The present study provides the largest transcriptome dataset for the class Florideophyceae and represents the first transcriptomic characterization of the seaweed Laurencia dendroidea. The presented numbers could be an overestimate of the contribution of L. dendroidea to the Florideophyceae database, since we worked with complex samples. Nevertheless, at least some of the sequencing projects in the Florideophyceae dbEST are also based on non-axenic field samples [51, 52], hampering the achievement of a more accurate estimate. Indeed, it is notable the presence of sequences deposited in this database that matched our bacterial sequences.
Recent advances in the field of algal genomics included only the complete sequencing of the nuclear genome of the microalgae Cyanidioschyzon merolae, Ostreococcus tauri, Chlamydomonas reinhardtii, and Cyanophora paradoxa and the brown macroalga Ectocarpus siliculosus. Moreover, EST projects have provided valuable information in the transcriptomic profile of some species of Rhodophyta [50, 51, 58–62] in the phylogenetic relationships among photosynthetic eukaryotes [63, 64] and have also unveiled genes involved in stress response [52, 65, 66] and in life phase differentiation [67–70].
The transcriptomic profile of L. dendroidea and its corresponding associated microbiome was closely similar among all the samples, regardless of their place of origin. Likewise, a previous study verified a higher similarity between bacterial populations from seaweeds of the same species sampled at different sites than between those from different species growing at the same habitat, emphasizing the specificity of this association . Our data reinforces these findings as we observed a high similarity in the taxonomic composition of the active microbiome associated with L. dendroidea in different sample sites.
Major groups of transcripts of L. dendroidea
The functional annotation of the transcripts revealed predominantly basic cellular metabolic pathways. In general, functions related to translation and protein synthesis, from amino acid precursors to post-translational modifications are the most abundantly expressed in the transcriptome of L. dendroidea. Besides, complete pathways for energy production were well represented, mainly related to the pyruvate dehydrogenase complex, electron transfer, thioredoxins, citric acid cycle and NADH dehydrogenase. The ESTs involved in carbohydrate transport and metabolism (mainly glycolysis, starch and sucrose metabolism, and pentose phosphate pathway), Cofactors, Vitamins, Prosthetic Groups, Pigments (including Folate and Pterines, Tetrapyrroles and Pyridoxine), RNA Metabolism (mainly RNA Processing and Modification) were among the most represented categories in the transcriptome of L. dendroidea. Other relevant features in this transcriptome are related to DNA replication, recombination and repair, which are important to the survival and growth of the seaweed, especially in the rocky-shore coastal environment where the organisms are subject to high UVB levels that causes serious damages to DNA . The ability to resist to UV-exposure influences the vertical distribution of seaweeds , and L. dendroidea typically grows in the lower midlittoral zone where UV-damage repair may be necessary. The same set of expressed sequences relevant in the transcriptome of L. dendroidea are among the most represented in the EST databases of Gracilaria gracilis, G. changii, G. tenuistipitata, Porphyra yezoensis[61, 67], P. haitanensis, Eucheuma denticulatum, Furcellaria lumbricalis, and Kappaphycus alvarezii, possibly indicating a general pattern of expression in red seaweeds.
Transcriptome of L. dendroidea-associated microbiome
Seaweeds are especially susceptible to epibiosis because they inhabit environments with strong competition for space , and release large amounts of organic compounds that induce the microbial colonization , but the interaction between seaweeds and their microbiomes is little known to the molecular level.
The functional analysis of the holobiont transcriptome revealed the expression of bacterial genes involved on cell motility and chemotaxis, for example the ESTs related to flagellum and CheY-like receiver domain which are important, respectively, for the recognition of the surface of the seaweed and the establishment of the biofilm [77, 78]. However, the relatively low abundance of these transcripts in comparison with the ones involved in extracellular polysaccharide synthesis suggests a mature biofilm with some level of detachment, possibly of dispersal cells . Transcripts coding for the enzyme S-adenosylmethionine synthetase, which participates in the synthesis of quorum sensing autoinducers, were also detected . Quorum sensing (QS) is a bacterial cell to cell communication mechanism based on the release and perception of signaling molecules such as oligopeptides, N-acyl homoserine lactones (AHL) and autoinducers that allow bacteria to monitor their own population density and to coordinate swarming, biofilm formation, stress resistance, and biosynthesis of toxins and secondary metabolites , and it exhibits an important role in the interactions between bacteria and their eukaryotic hosts. Several red seaweeds are able to inhibit bacterial QS signaling, such as Delisea pulchra and Ahnfeltiopsis flabelliformis, and a small inhibitory activity against QS signaling was previously detected in the ethyl acetate extract from a Laurencia sp. .
