Photosynthetic organisms in the intertidal zone: a focus on brown seaweeds
Not very many photosynthetic organisms have adapted to life in the intertidal zone. While the majority of organisms that inhabit the intertidal zone can follow the tides and avoid the stresses related to immersion and emersion, seaweeds are usually immobile and, in order to survive in the intertidal zone, have to rely mainly on cellular mechanisms to tolerate stress (Ocon 2007).
Probably the most abundant photosynthetic inhabitants of the intertidal zone are brown algae. Brown algae belong to the chromalveolate kingdom and have evolved to multicellularity independently from red and green algae, as well as the most commonly studied eukaryotic lineages – animals, fungi, or green plants (Baldauf 2003, Figure 2).
The phylogenetic distance of brown algae from other multicellular eukaryotes can be illustrated by a number of unique metabolic pathways and features. Brown algal plastids, for example, like those of all heterokonts, derive from a secondary endosymbiosis event with a red alga (Keeling 2004), and are surrounded by four membranes. Recent genomic analyses furthermore indicate that plastids derived from secondary endosymbiosis might have replaced an earlier green plastid in heterokonts (Moustafa et al. 2009), traces of which can still be found in heterokont genomes. Other unique features include the ability of brown algae to synthesize both C18 (plant-like) and C20 (animal-like) oxylipins (Ritter et al. 2008), their original cell wall composition and the associated pathways of cell wall synthesis (Kloareg & Quadrano 1988, Nyvall et al. 2003, Tonon et al. 2008), and the ability to accumulate high concentrations of iodine in their cell walls, a trait recently discovered in the kelp Laminaria digitata (Leblanc et al. 2006).
Brown algae are also of high ecological importance. One eminent example for this are kelp forests, which are formed mainly by brown algae of the order of Laminariales (Bartsch et al. 2008) and provide habitats for thousands of species. Most kelp forests are found in shallow coastal areas of temperate regions (Mann, 1973), but they have recently also been discovered in deep-water areas (>30 m) in tropical regions (Graham et al. 2007).
The economic interest of brown algae is related mainly to their use in the industrial production of polysaccharides (McHugh 2003). For example, alginate, a brown algal cell wall polysaccharide, is used both for pharmaceutical purposes (Tønnesen & Karlsen 2002) and in food industry (Jensen 1993). Another example is the production of the beta-1,3-glucan laminarin, which can act as an efficient stimulator of plant defense (Klarzynski et al. 2000).
Figure 2: The tree of eukaryotes (taken from the tree of life project http://tolweb.org/, Maddison et al. 2007)
Considering the economic and ecological importance of brown algae, it is essential to understand the mechanisms underlying the adaptation of these seaweeds to the intertidal zone. Understanding how an organism interacts with its natural environment is the first step towards understanding how (anthropogenic) changes will impact an organism, and the basis of deciphering complex interactions between an organism and its biotic surroundings. This is particularly true for brown algae, as, due to their phylogenetic distance from the green lineage including terrestrial plants, they might have developed very distinct mechanisms of stress tolerance.
Before summarizing what is known about the stress response in seaweed in section 1.3, and in spite of the phylogenetic distance of brown algae from terrestrial plants, the next section will give a brief overview of the stress response in land plants. There are several similarities between terrestrial plants and algae including brown algae (e.g. the fact that they are both sessile photosynthetic organisms with cell walls), and the approaches that were taken to study terrestrial plants have much inspired my work on Ectocarpus.
Abiotic stress tolerance in terrestrial plants
Approaches to studying stress tolerance in terrestrial plants
In terrestrial plants, numerous studies have already addressed the question of the mechanisms underlying the abiotic stress response, and research in this field was greatly accelerated by the availability of the Arabidopsis genome sequence (The Arabidopsis Genome Initiative 2000). Soon after the publication of the genome, a range of transcriptomic studies examined gene expression profiles in response to several stresses, starting with mechanical and biotic stress (Reymond et al. 2000), drought and cold stress (Seki et al. 2001), salt stress (Kawasaki et al. 2001), oxidative stress (Desikan et al. 2001), iron deficiency (Thimm et al. 2001), and high light stress (Rossel et al. 2002).
