Katherine W. Osteryoung

Chloroplast Division in Higher Plants Requires Members of Two Functionally Divergent Gene Families with Homology to Bacterial ftsZ

The division of plastids is critical for viability in photosynthetic eukaryotes, but the mechanisms associated with this process are still poorly understood. We previously identified a nuclear gene from Arabidopsis encoding a chloroplast-localized homolog of the bacterial cell division protein FtsZ, an essential cytoskeletal component of the prokaryotic cell division apparatus. Here, we report the identification of a second nuclear-encoded FtsZ-type protein from Arabidopsis that does not contain a chloroplast targeting sequence or other obvious sorting signals and is not imported into isolated chloroplasts, which strongly suggests that it is localized in the cytosol. We further demonstrate using antisense technology that inhibiting expression of either Arabidopsis FtsZ gene (AtFtsZ1-1 or AtFtsZ2-1) in transgenic plants reduces the number of chloroplasts in mature leaf cells from 100 to one, indicating that both genes are essential for division of higher plant chloroplasts but that each plays a distinct role in the process. Analysis of currently available plant FtsZ sequences further suggests that two functionally divergent FtsZ gene families encoding differentially localized products participate in chloroplast division. Our results provide evidence that both chloroplastic and cytosolic forms of FtsZ are involved in chloroplast division in higher plants and imply that important differences exist between chloroplasts and prokaryotes with regard to the roles played by FtsZ proteins in the division process.

ARC5, a cytosolic dynamin-like protein from plants, is part of the chloroplast division machinery

Chloroplast division in plant cells is orchestrated by a complex macromolecular machine with components positioned on both the inner and outer envelope surfaces. The only plastid division proteins identified to date are of endosymbiotic origin and are localized inside the organelle. Employing positional cloning methods in Arabidopsis in conjunction with a novel strategy for pinpointing the mutant locus, we have identified a gene encoding a new chloroplast division protein, ARC5. Mutants of ARC5 exhibit defects in chloroplast constriction, have enlarged, dumbbell-shaped chloroplasts, and are rescued by a wild-type copy of ARC5. The ARC5 gene product shares similarity with the dynamin family of GTPases, which mediate endocytosis, mitochondrial division, and other organellar fission and fusion events in eukaryotes. Phylogenetic analysis showed that ARC5 is related to a group of dynamin-like proteins unique to plants. A GFP–ARC5 fusion protein localizes to a ring at the chloroplast division site. Chloroplast import and protease protection assays indicate that the ARC5 ring is positioned on the outer surface of the chloroplast. Thus, ARC5 is the first cytosolic component of the chloroplast division complex to be identified. ARC5 has no obvious counterparts in prokaryotes, suggesting that it evolved from a dynamin-related protein present in the eukaryotic ancestor of plants. These results indicate that the chloroplast division apparatus is of mixed evolutionary origin and that it shares structural and mechanistic similarities with both the cell division machinery of bacteria and the dynamin-mediated organellar fission machineries of eukaryotes.

ARC6 is a J-domain plastid division protein and an evolutionary descendant of the cyanobacterial cell division protein Ftn2.

Replication of chloroplasts is essential for achieving and maintaining optimal plastid numbers in plant cells. The plastid division machinery contains components of both endosymbiotic and host cell origin, but little is known about the regulation and molecular mechanisms that govern the division process. The Arabidopsis mutant arc6 is defective in plastid division, and its leaf mesophyll cells contain only one or two grossly enlarged chloroplasts. We show here that arc6 chloroplasts also exhibit abnormal localization of the key plastid division proteins FtsZ1 and FtsZ2. Whereas in wild-type plants, the FtsZ proteins assemble into a ring at the plastid division site, chloroplasts in the arc6 mutant contain numerous short, disorganized FtsZ filament fragments. We identified the mutation in arc6 and show that the ARC6 gene encodes a chloroplast-targeted DnaJ-like protein localized to the plastid envelope membrane. An ARC6–green fluorescent protein fusion protein was localized to a ring at the center of the chloroplasts and rescued the chloroplast division defect in the arc6 mutant. The ARC6 gene product is related closely to Ftn2, a prokaryotic cell division protein unique to cyanobacteria. Based on the FtsZ filament morphology observed in the arc6 mutant and in plants that overexpress ARC6, we hypothesize that ARC6 functions in the assembly and/or stabilization of the plastid-dividing FtsZ ring. We also analyzed FtsZ localization patterns in transgenic plants in which plastid division was blocked by altered expression of the division site–determining factor AtMinD. Our results indicate that MinD and ARC6 act in opposite directions: ARC6 promotes and MinD inhibits FtsZ filament formation in the chloroplast.

