Cooperation of an external carbonic anhydrase and HCO3- transporter supports underwater photosynthesis in submerged leaves of the amphibious plant Hygrophila difformis.

BACKGROUND AND AIMS: HCO3- can be a major carbon resource for photosynthesis in underwater environments. Here we investigate the underlying mechanism of uptake and membrane transport of HCO3- in submerged leaves of Hygrophila difformis, a heterophyllous amphibious plant. To characterize these mechanisms, we evaluated the sensitivity of underwater photosynthesis to an external carbonic anhydrase (CA) inhibitor and an anion exchanger protein inhibitor, and we attempted to identify components of the mechanism of HCO3- utilization. METHODS: We evaluated the effects of the external CA inhibitor and anion exchanger protein inhibitor on the NaHCO3 response of photosynthetic O2 evolution in submerged leaves of H. difformis. Furthermore, we performed a comparative transcriptomic analysis between terrestrial and submerged leaves. KEY RESULTS: Photosynthesis in the submerged leaves was decreased by both the external CA inhibitor and anion exchanger protein inhibitor, but no additive effect was observed. Among upregulated genes in submerged leaves, two α-CAs, Hdα-CA1 and Hdα-CA2, and one ß-carbonic anhydrase, Hdß-CA1, were detected. Based on their putative amino acid sequences, the α-CAs are predicted to be localized in the apoplastic region. Recombinant Hdα-CA1 and Hdß-CA1 showed dominant CO2 hydration activity over HCO3- dehydration activity. CONCLUSIONS: We propose that the use of HCO3- for photosynthesis in submerged leaves of H. difformis is driven by the cooperation between an external CA, Hdα-CA1, and an unidentified HCO3- transporter.

Rewiring of hormones and light response pathways underlies the inhibition of stomatal development in an amphibious plant Rorippa aquatica underwater.

Land plants have evolved the ability to cope with submergence. Amphibious plants are adapted to both aerial and aquatic environments through phenotypic plasticity in leaf form and function, known as heterophylly. In general, underwater leaves of amphibious plants are devoid of stomata, yet their molecular regulatory mechanisms remain elusive. Using the emerging model of the Brassicaceae amphibious species Rorippa aquatica, we lay the foundation for the molecular physiological basis of the submergence-triggered inhibition of stomatal development. A series of temperature shift experiments showed that submergence-induced inhibition of stomatal development is largely uncoupled from morphological heterophylly and likely regulated by independent pathways. Submergence-responsive transcriptome analysis revealed rapid reprogramming of gene expression, exemplified by the suppression of RaSPEECHLESS and RaMUTE within 1 h and the involvement of light and hormones in the developmental switch from terrestrial to submerged leaves. Further physiological studies place ethylene as a central regulator of the submergence-triggered inhibition of stomatal development. Surprisingly, red and blue light have opposing functions in this process: blue light promotes, whereas red light inhibits stomatal development, through influencing the ethylene pathway. Finally, jasmonic acid counteracts the inhibition of stomatal development, which can be attenuated by the red light. The actions and interactions of light and hormone pathways in regulating stomatal development in R. aquatica are different from those in the terrestrial species, Arabidopsis thaliana. Thus, our work suggests that extensive rewiring events of red light to ethylene signaling might underlie the evolutionary adaption to water environment in Brassicaceae.

Transition From Proto-Kranz-Type Photosynthesis to HCO3 - Use Photosynthesis in the Amphibious Plant Hygrophila polysperma.

