Functional chloroplasts inside animal cells: exploring the photoprotective mechanisms.

Università di Pisa

Dipartimento di Scienze Agrarie, Alimentari e Agro-ambientali

Corso di Laurea Magistrale in

Biotecnologie vegetali e microbiche (LM-7)

Tesi di Laurea Magistrale

“Functional chloroplasts inside animal cells: exploring the

photoprotective mechanisms.”

Relatori: Prof.ssa Guidi Lucia

Dott.ssa Sònia Marisa Gonçalves da Cruz

Correlatore: Prof. Guglielminetti Lorenzo

Candidato: Morelli Luca

Abstract

Sacoglossa sea slugs feed on algae and maintained chloroplasts photosynthetically active inside their body. Photosynthetic organisms have evolved different ways to protect photosystem I and II (PSI and PSII respectively) from photodamage like adjusting light absorption, and the non-photochemical quenching (NPQ) processes but chloroplastic genome can encode only a small part of the proteins and pigments necessary for the protection against light stress (Edrhard et al. 2008). NPQ is related to the xantophyll cycle (XC): a reversible interconversion of zeaxanthin (Z) and violaxanthin (V) thank to the enzyme violaxanthin deepoxidase that converts violaxanthin to zeaxanthin (via antheraxanthin, A) and increases the depoxidation state of the XC pool. The zeaxanthin epoxidase catalyzes the reverse reaction. This work is divided into three parts. In the first experiment, Fv/Fm ratio (potential photochemical PSII efficiency) and rapid light curves (RLCs) were calculated in E. viridis and P. dendritica. The same was realised in E. timida and A. acetabulum. The Fv/Fm ratio and the RLCs remained constant regardless of the starvation period and of the morphotype except in P. dendritica. The second experiment was an analysis of the host-mediated photoprotection; B. hypnoides and of C. tomentosum were cultured under Low light (LL; 20 µmol of photons m-2 s-1) and High light (HL; 200 µmol of photons m-2 s-1). When the light curves showed a photoacclimation the animals were fed with the two algae The area of the dorsal surface of specimens of E. viridis and E. timida was measured via software after a fixed time of exposure to light before and after the feeding. Results suggested an influence of the acclimation light on the host-mediated photoprotection. The pigment composition of the animals and the algae was then analysed via HPLC. The pigment profile was maintained after one week of starvation in all the specimens except P. dendritica confirming the ability for a kleptoplastic animal not to digest the plastids immediately. An unknown carotenoid spotted only in E. viridis and E. timida could probably be implied to stabilize the chloroplast inside the animal cell. A build-up of the pigment trans-neoxanthin, that usually protect the LHCII from light, was observed in the HL acclimated organisms. An analysis of E. viridis treated with a Light-Stress-Recovery (LSR) allowed to identify this process as a long-term photoacclimation mechanism. The xantophyll cycle was analysed via HPLC in specimens of A. acetabulum and E. timida undergone the LSR protocol. The de-epoxidation state of the cycle [(A + Z)/(V + A + Z)] showed a normal cycle in the algal

sample and in the fed slug while in the starved animals was reported an inability to reconvert Z to V probably related to an excessive damage of the chloroplast, so unable to influence the gene expression. This work provides a series of starting point to proceed with a deeper analysis, relying also on molecular tools. These knowledges, opportunely integrated, would be a great support for all the engineering project involved into artificial photosynthesis.

Summary:

1

Introduction

6

1.1 The HULK project and its goals 6

1.2 The algae 7

1.3 The Chlorophyta 9

1.4 The Sacoglossan Opistobranchs 14

1.5 Kleptoplasty: how to rob a plastid 19

1.6 The photosynthetic machine: The chloroplast 22

1.7 Photosynthesis 25

1.8 The photoprotective mechanisms 32

1.9 The photobiology and its experimental measurements 39

2

Chapter 2: The first round with kleptoplasty

44 2.1 Introduction: the quantitative measurements of photosynthesis 44 2.2 Matherial and methods: Animal and algal material, system setting 46

2.3 Starvation experiment 47

2.4 Morphotypes comparison 47

2.5 Rapid light curves test 48

2.6 Fv/Fm measurements 49

2.7 Statistical analysis 49

2.8 Results 49

3

Chapter 3: How to face light with shadows: the

behavioural photoprotection

55

3.1 Introduction: host-mediated photoprotection 55

3.2 Animal and algal material, system setting 60

3.3 Fluorescence analysis 61

3.4 Exposure of dorsal photosynthetic area 62

3.5 High Light (HL) acclimation vs Low Light (LL) acclimation 63

3.6 Statistical analyses 63

4

Chapter 4: How to face light with colours: the

pigment-based machinery

76

4.1 Introduction: the photosynthetic pigments 76

4.2 Animal and algal material 80

4.3 Light-Stress-Recovery protocol (LSR) 80

4.4 Preparation of samples for the HPLC 81

4.5 Statistical analyses 82

4.6 Results 82

5

Discussion of the results

96

6

Conclusions and future remarks

113

Appendix A

114

Appendix B

116

Chapter 1:

Introduction

1.1 The HULK project and its goals

This master thesis was realised thank to collaboration with the University of Aveiro in Portugal and the CESAM (centre for marine and enviromental studies) in the context of the HULK project: “Functional chloroplasts inside animal cells, cracking the puzzle”. Was acknowledged support for laboratory work via R&D HULK project (PTDC/BIA-ANM/4622/2014 – POCI-01- 0145-FEDER- 016754) and the financial support to CESAM (UID/AMB/50017 – POCI-01- 0145-FEDER- 007638) by the Portuguese Foundation for Science and Technology (FCT; “Fundação para a Ciência e Tecnologia”), and FEDER (Fundo Europeu de Desenvolvimento Regional), within the Portugal 2020 Partnership Agreement and COMPETE 2020 (European Union).

Sacoglossa sea slugs feed on algae and sequester chloroplasts (kleptoplasts), which can be maintained photosynthetically active inside tubule cells of their digestive diverticula for different periods. This unique animal-plant association is far from being understood. It has been assumed that kleptoplast photosynthesis acts as a nutritional source during food depletion, even though little evidence exists on the putative translocation of photosynthates between plastids and host. Recent research has shown that photosynthesis might not be essential for animal survival in the absence of food, speculating that kleptoplasts act as food depots (Christa et al. 2014).

