High-throughput droplet microfluidics screening platform for selecting fast-growing and high lipid-producing microalgae from a mutant library.

Biofuels derived from microalgal lipids have demonstrated a promising potential as future renewable bioenergy. However, the production costs for microalgae-based biofuels are not economically competitive, and one strategy to overcome this limitation is to develop better-performing microalgal strains that have faster growth and higher lipid content through genetic screening and metabolic engineering. In this work, we present a high-throughput droplet microfluidics-based screening platform capable of analyzing growth and lipid content in populations derived from single cells of a randomly mutated microalgal library to identify and sort variants that exhibit the desired traits such as higher growth rate and increased lipid content. By encapsulating single cells into water-in-oil emulsion droplets, each variant was separately cultured inside an individual droplet that functioned as an independent bioreactor. In conjunction with an on-chip fluorescent lipid staining process within droplets, microalgal growth and lipid content were characterized by measuring chlorophyll and BODIPY fluorescence intensities through an integrated optical detection system in a flow-through manner. Droplets containing cells with higher growth and lipid content were selectively retrieved and further analyzed off-chip. The growth and lipid content screening capabilities of the developed platform were successfully demonstrated by first carrying out proof-of-concept screening using known Chlamydomonas reinhardtii mutants. The platform was then utilized to screen an ethyl methanesulfonate (EMS)-mutated C. reinhardtii population, where eight potential mutants showing faster growth and higher lipid content were selected from 200,000 examined samples, demonstrating the capability of the platform as a high-throughput screening tool for microalgal biofuel development.

OEP80, an essential protein paralogous to the chloroplast protein translocation channel Toc75, exists as a 70-kD protein in the Arabidopsis thaliana chloroplast outer envelope.

Toc75 and OEP80 are paralogous proteins found in the Viridiplantae lineages, and appear to have evolved from a protein in the outer membrane of an ancient cyanobacterium. Toc75 is known to act as a protein translocation channel at the outer membrane of the chloroplast envelope, whereas the exact function of OEP80 is not understood. In Arabidopsis thaliana, each protein is encoded by a single gene, and both are essential for plant viability from embryonic stages onward. Sequence annotation and immunoblotting data with an antibody against its internal sequence (αOEP80(325-337)) indicated that the molecular weight of OEP80 is ca. 80 kD. Here we present multiple data to show that the size of A. thaliana OEP80 is smaller than previously estimated. First, we prepared the antibody against a recombinant protein consisting of annotated full-length A. thaliana OEP80 with an N-terminal hexahistidine tag (αOEP80(1-732)). This antibody recognized a 70-kD protein in the A. thaliana chloroplast membrane fraction which migrated faster than the His-tagged antigen and the protein recognized by the αOEP80(325-337) antibody on SDS-PAGE. Immunoprecipitation followed by LC-MS/MS analysis confirmed that the 70-kD protein was encoded by the OEP80 cDNA. Next, we performed a genetic complementation assay using embryo-lethal oep80-null plants and constructs encoding OEP80 and its variants. The results revealed that the nucleotide sequence encoding the 52 N-terminal amino acids was not required for functional expression of OEP80 and accumulation of the 70-kD protein. The data also indicated that an additional C-terminal T7 tag remained intact without disrupting the functionality of OEP80, and was not exposed to the cytoplasmic surface of the chloroplast envelope. Finally, OEP80-T7 and Toc75 showed distinct migration patterns on blue native-PAGE. This study provides molecular tools to investigate the function of OEP80, and also calls for caution in using an anti-peptide antibody.

Functional diversification of thylakoidal processing peptidases in Arabidopsis thaliana.

