In vivo studies on the roles of Tic110, Tic40 and Hsp93 during chloroplast protein import

A multisubunit translocon of the inner envelope membrane, termed Tic, mediates the late stages of protein import into chloroplasts. Membrane proteins, Tic110 and Tic40, and a stromal chaperone, Hsp93, have been proposed to function together within the Tic complex. In Arabidopsis, single genes, atTIC110 and atTIC40, encode the Tic proteins, and two homologous genes, atHSP93-V and atHSP93-III, encode Hsp93. These four genes exhibited relatively uniform patterns of expression, suggesting important roles for plastid biogenesis throughout development and in all tissues. To investigate the roles played by these proteins in vivo, we conducted a comparative study of T-DNA knockout mutants for each Tic gene, and for the most abundantly expressed Hsp93 gene, atHSP93-V. In the homozygous state, the tic110 mutation caused embryo lethality, implying an essential role for atTic110 during plastid biogenesis. Homozygous tic110 embryos exhibited retarded growth, developmental arrest at the globular stage and a 'raspberry-like' embryo-proper phenotype. Heterozygous tic110 plants, and plants homozygous for the tic40 and hsp93-V mutations, exhibited chlorosis, aberrant chloroplast biogenesis, and inefficient chloroplast-import of both photosynthetic and non-photosynthetic preproteins. Non-additive interactions amongst the mutations occurred in double mutants, suggesting that the three components may cooperate during chloroplast protein import.     

Deletion of core components of the plastid protein import machinery causes differential arrest of embryo development in Arabidopsis thaliana

Among the genes that have recently been pinpointed to be essential for plant embryo development a large number encodes plastid proteins suggesting that embryogenesis is linked to plastid localized processes. However, nuclear encoded plastid proteins are synthesized as precursors in the cytosol and subsequently have to be transported across the plastid envelopes by a complex import machinery. We supposed that deletion of components of this machinery should allow a more general assessment of the role of plastids in embryogenesis since it will not only affect single proteins but instead inhibit the accumulation of most plastid proteins. Here we have characterized three Arabidopsis thaliana mutants lacking core components of the Toc complex, the protein translocase in the outer plastid envelope membrane, which indeed show embryo lethal phenotypes. Remarkably, embryo development in the atToc75-III mutant, lacking the pore forming component of the translocase, was arrested extremely early at the two-cell stage. In contrast, despite the complete or almost complete lack of the import receptors Toc34 and Toc159, embryo development in the a tToc33/34 and atToc132/159 mutants proceeded slowly and was arrested later at the transition to the globular and the heart stage, respectively. These data demonstrate a strict dependence of cell division and embryo development on functional plastids as well as specific functions of plastids at different stages of embryogenesis. In addition, our analysis suggest that not all components of the translocase are equally essential for plastid protein import in vivo.     

The Clp protease system; a central component of the chloroplast protease network

Intra-plastid proteases play crucial and diverse roles in the development and maintenance of non-photosynthetic plastids and chloroplasts. Formation and maintenance of a functional thylakoid electron transport chain requires various protease activities, operating in parallel, as well as in series. This review first provides a short, referenced overview of all experimentally identified plastid proteases in Arabidopsis thaliana. We then focus on the Clp protease system which constitutes the most abundant and complex soluble protease system in the plastid, consisting of 15 nuclear-encoded members and one plastid-encoded member in Arabidopsis. Comparisons to the simpler Clp system in photosynthetic and non-photosynthetic bacteria will be made and the role of Clp proteases in the green algae Chlamydomonas reinhardtii will be briefly reviewed. Extensive molecular genetics has shown that the Clp system plays an essential role in Arabidopsis chloroplast development in the embryo as well as in leaves. Molecular characterization of the various Clp mutants has elucidated many of the consequences of loss of Clp activities. We summarize and discuss the structural and functional aspects of the Clp machinery, including progress on substrate identification and recognition. Finally, the Clp system will be evaluated in the context of the chloroplast protease network. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.     