Genome, Functional Gene Annotation, and Nuclear Transformation of the Heterokont Oleaginous Alga Nannochloropsis oceanica CCMP1779

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.

A homologue of the bacterial cell division site-determining factor MinD mediates placement of the chloroplast division apparatus

BACKGROUND Chloroplast division in plant cells occurs by binary fission, yielding two daughter plastids of equal size. Previously, we reported that two Arabidopsis homologues of FtsZ, a bacterial protein that forms a cytokinetic ring during cell division, are essential for plastid division in plants, and may be involved in the formation of plastid-dividing rings on both the stromal and cytosolic surfaces of the chloroplast envelope membranes. In bacteria, positioning of the FtsZ ring at the center of the cell is mediated in part by the protein MinD. Here, we identified AtMinD1, an Arabidopsis homologue of MinD, and investigated whether positioning of the plastid-division apparatus at the plastid midpoint might involve a mechanism similar to that in bacteria. RESULTS Sequence analysis and in vitro chloroplast import experiments indicated that AtMinD1 contains a transit peptide that targets it to the chloroplast. Transgenic Arabidopsis plants with reduced AtMinD1 expression exhibited variability in chloroplast size and number and asymmetrically constricted chloroplasts, strongly suggesting that the plastid-division machinery is misplaced. Overexpression of AtMinD1 inhibited chloroplast division. These phenotypes resemble those of bacterial mutants with altered minD expression. CONCLUSIONS Placement of the plastid-division machinery at the organelle midpoint requires a plastid-targeted form of MinD. The results are consistent with a model whereby assembly of the division apparatus is initiated inside the chloroplast by the plastidic form of FtsZ, and suggest that positioning of the cytosolic components of the apparatus is specified by the position of the plastidic components.

Analysis of carotenoid biosynthetic gene expression during marigold petal development

Marigold (Tagetes erecta L.) flower petals synthesize and accumulate carotenoids at levels greater than 20 times that in leaves and provide an excellent model system to investigate the molecular biology and biochemistry of carotenoid biosynthesis in plants. In addition, marigold cultivars exist with flower colors ranging from white to dark orange due to ¿100-fold differences in carotenoid levels, and presumably similar changes in carbon flux through the pathway. To examine the expression of carotenoid genes in marigold petals, we have cloned the majority of the genes in this pathway and used these to assess their steady-state mRNA levels in four marigold cultivars with extreme differences in carotenoid content. We have also cloned genes encoding early steps in the biosynthesis of isopentenyl pyrophosphate (IPP), the precursor of all isoprenoids, including carotenoids, as well as two genes required for plastid division. Differences among the marigold varieties in the expression of these genes suggest that differences in mRNA transcription or stability underlie the vast differences in carotenoid synthesis and accumulation in the different marigold varieties.

Identification of cyanobacterial cell division genes by comparative and mutational analyses

We performed comparative and mutational analyses to define more comprehensively the repertoire of genes involved in cyanobacterial cell division. Genes ftsE, ftsI, ftsQ, ftsW, and (previously recognized) ftsZ, minC, minD, minE and sulA were identified as homologues of cell division genes of Gram‐negative and Gram‐positive bacteria. Transposon mutagenesis of Synechococcus elongatus PCC 7942 identified five additional genes, cdv1, cdv2, cdv3, ftn6 and cikA, involved in cell division. cdv1 encodes a presumptive periplasmic peptidyl‐prolyl cis‐trans isomerase. cdv2 has similarity to ylmF which, like divIVA, lies within the Gram‐positive bacterial ylm gene cluster whose members have functions associated with division. Conservation of other ylm genes in cyanobacteria suggests that cyanobacteria and Gram‐positive bacteria share specific division proteins. Two ylm homologues are also found in algal and plant genomes. cdv3 has low but significant similarity to divIVA, suggesting that minE and cdv3 both mediate division‐site determination in cyanobacteria. In contrast, Gram‐positive bacteria lack minE, and (Gram‐negative) proteobacteria lack divIVA. ftn6, of unknown function, and the circadian input kinase, cikA, are specific to cyanobacteria. In S. elongatus, unlike in other bacteria, FtsZ rings are formed at sites occupied by nucleoids. Thus, the division machinery of cyanobacteria differs in its composition and regulation from that of Gram‐negative and Gram‐positive bacteria.

