Common Evolutionary Origin of Starch Biosynthetic Enzymes in Green and Red Algae1
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J. Phycol. 41, 1131–1141 (2005) r 2005 Phycological Society of America DOI: 10.1111/j.1529-8817.2005.00135.x COMMON EVOLUTIONARY ORIGIN OF STARCH BIOSYNTHETIC ENZYMES IN GREEN AND RED ALGAE1 Nicola J. Patron and Patrick J. Keeling2 Canadian Institute for Advanced Research, Botany Department, University of British Columbia, 3529-6270 University Boulevard, Vancouver, BC, V6T 1Z4, Canada Plastidic starch synthesis in green algae and length and number of branches varying between or- plants occurs via ADP-glucose in likeness to pro- ganisms. The a-1,4-glucan chains are synthesized by karyotes from which plastids have evolved. In con- glycosyltransferases, which use uridine diphosphate trast, floridean starch synthesis in red algae (UDP)-glucose or ADP-glucose as the sugar donor proceeds via uridine diphosphate-glucose in sem- and a preexisting a-1,4-glucan chain as the acceptor. blance to eukaryotic glycogen synthesis and occurs Glycogen is localized in the cytoplasm of bacteria, fun- in the cytosol rather than the plastid. Given the gi, and animal cells. Both eukaryotic and prokaryotic monophyletic origin of all plastids, we investigated glycogens are always amorphous and never form the the origin of the enzymes of the plastid and cyto- crystalline granules characteristic of starch. Red algal solic starch synthetic pathways to determine wheth- starch, thought to be comprised purely of amylopectin er their location reflects their origin—either from chains (Marszalec et al. 2001, Yu et al. 2002), is cyto- the cyanobacterial endosymbiont or from the solic and is known as floridean starch, whereas in eukaryotic host. We report that, despite the com- green algae and plants, starch accumulates within the partmentalization of starch synthesis differing in plastid. green and red lineages, all but one of the enzymes of The eukaryotic ancestor of all plants, algae, and or- the synthetic pathways shares a common origin. ganisms derived from such acquired a plastid by the Overall, the pathway of starch synthesis in both lin- engulfment and subsequent retention of a cyanobac- eages represents a chimera of the host and end- terium (Gray and Spencer 1996). Descendants of this osymbiont glycogen synthesis pathways. Moreover, ancestor have given rise to a diversity of plastid-con- host-derived proteins function in the plastid in taining eukaryotes. Many genes have been transferred green algae, whereas endosymbiont-derived pro- from the endosymbiont to the nucleus: It is estimated teins function in the cytosol in red algae. This com- that as many as 18% of the genes in Arabidopsis are of plexity demonstrates the impacts of integrating plastid origin (Martin et al. 2002). It was long assumed pathways of host with those of both primary and that the protein products of all of these genes would be secondary endosymbionts during plastid evolution. targeted back to the organelle (Weeden 1981), but Key index words: amylose; carbohydrate; dinoflag- more recent work is calling the contribution of the ellate; endosymbiosis; floridean starch; glycogen; endosymbiont to the eukaryotic cell as a whole into plastid; starch question (Abdallah et al. 2000, Martin et al. 2002, Leis- ter 2003). Those that are redirected to the plastid are Abbreviations: AGPase, ADP-glucose pyropho- targeted using a transit peptide allowing them to fulfill sphorylase; BE, branching enzyme; GBSS, granule their original role in the plastid (Peltier et al. 2000). bound starch synthase; Glc-1-P, glucose-1-phos- Several plastidial reactions, however, have been re- phate; Glc-6-P, glucose-6-phosphate; G(S)S, glyco- placed by enzymes derived from the host (Fast et al. gen (starch) synthase; LSU, large subunit; PGM, 2001); similarly, some endosymbiont-derived proteins phosphoglucomutase; SS, soluble starch synthase; have been adopted by pathways in the cytoplasm SSU, small subunit; UDP, uridine diphosphate; (Brinkmann and Martin 1996). UGPase, UDP-glucose pyrophosphorylase Despite differences in structure between bacterial glycogen and plant starch, the plastidial location of plant starch and the use of ADP-glucose (as in bacte- Most living organisms accumulate either glycogen ria), rather than UDP-glucose, as a substrate (as in fun- or starch as energy storage polymers. Glycogen, amy- gi and animals) suggest that the plant pathway was lose, and amylopectin, the latter two of which comprise inherited from its prokaryotic endosymbiont. On the green algal and plant starch, are molecules of a-1,4- other hand, the use of ADP-glucose and the cytosolic linked glucose units with a-1,6-branch points, the location of starch in red algae have led to the sugges- tion that these algae and their derivatives use a novel pathway likely derived from the host (Nyvall et al. 1Received 10 March 2005. Accepted 17 August 2005. 