Photosynthesis in kleptoplasts can be sustained for several months after separation from its algal nucleus. Maintenance of functional plastids inside metazoan cells in the absence of the algal nucleus is puzzling, as several photosynthetic components display short lifespan. It has been hypothesized that algal nuclear genes transferred to the animal would support photosynthesis, but recent reports contradict this theory. (Wågele et al. 2011). Alternative hypotheses rely mostly on chloroplast robustness. Retention of functional kleptoplasts possibly results from a combination of several physical and molecular non-described mechanisms. In marine symbioses, the efficient use of light play a major role in keeping healthy associations. In Sacoglossa sea slugs, recent evidence indicates a major contribution of photoprotection mechanisms in the maintenance of photosynthetic activity in kleptoplasts (e.g. Jesus et al. 2010, Cruz et al. 2015). The aim of the study was to understand the role of kleptoplast in host metabolism and to determine what mechanisms are responsible for kleptoplast survival inside the animal cell. The principal questions are: do

photoprotective processes mitigate kleptoplast oxidative stress in Sacoglossa sea slugs thus contributing to kleptoplast longevity? What are the origins of these photoprotective mechanisms? To answer these questions the work was divided into three parts: the first was an investigation about the basic properites of a kleptoplastic organism to be able to set the extension and the limits of the following experiments. The second part was an investigation of the “Photoprotective behaviour”: the behaviour of the animals can be used to protect the chloroplast from the excessive oxidative stress avoiding excessive light or covering sensible part of the body. This aspect has been investigated by chlorophyll fluorescence analysis techniques and optical microscopy techniques. The third part was the analysis of the biochemical and physiological component of the photoprotection mechanisms in the algae that can be reproduced by the animal like the xantophyll cycle orthe accumulation of particular pigments. Non-photochemical quenching of chlorophyll a fluorescence (NPQ) was recorded upon light stress and during the recovery in which NPQ relaxation occurs.

1.2 The algae

Algae are studied by a branch of Biology called Phycology, term derived from the greek word “phycos” with the meaning of seaweed. The algae are tallophytes so they have not roots, stems or leaves. They have chlorophyll a as photosynthetic pigment and lack a sterile covering of cells around their reproductive cells. Algae are highly resilient and they can develop in many different enviroments but mainly they inhabit acquatic enviroments. Some of them can also be found in the snow or on bare rocks thanks to lichen associations. In most habitats, they function as the primary producers in the food chain, producing organic material from sunlight, CO2 and water by the photosynthetic process, producing also oxygen necessary for the metabolism of the consumer organisms. In fact a big percentual of the oxygen in the air is coming from the algae living in the ocean. Some algae can also be directly consumed by human and included in a diet; some of them are used to extract useful industrial compounds like carragenin or agar.

ü Structure of the algal cell and thallus

There are two basic types of cells in the algae, procaryotic and eucaryotic. Procariotic cells lack membrane-bounded organelles like plastids, mitochondria, nuclei, Golgi bodies and flagella and they are typical of the Cyanophyta (blue-green algae) and the

Prochlorophyta. The remainder of the algae are eucaryotic and have organelles. An eucaryotic cell is often surrounded by a cell wall composed of polysaccharides that are partially produced and secreted by the Golgi body. The plasma membrane called plasmalemma surrounds the remaining part of the cell. The algae have some locomotory organs like flagella or cilia that propel the cell through the medium where they are living.

In general, algal cell walls are made up of two components: the fibrilliar component, which is the skeleton of the wall, and the amorphous component: a matrix with fibrilliar component providing elasticity. The first is made mainly of cellulose a polimer of 1,4 linked β-D-glucose. In some siphonaceous green algae and in some Rhodophytae cellulose is replaced by mannan. In the phaeophytae is present a polimer of mannuronic acid residues called alginic acid and a polymer of fucose called fucoidin. Algae contain also plastids; the basic type of plastid is the chloroplast, which is capable of photosynthesis. A proplastid is a reduced plastid with few or no thylakoids and usually it develops in a chloroplast. A leucoplast or amyloplast is a colorless plastid that has become adapted to accumulate storage products. Some algae have the pirenoid, i.e. a differentiated region within the chloroplast composed of polypeptides with enzymatic properties as ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBiSCo) the key enzyme involved in CO2 assimilation (Salisbury and Floyd, 1978). This “organ” is considered a primitive evolutionary characteristic. Many motile algae or sexual cells of macroalgae (that are motile cells) have groups of carotenoid lipid globules that constitute an orange-red eyespot or stigma. These globules shade the photoreceptor, an area of the plasma membrane or chloroplast envelope containing specialized molecules (Melkonian and Robenek, 1980). For example in Chlamydomonas the photoreceptor consists of a chromophore (11-cis-retinol) linked to a protein embedded in the plasma membrane. When the alga is swimming through the medium the photoreceptor is shaded for various periods by the eyespot (normally made from lipid droplets near the photoreceptor) depending on the orientation of the algae. This shading is translated in a change of potential of the membrane thanks to a change in rhodopsin shape and this change of potential causes an influx of calcium ions into the cells: this affect the beating of the cells and the direction of the movement (Kamiya and Witman, 1984). A motile cell may swim toward light (positive phototaxis) or away from light (negative phototaxis).

The structure of the thallus is extremely different within the various species of algae: many algae are unicellular and can move by using flagella or by following the different streams of water always according the direction to their light preference and necessities. Some of them can also form colonies and, in this case, the algae are very near but there is not a total loss of indipendence. Other algae have a filamentous type of development. Filament results from cell division in the plane perpendicular to the axis of the filament and have cell chains consisting of daughter cells connected to each other by their end wall. Filaments can be simple like in Spirogyra (Chlorophyta) or can have false branching or true branching.

Another type is the Siphonocladous type: the algae with this cytomorphological design have multicellular thalli, with a basicalli mono-series filament, a branched or unbranched organization, composed of multinucleate cells because of incomplete cell divisions. Even if there are not clear, physical borders the cytoplasmic domains behave like independent entities like pseudocells.

The siphonous type instead consists of a single giant tubular cell containing thousands to millions of nuclei dividing by asynchronous mitosis. There are not cross-walls and often is taken the form of branching tubes. Examples of this habit are Bryopsis and Acetabularia sp. (Chlorophyta). Because of their structure siphonous algae are very vulnerable to injury but also very resilient to have the ability to regenerate the damage. The parenchymatous and pseudo parenchymatoud type consist of mostly macroscopic algae with tissue of undifferentiated cells and growth, which originates from a meristem with cell division in three dimensions. Often the various sheets are held together by mucilage. The last one is the palmelloid type. In this case, there are nonmotile and independent cells embedded with a commond mucilaginous matrix. Sometimes this type can be a stage of the life cycle of another alga, in certain enviromental conditions (Graham, 2012)

1.3 The Chlorophyta

The Chlorophyta, called also green algae, have chlorophylls a and b and form starch within the chloroplast usually in association with a pyrenoid. The chlorophyta thus differ from the rest of the eucaryotic algae because they form the storage product inside the chloroplast and not inside the cytosol. The chlorophyta are mainly freshwater

(Smith, 1955) but some orders are predominantly marine (Caulerpales, Dasycladales, Siphonocladales).

ü Cell structure and reproduction

In the Chlorophyta cell walls generally have cellulose as principal structural plysaccharide even though some microalgae for example have walls composed by glycoprotein as glycerol. Chloroplasts pigments are similar to those of higher plants: chlorophyll a and b are present and the main carotenoid is lutein. Exceptions are represented by siphonacean green algae that have siphonoxanthin. If some carotenoids are accumulated outside the chloroplast this is index of a color change in the alga that can become orange or red. The principal carotenoid in this case is beta-carotene or some keto derivatives (Graham, 2012).