Most of these data are deposited in public repositories in a standardized format according to the MIAME standards (minimum information about microarray experiments, Brazma et al. 2001). As of July 2009, Gene Expression Omnibus (Edgar et al. 2002), one of the largest public repositories available, already contained standardized information from over 300,000 hybridizations performed with different organisms, many of which were plants. This ever increasing amount of data now forms the basis for meta-analyses such as that of Benedict et al. (2006), who managed to identify a network of genes involved in cold signaling by re-analyzing a large number of publicly available datasets.
Gene expression is still a major element in studies of plant stress response (as can be seen from Table 1) although, after an initial “boom” of microarray studies, the scientific community became increasingly aware of the fact that transcriptomic regulation was only one several levels of regulation. Recent comparative studies have examined gene expression profiles along with protein abundance (Branco-Price et al. 2008), enzyme activity (Gibon et al. 2004), and metabolite concentration (Kaplan et al. 2007; Kempa et al. 2008). Although there was a general correlation between these measures and transcript abundance, several exceptions to this general trend were found. Especially in complex metabolic networks, it is very difficult to predict what a plant will do (metabolite changes, changes in enzyme activity) from what a plant “thinks” (transcription, Sweetlove 2008). Studies such as those of Branco-Price et al. (2008), Kaplan et al. (2008) and Kempa et al. (2008) have greatly contributed to our understanding of the stress response in terrestrial plants by highlighting the importance of additional levels of regulation.
Recently, transcriptomic and metabolomic studies have been extended to comparisons of the stress response between different ecotypes or similar species. The most prominent object of this type of study is Thellungiella halophila, a particularly (salt) stress resistant species, closely related to Arabidopsis thaliana (the average sequence identity is 92 %, Inan et al. 2004). The study of T. halophila has already yielded valuable information on the mechanisms underlying its stress tolerance, e.g. with respect to ion uptake and exclusion (reviewed in Amtmann 2009), and the development of further tools including the Thellungiella genome project (www.jgi.doe.gov/sequencing/why/50029.html) is currently underway.
The probably most important contribution to understanding the abiotic stress response in plants, however, comes from more targeted studies, as the analysis of changes in gene expression will only yield relevant information if complemented by functional studies of the genes and proteins in question. In Arabidopsis (and other terrestrial plants) a number of tools have facilitated a seemingly endless number of studies elucidating the function of specific genes or pathways. These tools include a large number of mutants (see e.g. Ito et al. 2005), as well as resources for gene silencing using RNAi (e.g. Hilton et al. 2004).
All together, these studies lead to a relatively complete (from a phycological point of view) picture of how plants perceive and respond to stress. In the following section, I will give a few examples illustrating some of the aspects of stress signaling and response in terrestrial plants, focusing on drought and saline stress.
Stress perception and signaling
Stress, and in particular saline and drought stress, is known to have several effects on plant cells that lead to the activation of stress signaling pathways, which include changes in cell turgor, to a certain extent changes in cell volume (Xiong & Zhu 2002), and in the worst case membrane damage and protein denaturation (Schluze et al. 2005). Membrane proteins such as receptor-like kinases, stretch-dependent ion (calcium) channels and redox-mediated systems (Urao et al. 1999, Kacperska 2004) have been identified as osmosensors which activate downstream signaling cascades (Figure 3).
With respect to (drought) stress signaling in terrestrial plants, a distinction is frequently made between abscisic acid (ABA)-dependent and ABA-independent pathways (Tuteja 2007). ABA is a C15 isoprenoid derived from the cleavage of C40 carotenoids (Nambara & Marion-Poll 2005). As can be seen from Table 1, ABA is a common subject in studies examining plant stress. ABA levels in plants increase in response to most abiotic stresses, but in particular in response to osmotic stress, and activate a number of other second messengers such as inositol triphosphate, reactive oxygen species, or phospholipid-based signals (Zhu 2002). ABA has been shown to be involved in processes such as stomatal closure, but also lipid synthesis and plant development. Although most commonly studied in plants, ABA has also been shown to be present in several metazoans, where it appears to play a protective role against injuries (reviewed in Wasilewsk et al. 2008), and in algae including brown algae, where it might be an inhibitor of growth (Schaffelke 1995).