PDV1 and PDV2 mediate recruitment of the dynamin-related protein ARC5 to the plastid division site.

During plastid division, the dynamin-related protein ACCUMULATION AND REPLICATION OF CHLOROPLASTS5 (ARC5) is recruited from the cytosol to the surface of the outer chloroplast envelope membrane. In Arabidopsis thaliana arc5 mutants, chloroplasts arrest during division site constriction. Analysis of mutants similar to arc5 along with map-based cloning identified PLASTID DIVISION1 (PDV1), an integral outer envelope membrane protein, and its homolog PDV2 as components of the plastid division machinery. Similar to ARC5, PDV1 localized to a discontinuous ring at the division site in wild-type plants. The midplastid PDV1 ring formed in arc5 mutants and the ARC5 ring formed in pdv1 and pdv2 mutants, but not in pdv1 pdv2. Stromal FtsZ ring assembly occurred in pdv1, pdv2, and pdv1 pdv2, as it does in arc5. Topological analysis showed that the large N-terminal region of PDV1 upstream of the transmembrane helix bearing a putative coiled-coil domain is exposed to the cytosol. Mutation of the conserved PDV1 C-terminal Gly residue did not block PDV1 insertion into the outer envelope membrane but did abolish its localization to the division site. Our results indicate that plastid division involves the stepwise localization of FtsZ, PDV1, and ARC5 at the division site and that PDV1 and PDV2 together mediate the recruitment of ARC5 to the midplastid constriction at a late stage of division.

The chloroplast small heat shock protein undergoes oxidation-dependent conformational changes and may protect plants from oxidative stress.

The nuclear-encoded chloroplast-localized Hsp21 is an oligomeric heat shock protein (Hsp), belonging to the protein family of small Hsps and alpha-crystallins. We have investigated the effects of high temperature and oxidation treatments on the structural properties of Hsp21, both in purified recombinant form and in transgenic Arabidopsis thaliana plants engineered to constitutively overexpress Hsp21. A conformational change was observed for the 300 kDa oligomeric Hsp21 protein during moderate heat stress (< or =40 degrees C) of Arabidopsis plants, as judged by a shift to lower mobility in non-denaturing electrophoresis. Similar changes in mobility were observed when purified recombinant Hsp21 protein was subjected to an oxidant. Exposure of Hsp21 protein to temperatures above 70 degrees C led to irreversible aggregation, which was prevented in presence of the reductant dithiothreitol. The transgenic plants that constitutively overexpressed Hsp21 were more resistant to heat stress than were wildtype plants when the heat stress was imposed under high light conditions. These results suggest that the physiological role of Hsp21 involves a response to temperature-dependent oxidative stress.

A unified nomenclature for Arabidopsis dynamin-related large GTPases based on homology and possible functions

Z. Hong1, S.Y. Bednarek2, E. Blumwald3, I. Hwang4, G. Jurgens5, D. Menzel6, K.W. Osteryoung7, N.V. Raikhel8, K. Shinozaki9, N. Tsutsumi10 and D.P.S. Verma1,∗ 1Department of Molecular Genetics and Plant Biotechnology Center, Ohio State University, Columbus, OH 43210, USA (∗author for correspondence; e-mail verma.1@osu.edu; 2Department of Biochemistry, University of Wisconsin, 433 Babcock Drive, Madison, WI 53706, USA; 3Department of Pomology, University of California, One Shields Ave., Davis, CA 95616, USA; 4Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang, 790-784, Korea; 5Lehrstuhl fur Entwicklungsgenetik, Universitat Tubingen, 72076 Tubingen, Germany; 6Zellbiologie der Pflanzen, Botanisches Institut, Universitat Bonn, Kirschallee 1, Bonn, 53115, Germany; 7Department of Plant Biology, 166 Plant Biology Building, Michigan State University, East Lansing, MI 48824, USA; 8Department of Botany and Plant Sciences and Center for Plant Cell Biology, University of California, Riverside, CA 92521, USA; 9Laboratory of Plant Molecular Biology, RIKEN Tsukuba Institute, 3-1-1 Koyadai, Tsukuba 305-0074, Japan; 10Laboratory of Plant Molecular Genetics, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan