Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high-value lipid products. First success in applying reverse genetics by targeted gene replacement makes Nannochloropsis oceanica an attractive model to investigate the cell and molecular biology and biochemistry of this fascinating organism group. Here we present the assembly of the 28.7 Mb genome of N. oceanica CCMP1779. RNA sequencing data from nitrogen-replete and nitrogen-depleted growth conditions support a total of 11,973 genes, of which in addition to automatic annotation some were manually inspected to predict the biochemical repertoire for this organism. Among others, more than 100 genes putatively related to lipid metabolism, 114 predicted transcription factors, and 109 transcriptional regulators were annotated. Comparison of the N. oceanica CCMP1779 gene repertoire with the recently published N. gaditana genome identified 2,649 genes likely specific to N. oceanica CCMP1779. Many of these N. oceanica–specific genes have putative orthologs in other species or are supported by transcriptional evidence. However, because similarity-based annotations are limited, functions of most of these species-specific genes remain unknown. Aside from the genome sequence and its analysis, protocols for the transformation of N. oceanica CCMP1779 are provided. The availability of genomic and transcriptomic data for Nannochloropsis oceanica CCMP1779, along with efficient transformation protocols, provides a blueprint for future detailed gene functional analysis and genetic engineering of Nannochloropsis species by a growing academic community focused on this genus.

Xyloglucans are the main hemicellulosic polysaccharides found in the primary cell walls of dicots and nongraminaceous monocots, where they are thought to interact with cellulose to form a three-dimensional network that functions as the principal load-bearing structure of the primary cell wall. To determine whether two Arabidopsis thaliana genes that encode xylosyltransferases, XXT1 and XXT2, are involved in xyloglucan biosynthesis in vivo and to determine how the plant cell wall is affected by the lack of expression of XXT1, XXT2, or both, we isolated and characterized xxt1 and xxt2 single and xxt1 xxt2 double T-DNA insertion mutants. Although the xxt1 and xxt2 mutants did not have a gross morphological phenotype, they did have a slight decrease in xyloglucan content and showed slightly altered distribution patterns for xyloglucan epitopes. More interestingly, the xxt1 xxt2 double mutant had aberrant root hairs and lacked detectable xyloglucan. The reduction of xyloglucan in the xxt2 mutant and the lack of detectable xyloglucan in the xxt1 xxt2 double mutant resulted in significant changes in the mechanical properties of these plants. We conclude that XXT1 and XXT2 encode xylosyltransferases that are required for xyloglucan biosynthesis. Moreover, the lack of detectable xyloglucan in the xxt1 xxt2 double mutant challenges conventional models of the plant primary cell wall.

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Xyloglucan (XyG) is the principal hemicellulose found in the primary cell walls of most plants. XyG is composed of a ␤-(1,4)-glucan backbone that is substituted in a regular pattern with xylosyl residues, which are added by at least one and likely two or three xylosyltransferase (XT) enzymes. Previous work identified seven Arabidopsis thaliana candidate genes, one of which (AtXT1) was shown to encode a protein with XT activity (Faik, A., Price, N. J., Raikhel, N. V., and Keegstra, K. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7797-7802). We expressed both AtXT1 and a second closely related gene, now called AtXT2, in insect cells and demonstrated that both have XT activity for cellopentaose and cellohexaose acceptor substrates. Moreover, we showed that cellohexaose was a significantly better acceptor substrate than cellopentaose. Product structural characterization showed that AtXT1 and AtXT2 preferentially added the first xylosyl residue to the fourth glucosyl residue from the reducing end of both acceptors. Furthermore, when the ratio of UDP-xylose to cellohexaose and the reaction time were increased, both AtXT1 and AtXT2 added a second xylosyl residue adjacent to the first, which generated dixylosylated cellohexaose. On the basis of these results, we concluded that AtXT1 and AtXT2 have the same acceptor specificities and generate the same products in vitro. The implications of these results for understanding in vivo XyG biosynthesis are considered.The primary wall surrounding plant cells is composed of cellulose, hemicellulose, pectin, and protein. Xyloglucan (XyG) 2 is the principal hemicellulose found in the primary cell walls of non-graminaceous plants, where it composes up to 25% of the cell wall. XyG is thought to link via hydrogen bonds to the surfaces of adjacent cellulose microfibrils, thereby forming three-dimensional cellulose-XyG networks that function as the principal load-bearing structure of the primary cell wall (1).XyG is composed of a backbone of 1,4-linked ␤-D-Glcp residues that are substituted in a regular pattern at O-6 with an ␣-D-Xylp residue (where "Xyl" is xylose) to form repeating subunits with specific xylosylation patterns that are highly conserved (2). Most plants have XyG with an XXXG-type repeating subunit (where "G" denotes an unsubstituted glucosyl residue, and "X" denotes a glucosyl residue substituted at O-6 with an ␣-D-Xylp residue; see Ref. 3 for XyG nomenclature). However, plants in Poaceae and Solanaceae have an XXGG-type repeating subunit and perhaps also an XXGGG-type repeating subunit (for reviews, see Refs. 4 -6). Further sugar substitution occurs primarily at specific xylosyl residues within the repeating subunit. These xylosyl residues are substituted at O-2 with a variety of glycosyl moieties, with the most common being ␤-DGalp (represented by "L" and found in many plant species), ␣-L-Araf (where "Ara" is arabinose; represented by "S" and found only in Poaceae and Solanaceae), and the disaccharide ␣-L-Fucp-(1,2)-␤-D-Galp (where "Fuc" is fucose; represented by "F")...