h under the same culture conditions (Kahara & Vermaat 2003).

Experimental design
A factorial design of 2 × 2 of light intensity and DIC level was used in this experiment: low light (LL, 90 µmol m −2 s −1 ) and high light (HL, 150 µmol m −2 s −1 ) intensities and low DIC (LC: no HCO 3 − addition) and high DIC (HC: 1 mM HCO 3 − addition) levels.These 4 combined treatments were marked as LL-LC, LL-HC, HL-LC, and HL-HC, respectively.For each treatment, 24 flasks (4 flasks each time × 6 times) with plants were used as replicates, and 12 flasks (2 flasks each time × 6 times) without plants were used to calibrate changes to the solution over time.At the beginning of the photoperiod (Time 0), 4 or 5 apical tips (about 0.5 g in fresh weight, without endogenous inorganic carbon) were weighed and transferred into 96 flasks (24 flasks × 4 treatments) containing 200 ml of the culture solution.In all, 144 conical flasks (96 flasks + [12 flasks × 4]) with a capacity of 250 ml were placed in 2 plant incubators with different light levels (each contained 72 flasks), with the same temperature conditions as for pre-culture.The addition of HCO 3 − was made by adding NaHCO 3 , and the pH of the culture solutions was adjusted to 8.2 by using 0.1 M NaOH (before being decanted into the flasks).

Experimental sampling and measurement
To study the utilization of DIC and calcification rate during the day and night, at various hours (30 min, 8 h, 12 h, 12.5 h, 20 h and 24 h after treatment), 20 ml of the solution were removed and stored at −20°C (10 ml for DIC analysis and 10 ml for ion content measurement).Four flasks with plants and 2 flasks without plants were used for each point of the daily cycle.The DIC content of the solution was measured using a total organic carbon analyser (O.I. analytical 1010).Analyses of cation content were performed using a Dionex DX100 ion chroma to graph equipped with a 25 µl sample loop, a cation-exchange column and a suppressed conductivity detection system.For separation, an ion Pac CG10A guard column (50 × 4 mm) was coupled to an IonPac CS10A analytical column (250 × 4 mm).A Dionex CSRS ultra selfregeneration suppressor was employed, and the 2.0 software was used for system control and data acquisition.All IC-related equipment was supplied by Dionex.
Pulse-amplitude modulated (PAM) chlorophyll fluorescence parameters were monitored using a submersible PAM fluorometer (diving-PAM; Walz).RLCs were generated for branchlets mounted in the chamber.According to the procedure of Ralph & Gademann (2005), RLCs were generated using a pre-installed software routine, where the actinic light was increased in 8 steps (plus initial quasidarkness measurement) beginning at an initial irradiance for a prescribed duration.To allow relative electron transport rate (rETR) to reach a plateau (over at least 2 points) and decline during the last light steps, 8 consecutively increasing actinic light intensities of 3,16,42,77,125,187,339 and 501 µmol m −2 s −1 were used.The first saturating pulse occurred after the quasi-darkness period.The second to ninth measurements occurred after a stepped actinic irradiation of 10 s duration, from 3 to 501 µmol photons m −2 s −1 .An RLC takes approximately 90 s and records fluorescence yield (F ), maxi mum fluorescence yield of a light-adapted leaf (F ' m ), effective quantum yield of PSII (ΦPSII), and ETR, which can be plotted as a function of photosynthetically active radiation (PAR) irradiance.To quantitatively compare RLCs under different treatments, they were described by several character para meters: α (photosynthetic rate in the light-limited region of RLC), Ek (minimum saturating irradiance) and rETRm (maximum rETR), determined by non-linear regression with the empirical equation proposed by Platt et al. (1980).

Calculation and analysis
DIC uptake by plants in each sample was calculated as the difference of DIC content in the solution with plants and the means of DIC content in 2 flasks without plants.A similar approach was used for Ca 2+ uptake.To avoid the effect of the pre-experiment fresh weight of plants, utilization of DIC at each sampling time was expressed as µmol C g −1 fresh weight, while calcification was expressed as µmol Ca g −1 fresh weight.To study the daily rhythm of calcification, calcification rate was defined as µmol Ca h −1 g −1 of fresh weight (Ca 2+ uptake ); similarly, utilization rate of DIC was expressed as µmol C h −1 g −1 of fresh weight (g) (DIC uptake ).
Regression curves were used to express the time course of DIC concentration in the solution.The effects of HCO 3 − addition and light increase on the Ca 2+ and DIC contents of the solution were tested using 2-way ANOVA.Pearson's correlation was used to analyse the relationship between utilization of DIC and DIC concentration, as well as calcification and DIC concentration in solution.Inverse regression curves were used to express the time course of Ca 2+ uptake .The correlation between DIC uptake and Ca 2+ uptake was calculated by linear regression.Nonlinear regression was used to determine the character parameters of RLC, and the effects of DIC addition and light increase on the above parameters were evaluated by 2-way ANOVA.All data were analysed using SPSS 13.0.