Thylakoidal processing peptidase (TPP) is responsible for removing amino-terminal thylakoid-transfer signals from several proteins in the thylakoid lumen. Three TPP isoforms are encoded by the nuclear genome of Arabidopsis thaliana. Previous studies showed that one of them termed plastidic type I signal peptidase 1 (Plsp1) was necessary for processing three thylakoidal proteins and one protein in the chloroplast envelope in vivo. The lack of Plsp1 resulted in seedling lethality, apparently due to disruption of proper thylakoid development. The physiological roles of the other two TPP homologs remain unknown. Here we show that the three A. thaliana TPP isoforms evolved to acquire diverse functions. Phylogenetic analysis revealed that TPP may have originated before the endosymbiotic event, and that there are two groups of TPP in seed plants: one includes Plsp1 and another comprises the other two A. thaliana TPP homologs, which are named as Plsp2A and Plsp2B in this study. The duplication leading to the two groups predates the gymnosperm-angiosperm divergence, and the separation of Plsp2A and Plsp2B occurred after the Malvaceae-Brassicaceae diversification. Quantitative reverse transcription-PCR assay revealed that the two PLSP2 genes were co-expressed in both photosynthetic tissues and roots, whereas the PLSP1 transcript accumulated predominantly in photosynthetic tissues. Both PLSP2 genes were expressed in the aerial parts of the plsp1-null mutant at levels comparable to those in wild-type plants. The seedling-lethal phenotype of the plsp1-null mutant could be rescued by a constitutive expression of Plsp1 cDNA but not by that of Plsp2A or Plsp2B. These results indicate that Plsp1 and Plsp2 evolved to function differently, and that neither of the Plsp2 isoforms is necessary for proper thylakoid development in photosynthetic tissues.

Indispensable Roles of Plastids in Arabidopsis thaliana Embryogenesis.

The plastid is an organelle vital to all photosynthetic and some non-photosynthetic eukaryotes. In the model plant Arabidopsis thaliana, a number of nuclear genes encoding plastid proteins have been found to be necessary for embryo development. However, the exact roles of plastids in this process remain largely unknown. Here we use publicly available datasets to obtain insights into the relevance of plastid activities to A. thaliana embryogenesis. By searching the SeedGenes database (http://www.seedgenes.org) and recent literature, we found that, of the 339 non-redundant genes required for proper embryo formation, 108 genes likely encode plastid-targeted proteins. Nineteen of these genes are necessary for development of preglobular embryos and/or their conversion to globular embryos, of which 13 genes encode proteins involved in non-photosynthetic metabolism. By contrast, among 38 genes which are dispensable for globular embryo formation but necessary for further development, only one codes for a protein involved in metabolism. Products of 21 of the 38 genes play roles in plastid gene expression and maintenance. Examination of RNA profiles of embryos at distinct growth stages obtained in laser-capture microdissection coupled with DNA microarray experiments revealed that most of the identified genes are expressed throughout embryo morphogenesis and maturation. These findings suggest that metabolic activities are required at preglobular and throughout all stages of embryo development, whereas plastid gene expression becomes necessary during and/or after the globular stage to sustain various activities of the organelle including photosynthetic electron transport.

Two evolutionarily conserved essential beta-barrel proteins in the chloroplast outer envelope membrane.

Chloroplasts are organelles specific to photosynthetic eukaryotes that support the lives of most organisms on earth. Chloroplasts were derived from an ancient cyanobacterium by endosymbiosis, and one characteristic shared between them and extant cyanobacteria is the presence of beta-barrel proteins in the outer membrane. These integral membrane proteins are also found in the outer membranes of proteobacteria and mitochondria. In particular, a group of homologous beta-barrel proteins called BamA homologs are present in all Gram-negative bacteria and the endosymbiotic organelles, i.e., chloroplasts and mitochondria. It was recently revealed that, in both proteobacteria and mitochondria, there is a single essential BamA homolog that mediates beta-barrel protein assembly. In a chloroplast, there are two distinct BamA homologs, Toc75 and OEP80, which diverged early in the evolution of chloroplasts from their common ancestor with extant cyanobacteria. Recent genetic studies demonstrated that each of these proteins is indispensable for viability of plants although neither has been shown to be involved in beta-barrel protein assembly. Toc75 catalyzes import of nuclear-encoded precursor proteins, a process that is not required for bacteria, whereas the molecular function of OEP80 remains elusive. Establishment of a protein import apparatus was required to facilitate the transfer of genes from the endosymbiont to the host cell nucleus. Hence, we propose that the gene duplication giving rise to the two essential BamA homologs was a prerequisite for the successful conversion of the cyanobacterial endosymbiont into the chloroplast. Consequently, continued study of these two chloroplast proteins should advance our understanding of endosymbiosis and evolutionarily conserved proteins in general.

