Chlorophyll-binding proteins revisited--a multigenic family of light-harvesting and stress proteins from a brown algal perspective

Abstract

Background: Chlorophyll-binding proteins (CBPs) constitute a large family of proteins with diverse functions in both light-harvesting and photoprotection. The evolution of CBPs has been debated, especially with respect to the origin of the LI818 subfamily, members of which function in non-photochemical quenching and have been found in chlorophyll a/c-containing algae and several organisms of the green lineage, but not in red algae so far. The recent publication of the Ectocarpus siliculosus genome represents an opportunity to expand on previous work carried out on the origin and function of CBPs.

Results: The Ectocarpus genome codes for 53 CBPs falling into all major families except the exclusively green family of chlorophyll a/b binding proteins. Most stress-induced CBPs belong to the LI818 family. However, we highlight a few stress-induced CBPs from Phaeodactylum tricornutum and Chondrus crispus that belong to different sub-families and are promising targets for future functional studies. Three-dimensional modeling of two LI818 proteins revealed features common to all LI818 proteins that are likely to interfere with their capacity to bind chlorophyll b and lutein, but may enable binding of chlorophyll c and fucoxanthin. In the light of this finding, we examined the possibility that LI818 proteins may have originated in a chlorophyll c/fucoxanthin containing organism and compared this scenario to three alternatives: an independent evolution of LI818 proteins in different lineages, an ancient origin together with the first CBPs, before the separation of the red and the green lineage, or an origin in the green lineage and a transfer to an ancestor of haptophytes and heterokonts during a cryptic endosymbiosis event.

Conclusions: Our findings reinforce the idea that the LI818 family of CBPs has a role in stress response. In addition, statistical analyses of phylogenetic trees show an independent origin in different eukaryotic lineages or a green algal origin of LI818 proteins to be highly unlikely. Instead, our data favor an origin in an ancestral chlorophyll a/c-containing organism and a subsequent lateral transfer to some green algae, although an origin of LI818 proteins in a common ancestor of red and green algae cannot be ruled out.

Figure 1

Phylogenetic tree of CBPs. Red stars next to the sequence name indicate genes that have been shown to be induced in response to stress. Only selected confidence values were plotted (PhyML bootstrap, PhyML Approximate Likelihood test, and MrBayes posterior probabilities respectively, with dash indicating no support by MrBayes). Dotted lines were used to indicate groups according to current naming conventions, but which are poorly resolved in our phylogeny (see text). Abbreviations: Ath = Arabidopsis thaliana, Bna = Bigelowiella natans, Ccr = Chondrus crispus, Cme = Cyanidioschyzon merolae, Cne = Chaetoceros neogracile, Cre = Chlamydomonas reinhardtii, Ccy = Cyclotella meneghiniana, Ehu = Emiliania huxleyi, Esi = Ectocarpus siliculosus, Fse = Fucus serratus, Fve = Fucus vesiculosus, Gch = Gracilaria changii, Gth = Guillardia theta, Iga = Isochrysis galbana, Kmi = Karlodinium micrum, Mev = Mesostigma viride, Msp = Micromonas sp. RCC299, Msq = Micromonas sp. CCMP490, Osp = Ostreococcus sp. RCC809, Ota = Ostreococcus tauri, Plu = Pavlova lutheri, Ppa = Physcomitrella patens, Psa = Pisum sativum, Psi = Picea sitchensis, Ptr = Phaeodactylum tricornutum, Pye = Porphyra yezoensis, Rsp = Rhodomonas sp. CS24, Sco = Scenedesmus obliquus, Tps = Thalassiosira pseudonana, Vca = Volvox carteri.

Figure 2

Structural comparison of the crystallized CAB from spinach with the 3D models of the LI818 protein Cre_23 from Chlamydomonas reinhardtii and of the stress-induced LHC protein Esi_02 from Ectocarpus siliculosus. Stereo ribbon representation of the crystal structure of the spinach CAB (PDB code: 1RWT) (A), and of the modeled proteins Cre_23 (B) and Esi_02 (C).

Figure 3

Structure-based sequence alignment of the crystallized spinach CAB (code 1RWT) with proteins belonging to the LI818 clade. The secondary structure of the spinach CAB is shown above the alignment. Conserved amino acids highlighted by a red background are identical and those in red letters are similar. Alpha helices are represented as helices, and β-turns are marked with TT. Blue triangles indicate the conserved residues involved in the binding of chlorophyll a molecules. The green star shows the conserved glutamate in LI818-like proteins, predicted to preclude the binding of Chlb 607 observed in the spinach CAB. The colored frames indicate the three subgroups of helix α2 within the LI818 subfamilies.

Figure 4

Comparison of the binding site of chlorophyll b and lutein molecules. (A) Stereo representation of the superimposition of the crystallized spinach CAB (green) and the modeled LI818 protein Cre_23 from Chlamydomonas reinhardtii (cyan). (B) Stereo representation of the superimposition of the crystallized spinach CAB (green) and the modeled LHC Esi_02 (brown). The view is a zoom on the region comprising the helix α2. The chlorophyll and lutein molecules bound to the spinach CBP are represented in balls and sticks, with the CPK color code.

Figure 5

Schematic representation of the possible evolution of CBPs from Chl a/b-, Chl a-, and Chl a/c-containing organisms. A: Simplified representation of the nuclear phylogeny of photosynthetic organisms according to Sanchez-Puerta and Delwiche [70,71]. Hypotheses 1, 2, 3, and 4 as well as blue arrows indicate possible origins of LI818 proteins (see B and text). Orange arrows designate possible horizontal gene transfers according to hypotheses 3 and 4. Names marked with "*" indicate branches with known LI818 proteins. PE = primary endosymbiosis, SE = secondary endosymbiosis, TE = tertiary endosymbiosis. B: Different hypotheses regarding the position of LI818 proteins and associated p-values using the Approximately Unbiased test (AU) and the Weighted Shimodaira-Hasegawa test (WSH). "Lost in" indicates lineages in which, based on currently available sequence data, we would assume LI818 proteins to have been lost according to these hypotheses. Hypothesis 1 assumes that LI818 originated independently in green plants and chlorophyll a/c-containing organisms; Hypothesis 2 assumes that LI818 proteins evolved before the separation of the red- and green lineage; Hypothesis 3 assumes a transfer of genes from the green lineage to a common ancestor of heterokonts and haptophytes (Chl a/c) during the cryptic endosymbiosis event proposed by Moustafa et al. [28]; Hypothesis 4 assumes a horizontal gene transfer from an ancestral chlorophyll a/c-containing organism to an early member of the green lineage.