Effect of plants on DIC concentration in the solution
As expected, plant photosynthesis and calcification significantly affected the DIC concentration of the solution.The variation of DIC concentration in the solution across sampling times for all treatments could be fitted to a quadratic equation, i.e.DIC concentration sharply decreased during the light period but slowly increased during the dark period (Fig. 1).
The results of 2-way ANOVA revealed that both light levels and the addition of HCO 3 − significantly af fected the contents of DIC at 12 and 24 h sampling times (Table 1).The largest change in DIC was found in LL-HC at 12 h and in HL-HC at 24 h (Fig. 1).Addition of HCO 3 − significantly de creased the pH of the solution, therefore ΔpH at low HCO 3 − levels was higher than that at high HCO 3 − levels (Table 1).

Variation in DIC utilization and calcification
of Chara thalli DIC utilization (µmol C g −1 ) followed a negative quadratic equation with sampling time (Fig. 2).Pearson's correlation showed that DIC utilization (µmol C g −1 ) was significantly negatively related to DIC concentration for each treatment, whereas significant correlation between calcification (µmol Ca g −1 ) and DIC concentration only occurred under low light conditions (Table 2).The highest calcification rate (Ca 2+ uptake ) was found at 0.5 h, with sharp decreases in Ca 2+ uptake at all subsequent time points for all treatments.An inverse regression demonstrated a significant de crease in Ca 2+ uptake over time (Fig. 3).HL decreased Ca 2+ uptake , whereas the addition of HCO 3 − increased Ca 2+ uptake .The calcification rate was linearly related to the utilization rate of DIC for all treatments (Fig. 4).Ratio of calcification rate to utilization of DIC rate, i.e. slopes of lines shown in Fig. 4, was higher at HC than at LC, while it was lower at HL than that at LL levels (Table 1, Fig. 4).

Responses of chlorophyll fluorescence to light levels and addition of HCO 3 −
All plots of ETR as a function of irradiance showed the classical shape of a photosynthesis-irradiance (P-E) curve, with a linear rise in the light-limited region followed by a plateau, where PAR is above Ek and the photosynthetic pathway becomes limited (Fig. 5a).Both HL and HC significantly increased rETRm and Ek (minimum saturating irradiance).There was a significant inter action on rETRm be tween light level and DIC addition (Table 3).The highest rETRm was found in HL-HC (Fig. 5b).

Daily rhythm of the calcification rate
To date, no experiment under constant light intensity has addressed the daily rhythm of calcification of Chara thalli.Although many studies have been performed on coral calcification, revealing non-constant daily patterns of calcification rate, the results exhibited many specific-species differences (e.g.Moya et al. 2006, Al-Horani et al. 2007).Here, we found that under the constant light intensities of 90 and 150 µmol photons m −2 s −1 , the calcification rate of Chara thalli showed a continuous decrease during a 24 h period (light:dark, 12:12 h) at both DIC levels tested (Fig. 3).According to McConnaughey (1998), the calcification rate of Chara thalli is mainly affected by light intensity and Ca 2+ and DIC concentrations in the solution.In the present study, to avoid the effect of Ca 2+ and Mg 2+ contents on calcification, Ca 2+ content in the solution was above 60 mg l −1 , while Mg cation rate should be ascribed to a restriction in DIC concentration.This is further supported by a significant negative Pearson's correlation between calcification and DIC concentration under LL conditions and the much lower calcification rate found in the HL-LC treatment (Fig. 3).Our finding that external DIC concentration significantly af fects the calcification rate of Chara thalli differs from the findings of previous studies that reported endogenous (plant body) inorganic carbon being used in the calcification of Chara (McConnaughey 1991).In addition, many studies of coral have shown that calcification is independent of exogenous DIC concentration (e.g.Furla et al. 2000).However, this independence of calcification on DIC content in the solution can be explained by the presence of sufficient inorganic carbon within the plants.In this study, endogenous inorganic carbon was exhausted during pre-treatment.Thus, external HCO 3 − showed a significant effect on the calcification rate of the plants.