The Omp85-related chloroplast outer envelope protein OEP80 is essential for viability in Arabidopsis.

beta-Barrel proteins of the Omp85 (Outer membrane protein, 85 kD) superfamily exist in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Prominent Omp85 proteins in bacteria and mitochondria mediate biogenesis of other beta-barrel proteins and are indispensable for viability. In Arabidopsis (Arabidopsis thaliana) chloroplasts, there are two distinct types of Omp85-related protein: Toc75 (Translocon at the outer envelope membrane of chloroplasts, 75 kD) and OEP80 (Outer Envelope Protein, 80 kD). Toc75 functions as a preprotein translocation channel during chloroplast import, but the role of OEP80 remains elusive. We characterized three T-DNA mutants of the Arabidopsis OEP80 (AtOEP80) gene. Selectable markers associated with the oep80-1 and oep80-2 insertions segregated abnormally, suggesting embryo lethality of the homozygous genotypes. Indeed, no homozygotes were identified among >100 individuals, and heterozygotes of both mutants produced approximately 25% aborted seeds upon self-pollination. Embryo arrest occurred at a relatively late stage (globular embryo proper) as revealed by analysis using Nomarski optics microscopy. This is substantially later than arrest caused by loss of the principal Toc75 isoform, atToc75-III (two-cell stage), suggesting a more specialized role for AtOEP80. Surprisingly, the oep80-3 T-DNA (located in exon 1 between the first and second ATG codons of the open reading frame) did not cause any detectable developmental defects or affect the size of the AtOEP80 protein in chloroplasts. This indicates that the N-terminal region of AtOEP80 is not essential for the targeting, biogenesis, or functionality of the protein, in contrast with atToc75-III, which requires a bipartite targeting sequence.

Two distinct Omp85 paralogs in the chloroplast outer envelope membrane are essential for embryogenesis in Arabidopsis thaliana.

Homologs of a bacterial beta-barrel protein, Omp85, ubiquitously exist in the outer membranes of Gram-negative bacteria, mitochondria and chloroplasts. Those in non-photosynthetic bacteria and mitochondria are responsible for beta-barrel protein sorting to the outer membranes, and thus are essential for viability of the organisms. There are two distinct Omp85 homologs in chloroplasts of the model plant, Arabidopsis thaliana. One of them, Toc75, functions as the main protein import translocation channel, and was shown to be indispensable from a very early stage of embryogenesis. By contrast, the role of another homolog, OEP80, remains elusive. Recently, we showed that disruption of the OEP80 gene causes embryo abortion in A. thaliana at a stage later than that affected by TOC75 knockout. This indicates that the two chloroplastic Omp85 homologs are both essential for viability of plants from very early stages of development, but may have distinct functions. Defining the functional and evolutionary relationships of Toc75 and OEP80 by further studies should advance our understanding of the importance of plastids during embryogenesis, as well as that of the molecular details of plastid biogenesis.

Cloning, expression, characterization, and role in autocrine cell growth of cell surface retention sequence binding protein-1.

Cell surface retention sequence binding protein-1 (CRSBP-1) is a cell surface binding protein for the cell surface retention sequence (CRS) motif of the v-sis gene product (platelet-derived growth factor-BB). It has been shown to be responsible for cell surface retention of the v-sis gene product in v-sis-transformed cells (fibroblasts) and has been hypothesized to play a role in autocrine growth and transformation of these cells. Here we demonstrate that the CRSBP-1 cDNA cloned from bovine liver libraries encodes a 322-residue type I membrane protein containing a 23-residue signal peptide, a 215-residue cell surface domain, a 21-residue transmembrane domain, and a 63-residue cytoplasmic domain. CRSBP-1 expressed in transfected cells is an approximately 120-kDa disulfide-linked homodimeric glycoprotein and exhibits dual ligand (CRS-containing growth regulators (v-sis gene product and insulin-like growth factor binding protein-3, IGFBP-3) and hyaluronic acid) binding activity. CRSBP-1 overexpression (by stable transfection of cells with CRSBP-1 cDNA) enhances autocrine loop signaling, cell growth, and tumorigenicity (in mice) of v-sis-transformed cells. CRSBP-1 expression also enhances autocrine cell growth mediated by IGFBP-3 in human lung carcinoma cells (H1299 cells), which express very little, if any, endogenous CRSBP-1 and exhibits a mitogenic response to exogenous IGFBP-3, stably transfected with IGFBP-3 cDNA. However, CRSBP-1 overexpression does not affect growth of normal and transformed cells that do not produce these CRS-containing growth regulators. These results suggest that CRSBP-1 plays a role in autocrine regulation of cell growth mediated by growth regulators containing CRS.