Chloroplasts are surrounded by a double membrane chloroplast envelope and there is not chloroplast endoplasmic reticulum while the thylacoids are stacked into bands of three to five unities. Some species show amyloplasts while the chloroplast DNA is usually partially looped and is present in a zone of the 70S ribosome free of the stroma. In Acetabularia is shown that only 20-25% of the chloroplast have DNA (Woodcock and Bogorad, 1970). The starch usually is synthesized in the chloroplast as in higher plants.

The reproduction can be asexual or sexual, thus involving the gamete formation or not. There are various types of asexual reproduction; the simplest one is the fragmentation of colonies into two or more part. Some algae are more predisposed to this type of reproduction and every new part become a new colony. Commonly zoosporogenesis also occurs: zoospores are produced in vegetative cells and usually by the younger parts of the algal filament. There are different typologies of spores: aplanospores which are nonflagellated and have a wall distinct from the parent cell wall; autospores are similar to the other ones but have the same shape as the parent cell. Coenobia are colonies with a definite number of cells organized in a specific manner.

Sexual reproduction: in the Chlorophyceae it may be isogamous, anisogamous or oogamous. Gametes are usually specialized cells and not only vegetative cells. Sometimes gametogenesis is induced by enviromental factors while in some other organisms the presence of two different strains is necessary. In this case vegetative cells begin to secrete a substance that initiates sexual differentiation in competent cells of the opposite sex. In isogamous species, instead sexually different gametes meet at random and immediately adhere for an agglutination reaction. At the beginning, the two cells

conserve the flagells but it is released during the mating process. After the fusion of gametes (syngamy), meiosis occurs in the zygotes of various species of alga.

ü Codium tomentosum

Codium tomentosum is an alga belonging to the division of Chlorophyta, class, Bryopsidophyceae, order, Bryopsidales, family, Codiuaceae and it is usually called “Spongeweed”. It is a spongy, repeatedly dichotomously branched green alga without any flattening at the forks of the branches. It lives in the marine enviroment mainly on the Portuguese, English and Galician coast. It lives sticked to rock and other sessile organisms like molluscs. Codium occurs from the low tide mark up to 70 m depth in tropical and temperate marine waters. It is very resilient and for this reason from its introduction, it has become a pest in oyster beds. The thallus has a crustose prostrate portion that bears several cylindrical dichotomously branched shoots. These bodies have a central medulla composed of colorless filaments that give rise to thick branchlets called utricles (Figure 1).

Figure 1: Particular of utricles of Codium tomentosum (Brad Scott 2015).

They surround the medulla and have a layer of cytoplasm around a large vacuole. There is a differencial distribution: the discoid chloroplasts are in the outer part of the cytosol while the nuclei are in the interior part. The filaments of the medulla are divided in places by walls. From the utricles of the diploid thallus are produced dark green female and brown male gametangia. The gametes are formed meiotically and are released from the apical portion of the gametangium that breaks releasing a gelatinous substance with a central canal for the mobility of the gametes that at the beginning are transported passively but after some time extrude the flagella. The male gamete after will lose its flagella and the propel of the zygote will be up only to the flagella of the female gamete (Lee, 1980).

Whole plants of Codium are able to fix nitrogen thanks to association with some bacteria like Azotobacter on the surface of the alga (Head and Carpenter, 1975). The bacterium uses the glucose secreted by the alga at the rate of 0.7 to 1.3 mg of glucose per gram of dry weight per hour and in turn, it fixes nitrogen. During winter, months when the availability of dissolved inorganic nitrogen is at its highest Codium accumulates reserves of nitrogen, which will be utilized in deficiency periods (Hanisak, 1979). The period of maximum carbon fixation, pigment content and chloroplast size occurs during early winter when the conditions are stable and there is not competition with other algae. In summer because of the drying of intertidal spaces Codium develops less. The alga is implied in several symbiotic relationship expecially with some molluscs that can take advantage of the chloroplasts of this alga to develop a phenomenon called kleptoplasty. Being a coenocytic alga is a crucial factor for this to happen because, lacking septa the ingestion of the cytoplasm is facilitated.

ü Bryopsis hypnoides

Bryopsis hypnoides is an alga belonging to the division of Chlorophyta, class Chlorophyceae, order Bryopsidales, family Briopsidaceae, genus Bryopsis. Lamouroux described it for the first time in 1809 in the “Cahiers be Biologie marine”. It develops in filamentous tufts and can grow up to 10 cm tall. It branches following an irregular pattern and only the primary axis is much branched. (Figure 2)

Figure 2: Particular of the branch of Bryopsis hypnoides (Sjøtun, 2006).

Fronds decrease in diameter with each successive division; branchlets forms irregularly, undifferentiated from axes, constricted at base. Rhizoidal system is fibrous. The colour is a dark and dull green. It is very common near freshwater and nutrient rich outputs so it is easy to find in Atlantic Ocean, Australia, Mediterranean and Caribbean Sea where it attaches to hard surfaces such as basalt, rocks or rubble forming delicate fronds, which move with currents. Main axes is 65-140 µm diameter, branchlets are

40-80 µm diameter. Apices are rounded. Vegetative pennae function as the gametangia. Plants are dioecious, with male plants becoming yellowish-green and female plants turning dark green. Gametangia develop from the laterals pinnae and release anisogametes into the water. From the fertilization, originate Planozygotes that attacked to the substrate and grow in tiny filaments with a single large nucleus. At this point nuclear division occurs forming many zoospores that regenerate the vegetative thallus (van den Hoek et al. 1995).

Bryopsis hypnoides is usually a small part of the biodiversity of a marine ecosystem because it never becames excessively big in its develop but, like most green algae Bryopsis is very opportunistic in eutrophic conditions so that when the concentration of nitrate in water is very high, its development increases drastically resulting in an higher biomass and an enviromental invasion. For this reason Bryopsis species are potentially invasive and some of them can result in generating toxic substances.

ü Acetabularia acetabulum

Acetabularia acetabulum is an alga belonging to the division Clorophyta, class Ulvophyceae, order Dasycladales, family Polyphysaceae. It is commonly known as “Mermaid’s wine glass”. Typically, it grows in large clumps covering rocks. A not ramified stalk anchored to the substrate with a rhyzoid composes it. During its growth, this alga develops whorls of lateral branches that progressively degenerate with the increase of the lenght leaving a ringo of scars on the stalk. The cap is formed of adherent branches called cap rays and these are actually gametophores. This alga is, in reality, composed just by one enormous cell. After the development of the cap, numerous secondary nuclei stream in the gametophores imparting a dark green color. (Figure 3).

Every section of the gametophore is divided by the others thank to a septum that breaks to release the cysts when they reach maturity. Gametes are lately released from the cysts and they swim to find other gametes and to mate by agglutination. Zygotes are then negatively phototactic and settle on the substrate to develop the remaining structures. This alga lives in the warm water of shallow lagoons. The thallus is often calcified except when Acetabularia is growing in warm stagnant waters. In that case, the calcification is degraded and the stalk is free. This alga is the favourite one for the studies of morphogenetic since the first approach of Haemmerling in 1930. The main advantage is related to the position of the nucleus that, during the vegetative phase, is in the rhizoids. It is possible to remove the rhizoids with the corrispondent nucleus and to graft it onto cut parts of another Acetabularia allowing thus the nucleus to influence the morphology of the new alga (Haemmerling et al. 1963). This organism can show color variations related with its light acclimation state because of a pigment shift. (Costa et al. 2012). The capacity of the cell for morphogenesis is related to the content of substances produced by the nucleus: they accumulate in the cytoplasm and if the nucleus is removed, can control morphogenesys. These susbtances are in a gradient inside the algal body, the maximum quantity near the tip and the least near the base. The information necessary for the production of these substances is contained in the genes of the nucleus and is carried to the cytoplasm thanks to mRNAs that migrate from the stalk to the cap. These mRNAs can dominate not only the morphogenesis but other functions like the circadian rhytms, the chloroplast sizes, the polysaccharide content of the alga (van den Driessche, 1970).

1.4 The Sacoglossan Opisthobranchs

Sacoglossa are commonly known as “sap-sucking sea slugs” because of their feeding behaviour are a clade of sea slugs: marine gastropod mollusks. They live by ingesting the cellular content of algae thanks to their particular mouth anatomy. Some sacoglossans simply digest the fluid and the cellular content, which they suck from the algae, but in some other species the slugs sequester and utilize chloroplasts from the algae they eat integrating them in their tissues. This phenomenon is called kleptoplasty and can be found in some single-celled protozoa. Because the chloroplasts stay functional inside the animal performing the photosynthesis, the slugs have the title of the "solar-powered sea slugs", and this makes them unique among metazoa. The

Sacoglossa are divided into two clades according to the presence of the shell. The shelled families belong to the clade Oxynoacea while the shell-less families belong to the Plakobranchacea.

ü The feeding habit

All sacoglossans are distinguished from related groups for the structure of their radula. The radula is usually a membranous belt placed on the floor of the buccal cavity over an elongated, muscular and cartilaginous mass called odontophore. The radula usually bears transverse rows of theeth and arises from a deep outpocket called: radula sac. The odontophore can be projected out of the mouth and the radula can move to some extent over the odontophore. When this one is projected out of the mouth the changing tension causes the radula belt to flatten and this fact makes the theeth to erect. The theet curve posteriorly and the scraping causes a gradual loss of membrane and teeth at the anterior end which is compensated by the secretion of new theeth at the posterior end. In sacoglossans, the structure of the radula is slightly different from this general overview: there is the presence of a single row of teeth on the radula adapted for the suctorial feeding habits of the group. The uniseriate radula of the sacoglossa is composed of an ascending limb that correspond to the radular sac of the other gastropods, a long ventral limb surrounded by muscels and a posterios sac called “Ascus” where the teeth are stored packed or rolled (Thompson, 1976; Gasgoigne, 1977; Jensen, 1991). This structure is uniform within the Sacoglossa so the piercing-sucking feeding method is ubiquitous within the order. Because the algae used as food exhibit a wide range of charactheristics like toxic substances or heavy calcification there are some methods of dietary specialization in the animals: some of them can develop a morphological constraint imposed by certain food plants while others can exhibith a great plasticity. Sacoglossan teeth have a large, robust base with cospicous articulation knobs (Gascoigne, 1977) and a cusp that typically can have three basic morphological types:

- Broadly triangular cusps with or without denticles along with lateral margins. - Blade shaped cusps with lateral or median denticles.

- Sabot-shaped teeth with denticles along the lateral margins but this type are almost exclusive of the Stiligeriadeae.

Denticles are usually common in the animals that feed on algae able to calcify. In the species able to change diet, like Elysia viridis can be observed an high level of plasticity in form and dimension of teeth that can be related with the food source. Species isolated from Chaetomorpha for example have smaller teeth with almost straight, denticulate

cutting edges compared to the ones of the animals collected on Codium that have teeth of almost the same width of the utricles. When an animal is transferred from a food source to another there is a learning span of at least 2 weeks and during this time the morphology of teeth is not distinguishable from the one connected to the previous food source (Jensen et al. 1994). This learning process involves an increased efficiency in the process of piercing the algal cell, of sucking out the sap and of grasping the food (Jensen 1989). The feeding process can be divided into two phases: a “rasping” phase and a “sucking” phase. In the first one the teeth are active into piercing the cell wall, in the latter the muscular pharynx pumps in the cell sap of the food plant. Some Sacoglossans sometimes reverse the flow of alga cytoplasm returning the sucked material in a process called “Buccal regurgitation”. This process in a similar way to what happens in some blood-sucking insects is used to mix the algal content to the saliva to decrease the viscosity and eliminate eventual deposits of polysaccharidic substances (Jensen 1981, Gascoigne 1983). The animals used for this study are three sacoglossans specimens: Elysia viridis, Elysia timida and Placida dendritica.

ü Elysia viridis

Elysia viridis is a small to medium sized species of green sea slug (Figure 4) It is a marine Ophistobranch in the family Plachobranchidae. Even if there is a strong similarity between this sea slug and a nudibranch this animal is a Sacoglossa, so it is not so near to that clade of Gasteropods.

Figure 4: Image of Elysia viridis (Cochu, 2008).

The species lives in the northeastern Atlantic, from Norway to the Mediterranean Sea and can be found in the intertidal zone to a depth of about 5 m. The body is almost cylindrical and can grow up to 30 mm in lenght. The body is bright

green or brown according to its feeding state and its age and has iridiscent spots along its side. There are also two wing-like flaps extending along the body, which are usually folded called parapodia.

Usually the rhinophores (chemosensorial organs positioned on the surface of the body of sea slugs and nudibranchs used as scent or taste receptors) are usually rolled. This species has a wide range of algal food sources but it is mainly distributed on Codium spp. The most important characteristic from the algal structure point of view is the lack of septation. This makes the alimentation process easier. The chloroplasts are not digested and are instead stored and distributed into the digestive diverticular glands that are spread throughout the all body. The dorsal side of the animal is the part where the distance between the plastid and the outer enviroment is more reduced making it a very sensible part.

ü Elysia timida

Elysia timida (Risso, 1818) is a species of marine opistobranch gastropod mollusc (Figure 5).

Figure 5: Image of Elysia timida (Heurtaux, 2002).

It is mainly white with an high concentration of diverticular glands in the central part of the body that tooks the color from the algal food defining in this way various morphotypes. It feeds mainly on Acetabularia acetabulum but sometimes (like during the periods of maximum calcification of Acetabularia) it can shift feeding habits eating some brown algae like Padina pavonica (Ros et al. 1985). E. timida keeps the chloroplasts intact inside its body and uses them to do photosynthesis. This species occurs in the Mediterranian Sea, the Atlantic Ocean and in the Caribbean Sea living in shallow, well-lit, litoral habitat (Bouchet 1984, Thompson 1988). Studies of this

photosynthetic sacoglossan (Rahat et. al 1979) have demonstrated that the surfaces of its parapodia respond to the prevailing light intensity in such a way to ensure optimal exposure to light of its symbiotic chloroplasts.

One of the main differences with other sacoglossan specimens like E. viridis is in the development. Usually three distinct types of development can be recognized in the opisthobranchs: species with planktotrophic veliger larvae; species with lecithotrophic larvae and species with direct development, in which the veliger structures are recapitulated inside the egg, before hatching (Thompson, 1967). The sacoglossan elysioid molluscs were found to belong to the first type (Hamatani, 1960; Reid, 1964; Greene, 1968; Hagerman, 1970). E. timida, however, differs in its development from these elysioids. Because it shows a direct development hatching from the egg a “baby” E. timida that immediately saps the cytoplasm of Acetabularia and begin to grow completing its maturity cycle in less than three months. This characteristic contributes to use E. timida as an experimental model because of the possibility to have it growing in lab conditions.

ü Placida dendritica

Placida dendritica is a species of tiny sea slugs, it belongs to the class of Gastropoda and to the clade of Sacoglossa, the family is Limapontiidae. This species is highly common in the Portuguese territory. Like others Sacoglossa P. dendritica is able to pierce the surface of coenocytic algae to suck the content; however it is not able to retain chloroplasts for a long time and when they are inside the animal body are not completely functional but conserve only a residual activity because not all the chloroplasts inside the animal are undamaged but some of them are partially digested. The form of the body is very different from the one of the Elysiidae. Rhinophores are solid or rolled, not perfoliate with longitudinal ridges or vertical pinnate plumes (Figure

6) and cannot be retracted. The dorsum is covered by outgrowths called cerata (one in a

series or bundle of repiratory projections on the dorsal surface of a snail with reduced or no shell. Usually it contains outpocketings of the digestive gland.) An adult has up to 50 cerata and rhinophores but no parapodia. The P. dendritica is typically smaller than Elysia reaching a maximum total lenght of 1.4 cm but usually stopping at 0.8 cm. They have a static behaviour compared to other sea slugs often covering the thallus of the algal food with mucus and disgregating the alga. This slug is used as an experimental control for all the experiments where is needed a sea slug with “solar panels” but without “solar power”.

Figure 6: Image of Placida denritica (Rudman 2007).

1.5 Kleptoplasty: how to rob a plastid

Sacoglossan-algal relationship is clearly parallel to the plant-herbivore coevolutionary relationship of terrestrial enviroments (Clark and De Freese 1987). This retention of chloroplasts has been described as “chloroplast symbiosis” by various authors but several have been in disagree with the term (Taylor 1968, Blackbourn et al. 1973) wanting to change it with “Kleptoplasty” (Gilyarov 1983, Waugh and Clark 1986). These plastids are effectively stolen or borrowed organelles and their existance inside the animals is finite and non-reproductive. For this reason, the symbiosis appears to be slightly accidental but it is still relevant because it develops an active photosynthesis process: significantly higher rates of carbon fixation in light than in the dark, detectable synthesis of intermediates and products of the photosynthetic carbon fixation pathway, and oxygen evolution. The slugs seem to be plants and Brüel first recognized the true basis for their color (algal chloroplasts sequestered in tubules of the digestive gland) in 1914.

Clark et al. (1978) have proposed six stages in evolution of functional kleptoplasty: - Non-retention: animal feeds on algal food as potential donor of plastids but they

are digested almost immediately after the phagocytosis and the diverticola do not contain any algal pigment.

- Short-term non-functional retention: animal is pigmented for at least a few hours but no photosynthate is produced.

- Medium-term non-functional retention: structurally intact plastids last at least 24 hours but no photosynthetic activity can be demonstrated (es: P. dendritica).

- Short-term functional retention: animal exhibits photosynthesis in field enviroment but the plastids are rapidly digested and the functionality decreases when the animal is removed from the field.

- Medium-term functional retention: photosynthesis persists for more than 24 hours but ceases after some weeks in starvation.

- Long-term functional retention: photosynthesis lasts more than a week.

This classification defines a pattern of evolution of kleptoplasty. This initial stage in plastid retention probably functioned in nutritional homocromy (cryptic coloration based on retention of prey pigmentation) allowing a durable and accurate retention of chloroplasts. Long-term functional plastids appear in the most primitive representatives of the Elysioidea and Stiligeroidea and persist through advanced taxa but some advanced species often do not retain functional plastids because the food (Cladophorales and Seagrasses) is unsuitable and the chloroplasts are degraded during the feeding process. Sacoglossan have anatomical, physiological and behavioral adaptations to optimize benefits from kleptoplasty. For example, the digestive gland morphology varies with degree of kleptoplastid funtionality. Usually there is a drift from a holohepatic disposition of the digestive glands (digestive glands are packed in a single-block structure) to a cladohepatic one (digestive glands are branched throughout the body tissues) (Clark and Busacca 1978). Branching of diverticula into the surface tissues enhances the homochromic values of kleptoplastids and is a crytical support for plastids functionality because it increases the surface area for light absorption. All species with funtional plastids are cladohepatic to some degrees and extension of branches increases with level of photosynthate production because these products stimulate diverticular development and branching. In the members of the Elysiidae the entire dorsal surface is permeated with diverticula and the surface area for light absorption is maximized through modification of shape like the formation of parapodia (flaplike extensions of the dorsal body). Phototaxic behavior varies with plastids retention: animals that not retain functional plastids are photophobic while species with functional chloroplasts actively orient to light (Fraenkel 1927) and unfold their parapodia in optimal light intensities (Monselise and Rahat 1980) selecting often an intensity of light lower than the optimum probably to avoid excessive damages to the chloroplast. Inside the slugs, the chloroplasts are confined to the digestive cells of the hepatopancreas. The plastids occur in the cells of the “end bulb” of the tubules and in the cells lining the interconnecting ducts.

The biochemical interaction between the symbiotic chloroplasts and their host cells has been studied in only two species of sacoglossan. For many years E. viridis was the best example for the animal kleptoplasty but its veliger larva stage makes difficult to raise it in captivity so it was gradually juxtaposed by E. timida and E. chlorotica. E. viridis can sustain its symbiotic plastids thanks to the products of photosynthesis for a few months but loses weight during starvation periods and some photosynthetic activity (at least -47% after 2.5 months without food) (Evertsen and Johnsen 2009) is lost also because of plastid degradation. Some studies demonstrated the capacity of kleptoplasts to perfom CO2 fixation and that this carbon is important for the metabolism of the mollusc even if the effective pathway followed by this methabolite is yet unknown. (Kawaguti, 1965). The ability to mantain the photosynthetic activity without the support of an algal nucleus is interesting because the chloroplastic genome is able to encode only a small part of the proteins considered necessary for photosynthesis (Eberhard et al. 2008). One of the various hypothesis elaborated is the one of the horizontal gene transfer from the algal nucleus to the sea slug. The base of communication between the animal and the plastid is almost unknown but the main idea is that the communicative process is based on biochemical interactctions between kleptoplasts and the animal host cells (Rumpho et al. 2007). These processes usually involve intercompartmental crosstalk between the plastid and the nucleus: both anterograde (from the nucleus to the plastid) and anterograde (from the plastid to the nucleus) signalling and many photosynthetic proteins and enzymes are directly related to the nucleus and targeted to the chloroplast with the nuclear-encoded proteins able to manipulate the expression of plastid encoded genes. In addition, some of the methabolic machinery that equipped the ancestral green cyanobacterium progenitor were lost during the evolution inside the animal cells. For all these reasons, the crosstalk is particularly important also to regulate some mechanisms and processes related to the interaction between the light and the animal like the photoacclimation and the short-term and long-term photoprotection (Serodio et al. 2014).

