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Mechanisms for carbon uptake in the small-bodied Halophila johnsonii and large-bodied Thalassia testudinum were compared using photosynthesis measurements (oxygen flux) with, and without, the extracellular carbonic anhydrase inhibitor acetazolamide (AZ) and TRIS buffer. Our results indicated T. testudinum and H. johnsonii both utilize external membrane-bound carbonic anhydrase to facilitate dehydration of HCO3− into CO2(aq). T. testudinum also utilizes an active proton (H+) pump to create localized H+ gradients within the boundary layer and it derives a larger portion of photosynthetic carbon from bicarbonate than H. johnsonii. Stable carbon isotope composition (δ13C) of T. testudinum and H. johnsonii leaves under high and reduced irradiance were also compared to evaluate photosynthetic carbon use. Isotope ratios of H. johnsonii were significantly more negative than T. testudinum at a high-light field site (−10.44 ± 0.26‰ vs −9.25 ± 0.25‰). In contrast, there was no significant difference in isotope ratios between species for plants maintained in a greenhouse under reduced photosynthetically active radiation. This suggests photosynthesis of T. testudinum is more carbon limited or more reliant on bicarbonate for photosynthesis than H. johnsonii under high-light conditions. Although photosynthesis of both species is expected to increase in response to greater CO2(aq) availability, ocean acidification and its CO2(aq) enrichment of seawater may have greater benefits for H. johnsonii compared to T. testudinum, whose photosynthesis relies more heavily on HCO3− use.

Plants of the threatened seagrass, Halophila johnsonii Eiseman, from a riverine and marine population were exposed to a series of salinity treatments within mesocosms. Survival and maximum photochemical efficiencies of PSII (Fv/Fm) were measured in response to varied frequency, duration, and amplitude of hyposalinity exposures. Both populations exhibited high survival after two cycles of short, pulsed hyposalinity treatments (100% and 89%, respectively). However, two cycles of long pulses of low salinity resulted in 100% mortality for marine H. johnsonii and >50% mortality for riverine plants. After two cycles of gradual salinity reduction to a salinity of 5, survival for marine and riverine plants was also low (22% and 33%, respectively). Fv/Fm values of riverine H. johnsonii were relatively high (0.65-0.70) after a single short pulse or gradual reduction to salinity of 10. Fv/Fm values of marine plants were lower in these two treatments and exhibited greater declines following a single, long pulse to a salinity of 10 or after a single gradual reduction to a salinity of 5. Fv/Fm values showed riverine plants were also more resistant to repeated pulses of moderate low salinity than marine plants. Our results indicate wide tolerances of H. johnsonii to short pulses of hyposalinity, but suggest that repeated or prolonged hyposalinity stress at near-tolerance salinity levels can have additive effects on photosynthetic health and survival. Differences in resilience of marine and riverine H. johnsonii populations are consistent with previous suggestions that estuarine ecophenes are more tolerant to hyposalinity than marine populations.

article i nfo The threatened seagrass Halophila johnsonii contains UV-absorbing flavonoids localized in the cuticle and cy- tosol of epidermal cells in both abaxial and adaxial leaf surfaces. Previous studies indicate that these com- pounds exhibit variations in response to salinity stress and may have physiological roles other than UV protection. In this study, controlled light and salinity mesocosm experiments were performed to investigate effects of salinity variation (35, 25 and 15) with or without light reduction in order to elucidate possible physiological roles of flavonoids in H. johnsonii. Response variables were measured over short (one day) and extended (three week) time periods and included flavonoid content and localization, Trolox equivalent antioxidant capacity (TEAC), and chlorophyll fluorescence parameters. Confocal laser scanning microscopy (CLSM) indicated that distribution of flavonoids in the cuticle and cytosol of abaxial and adaxial leaf epider- mal cells did not change in response to the salinity or light treatments. Likewise, salinity and light did not have a significant effect on total flavonoid content overall, although within day 1, flavonoid content was signifi- cantly higher among shade treatments. Among salinities, TEAC values were significantly higher for salinity 15 at 14 and 21 days; TEAC values were similar between shaded and unshaded treatments. Neither salinity nor light had significant treatment effects on individual photosynthetic parameters, but a reduction of salinity and light did have a significant interactive effect as rapid light curves (RLCs) for salinity 15 and 25 shade treatments were significantly lower than all other treatments. Flavonoids were significantly correlated with TEAC values, suggesting a possible antioxidant function for cytosolic flavonoids in H. johnsonii. However, a sunscreen role for cuticular flavonoids inthis species wasnot supportedastotal flavonoidcontent tendedtobe higher inshaded plants and intracellular flavonoid distribution was unaffected by light conditions.

a b s t r a c t In this study, flavonoid localization, content and total antioxidant capacity in leaves of subtidal Halophila decipiens were compared to intertidal and subtidal Halophila johnsonii. H. johnsonii leaves had significantly higher flavonoid content (3.5 and 3.8 nmol quercetin equivalent mm −2 leaf for intertidal and subtidal H. johnsonii, respectively) and antioxidant capacity (101.7 and 224.2 nmol Trolox equivalent mm−2 leaf for intertidal and subtidal H. johnsonii, respectively) than H. decipiens leaves (1.4 nmol quercetin equivalent mm−2 leaf and 21.0 nmol Trolox equivalent mm−2 leaf). Flavonoid content did not significantly differ between intertidal and subtidal H. johnsonii, however, antioxidant capacity was significantly higher in subtidal plants. Confocal laser scanning microscopy of fresh leaf cross sections indicated that both species contained flavonoids in the cuticle, but only H. johnsonii contained intracellular flavonoids. Intracellular flavonoids are better situated to perform antioxidant functions in planta. These results suggest that flavonoid compounds in H. johnsonii are capable of sunscreen and antioxidant functions while an antioxidant role for flavonoids within H. decipiens is not supported.