Nucleus-encoded mRNAs for chloroplast proteins GapA, PetA, and PsbO are trans-spliced in the flagellate Euglena gracilis irrespective of light and plastid function.

Euglena gracilis is a fresh-water flagellate possessing secondary chloroplasts of green algal origin. In contrast with organisms possessing primary plastids, mRNA levels of nucleus-encoded genes for chloroplast proteins in E. gracilis depend on neither light nor plastid function. However, it remains unknown, if all these mRNAs are trans-spliced and possess spliced leader sequence at the 5'-end and if trans-splicing depends on light or functional plastids. This study revealed that polyadenylated mRNAs encoding the chloroplast proteins glyceraldehyde-3-phosphate dehydrogenase (GapA), cytochrome f (PetA), and subunit O of photosystem II (PsbO) are trans-spliced irrespective of light or plastid function.

Variability of wax ester fermentation in natural and bleached Euglena gracilis Strains in response to oxygen and the elongase inhibitor flufenacet.

Euglena gracilis is able to synthesize adenosine triphosphate under anaerobic conditions through a malonyl-independent fatty acid synthesis leading to wax ester fermentation. Mitochondrial fatty acid synthesis uses acetyl-CoA and propionyl-CoA as C2- and C3-donors for de novo synthesis of even- and odd-numbered fatty acids, respectively. Euglena's wax ester fermentation has only been described in the E. gracilis strain 1224-5/25 Z. Here we investigate eight E. gracilis strains isolated in 1932-1958 from different localities in Europe and two bleached substrains of E. gracilis 1224-5/25, obtained by treatment with streptomycin and ofloxacin, and examine their anaerobic growth, wax ester fermentation, and wax ester composition. Under ambient oxygen levels, all strains accumulated wax esters in concentrations between 0.3% and 3.5% of the dry weight, but the strains revealed marked differences in wax ester accumulation with respect to anaerobic growth. Most fermenting strains tested showed increased wax ester synthesis under anaerobic conditions as well as the increased synthesis of odd-numbered fatty acids and alcohols suggesting an activation of the mitochondrial fatty acid biosynthesis pathway. Addition of the elongase inhibitor flufenacet to the growth medium specifically reduced the accumulation of odd-numbered fatty acids and alcohols and tended to increase the overall yield of anaerobic wax esters.

Expression of nucleus-encoded genes for chloroplast proteins in the flagellate Euglena gracilis.

Reverse transcription PCRs (RT-PCRs), real-time RT-PCRs and microarrays containing 50-mer oligonucleotides representing nucleus-encoded genes for chloroplast proteins from Euglena gracilis were used to compare light- and dark-grown wild-type mRNA levels to those of light- and dark-grown E. gracilis stable white mutant strains W(gm)ZOflL, W3BUL and W10BSmL. The analyses revealed no light-dependent regulation of mRNA levels. Moreover, the mRNA levels of most genes were unchanged in all white mutants in comparison with wild-type. These results suggest that mRNA levels of nucleus-encoded genes for chloroplast proteins in E. gracilis do not depend on either light or plastid function.

Protein trafficking to the complex chloroplasts of Euglena.

Proteins are delivered to Euglena chloroplasts using the secretory pathway. We describe analytical methods to study the intracellular trafficking of Euglena chloroplast proteins and a method to isolate preparative amounts of intact import competent chloroplasts for biochemical studies. Cells are pulse labeled with 35S-sulfate and chased with unlabeled sulfate allowing the trafficking and posttranslational processing of the labeled protein to be followed. Sucrose gradients are used to separate a 35S-labeled cell lysate into cytoplasmic, endoplasmic reticuum (ER), Golgi apparatus, chloroplast and mitochondrial fractions. Immunoprecipitation of each gradient fraction allows identification of the intracellular compartment containing a specific 35S-labeled protein at different times after synthesis delineating the trafficking pathway. Because sucrose gradients cannot be used to isolate preparative amounts of highly purified chloroplasts for biochemical characterization, a preparative high-yield procedure using Percoll gradients to isolate highly purified import competent chloroplasts is also presented.

A small portion of plastid transcripts is polyadenylated in the flagellate Euglena gracilis.