The taxonomic analysis of the transcriptome showed Bacteria as the dominant active group in the microbiome of L. dendroidea, with Cyanobacteria and Proteobacteria as the most represented bacterial phyla. These groups were also verified as predominant in the evaluation of the microbial diversity associated with four functional groups of seaweeds through metagenomics .
Among the cyanobacterial transcripts associated with the thalli of L. dendroidea, the Chroococcales, Oscillatoriales and Nostocales were the dominant orders, all of them comprising nitrogen fixing species. In a previous study, Phlips and Zeman  reported the occurrence and the nitrogen fixing activity of epiphytic forms of Oscillatoria associated to Sargassum thalli. Nitrogen can be the limiting nutrient in coastal ecosystems  and under this situation, nitrogen fixing cyanobacteria may be favored and gain in growth and reproductive success. In fact, Hoffman  pointed that despite their important contribution to benthic primary production, the main role of Cyanobacteria in the tropical marine ecosystems appears to be as nitrogen fixers. However, no sequences related to nitrogen fixation were observed in our data. This is expected since our data clearly indicates an oxygenic environment, and the nitrogenase expression is inhibited by oxygen . Our samples, collected near the peak of photosynthetic activity (right before midday) should have a very low expression of this nitrogenase . In fact, the most abundant cyanobacteria genus were Synechococcus and Cyanothece, which together with Lyngbya and Synechocystis were previously reported to rely on temporal separation between photosynthesis and nitrogen fixation, the last occurring mainly at night [90, 91]. Further studies on the diel variation of the transcriptome profile could verify this hypothesis.
Analyzing the functional relative contribution of specific domains, we noticed a higher involvement of Bacteria in the Amino acid metabolism, except for the biosynthesis of glutamate, more represented in eukaryotes. Such situation was reported for Rhizobium nodules, where plants provide glutamate and a carbon source and in turn the nitrogen fixing Bacteria provide ammonium and amino acids such as alanine and aspartate for asparagine biosynthesis in the plant cytosol . Although specialized mechanisms like nodules are not known in red algae, our data suggests a similar interaction between the seaweed and the associated microbiome, involving the exchange of nitrogen compounds.
Proteobacteria was the second largest active group with assigned sequences mostly to the classes Gammaproteobacteria and Alphaproteobacteria. The higher abundance of these classes was previously reported for the surface microbiome of the macroalgae Ulva australis and Laminaria hyperborean, through denaturing gradient gel electrophoresis (DGGE) analysis. Predominantly heterotrophs, these groups would be opportunists, exploring an oxic productive environment . The high prevalence of aerobic and aerotolerant groups reflects a photosynthesizing environment, also noted by Barott et al.. The predominance of respiration over fermentative metabolism in the holobiont transcriptomic profile reinforces these findings. The aerobic metabolism generates reactive oxygen species (ROS)  that can damage DNA, lipids, and proteins . In order to cope with oxygen toxicity and grow in aerobic conditions, Bacteria expressed genes correlated to oxidative stress, such as Superoxide dismutase, Glutaredoxins and Alkyl hydroperoxide reductase , and also stress related chaperones such as GroEL, DnaJ and DnaK [99, 100].
Transcripts associated to photosynthesis and to the biosynthesis of carbohydrate reserves, such as starch, were more represented in eukaryotes, which indicate an important role of L. dendroidea in the primary production of the holobiont, generating carbon in excess to its immediate demand. The typical starch from Rhodophyta is called floridean starch and it shows structural similarities with starch granules from higher plants except for the lack of amylose in most of the species . On the other hand the Bacteria contributed more to Carbohydrate and Lipid Transport and Metabolism, and to Energy Production and Conversion, standing out genes related to glycolysis and also to lipid and polysaccharide breakdown, reinforcing the role of Bacteria as consumers of organic matter in this holobiont .