Hygrophila polysperma is a heterophyllous amphibious plant. The growth of H. polysperma in submerged conditions is challenging due to the low CO2 environment, increased resistance to gas diffusion, and bicarbonate ion (HCO3 -) being the dominant dissolved inorganic carbon source. The submerged leaves of H. polysperma have significantly higher rates of underwater photosynthesis compared with the terrestrial leaves. 4,4'-Diisothiocyanatostilbene-2,2'-disulfonate (DIDS), an anion exchanger protein inhibitor, and ethoxyzolamide (EZ), an inhibitor of internal carbonic anhydrase, repressed underwater photosynthesis by the submerged leaves. These results suggested that H. polysperma acclimates to the submerged condition by using HCO3 - for photosynthesis. H. polysperma transports HCO3 - into the leaf by a DIDS-sensitive HCO3 - transporter and converted to CO2 by carbonic anhydrase. Additionally, proteome analysis revealed that submerged leaves accumulated fewer proteins associated with C4 photosynthesis compared with terrestrial leaves. This finding suggested that H. polysperma is capable of C4 and C3 photosynthesis in the terrestrial and submerged leaves, respectively. The ratio of phosphoenol pyruvate carboxylase to ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the submerged leaves was less than that in the terrestrial leaves. Upon anatomical observation, the terrestrial leaves exhibited a phenotype similar to the Kranz anatomy found among C4 plants; however, chloroplasts in the bundle sheath cells were not located adjacent to the vascular bundles, and the typical Kranz anatomy was absent in submerged leaves. These results suggest that H. polysperma performs proto-Kranz type photosynthesis in a terrestrial environment and shifts from a proto-Kranz type in terrestrial leaves to a HCO3 - use photosynthesis in the submerged environments.

Gibberellic acid induces non-Kranz anatomy with C4-like biochemical traits in the amphibious sedge Eleocharis vivipara.

MAIN CONCLUSION: Gibberellic acid induces photosynthetic tissues with non-Kranz anatomy and C4-like biochemical traits in terrestrial-form plants of Eleocharis vivipara. This suggests that the structural and biochemical traits are independently regulated. The amphibious leafless sedge, Eleocharis vivipara Link, develops culms (photosynthetic organs) with C4-like traits and Kranz anatomy under terrestrial conditions, and C3 traits and non-Kranz anatomy under submerged conditions. The conversion from C3 mode to C4-like mode in E. vivipara is reportedly mediated by abscisic acid. Here, we investigated the effects of gibberellic acid (GA) on the differentiation of anatomical and photosynthetic traits because GA is involved in heterophylly in aquatic plants. When 100 µM GA was sprayed on terrestrial plants, the newly developed culms had non-Kranz anatomy in the basal part and Kranz-like anatomy in the upper part. In the basal part, the mesophyll cells were well developed, whereas the Kranz (bundle sheath) cells were reduced and contained few chloroplasts and mitochondria. Stomatal frequency was lower in the basal part than in the upper part. Nevertheless, these tissues had abundant accumulation and high activities of C4 photosynthetic enzymes and had C4-like δ13C values, as seen in the culms of the terrestrial form. When submerged plants were grown under water containing GA-biosynthesis inhibitors (uniconazole or paclobutrazol), the new culms had Kranz anatomy. The culms developed under paclobutrazol had the C3 pattern of cellular accumulation of photosynthetic enzymes. These data suggest that GA induces production of photosynthetic tissues with non-Kranz anatomy in terrestrial plants of E. vivipara, without concomitant expression of C3 biochemical traits. The data also suggest that the differentiation of C4 structural and biochemical traits is regulated independently.

Photosynthetic acclimation of terrestrial and submerged leaves in the amphibious plant Hygrophila difformis.

