Red seaweeds are key components of coastal ecosystems and are economically important as food and as a source of gelling agents, but their genes and genomes have received little attention. Here we report the sequencing of the 105-Mbp genome of the florideophyte Chondrus crispus (Irish moss) and the annotation of the 9,606 genes. The genome features an unusual structure characterized by gene-dense regions surrounded by repeat-rich regions dominated by transposable elements. Despite its fairly large size, this genome shows features typical of compact genomes, e.g., on average only 0.3 introns per gene, short introns, low median distance between genes, small gene families, and no indication of large-scale genome duplication. The genome also gives insights into the metabolism of marine red algae and adaptations to the marine environment, including genes related to halogen metabolism, oxylipins, and multicellularity (microRNA processing and transcription factors). Particularly interesting are features related to carbohydrate metabolism, which include a minimalistic gene set for starch biosynthesis, the presence of cellulose synthases acquired before the primary endosymbiosis showing the polyphyly of cellulose synthesis in Archaeplastida, and cellulases absent in terrestrial plants as well as the occurrence of a mannosylglycerate synthase potentially originating from a marine bacterium. To explain the observations on genome structure and gene content, we propose an evolutionary scenario involving an ancestral red alga that was driven by early ecological forces to lose genes, introns, and intergenetic DNA; this loss was followed by an expansion of genome size as a consequence of activity of transposable elements.T he red algae, together with the glaucophytes and the Chloroplastida, are members of the Archaeplastida, the phylogenetic group formed during the primary endosymbiosis event that gave rise to the first photosynthetic eukaryote. Red algal genomes, both plastid and nuclear, also contributed, via secondary endosymbiosis, to several other eukaryotic lineages, including

Solid semi-crystalline starch and hydrosoluble glycogen define two distinct physical states of the same type of storage polysaccharide. Appearance of semi-crystalline storage polysaccharides appears linked to the requirement of unicellular diazotrophic cyanobacteria to fuel nitrogenase and protect it from oxygen through respiration of vast amounts of stored carbon. Starch metabolism itself resulted from the merging of the bacterial and eukaryote pathways of storage polysaccharide metabolism after endosymbiosis of the plastid. This generated the three Archaeplastida lineages: the green algae and land plants (Chloroplastida), the red algae (Rhodophyceae), and the glaucophytes (Glaucophyta). Reconstruction of starch metabolism in the common ancestor of Archaeplastida suggests that polysaccharide synthesis was ancestrally cytosolic. In addition, the synthesis of cytosolic starch from the ADP-glucose exported from the cyanobacterial symbiont possibly defined the original metabolic flux by which the cyanobiont provided photosynthate to its host. Additional evidence supporting this scenario include the monophyletic origin of the major carbon translocators of the inner membrane of eukaryote plastids which are sisters to nucleotide-sugar transporters of the eukaryote endomembrane system. It also includes the extent of enzyme subfunctionalization that came as a consequence of the rewiring of this pathway to the chloroplasts in the green algae. Recent evidence suggests that, at the time of endosymbiosis, obligate intracellular energy parasites related to extant Chlamydia have donated important genes to the ancestral starch metabolism network.

Plants contain two types of ␣ (1 → 6) glucan hydrolase (starch-debranching enzyme [DBE]). Mutations that affect the pullulanase-type DBE have not been described, although defects in isoamylase-type DBE, known in many plant species, indicate a function in starch biosynthesis. We describe a null mutation of a pullulanase-type DBE gene, a Mutator insertion in maize Zpu1 . Plants homozygous for the zpu1-204 mutation are impaired in transient and storage starch degradation. Thus, hydrolytic activity of pullulanase-type DBE contributes to starch catabolism. Developing zpu1-204 endosperm accumulates branched maltooligosaccharides not found in the wild type and is deficient in linear maltooligosaccharides, indicating that the pullulanase-type DBE functions in glucan hydrolysis during kernel starch formation. Furthermore, in a background deficient in isoamylase-type DBE, zpu1-204 conditions a significant accumulation of phytoglycogen in the kernel that is not seen in the wild type. Therefore, pullulanase-type DBE partially compensates for the defect in isoamylase-type DBE, suggesting a function during starch synthesis as well as degradation.