Despite the beneficial or neutral interaction processes depicted here between L. dendroidea and its microbiome, some bacteria may also offer threats to the health and survival of seaweeds in their natural environment . As such, defense mechanisms, such as the aforementioned secondary compounds of L. dendroidea, may have been evolutionarily selected. The expression of vanadium-dependent bromoperoxidases, involved on the halogenation and cyclization of terpenes in Rhodophyta , was detected in the transcriptomic profile of L. dendroidea. Additionally the previously reported increase on the bromination activity of red algae in response to infection signals, such as agar oligosaccharide , indicates an important role of this enzyme in the chemical defense of Rhodophyta.
Terpenoid biosynthesis in the holobiont
The biosynthesis of terpenoid backbones provides precursors for the biosynthesis of diverse compounds that display relevant roles in plant and algal physiology . The identified genes are involved in important steps for the biosynthesis of the building blocks dimethylallyl diphosphate, isopentenyl diphosphate and the higher-order building blocks geranyl diphosphate, farnesyl diphosphate and geranylgeranyl diphosphate, which are the precursors of monoterpenoids (C10), sesquiterpenoids (C15), and diterpenoids (C20), respectively . The subsequent addition of isoprene units leads to the biosynthesis of sterols (isoprenoids with a C30 backbone) which are components of cell membranes; carotenoids (C40) and chlorophylls (with a C20 isoprenoid side-chain) that act as photosynthetic pigments; and plastoquinone, phylloquinone and ubiquinone (with long isoprenoid side-chains) that participate in electron transport systems for respiration or photosynthesis . Terpenoid backbones are also required for the biosynthesis of N-glycans, important components for the proper folding of proteins in eukaryotic cells . The biosynthesis of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), the central intermediates in the biosynthesis of isoprenoids, occur through two different pathways in plants, one dependent (MVA) and other independent of mevalonate (DOXP/MEP). The mevalonate (MVA) pathway, located in the cytosol, is responsible for the production of sterols, triterpenes and some sesquiterpenes . The MVA-independent pathway operates in plastids and provides the precursors to monoterpenes, diterpenes, certain sesquiterpenes, carotenoids and the side chains of chlorophyll and plastoquinone . This division between isoprenoids derived from plastids and cytoplasm was also observed in red algae [111, 112]. Despite the occurrence of both biosynthetic routes in Rhodophyta, this study found only transcripts associated with the mevalonate-independent pathway. Furthermore, three transcripts were identified containing the terpene synthase family metal-binding domain , representing new possible targets for further functional clarification. Phylogenetic reconstruction based on genes of terpene synthases was attempted, using the fragments (50–310 amino acids) we obtained from our whole transcriptome strategy (data not shown). However, it is difficult to infer a phylogenetic relationship among taxonomic groups using the gene fragments of this pathway because, in nearly all cases, the bootstrap support for the branches is low when homologous sequences were available for analysis. Nevertheless, it is notable that in most cases, the sequences from L. dendroidea holobiont and other red algae cluster together with a relatively high bootstrap support.
These findings associated to the reconstruction of a complete pathway for the biosynthesis of terpenoid backbones in L. dendroidea are important steps to enable the heterologous biosynthesis of terpenes of interest, such as (-)-elatol, in genetically modified organisms. The molecular engineering of Escherichia coli and Saccharomyces cerevisiae has recently allowed the use of these microorganisms as cell factories to synthesize plant terpenes such as the antimalarial drug artemisinin [113, 114], opening up new avenues for the scalable biosynthesis of terpenoid compounds. Our research provides a comparative basis for prospecting more specific terpene synthases genes for (-)-elatol and other commercially relevant terpenes, which could be explored in cell factories. This could be accomplished through the use of high producing strains of L. dendroidea under favorable conditions.