Interactions between the cell cycle and embryonic patterning in Arabidopsis uncovered by a mutation in DNA polymerase epsilon

Pattern formation and morphogenesis require coordination of cell division rates and orientations with developmental signals that specify cell fate. A viable mutation in the TILTED1 locus, which encodes the catalytic subunit of DNA polymerase epsilon of Arabidopsis thaliana, causes a lengthening of the cell cycle by approximately 35% throughout embryo development and alters cell type patterning of the hypophyseal lineage in the root, leading to a displacement of the root pole from its normal position on top of the suspensor. Treatment of preglobular and early globular stages, but not later stage, embryos with the DNA polymerase inhibitor aphidicolin leads to a similar phenotype. The results uncover an interaction between the cell cycle and the processes that determine cell fate during plant embryogenesis.     

The function and diversity of plastid protein import pathways: a multilane GTPase highway into plastids

The photosynthetic chloroplast is the hallmark organelle of green plants. During the endosymbiotic evolution of chloroplasts, the vast majority of genes from the original cyanobacterial endosymbiont were transferred to the host cell nucleus. Chloroplast biogenesis therefore requires the import of nucleus-encoded proteins from their site of synthesis in the cytosol. The majority of proteins are imported by the activity of Toc and Tic complexes located within the chloroplast envelope. In addition to chloroplasts, plants have evolved additional, non-photosynthetic plastid types that are essential components of all cells. Recent studies indicate that the biogenesis of various plastid types relies on distinct but homologous Toc-Tic import pathways that have specialized in the import of specific classes of substrates. These different import pathways appear to be necessary to balance the essential physiological role of plastids in cellular metabolism with the demands of cellular differentiation and plant development.     

Development and germination of pecan somatic embryos derived from liquid culture

Aggregates of globular and pre-globular stage somatic embryos from suspension cultures of pecan (Carya illinoensis Koch) were cultured on solidified media for embryo development. Embryo aggregates and pre-globular stage embryo masses were given various treatments to further ontologic development. A 2- to 4-wk mild dehydration of the embryo aggregates suppressed recurrent embryogenesis, promoted development of globular embryos into cotyledonary stage embryos, and enhanced plant development beyond germination. Fine embryogenic tissue masses filtered from suspension formed cotyledonary-staged embryos when the collection filters were plated on solified medium. The embryogenic capacity of preglobular stage embryo masses was compared between media supplemented with varying concentrations of polyethylene glycol (molecular weight 8 000) vs. filter overlays. The filter paper overlays were not necessary for embryo development. An inverse relationship was found between the number of embryos that developed and the concentration of polyethylene glycol in the medium. However, this relationship was reversed for ability of embryos to germinate and develop into a plant.     

Plastid genes in a non-photosynthetic dinoflagellate

Dinoflagellates are a diverse group of protists, comprising photosynthetic and heterotrophic free-living species, as well as parasitic ones. About half of them are photosynthetic with peridinin-containing plastids being the most common. It is uncertain whether non-photosynthetic dinoflagellates are primitively so, or have lost photosynthesis. Studies of heterotrophic species from this lineage may increase our understanding of plastid evolution. We analyzed an EST project of the early-diverging heterotrophic dinoflagellate Crypthecodinium cohnii looking for evidence of past endosymbiosis. A large number of putative genes of cyanobacterial or algal origin were identified using BLAST, and later screened by metabolic function. Phylogenetic analyses suggest that several proteins could have been acquired from a photosynthetic endosymbiont, arguing for an earlier plastid acquisition in dinoflagellates. In addition, intact N-terminal plastid-targeting peptides were detected, indicating that C. cohnii may contain a reduced plastid and that some of these proteins are imported into this organelle. A number of metabolic pathways, such as heme and isoprenoid biosynthesis, seem to take place in the plastid. Overall, these data indicate that C. cohnii is derived from a photosynthetic ancestor and provide a model for loss of photosynthesis in dinoflagellates and their relatives. This represents the first extensive genomic analysis of a heterotrophic dinoflagellate.     