1999, Viola et al. 2001). There is little direct evidence 2Author for correspondence: e-mail pkeeling@interchange.ubc.ca. for the nature of the starch biosynthesis pathway in red 1131 1132 NICOLA J. PATRON AND PATRICK J. KEELING algae and until recently virtually no molecular data. which have been shown to have residual PGM activi- However, the recent sequencing of two red algal ty in addition to their primary functions. In humans, genomes (Matsuzaki et al. 2004, Michigan State Uni- three loci for PGM were found and named PGM1, versity Galdieria Database http://genomics.msu.edu/ PGM2, and PGM3. Phosphoglucomutase 1 is a true galdieria_public) and the accumulation of Expressed PGM, whereas PGM2 is a phosphoribomutase (Ninfali Sequence Tag (EST ) and genomic data from several et al. 1984) and PGM3 is an N-acetylglucosamine- algal groups with plastids derived from red algae phosphate mutase (Pang et al. 2002). Two isoforms of (Armbrust et al. 2004, Patron et al. 2004b) now allow PGM are present in plants, one located in the cytosol us to investigate the evolutionary origins of their starch and the other in the chloroplast stroma (Mu¨hlbach and biosynthetic pathway. Here, we investigate the origins Schnarrenberger 1978). The plastid PGM is required of these enzymes by examining their molecular phylo- for starch synthesis to store net photosynthate during geny to determine whether the plant and algal proteins the day (Dietz 1987, Periappuram et al. 2000). Defi- were inherited from the cyanobacterial endosymbiont ciency in the plastid PGM activity resulted in a ‘‘starch- or from the eukaryotic host. Particular attention is paid less’’ phenotype in Arabidopsis (Kofler et al. 2000). to the red algae, because little is known about starch Phosphoglucomutases of all eukaryotes and pro- synthesisinanyrhodophyte,andtheoriginoftheen- karyotes are relatively well conserved at the primary zymes may resolve whether their pathway is similar to amino acid level, and homologues from all sampled that found in the green plastid lineage or whether it taxa were readily alignable. Prokaryotes form a mod- evolved independently and is novel. erately supported clade (82 Maximum Likelihood [ML] and 54 Weighbor [WBR] bootstrap support), MATERIALS AND METHODS Metazoan, fungal, chlorophyte-plastid, and chloro- phyte-cytosolic forms also separate into well-supported Protein sequences were obtained from GenBank and from the Cyanidioschyzon merolae (http://merolae.biol.s.u-tokyo.ac.jp/) clades, but there is no support for their position or or- and Galdieria sulphuraria (http://genomics.msu.edu/galdieria/ der within the tree (Fig. 1). It is not possible to say con- funding.html) genome projects and from an in-house Hetero- clusively if the proteins found in red and green algae capsa triquetra EST project (http://amoebidia.bcm.umontreal.ca/ and plants share a common ancestry or whether they public/pepdb/agrm.php). are of cyanobacterial or eukaryotic origin, although the Protein alignments were made using Clustal X and manu- support for the prokaryotic branch suggests they are ally edited. All ambiguous sites of the alignments were re- more likely eukaryotic. The red alga Cyandioschyzon me- moved from the data set for phylogenetic analyses. The alignment data are available on request. Protein maximum rolae encodes three PGMs, one of which is most similar likelihood analyses used PhyML (Guindon and Gascuel 2003) to a chloroplast isoform from spinach (data not shown). with input trees generated by BIONJ, the JTT model of amino Both the C. merolae and spinach isoforms are highly di- acids substitution, proportion of variable rates estimated from vergent, and a reduced character set had to be used to the data, and nine categories of substitution rates (eight vari- analyze the data with these sequences (data not shown), able and one invariable). One hundred bootstrap trees were which gave a tree with even less resolution and no con- calculated with PhyML with four gamma correction categories. For distance analyses, gamma-corrected distances were calcu- clusion to the origin of these isoforms. The amino- lated by TREE-PUZZLE 5.0 using the WAG substitution terminus of this C. merolae protein is extended by com- matrix with eight variable rate categories and invariable sites. parison to prokaryotic enzymes, but the extension does Trees were inferred by weighted neighbor joining using not have any of the characteristics of a transit peptide, WEIGHBOR 1.0.1a. Bootstrap resampling was performed us- such as frequent hydroxylated residues. The C. merolae ing PUZZLEBOOTwith rates and frequencies estimated using sequence that branches between the prokaryotes and TREE-PUZZLE 5.0. the eukaryotes has a similar amino-terminal extension, but there is no support for the positions of either this or RESULTS the other rhodophyte sequences within the tree. Given Phosphoglucomutase. Phosphoglucomutase (PGM; that floridean starch is cytosolic, there is no expectation EC 2.7.5.1) catalyzes the interconversion of glucose- for plastid isoforms of PGM for the purpose of starch 1-phosphate (Glc-1-P) and glucose-6-phosphate (Glc- production. However, other plastid pathways, such as 6-P) at a pivotal metabolic trafficking point that con- fatty acid and amino acid biosynthesis, also require Glc- trols the synthesis and degradation of carbohydrates.