Dark calcification
According to the trans-calcification models proposed by McConnaughey & Whelan (1997), Chara calcification does not require light.However, to date no experimental study has provided direct data on dark calcification of Chara.In the dark, plant photosynthesis ceases completely and CO 2 is produced by respiration; therefore, DIC uptake of the plants (calculated as the difference of DIC content in the solution with plants and the means of DIC content in 2 flasks without plants in the present study) from photo synthesis during the dark period would decrease re sulting in reduced utilization of DIC.In other words, the increased utilization of DIC in the dark must result from calcification, as observed in HL-HC (Fig. 2).In our study, the calculation of the utilization of DIC did not take CO 2 produced by dark respiration into consideration.Therefore, the utilization rate of DIC by calcification in darkness was underestimated.Although light is generally not considered to be necessary for calcification, higher calcification rates have been found in the light than in the dark for calcareous plants.This phenomenon is termed light-enhanced calcification and has already been de scribed in some taxonomic groups, including scler acti nian corals (e.g.Furla et al. 2000, Al-Horani et al. 2003, 2007), coccolithophorids (e.g.Nimer & Merrett 1992, 1993), and Chara spp.(McCon naughey & Falk 1991, Wang 2008).However, in our study, the calcification rate of Chara thalli under LL was higher than that under HL (Fig. 3).Light-enhanced calcification occurs through enhanced O 2 production (Rinkevich & Loya 1984) or pH change (Moya et al. 2006).For Chara plants, calcification accompanies the photosynthetic utilization of HCO 3 − ; therefore, calcification competes with photosynthesis for available DIC.The possible explanation for our finding that the calcification rate is lower under HL might be that, under LC, calcification is limited by the competitive capability of photosynthesis for DIC.This could also explain the higher calcification rate found in HL-HC than in HL-LC.

Dependence of calcification on the utilization of DIC
Here, we identified a linear correlation between calcification rate and the utilization rate of DIC (Fig. 3).Although a similar result has been reported in a coccolithophorid, the level of interaction between photosynthesis and calcification was taxondependent (Nimer & Merrett 1992).We reported that a high competition for DIC between photosynthesis and calcification exists in Chara thalli (Wang et al. 2013), whereas no such competition was found in Stylo phora pistillata microcolonies (McConnaughey & Falk 1991, Furla et al. 2000).In the present study, the utilization of DIC resulted from photosynthesis and calcification of plants.We speculate that the high dependence of calcification on the utilization of DIC in Chara thalli was primarily related to the ratio of calcification to photosynthesis (C:P).A consequence of this is that any increase or decrease in the C:P ratio by HCO 3 − addition causes a corresponding change in the calcification to DIC utilization ratio (Wang et al. 2013).According to our results, high light intensity and increased HCO 3 -likely affect the ratio of calcification to the utilization of DIC by affecting the C:P ratio.
Owing to the sound understanding of the relationships between fluorescence parameters and photosynthetic electron transport in vivo (Maxwell & Johnson 2000, Baker 2008), RLCs of chlorophyll fluorescence can provide direct evidence about the effects of light level and additional HCO 3 − on photosynthesis in Chara thalli.In the present study, both light increase and addition of HCO  Data are mean ± SD (n = 4).Treatments are defined in Fig. 1 the ETRm and Ek of RLCs (Fig. 5), indicating that light and external DIC in creased the photosynthetic rate and adaptive capacity of the plant to a high light level, which has been reported for some seagrasses (Björk et al. 1997, Schwarz et al. 2000).Our finding of a significant positive interaction on ETRm between light and HCO 3 − levels indicated that the photosynthetic performance of Chara thalli was the highest under both HL and HC.
Calcification in Chara thalli does not require light (McConnaughey & Whelan 1997).Therefore, light increased the utilization of DIC by photosynthesis, resulting in decreased DIC utilization by calcification.Thus, the ratio of calcification to the utilization of DIC decreased at HL relative to LL (Fig. 4, Table 1).With HCO 3 − addition, both photosynthesis (Fig. 5) and calcification rate in creased and resulted in increased utilization of DIC (Fig. 4).However, extant carbon added as HCO 3 − was preferentially incorporated into calcite (Sikes et al. 1980).This suggested a higher C:P ratio, and correspondingly, a higher ratio of calcification to the utilization of DIC for LL-HC and HL-HC treatments (Fig. 4).Results from pH-drift experiments revealed that higher HCO 3 − -alkalinity suggested a stronger buffer capability (Kahara & Vermaat 2003).Our finding that there is a significant decrease in the pH of the solution in response to additional HCO 3 − is in line with the findings of the pH-drift experiments.This is also supported by the finding that external HCO 3 − inhibited the increase in solution pH by increasing calcification of calcareous plants, as reported by Wang et al. (2013).

CONCLUSION
Here, we showed that when endogenous inorganic carbon is exhausted, addition of HCO 3 − significantly promoted the calcification rate of Chara thalli.Calcification of C. vulgaris was primary restrained by HCO 3 − alkalinity in an open 24 h experiment.Further more, calcification of Chara thalli was dependent on the utilization of DIC; we propose that this dependence is determined by the ratio of calcification to photosynthesis affected by light level and DIC level.Therefore, calcification or CaCO 3 encrustations of Chara might be a useful indication of water chemistry in the field.

Table 1 .
F-values and significance of 2-way ANOVA of the effects of dissolved inorganic carbon (DIC) and light level on the ratio of calcification to utilization of DIC, increased pH and variation in DIC both at 12 and 24 h for Chara vulgaris thalli.***p < 0.001, **p < 0.01, *p < 0.5, ns: p > 0.5