One of the first elements of the ABA-independent signaling described in detail was the dehydration responsive element (DRE) in the cis-terminal region of RD29A, a gene strongly induced in response to drought and salt stress (Yamaguchi-Shinozaki & Shinozaki 1994). Other genes are the Arabidopsis thaliana SHAGGY-related protein kinase (AtSK) and the calcium-dependent protein kinase (CDPK), which are thought to be activated directly by osmotic stress or by Ca2+ respectively, or the CBF4 transcription factor (Boudsocq & Lauriere 2005).
General mechanisms of response
These ABA-dependent and ABA-independent pathways, and several other mechanisms, coordinate the actual abiotic stress response, which, according to Vinocur and Altman (2005), consists of four essential parts: synthesis of compatible osmolytes (in the case of osmotic stress), synthesis of chaperones, activation of detoxification mechanisms, and activation of water and ion transporters. In addition, a 5th element, the regulation of growth, has been attributed increasing importance (Kovtun et al. 2000, Achard et al. 2008, Figure 4). In the following section, I will give a brief overview of a few of the mechanisms known for each of these five parts in terrestrial plants.
Synthesis of compatible osmolytes
Compatible osmolytes, i.e. non-perturbing and osmotically active substances, are commonly employed in all domains of life to control water balance, in particular in response to different salinities or to drought (Yancey et al. 1982). Two prominent examples of compatible osmolytes in terrestrial plants are glycine-betaine and proline, both of which have been shown to be very abundant in halophytes and to correlate with salt tolerance (reviewed in Hellebust 1976). Mannitol seems to be less important in terrestrial plants, although artificially increased mannitol levels have been shown to enhance the stress tolerance in tobacco (Tarczynski et al. 1993). Many substances, deemed compatible osmolytes, might also play a more direct role in the stress response as osmoprotectors, making it difficult to distinguish between the osmotic and the protective effect of a compound. Glycine-betaine concentrations, for example, are highest in chloroplasts, where glycine-betaine plays an important role in maintaining photosynthetic activity by stabilizing the thylakoid membrane (Robinson & Jones 1986). Proline also was shown to be an efficient hydroxyl radical scavenger (Smirnoff & Cumbes 1989). In many ways, these functions resemble those of chaperone proteins, some of which will be mentioned in section 1.2.6.
Activation of water and ion transporters
In the case of salt stress, one mechanism that is of particular importance for restoring homeostasis and growth are transporters (reviewed in Blumwald 2000, Munns & Tester 2008). As high intracellular Na+ concentrations are toxic for most plants, salt tolerance is often achieved by actively extruding Na+ from the cell. Two enzymes seem to be vital for this process: H+-ATPases, which create an H+ gradient over a membrane, and Na+/H+ antiporters, which use this gradient to transport Na+. This transport can either be directed out of the cell or into specific cell compartments such as the vacuole, where salts will have fewer or no toxic effects.
Synthesis of chaperones
Molecular chaperones (reviewed in Boston et al. 1996, Wang et al. 2004) are classically considered proteins that are able to bind to many other proteins and assist correct protein folding and maturation. Chaperones consequently also play a role in stabilizing the correct protein conformation, in particular under stress conditions, where their synthesis is frequently increased. Families of common (plant) chaperones include the calmodulin-like protein (CLP) family, the heat shock protein (HSP) 90 family, the HSP70 family, calnexins, and small HSPs, many of which are in turn associated to co-chaperones such as DNAJ/HSP40, glucose regulated protein (GPR) E, or chaperonin (CPN) 10.