Ovule fates in Epilobium obcordatum (Onagraceae)

We investigated the fates of ovules following self- and cross-pollination of the variably self-sterile Epilobium obcordatum by observing thin sections of ovaries 4, 5, and 10 d postpollination (PP). Three types of unfertilized ovules were observed: apparently normal ovules not entered by pollen tubes, nonfunctional ovules with a nucellus but lacking an embryo sac, and empty ovules with integuments but lacking a nucellus. The average frequencies for all three types were not different for selfs and crosses. Fertilization frequencies did not differ among pollination treatments, averaging 61.4% of the total sample of 4 and 5 d PP ovules. The phenotypes of failing postfertilization ovules at 4 and 5 d PP included those containing preglobular and globular embryos lacking endosperm, ovules with endosperm but no embryo present, and failing embryos in the presence of endosperm. Self- and cross-fertilized 10 d PP ovules also possessed heart-stage and torpedo-stage failing embryos. Early embryo failures (5 d PP) were distributed throughout the length of the ovary, as expected if failures are the consequence of factors intrinsic to each ovule. The sizes of normally developing self- and cross-fertilized embryos were not different at 4 d PP. The 5 d PP cross embryos were larger than selfs. A comparison of 10 d samples was not possible due to attrition of self embryos. Although ovular position did not affect the probability of early embryo failure, it did affect the size of normally developing embryos at 4, 5, and 10 d PP, with embryos at the stylar end larger than those at the basal end.     

A Genomic and Phylogenetic Perspective on Endosymbiosis and Algal Origin

Accounting for the diversity of photosynthetic eukaryotes is an important challenge in microbial biology. It has now become clear that endosymbiosis explains the origin of the photosynthetic organelle (plastid) in different algal groups. The first plastid originated from a primary endosymbiosis, whereby a previously non-photosynthetic protist engulfed and enslaved a cyanobacterium. This alga then gave rise to the red, green, and glaucophyte lineages. Algae such as the chlorophyll c-containing chromists gained their plastid through secondary endosymbiosis, in which an existing eukaryotic alga (in this case, a rhodophyte) was engulfed. Another chlorophyll c-containing algal group, the dinoflagellates, is a member of the alveolates that is postulated to be sister to chromists. The plastid in these algae has followed a radically different path of evolution. The peridinin-containing dinoflagellates underwent an unprecedented level of plastid genome reduction with the ca. 16 remaining genes encoded on 1–3 gene minicircles. In this short review, we examine algal plastid diversity using phylogenetic and genomic methods and show endosymbiosis to be a major force in algal evolution. In particular, we focus on the evolution of targeting signals that facilitate the import of nuclear-encoded photosynthetic proteins into the plastid.     

Versatile roles of Arabidopsis plastid ribosomal proteins in plant growth and development

A lack of individual plastid ribosomal proteins (PRPs) can have diverse phenotypic effects in Arabidopsis thaliana, ranging from embryo lethality to compromised vitality, with the latter being associated with photosynthetic lesions and decreases in the expression of plastid proteins. In this study, reverse genetics was employed to study the function of eight PRPs, five of which (PRPS1, ‐S20, ‐L27, ‐L28 and ‐L35) have not been functionally characterised before. In the case of PRPS17, only leaky alleles or RNA interference lines had been analysed previously. PRPL1 and PRPL4 have been described as essential for embryo development, but their mutant phenotypes are analysed in detail here. We found that PRPS20, ‐L1, ‐L4, ‐L27 and ‐L35 are required for basal ribosome activity, which becomes crucial at the globular stage and during the transition from the globular to the heart stage of embryogenesis. Thus, lack of any of these PRPs leads to alterations in cell division patterns, and embryo development ceases prior to the heart stage. PRPL28 is essential at the latest stages of embryo–seedling development, during the greening process. PRPS1, ‐S17 and ‐L24 appear not to be required for basal ribosome activity and the organism can complete its entire life cycle in their absence. Interestingly, despite the prokaryotic origin of plastids, the significance of individual PRPs for plant development cannot be predicted from the relative phenotypic severity of the corresponding mutants in prokaryotic systems.     