Euglena gracilis possesses secondary plastids of green algal origin. In this study, E. gracilis expressed sequence tags (ESTs) derived from polyA-selected mRNA were searched and several ESTs corresponding to plastid genes were found. PCR experiments failed to detect SL sequence at the 5'-end of any of these transcripts, suggesting plastid origin of these polyadenylated molecules. Quantitative PCR experiments confirmed that polyadenylation of transcripts occurs in the Euglena plastids. Such transcripts have been previously observed in primary plastids of plants and algae as low-abundance intermediates of transcript degradation. Our results suggest that a similar mechanism exists in secondary plastids.

A possible role for short introns in the acquisition of stroma-targeting peptides in the flagellate Euglena gracilis.

The chloroplasts of Euglena gracilis bounded by three membranes arose via secondary endosymbiosis of a green alga in a heterotrophic euglenozoan host. Many genes were transferred from symbiont to the host nucleus. A subset of Euglena nuclear genes of predominately symbiont, but also host, or other origin have obtained complex presequences required for chloroplast targeting. This study has revealed the presence of short introns (41-93 bp) either in the second half of presequence-encoding regions or shortly downstream of them in nine nucleus-encoded E. gracilis genes for chloroplast proteins (Eno29, GapA, PetA, PetF, PetJ, PsaF, PsbM, PsbO, and PsbW). In addition, the E. gracilis Pbgd gene contains two introns in the second half of presequence-encoding region and one at the border of presequence-mature peptide-encoding region. Ten of 12 introns present within presequence-encoding regions or shortly downstream of them identified in this study have typical eukaryotic GT/AG borders, are T-rich, 45-50 bp long, and pairwise sequence identities range from 27 to 61%. Thus single recombination events might have been mediated via these cis-spliced introns. A double crossing over between these cis-spliced introns and trans-spliced introns present in 5'-UTRs of Euglena nuclear genes is also likely to have occurred. Thus introns and exon-shuffling could have had an important role in the acquisition of chloroplast targeting signals in E. gracilis. The results are consistent with a late origin of photosynthetic euglenids.

Homologous and heterologous reconstitution of Golgi to chloroplast transport and protein import into the complex chloroplasts of Euglena.

Euglena complex chloroplasts evolved through secondary endosymbiosis between a phagotrophic trypanosome host and eukaryotic algal endosymbiont. Cytoplasmically synthesized chloroplast proteins are transported in vesicles as integral membrane proteins from the ER to the Golgi apparatus to the Euglena chloroplast. Euglena chloroplast preprotein pre-sequences contain a functional N-terminal ER-targeting signal peptide and a domain having characteristics of a higher plant chloroplast targeting transit peptide, which contains a hydrophobic stop-transfer membrane anchor sequence that anchors the precursor in the vesicle membrane. Pulse-chase subcellular fractionation studies showed that (35)S-labeled precursor to the light harvesting chlorophyll a/b binding protein accumulated in the Golgi apparatus of Euglena incubated at 15 degrees C and transport to the chloroplast resumed after transfer to 26 degrees C. Transport of the (35)S-labeled precursor to the chlorophyll a/b binding protein from Euglena Golgi membranes to Euglena chloroplasts and import into chloroplasts was reconstituted using Golgi membranes isolated from 15 degrees C cells returned to 26 degrees C. Transport was dependent upon extra- and intrachloroplast ATP and GTP hydrolysis. Golgi to chloroplast transport was not inhibited by N-ethylmaleimide indicating that fusion of Golgi vesicles to the chloroplast envelope does not require N-ethylmaleimide-sensitive factor (NSF). This suggests that N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are not utilized in the targeting fusion reaction. The Euglena precursor to the chloroplast-localized small subunit of ribulose-1,5-bisphosphate carboxylase was not imported into isolated pea chloroplasts. A precursor with the N-terminal signal peptide deleted was imported, indicating that the Euglena pre-sequence has a transit peptide that functions in pea chloroplasts. A precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase with the hydrophobic membrane anchor and the pre-sequence region C-terminal to the hydrophobic membrane anchor deleted was imported localizing the functional transit peptide to the Euglena pre-sequence region between the signal peptidase cleavage site and the hydrophobic membrane anchor. The Euglena precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase and the deletion constructs were not post-translationally imported into isolated Euglena chloroplasts indicating that vesicular transport is the obligate import mechanism. Taken together, these studies suggest that protein import into complex Euglena chloroplasts evolved by developing a novel vesicle fusion targeting system to link the host secretory system to the transit peptide-dependent chloroplast protein import system of the endosymbiont.