The photoacclimation is one of the first responses to changes in light enviroment and it implies a series of adjustments in various components of the photosynthetic apparatus to regulate excitation pressure and maintaining carbon fixation while avoiding photo-oxidative stress. In algae, photoacclimation includes changes and modelling of the reaction centres of photosystems (RCI and RCII), photosynthetic pigments, photosystem II (PSII) stoichiometry, degradation and synthesis of antenna proteins. In

hospite is difficult to predict the ability of the kleptoplast to undergo this series of adaptation mainly because nuclear genes encode many of the genes that regulate this series of mechanisms. Since the beginning, research on kleptoplast photosynthesis has predominantly focused on the provision of photosynthates to the host. It seems that the host animals receive a benefit from hosting the kleptoplasts and obtain a significant amount of carbon from photosynthesis (almost the 60% of the carbon imput is derived from the photosynthesis of the plastid (Raven et al. 2001). In addition, the plastid gains benefits from the association even if the lifespan is much shortened. The photosynthetic performances of the kleptoplasts in fact appears to be superior to corresponding chloroplasts of their original algal cells probably because of the increased light exposure inside the branched digestive system or because of some improved photoprotection mechanisms provided combining mechanical and biochemical mechanisms. Another way for the animal to enhance the photosynthesis inside its cells is throught the inhibition of photorespiration because the intensity of this process is dependent on the CO2 to O2 ratio at the RuBisCO site. Inside the host cell there is a great consumption of O2 as long as a great availability of CO2 generated by the cellular functions.

1.6 The photosynthetic machine: The chloroplast

In green algae and in higher plants the chlorophyll is contained in a cellular plastid called chloroplast. In leaves, chloroplasts occupy about 8% of the total cell volume and are usually saucer-shaped bodies 4 to 10µm in diameter and 1 µm in thickness with an outer membrane separating them from the rest of the cytosol. Internally the chloroplast is comprised of a system of lamellae or flattened thylacoids, which are organized in stacks in regions known as grana. The grana are enveloped in a colourless matrix called stroma and all the chloroplast is surrounded by a double membrane (Figure 7).

The surfaces thus exposed show the distribution of chlorophyll-protein complexes seen as particles and mainly the PSII oxygen evolving complexes are predominant in the grana while the photosystem I (PSI) is mainly distributed in the stroma lamellae. The space between the thylakoid double membranes is called lumen. During the photosynthetic electron transport, protons are transferred to the lumenal space and used in ATP synthesis by the ATP synthase. The membrane are half lipid and half protein in their chemical composition: the proteins catalyze the enzyme reactions,

give mechanical strenght to the membrane and are associated with the principal photosynthetic pigments like Chl a and b.

Figure 7: Representation of the chloroplast structure (Taiz and Zeiger 2013).

The thylakoids of green algae are arranged in a very similar manner as those of the higher plants; they tend to be long and extend from one end of the chloroplast to the other. Adjacent thylakoids form stacked membrane regions that at a supramolecular level are very difficult to distinguish from the stacked grana membranes of higher plants (Staehelin et al. 1977). The fact that all green algae and higher plants possess stacked membrane regions but that the arrangement of these stacked regions is quite variable suggests that the thylakoid membrane stacking is very important from the functional point of view but the specific arrangement much less so.

ü The photosystem II

The photosystem II is that part of the oxygenic photosynthetic apparatus, which catalyzes the light induced oxidation of water, coupled to the reduction of plastoquinone. During the process, four protons are transferred across the thylacoid membrane from the stromal side to the lumenal side, which are utilized in ATP synthesis. PSII is located within the thylakoid membrane in close association with lipids (Figure 8) In the PSII there are three functional parts: an antenna system consisting of pigment-protein complexes, a reaction centre containing the primary reactants, plastoquinones and the water oxidizing complex and a regulatory cap made up of polypeptides bound to the lumenal surface of the thylakoid membrane.

Figure 8: Structure of the photosystem II (Taiz and Zeiger 2013).

The light-harvesting antenna can be divided into proximal and distal antennae. The proximal surrounds the P680 core and is tightly coupled to it. It consists of two pigment-protein complexes, CP47 and CP43 that contains Chl a and β-carotene bound to the apoproteins but no Chl b. Excitation energy cught by th antenna is transferred to P680 from the proximal complex. The distal is mainly composed of light harvesting complexes (LHC II) and contains chlorophyll a and b as well as xantophylls. The regulatory cap is comprised of hydrophilic extrinsic proteins that can act as a barrier between the complex for the oxidation of water and the lumenal phase (Taiz, 2013).

The PSII reaction centre can be subdivided into the light- absorption and electron transfer segments and the water oxidizing-complex. The core is the minima unit able to do light-induced charge separation and electron transport but it is unable to evolve O2. The core is composed by 4 to 6 molecules of Chl a, 2 molecules of pheophytin (Pheo), 2 molecules of β-carotene, 1 D1-D2 heterodimer proteins, the CP47 and CP43 proteins, cytochrome b559 and the 10 kDa polypeptide per P680. The P680 and Pheo are believed to be bound to the D2-D1 proteins. The family of proteins with low molecular weight represents the D1 protein (also known as PsbA); in higher plants, the N-terminal residues of both proteins (D1 and D2), which are exposed to the stromal surface, can be reversibly phosphorylated. After insertion in the membrane, the C-terminal of the D1 protein is cleaved by a protease to have the mature protein. This processing is essential for the assembly of a functional 4-atom manganese cluster located on the lumenal surface of the D1 and D2 proteins. In addition to the Mn cluster, the D1/D2 core binds to various cofactors, including: two Pheo molecules, only one of

which is phytochemically active, non-haem iron, and two quinones, Qa (bound to D2) and Qb (bound to D1). Upon light excitation, an electron is transferred from the primary donor (chlorophyll a) via intermediate acceptor pheophytin to the primary quinone Qa, then to the secondary quinone Qb. At the oxidising side of PSII, a redox-active residue in the D1 protein reduces P680. The oxidised tyrosine then withdraws electrons from a manganese cluster, which in turn withdraws electrons from water, leading to the splitting of water and the formation of molecular oxygen. PSII thus provides a source of electrons that can be used by PSI to produce the reducing power (NADPH) required to convert CO2 to glucose (Taiz, 2013).