Plastid translation and transcription genes in a non-photosynthetic plant: intact, missing and pseudo genes

The non-photosynthetic, parasitic flowering plant Epifagus virginiana has recently been shown to contain a grossly reduced plastid genome that has lost many photosynthetic and chloro-respiratory genes. We have cloned and sequenced a 3.9 kb domain of plastid DNA from Epifagus to investigate the patterns of evolutionary change in such a reduced genome and to determine which genes are still present and likely to be functional. This 3.9 kb domain is colinear with a 35.4 kb region of tobacco chloroplast DNA, differing from it by a minimum of 11 large deletions varying in length from 354 bp to 11.5 kb, as well as by a number of small deletions and insertions. The nine genes retained in Epifagus encode seven tRNAs and two ribosomal proteins and are coextensive and highly conserved in sequence with homologs in photosynthetic plants. This suggests that these genes are functional in Epifagus and, together with evidence that the Epifagus plastid genome is transcribed, implies that plastid gene products play a role in processes other than photosynthesis and gene expression. Genes that are completely absent include not only photosynthetic genes, but surprisingly, genes encoding three subunits of RNA polymerase, four tRNAs and one ribosomal protein. In addition, only pseudogenes are found for two other tRNAs. Despite these defunct tRNA genes, codon and amino acid usage in Epifagus protein genes is normal. We therefore hypothesize that the expression of plastid genes in Epifagus relies on the import of nuclear encoded tRNAs and RNA polymerase from the cytoplasm.     

New Embryo Lethals in Arabidopsis thaliana: Basic Genetic and Morphological Study

Six different mutations with defects in immature seed development have been identified during screening of a T-DNA collection of Arabidopsis thaliana. The mutations were confirmed to be monogenic and recessive-lethal by genetic analysis. Mutant embryos were blocked in certain steps in the process necessary for embryo viability and development, and therefore they belong to the embryo-lethal class of mutants. The genetic and morphological studies of T-DNA mutations affecting embryo development are presented. The youngest embryos with a defect were observed at the globular stage in the VIII-64 mutation. Externally located cells, precursor of the protoderm, were characterised by abnormal cell division. VIII-41 mutation with a defect at the late globular stage was arrested at the globular-heart stage transition. VIII-111 mutation showed defect at heart stage of embryogenesis with atypical development of cotyledon primordia. The defect was associated with abnormal pattern of cell division constituting the precursor of the shoot apical meristem. In VIII-82 mutation defect in torpedo stage with asymmetric cotyledons was observed. Cotyledon stage of embryos and chlorophyll defect were observed in VIII-75 mutant. Abnormal suspensor consisting of two columns of cells was observed in 280-4-4 mutation. Newly identified embryo-lethals can serve as starting material for more detailed genetic and molecular studies.     

Retrograde signaling and plant stress: plastid signals initiate cellular stress responses

Retrograde signaling coordinates the expression of nuclear genes encoding organellar proteins with the metabolic and developmental state of the organelle. These plastid signals are essential not only for coordinating photosynthetic gene expression in both the nucleus and in the chloroplasts but also for mediating plant stress responses. The chloroplasts therefore act as sensors of environmental changes and complex networks of plastid signals coordinate cellular activities and assist the cell during plant stress responses. Recent work suggests that information from both cytosolic-signaling and plastid-signaling networks must be integrated for the plant cell to respond optimally to environmental stress.     

Expression and downregulation of WNT signaling pathway genes in rhesus monkey oocytes and embryos

Mammalian WNT genes encode secreted glycoproteins that are conserved homologues of the Drosophila Wingless gene, which plays a crucial role in Drosophila development. Recently, WNT pathway signaling has been implicated in ovarian development, oogenesis, and early development. We sought to evaluate whether these genes may contribute to the formation of healthy human oocytes or embryos, and whether the expression of these genes could provide informative markers of human oocyte and embryo quality. To do this, we employed the primate embryo gene expression resource (PREGER; www.preger.org) to examine expression of mRNAs encoding 38 components of the WNT signaling pathway in rhesus monkey oocytes and embryos as a nonhuman primate model. We observed considerable conservation between rhesus monkey and mouse of expression of WNT, FZD, and effector gene mRNAs, and a generalized downregulation of genes encoding key components of the WNT signaling pathway during preimplantation development. Our results support a role for WNT signaling during oocyte growth or maturation, but not during preimplantation development. Additionally, we observed differences between in vitro cultured and in vivo developing blastocysts, indicating possible effects of culture on WNT signaling during the peri-implantation period.