Hygrophila difformis, a heterophyllous amphibious plant, develops serrated or dissected leaves when grown in terrestrial or submerged conditions, respectively. In this study, we tested whether submerged leaves and ethylene-induced leaves of the heterophyllous, amphibious plant H. difformis have improved photosynthetic ability under submerged conditions. Also, we investigated how this amphibious plant photosynthesizes underwater and whether a HCO3 - transport system is present. We have analysed leaf morphology, measured underwater photosynthetic rates and HCO3 - affinity in H. difformis to determine if there are differences in acclimation ability dependent on growth conditions: terrestrial, submerged, terrestrial treated with ethylene and submerged treated with an ethylene inhibitor. Moreover, we measured time courses for changes in leaf anatomical characteristics and underwater photosynthesis in terrestrial leaves after submersion. Compared with the leaves of terrestrially grown plants, leaf thickness of submerged plants was significantly thinner. The stomatal density on the abaxial surface of submerged leaves was also reduced, and submerged plants had a significantly higher O2 evolution rate. When the leaves of terrestrially grown plants were treated with ethylene, their leaf morphology and underwater photosynthesis increased to levels comparable to those of submerged leaves. Underwater photosynthesis of terrestrial leaves was significantly higher by 5 days after submersion. In contrast, leaf morphology did not change after submergence. Submerged leaves and submerged terrestrial leaves were able to use bicarbonate but submerged terrestrial leaves had an intermediate ability to use HCO3 - that was between terrestrial leaves and submerged leaves. Ethoxyzolamide, an inhibitor of intracellular carbonic anhydrase, significantly inhibited underwater photosynthesis in submerged leaves. This amphibious plant acclimates to the submerged condition by changing leaf morphology and inducing a HCO3 - utilizing system, two processes that are regulated by ethylene.

Resurrection and typification of Elatine campylosperma (Elatinaceae), a long-forgotten waterwort species.

The name Elatine campylosperma Seub. is generally treated as one of the synonyms of E. macropoda Guss. However, recent morphological, phylogenetic and karyological studies indicate that this judgement should be revised. In the present paper we typify the name E. campylosperma, review its taxonomic history and provide a thorough description, with compilation of previously published data and our new measurements from in vitro cultures. Based on our herbarium survey, we outline its Atlantic-Mediterranean distribution area (Spain, France, Italy, Greece, Turkey and Algeria). Habitat preferences are summarized from our field observations, water quality measurements and the label information of the herbarium specimens examined. Intact E. campylosperma seeds were found in faecal samples of the Eurasian Coot (Fulica atra L.) in southern Spain and two of them were germinated, suggesting that E. campylosperma has a capacity for long distance dispersal via endozoochory.

Shifting effects of physiological integration on performance of a clonal plant during submergence and de-submergence.

BACKGROUND AND AIMS: Submergence and de-submergence are common phenomena encountered by riparian plants due to water level fluctuations, but little is known about the role of physiological integration in clonal plants (resource sharing between interconnected ramets) in their adaptation to such events. Using Alternanthera philoxeroides (alligator weed) as an example, this study tested the hypotheses that physiological integration will improve growth and photosynthetic capacity of submerged ramets during submergence and will promote their recovery following de-submergence. METHODS: Connected clones of A. philoxeroides, each consisting of two ramet systems and a stolon internode connecting them, were grown under control (both ramet systems untreated), half-submerged (one ramet system submerged and the other not submerged), fully submerged (both ramet systems submerged), half-shaded (one ramet system shaded and the other not shaded) and full-shaded (both ramet systems shaded) conditions for 30 d and then de-submerged/de-shaded for 20 d. The submerged plants were also shaded to very low light intensities, mimicking typical conditions in turbid floodwater. KEY RESULTS: After 30 d of submergence, connections between submerged and non-submerged ramets significantly increased growth and carbohydrate accumulation of the submerged ramets, but decreased the growth of the non-submerged ramets. After 20 d of de-submergence, connections did not significantly affect the growth of either de-submerged or non-submerged ramets, but de-submerged ramets had high soluble sugar concentrations, suggesting high metabolic activities. The shift from significant effects of integration on both submerged and non-submerged ramets during the submergence period to little effect during the de-submergence period was due to the quick recovery of growth and photosynthesis. The effects of physiological integration were not found to be any stronger under submergence/de-submergence than under shading/de-shading. CONCLUSIONS: The results indicate that it is not just the beneficial effects of physiological integration that are crucial to the survival of riparian clonal plants during periods of submergence, but also the ability to recover growth and photosynthesis rapidly after de-submergence, which thus allows them to spread.