Under the endosymbiont hypothesis, over a billion years ago a heterotrophic eukaryote entered into a symbiotic relationship with a cyanobacterium (the cyanobiont). This partnership culminated in the plastid that has spread to forms as diverse as plants and diatoms. However, why primary plastid acquisition has not been repeated multiple times remains unclear. Here, we report a possible answer to this question by showing that primary plastid endosymbiosis was likely to have been primed by the secretion in the host cytosol of effector proteins from intracellular Chlamydiales pathogens. We provide evidence suggesting that the cyanobiont might have rescued its afflicted host by feeding photosynthetic carbon into a chlamydiacontrolled assimilation pathway.

Starch production in all plants examined is altered by mutations of isoamylase-type starch-debranching enzymes (DBE), although how these proteins affect glucan polymer assembly is not understood. Various allelic mutations in the maize (Zea mays) gene sugary1 (su1), which codes for an isoamylase-type DBE, condition distinct kernel phenotypes. This study characterized the recessive mutations su1-Ref, su1-R4582::Mu1, and su1-st, regarding their molecular basis, chemical phenotypes, and effects on starch metabolizing enzymes. The su1-Ref allele results in two specific amino acid substitutions without affecting the Su1 mRNA level. The su1-R4582::Mu1 mutation is a null allele that abolishes transcript accumulation. The su1-st mutation results from insertion of a novel transposon-like sequence, designated Toad, which causes alternative pre-mRNA splicing. Three su1-st mutant transcripts are produced, one that is nonfunctional and two that code for modified SU1 polypeptides. The su1-st mutation is dominant to the null allele su1-R4582::Mu1, but recessive to su1-Ref, suggestive of complex effects involving quaternary structure of the SU1 enzyme. All three su1-alleles severely reduce or eliminate isoamylase-type DBE activity, although su1-st kernels accumulate less phytoglycogen and Suc than su1-Ref or su1-R4582::Mu1 mutants. The chain length distribution of residual amylopectin is significantly altered by su1-Ref and su1-R4582::Mu1, whereas su1-st has modest effects. These results, together with su1 allele-specific effects on other starchmetabolizing enzymes detected in zymograms, suggest that total DBE catalytic activity is the not the sole determinant of Su1 function and that specific interactions between SU1 and other components of the starch biosynthetic system are required.

