An Efficient Method for the Plating of Haploid and Diploid Emiliania huxleyi on Solid Medium1.

Emiliania huxleyi is a globally important coccolithophore and one of the most successful eukaryotic organisms in the modern oceans. Despite a large body of work on this organism, including the sequencing of its genome, the tools required for forward and reverse functional genetic studies are still undeveloped. Here we present an optimized method for the clonal isolation of E. huxleyi by plating on solid medium. We demonstrate the utility of this method for a variety of strains including haploid, calcifying-diploid, and noncalcifying diploid strains. We show that, in contrast to previous studies, no changes in cell ploidy status occur when the cells are plated. Our method will greatly aid attempts to elucidate the genetic basis of the remarkable physiology of E. huxleyi by forward and reverse genetic approaches.

Plastidial thioredoxin z interacts with two fructokinase-like proteins in a thiol-dependent manner: evidence for an essential role in chloroplast development in Arabidopsis and Nicotiana benthamiana.

Here, we characterize a plastidial thioredoxin (TRX) isoform from Arabidopsis thaliana that defines a previously unknown branch of plastidial TRXs lying between x- and y-type TRXs and thus was named TRX z. An Arabidopsis knockout mutant of TRX z had a severe albino phenotype and was inhibited in chloroplast development. Quantitative real-time RT-PCR analysis of the mutant suggested that the expressions of genes that depend on a plastid-encoded RNA polymerase (PEP) were specifically decreased. Similar results were obtained upon virus-induced gene silencing (VIGS) of the TRX z ortholog in Nicotiana benthamiana. We found that two fructokinase-like proteins (FLN1 and FLN2), members of the pfkB-carbohydrate kinase family, were potential TRX z target proteins and identified conserved Cys residues mediating the FLN-TRX z interaction. VIGS in N. benthamiana and inducible RNA interference in Arabidopsis of FLNs also led to a repression of PEP-dependent gene transcription. Remarkably, recombinant FLNs displayed no detectable sugar-phosphorylating activity, and amino acid substitutions within the predicted active site imply that the FLNs have acquired a new function, which might be regulatory rather than metabolic. We were able to show that the FLN2 redox state changes in vivo during light/dark transitions and that this change is mediated by TRX z. Taken together, our data strongly suggest an important role for TRX z and both FLNs in the regulation of PEP-dependent transcription in chloroplasts.

Elimination of a group II intron from a plastid gene causes a mutant phenotype.

Group II introns are found in bacteria and cell organelles (plastids, mitochondria) and are thought to represent the evolutionary ancestors of spliceosomal introns. It is generally believed that group II introns are selfish genetic elements that do not have any function. Here, we have scrutinized this assumption by analyzing two group II introns that interrupt a plastid gene (ycf3) involved in photosystem assembly. Using stable transformation of the plastid genome, we have generated mutant plants that lack either intron 1 or intron 2 or both. Interestingly, the deletion of intron 1 caused a strong mutant phenotype. We show that the mutants are deficient in photosystem I and that this deficiency is directly related to impaired ycf3 function. We further show that, upon deletion of intron 1, the splicing of intron 2 is strongly inhibited. Our data demonstrate that (i) the loss of a group II intron is not necessarily phenotypically neutral and (ii) the splicing of one intron can depend on the presence of another.

Knockout of the plastid RNase E leads to defective RNA processing and chloroplast ribosome deficiency.

Ribonuclease E (RNase E) represents a key enzyme in bacterial RNA metabolism. It plays multifarious roles in RNA processing and also initiates degradation of mRNA by endonucleolytic cleavage. Plastids (chloroplasts) are derived from formerly free-living bacteria and have largely retained eubacterial gene expression mechanisms. Here we report the functional characterization of a chloroplast RNase E that is encoded by a single-copy nuclear gene in the model plant Arabidopsis thaliana. Analysis of knockout plants revealed that, unlike in bacteria, RNase E is not essential for survival. Absence of RNase E results in multiple defects in chloroplast RNA metabolism. Most importantly, polycistronic precursor transcripts overaccumulate in the knockout plants, while several mature monocistronic mRNAs are strongly reduced, suggesting an important function of RNase E in intercistronic processing of primary transcripts from chloroplast operons. We further show that disturbed maturation of a transcript encoding essential ribosomal proteins results in plastid ribosome deficiency and, therefore, provides a molecular explanation for the observed mutant phenotype.

High-level expression of a suite of thermostable cell wall-degrading enzymes from the chloroplast genome.

The biological conversion of plant biomass into fermentable sugars is key to the efficient production of biofuels and other renewable chemicals from plants. As up to more than 90% of the dry weight of higher plants is fixed in the cell wall, this will require the low-cost production of large amounts of cell wall-degrading enzymes. Transgenic plants can potentially provide an unbeatably cheap production platform for industrial enzymes. Transgene expression from the plastid genome is particularly attractive, due to high-level foreign protein accumulation in chloroplasts, absence of epigenetic gene silencing and improved transgene containment. Here, we have explored the potential of transplastomic plants to produce large amounts of thermostable cell wall-degrading enzymes from the bacterium Thermobifida fusca. We show that a set of four enzymes that are required for efficient degradation of cellulose (and the hemicellulose xyloglucan) could be expressed successfully in transplastomic tobacco plants. However, overexpression of the enzymes (to between approximately 5 and 40% of the plant's total soluble protein) resulted in pigment-deficient mutant phenotypes. We demonstrate that the chloroplast-produced cellulolytic enzymes are highly active. Although further optimization is needed, our data indicate that transgenic plastids offer great potential for the production of enzyme cocktails for the bioconversion of cellulosic biomass.