1.7 Photosynthesis

The photosynthesis is a ubiquitous process being realised by terrestrial and marine organisms. One of the main differences between these two categories is the medium where they are developing; in the aquatic environment, many biochemical processes are influenced by the chemical properties of water. For example, the rate of diffusion of oxygen in water is 10.000 times slower in water than in the air, so the diffusional supply of CO2 to the plant surface is restricted in water because there is always a layer of water surrounding the plant surface, which this gas must pass by diffusion. This layer influences also the diffusion of O2 generated by photosynthesis. Light is also influenced by the acquatic medium. Plants use the 400-700 nm wavelenght band for photosynthesis that is called PAR i.e. photosynthetically active radiation. The energy of a photon is inversely proportional to its wavelenght (λ) according to the general relationship E=hc/ λ where h is the Planck’s constant (6.63x10-34 J s-1), c is the speed of light and λ is the wavelenght. The irradiance unit when referring to plant utilisation of light is µmol photons m-2 s-1.

In water, the spectral distribution of PAR reaching a plant is different from that on land because water attenuates the light intensity and the attenuation with depth is wavelenght dependent. The two main characteristics that determine the quantity and quality of irradiance are absorption and scattering. The first one is the property of the water molecules to absorb photons according to their energy: red photons are low energy photons and so are more readily absorbed than blue ones. Scatter is another property and is related to particles suspended in the water column: they attenuate light in water and affetct short-wavelenght photons that are “bounced off” by the particles

(this is particularly evident in the color pattern of the turbid waters). The zone that extends vertically from the sea surface down to the depht where enough light remains is called the euphotic zone, the irradiance at the deep end of this zone is usually set to 1% of the surface irradiance and this parameter deeply affects the ecology of the marine organisms according with their photosynthate necessities.

The salinity is another very important parameter for the marine photosynthesis. Mid-oceanic waters contain 3.1-3.8% of salts mostly represented by NaCl. For photosynthesis, there are two major effects of salts. First, salty waters usually contain less dissolved CO2 than freshwater at a given temperature and this fact may affect the acquisition of inorganic carbon (Ci). Secondly, salts negatively affect the activity of enzymes involved in the Calvin cycle of CO2 assimilation and reduction so the salinity has to be mantained low in the cells and their chloroplasts. This is usually accomplished by restricting the inflow of ions at the plasma membrane level, by accumulating compatible organic solutes like proline or sugars and by relying on an efficient active removal of specific salts that penetrate into the cells. The intracellular concentration of ions in this way is kept at 120mM for the Na and 80mM for the Cl instead of 500mM for both ions; those concentrations affected the activity of key enzymes like RuBiSCo more than the salinity of seawater.

Looking at the general equation of the photosynthesis where the energy of light is used to form energy-rich organic compounds by reducing CO2 to carboihydrates thanks to electrons from H2O (Figure 9) it is logical to think that the accumulation of the O2 as a final product should inhibit the entire process. This is the case for most terrestrial plants but not for marine ones (Beer, 2014).

The process of photosynthesis can be separed into two parts: the light reactions in which light energy is converted into chemical energy bound in the molecule ATP and reducing power is formed as NADPH and the biochemical in which ATP and NADPH are used to reduce CO2 to sugars (Calvin cycle).

To make the photosynthesis work plants need to harvest light. This is done thank to the photosynthetic pigments: they not only convert the energy of absorbed photons to heat but also the photon energy into a flow of electrons used to provide chemical energy to reduce CO2 to carbohydrates. Chlorophyll is the major photosynthetic pigment and chlorophyll a is present in all plants, with a highly hydrophobic molecule with a molecular weight of about 900. One part is made by four pyrrole rings held together by a magnesium atom to form a large porphyrin-like ring structure while a hydrocarbon chain forms the “tail” used to anchor the molecule to the thylacoid lipidic membrane thanks to its lipophilic nature. The “head” structure makes it easy for the pigment to absorb red and blue light but not as easy to absorb green light so that the color is reflected and the plants look green. Furthermore, the molecular structure of the chlorophyll makes it amenable to become excited by the photons that it absorbs and the subsequently return to the original level dissipating the excess of energy through different ways: resonant energy transfer, heat dissipation, fluorescence emission and photoxidation (see below for detailed description). The differences in the head part of the various chlorophylls (a, b, c and d) influence the absorption spectra in the blue and red absorbing range. In addition to chlorophyll, all plants contain carotenoids; some of them are ubiquitous like β-carotene while others like fucoxanthin and peridinin are found only in particular groups of algae. The fucoxanthin for example is the major carotenoid in the group of Chromophyte. These pigments are mainly used to fill the “green window” generated by the chlorophyll non-absorbance in that band extending in this way the range of light spectrum that could be used.

When a photosynthetic pigment molecule absorbs a photon, one electron is moved an orbital farther away from the nucleus of one of its atoms and the molecule become “energised”. The electron is, in fact, pushing or knocking the electron energetically superior to an outer orbital trasferring in this way the energy to the subsequently excited molecule. In this state, however the chlorophyll is very unstable and the electron returns to the previous energy state very quickly and spontaneously in a process called de-excitation. The energy is mainly released as heat but a part is utilized

to drive the photochemistry of photosynthesis. Among different de-excitation pathway, a small portion of excitation energy is re-emitted as light in a process defined fluorescence. While high-energy photons knock chlorophyll-molecule electrons to a higher energetic level than lower energy photons, energy that causes fluorescence is released only from red-photon excitation state (also called low excited state). The fact that photosynthesis occurs only from one critical energy dissipation excitation level is the proof that the quantum yield of the overall photosynthetic process is the same whether caused by high-energy blue photons or low energy red photons.

There is an inverse correlation between fluorescence yield and photosynthetic yield (Beer, 2014): the amount of fluorescence generated per photon absorbed by chlorophyll and the amount of photosynthesis performed per photon are inversally proportioned. Energy transfer refers to the transfer of excitation energy from one photosynthetic pigment molecule to another, i.e. photosynthetic antenna, until the energy reaches the reaction centre. Here a primary electron transfer starts a flow of electrons from water, which is the ultimate electron donor towards ferrodoxin, which is the ultimate acceptor in the chloroplastic electron transport flow. A photosynthetic antenna is comprised of up to several hundred pigments molecules surrounding a reaction centre and arranged within the thylakoid membrane to channel the energy absorbed from the photons. This energy is called resonance transfer and there is almost no energy loss in heat or other. Thus, energy is funnelled throughout the antenna with its various pigments tilli t reaches the reaction centre at its core the direction is always from the pigment able to absorb at shorter wavelenghts to thos that absorb at longer wavelenghts. A typical cascade of energy flow between pigments in an antenna is:

Carotenoids à chlorophyll b à chlorophyll a à reaction centre

Even if the energy is bounced from one pigment to the next, primary electron transfer occurs only in the reaction centres where special molecules of chlorophyll a have the ability to transfer their electrons out of the molecule to an electron acceptor reducing it. While doing so those reaction centres chlorophylls become positively charged ions and act like oxidants as they regain electrons from a donor. The reason why are present a lot of differen photosynthetic molecules and not all of them are of the kind that can perform electron transfer is because the pigments that constitutes the reaction centre can absorb only few photons per seconda the photon flux of full sunlight. In addition and more importantly, the chl a molecules do not readily absorb a large portion of the solar spectrum and, involving other pigments, the utilization of the