Activation of detoxification mechanisms
Abiotic stress is often related to the production of oxygen radicals that play an important role in cell signaling, but which at the same time are cytotoxic and need to be detoxified (Mittler et al. 2004). Plant cells contain a repertoire of ROS scavenging enzymes such as superoxide dismutases (Monk et al. 1989), catalases, glutathione peroxidases (Halliwell 1974), ascorbate peroxidases (Asada 1992), or peroxiredoxins (Wood et al. 2003), which, in most cases, catalyze the reaction of reactive oxygen species with antioxidants such as ascorbic acid and glutathione. These antioxidants are regenerated, mainly using NAD(P)H, by enzymes such as glutathione reductases or monodehydroascorbate reductases. Furthermore, several other compounds have been shown to exhibit antioxidant properties independently of the ROS scavenging enzymes. These include lipid-related substances like carotenoids (Burton & Ingold 1984, Liebler & McClure 1996), but also the putative compatible osmolytes sorbitol, mannitol, myo-inositol and proline (Smirnoff & Cumbes 1989).
A comprehensive description of the role of only ROS scavenging enzymes and antioxidants alone would be enough information to fill an entire thesis; however they only represent a part of the detoxification mechanisms known in plants. Depending on the type of stress, other detoxification mechanisms are activated. Some examples are ferritins (detoxification of heavy metals), gluthathione S-transfereases (removal of peroxidised lipids and xenobiotics), ubiquitination and protein degradation (removal of damaged proteins), or DNA repair.
Inhibition of growth
The last aspect of plant stress tolerance I would like to mention has received comparatively little attention in the literature: the regulation of growth. Growth is the fifth most common word in abstracts related to plant stress (Table 1) and, by definition, is negatively affected by stress (see figure 5 for an example). However, it is oftentimes forgotten that the inhibition of growth is based on complex regulatory mechanisms, which are essential for survival. One example for this regulation was given by Kovtun et al. (2000), who demonstrated that the Arabidopsis mitogen-activated protein kinase ANP1, which is activated by H2O2 and initiates several phosphorylation cascades related to the stress response, also inhibited the effect of auxin, an important growth hormone in plants. More recently, more complex connections between growth and stress signaling have been discovered. Again in Arabidopsis, the changes in expression of a transcriptional regulator controlling genes involved in the response to cold stress, the CBF1/DREB1b gene, were shown to have a positive effect on the accumulation of DELLA protein, an important repressor of plant growth (Achard et al. 2008). Under normal conditions the DELLA protein is rapidly targeted for degradation by the activity of a specific SCF ubiquitin ligase. However, this targeting requires binding of the gibberelin receptor GID1 and biologically active gibberelin. CBF1 regulates the concentration of biologically active gibberellin via the activation of gibberellin oxidase genes, thus decreasing the degradation of the DELLA protein and inhibiting growth.
These two examples are only a small part of the molecular bases underlying the crosstalk between stress signaling and regulation of growth, and much research will still be required before this part of plant life will be fully understood.
Figure 5: Effects of abiotic stress on plant growth (photo courtesy of René Bürgi)
Stress tolerance in seaweeds
The first section of this introduction (1.1) demonstrated that seaweeds live in a unique habitat with high levels of abiotic stress, and the previous section (1.2) illustrated that terrestrial plants possess several mechanisms for tolerating abiotic stresses, and that these mechanisms have been relatively well studied. In this third section, I will give an overview of what is known about specific mechanisms of abiotic stress tolerance in seaweeds. Although most of the work regarding stress in intertidal seaweeds aims towards understanding the distribution of different seaweeds in the intertidal zone (reviewed in Davison and Pearson 1996), a few studies relate the tolerance towards a particular stress to an underlying mechanism such as the activation of certain detoxification pathways. As the focus of my thesis is on brown algae, I will concentrate on this lineage, but also cite some results obtained for the closely related diatoms as well as other seaweeds.
Stress perception and signaling
Very little is known about stress perception and signaling, although several hypotheses exist. In response to osmotic stress, for example, it is generally believed that these changes are perceived by the cell via sensors probably located in the cell membrane (Kirst 1989). Observations in the brown alga Fucus serratus also demonstrate that hyperosmotic stress induces ROS production at the plasma membrane and cytosolic Ca2+ release, which in turn is responsible for a second oxidative burst in the mitochondrion (Coelho et al. 2002). These early events may form the starting point for subsequent signaling cascades that coordinate the stress response.