Functions of isoamylase-type starch-debranching enzyme (ISA) proteins and complexes in maize (Zea mays) endosperm were characterized. Wild-type endosperm contained three high molecular mass ISA complexes resolved by gel permeation chromatography and native-polyacrylamide gel electrophoresis. Two complexes of approximately 400 kD contained both ISA1 and ISA2, and an approximately 300-kD complex contained ISA1 but not ISA2. Novel mutations of sugary1 (su1) and isa2, coding for ISA1 and ISA2, respectively, were used to develop one maize line with ISA1 homomer but lacking heteromeric ISA and a second line with one form of ISA1/ISA2 heteromer but no homomeric enzyme. The mutations were su1-P, which caused an amino acid substitution in ISA1, and isa2-339, which was caused by transposon insertion and conditioned loss of ISA2. In agreement with the protein compositions, all three ISA complexes were missing in an ISA1-null line, whereas only the two higher molecular mass forms were absent in the ISA2-null line. Both su1-P and isa2-339 conditioned near-normal starch characteristics, in contrast to ISA-null lines, indicating that either homomeric or heteromeric ISA is competent for starch biosynthesis. The homomer-only line had smaller, more numerous granules. Thus, a function of heteromeric ISA not compensated for by homomeric enzyme affects granule initiation or growth, which may explain evolutionary selection for ISA2. ISA1 was required for the accumulation of ISA2, which is regulated posttranscriptionally. Quantitative polymerase chain reaction showed that the ISA1 transcript level was elevated in tissues where starch is synthesized and low during starch degradation, whereas ISA2 transcript was relatively abundant during periods of either starch biosynthesis or catabolism.Starch biosynthesis is a central function in plant metabolism that is accomplished by a multiplicity of conserved enzymatic activities. Two known activities are starch synthase, which catalyzes the polymerization of glucosyl units into a(1/4)-linked "linear" chains, and starch-branching enzyme, which catalyzes the formation of a(1/6) glycoside bond branches that join linear chains. Acting together, the starch synthases and starch-branching enzymes assemble the relatively highly branched polymer amylopectin, with approximately 5% of the glucosyl residues participating in a (1/6) bonds, and the lightly branched molecule amylose. Amylopectin and amylose assemble into semicrystalline starch granules, which in land plants and green algae are located in plastids.A third activity necessary for normal starch biosynthesis is provided by starch-debranching enzyme (DBE), which hydrolyzes a(1/6) linkages. Two DBE classes have been conserved separately in plants (Beatty et al., 1999). These are referred to here as pullulanase-type DBE (PUL) and isoamylase-type DBE (ISA), based on similarity to prokaryotic enzymes with particular substrate specificity. ISA function in starch production is implied from genetic observations that mutations typically result in reduced ...

Mutations in the maize gene sugary2 ( su2 ) affect starch structure and its resultant physiochemical properties in useful ways, although the gene has not been characterized previously at the molecular level. This study tested the hypothesis that su2 codes for starch synthase IIa (SSIIa). Two independent mutations of the su2 locus, su2-2279 and su2-5178 , were identified in a Mutator -active maize population. The nucleotide sequence of the genomic locus that codes for SSIIa was compared between wild type plants and those homozygous for either novel mutation. Plants bearing su2-2279 invariably contained a Mutator transposon in exon 3 of the SSIIa gene, and su2-5178 mutants always contained a small retrotransposon-like insertion in exon 10. Six allelic su2 (-) mutations conditioned loss or reduction in abundance of the SSIIa protein detected by immunoblot. These data indicate that su2 codes for SSIIa and that deficiency in this isoform is ultimately responsible for the altered physiochemical properties of su2 (-) mutant starches. A specific starch synthase isoform among several identified in soluble endosperm extracts was absent in su2-2279 or su2-5178 mutants, indicating that SSIIa is active in the soluble phase during kernel development. The immediate structural effect of the su2 (-) mutations was shown to be increased abundance of short glucan chains in amylopectin and a proportional decrease in intermediate length chains, similar to the effects of SSII deficiency in other species.

Eukaryotic cells are composed of a variety of membrane-bound organelles that are thought to derive from symbiotic associations involving bacteria, archaea, or other eukaryotes. In addition to acquiring the plastid, all Archaeplastida and some of their endosymbiotic derivatives can be distinguished from other organisms by the fact that they accumulate starch, a semicrystalline-storage polysaccharide distantly related to glycogen and never found elsewhere. We now provide the first evidence for the existence of starch in a particular species of single-cell diazotrophic cyanobacterium. We provide evidence for the existence in the eukaryotic host cell at the time of primary endosymbiosis of an uridine diphosphoglucose (UDP-glucose)-based pathway similar to that characterized in amoebas. Because of the monophyletic origin of plants, we can define the genetic makeup of the Archaeplastida ancestor with respect to storage polysaccharide metabolism. The most likely enzyme-partitioning scenario between the plastid's ancestor and its eukaryotic host immediately suggests the precise nature of the ancient metabolic symbiotic relationship. The latter consisted in the export of adenosine diphosphoglucose (ADP-glucose) from the cyanobiont in exchange for the import of reduced nitrogen from the host. We further speculate that the monophyletic origin of plastids may lie in an organism with close relatedness to present-day group V cyanobacteria.