“excluded” wavelenghts is more efficient. In some cases more photon energy is received by a plant than can be used for the photosynthetic process higher is the probability to have photoinhibition or photodamage. There are two types of reaction centres: one associated with PSII fed by energy funneled through the antennae complexes of that photosystem and one associated with PSI fed by its own antennae. While (as described in the previous section) the pair of chlorophyll constituting the PSII are called P680 the special pair related to PSI is called P700. When photons excite these pigments, electrons are knocked out of P680 and P700 to form respectively P680+ and P700+. The primary acceptor of electrons released by the reaction centre of PSII is pheophytin a compound with a higher redox potential than water so only the excited P680 can reduce it. H2O is the ultimate electron donor to P680 so once the electron is knocked out the remaining ion P680+ can be re-reduced to P680 only by drawing an electron from water. In PSI plastocyanin is the electron donor to P700 and a chlorophyll a molecule, is the primary electron acceptor in this photosystem and is reduced by the energised P700+_{. After this compound, other different electron carriers (phylloquinone,} Fe-S centres) transfer electrons until they reach the final acceptor of electrons, the ferrodoxin. Boosting electrons from water to ferrodoxin the reaction centres are able to bring about the reduction of NADP+ to form NADPH.

The electron pathway starts from a series of complexes containing various forms of manganese ions that are involved into the water splitting reactions as chloride and calcium ions. H2O is splitted into electrons, protons and molecular oxygen (this oxygen is a by-product of photosynthesis that could be used to measure the photosynthetic rate). Through PSII electrons reduce the first electron acceptor pheophytin, this one, once reduced becomes a reductant compound able to reduce the first of two plastoquinone (PQ) molecules, tightly bound at the site QA of D1 protein in the PSII. Then, via an iron atom, an electron is transferred to the next PQ at the site QB. Both PQs require two electrons for their complete reduction; at the QA site, PQ undergoes to a single reduction event to the semiquinone state before being re-oxidized by the PQ at QB site. Two successive reductions occur that fully reduce PQ at QB site, which, for its reduction, requires also two protons from the stromal side of the membranes and forms PQH2 that leaves PSII and diffuses in the lipid bilayer, representing a mobile carrier of protons and electrons. A new molecule of PQ (in oxidized form) replaces this plastoquinone in the QB site. Reduced plastoquinone transfers the electrons to another major protein complex: the cythochrome b6/f complex, which transfers them to plastocyanin, which is

the final acceptor of electrons of PSII. The plastocyanin is the the direct electron dono to PSI and when the reaction centres of PSI are energetically excited the P700+ acts like an oxidant and draws electrons from plastocyanin starting the previously described chain of reactions that ends with the obtaining of reduced ferredoxin. Recued ferredoxin finally can reduced NADP+ to NADPH or donates electron to other pathways. Because the electrons follow this “zig-zag” pathway between compounds with various redox potentials, this path is termed the “Z-scheme” (Figure 10).

Figure 10: Electron transport rate in thylakoid membrane (Buchanan 2000).

This NADPH coupled with the ATP generated thanks to the ATP synthase is used to reduce CO2 to carbohydrates.

The CO2 fixation is realised via the Calvin cycle and thanks to a primary enzyme called RuBisCO (ribulose-bisphosphate carboxylase/oxygenase) that can fix both CO2 and O2. When RuBisCO catalyzes the oxygenation reaction, the phenomenon is called photorespiration and it is a consequence of the low concentration of CO2 and high concentration of O2 compared with RuBisCO’s relative affinity. To reduce CO2 to sugars the NADPH and ATP formed in the light reactions are used is a series of chemical reactions taking place in the chloroplast stroma: carbon dioxide in the stroma is fixed onto a 5-carbon sugar called ribulose-bisphosphate (RuBP) in order to form two molecules of phosphoglyceric acid (PGA). These molecules are then energised by the ATP and reduced by NADPH to form a 3-carbon phosphorylated sugar (Glyceraldehyde 3-phosphate) in equilibrium with its isomer dyhydoxyacetone-phosphate. A part (1/6) of these triose-phosphate (TP) products will leave the cycle while the rest is needed in order to re-generate RuBP molecules in a regenerative part of the cycle. To complete this biochemycal cycle are necessaries 2 NADPH and 3 ATP for

each CO2 fixed and reduced with 3 CO2 required for every TP leaving the cycle to form other sugars, 6 NADPH and 9 ATP for each such TP. The regeneration phase is complex: in principle 5 molecules of TP are rearranged to form 3 molecules of RuBP. The RuBisCO enzyme implied in the first part of the Calvin cycle is considered the bottleneck for photosynthetic production. This enzyme is big, slow acting because of its low affinity for CO2 and plants have to use mechanisms to cope with this low affinity by using mechanisms to concentrate carbon dioxide. During the evolution, two mechanisms are the C4 and CAM (crassulacean acid metabolism) cycle. The first one is so called because the first stable product is not a 3 carbons PGA but malic acid or aspartic acid, which are 4-carbon, compounds. In this biochemical cycle, CO2 enters the mesophyll cells, close to the atmosphere and is fixed in cytosol onto 3-carbon molecule phosphoenolpyruvate (PEP) by the enzyme PEP-carboxylase. The first compound formed by this carboxylation is oxaloacetic acid, another 4-carbons compound that is immediately reduced to malate thank to the malate dehydrogenase. This compound is traslocated to bundle sheat cells located in the middle of the leaf where it is decarboxylated to free the CO2 originally fixed in the PEP, this carbon dioxide enters then in the Calvin cycle to be refixed and reduced. This is a mechanism to concentrate CO2 mainly because the PEP-carboxylase does not have an oxygenase function excluding the competition for the active site of the RuBisCO. The CAM cycle on the other hand is very similar to the C4 cycle but the fixation via PEP-case is carried out during the night when the stomata of these plants are open. Photosynthesis cannot be performed and the malate is stored in the vacuoles. During the day, the malate is moved to the cytosol where it is decarboxylated to form CO2 and pyruvate and the first one is then carboxylated via RuBisCO and reduced in the Calvin cycle.

For all these reasons, the succesful photosynthesis of marine plants depends on efficient fixation of CO2 in the first steps of the cycle. Marine plants can use only the carbon dioxide dissolved in oceanic water and this is a limiting factor given that the diffusivity of solutes such as CO2 in liquid media is some 4 orders of magnitude lower than in air. There is no average higher affinity for carbon dioxide in macroalgae being the average Km(CO2) of RuBisCO 50µM (macroalgae could perform half of their photosynthetic capacity at 50µM CO2) and only a little amount of species use mechanisms of nightly concentration comparable to those of CAM land species. The CO2 concentrating mechanisms (CCM) of marine macroalgae are so dependent on HCO3- utilization, which brings about higher concentration of carbon dioxide near