In the red alga C. crispus, exposure to methyl jasmonate, an important hormone involved in the abiotic and biotic stress response in terrestrial plants (Creelman & Mullet 1997) enhanced both the synthesis of other oxylipins (Bouarab et al. 2004) and the expression of genes with putative functions in stress response (Collén et al. 2006a). The same is true for brown algae (Laminariales), where oxylipin synthesis of both plant-like (C18) and animal-like (C20) oxylipins was stimulated in response to methyl jasmonate treatment (Küpper et al. 2009) and in response to copper stress (Ritter et al. 2008). Even though no experimental evidence of methyl jasmonate production exists in either red or brown seaweeds, these results suggest a possible stress signaling role of both methyl jasmonate and other oxylipins, as reported for terrestrial plants (Blechert et al. 1995) and animals (Funk 2001).
In diatoms, a calcium- and nitric oxide based signaling mechanism has been revealed in response to treatments with polyunsaturated aldehydes (Vardi et al. 2006). More recently, Vardi et al. (2008) identified a chloroplastic nitric oxide-associated protein (PtNOA) and demonstrated that the over-expression of this gene increases nitric oxide production in several organisms as well as the sensitivity of diatoms to polyunsaturated aldehydes, thus further supporting the role of nitric oxide in stress signaling in diatoms. To my knowledge, no studies so far have demonstrated a signaling role of nitric oxide in brown algae1.
Similarly to polyunsaturated aldehydes in diatoms, which are produced upon cell death, in brown algae, oligoguluronates, i.e. oligomeric degradation products of alginate, a brown algal cell wall polysaccharide, are produced as a direct result of grazing. In L. digitata, treatment with oligoguluronates was shown to elicit a strong oxidative burst with a parallel efflux of potassium (Küpper et al. 2001). ROS formation is likely to occur via the activity of an oxidase with a flavoprotein subunit, and signaling events involved in this process probably include phospholipase A2 as well as calcium and possibly chloride channels. Although several studies have examined theses events, the precise signaling pathways involved still remain unknown (Cosse et al. 2007).
In response to saline stress, signaling events such as the ones described above need to coordinate the adaptation to rapid changes in cell turgor and / or size (Figure 6), changed ion concentration, and in ion- and water exchange. Very high salinities have been shown to cause plasmolysis, i.e. the detachment of cell membrane from the cell wall due to a loss of water within the cell. Very low salinities, on the other hand, could cause an influx of water that could eventually damage the cell or even cause it to burst (even if the latter is rather unlikely, considering the solid cell walls). Although the sensory mechanisms underlying the saline stress response are yet unknown, several reactions to saline stress can be observed within the cell.
As maintaining a strong osmotic difference between intracellular and extracellular medium is costly, algae generally adapt their intracellular concentration of osmotically active compounds. These might either be inorganic (i.e. ions) or organic compatible osmolytes. The cost of producing compatible osmolytes, along with the effect of increased ion concentrations, were often thought to be the reasons for a reduction of algal growth in response to stress (Kirst 1989 and references therein). Mannitol is considered the main osmolyte in brown (Reed et al. 1985, Thomas & Kirst 1991) and in several species of red algae (Mostaert et al. 1995, Eggert et al. 2007), some of which also actively exclude sodium in response to hypersaline stress (Mostaert et al. 1995).
Table of contents :
1.1 The intertidal zone, a unique changing habitat with many stressors
1.2 Abiotic stress tolerance in terrestrial plants
1.3 Stress tolerance in seaweeds
1.4 The new model Ectocarpus siliculosus
1.5 Objectives and approach
2. Primary metabolism
2.2 Primary metabolism in E. siliculosus
3. Short-term response to abiotic stress
3.2 Transcriptomic response to short-term saline and oxidative stress
3.3 Metabolite changes in response to short-term saline and oxidative stress
3.4 Stress-responsive fucoxanthin chlorophyll a/c binding proteins
4. Long-term adaptation to different salinities
4.2 Comparative genome hybridization of Ectocarpus strains
4.3 The Ectocarpus freshwater strain
5. Conclusion and outlook
6.1 Appendix 1: Oral presentations
6.2 Appendix 2: Posters
6.3 Appendix 3: Normalisation genes in Ectocarpus.
6.4 Appendix 4