International plant molecular biology: a bright future for green science

A new study in this issue of Genome Biology sheds light on why some pseudogenes persist in rodent, and other mammalian, genomes. Please see related Research article by Marques et al http://genomebiology.com/2012/13/11/R102     

Shopping for plastids

Recent suggestions that endosymbionts in a diatom and an amoeba represent independent origins of plastids from those in plants and algae raise again the question of how many times plastids have evolved. In this Opinion article, we review the evidence for a single origin or multiple origins of primary plastids. Although the data are widely taken as supporting a single origin, we stress the assumptions underlying that view, and argue for a more cautious interpretation. We also suggest that the implicit view of plastids being acquired from single ancestors at a single point (or points) in time is an over-simplification.     

Ketocarotenoid biosynthesis outside of plastids in the unicellular green alga Haematococcus pluvialis

The carotenoid biosynthetic pathway in algae and plants takes place within plastids. In these organelles, carotenoids occur either in a free form or bound to proteins. Under stress, the unicellular green alga Haematococcus pluvialis accumulates secondary carotenoids, mainly astaxanthin esters, in cytoplasmic lipid vesicles up to 4% of its dry mass. It is therefore one of the favored organisms for the biotechnological production of these antioxidative compounds. We have studied the cellular localization and regulation of the enzyme beta-carotene oxygenase in H. pluvialis that catalyzes the introduction of keto functions at position C-4 of the beta-ionone ring of beta-carotene and zeaxanthin. Using immunogold labeling of ultrathin sections and Western blot analysis of cell fractions, we discovered that under inductive conditions, beta-carotene oxygenase was localized both in the chloroplast and in the cytoplasmic lipid vesicles, which are (according to their lipid composition) derived from cytoplasmic membranes. However, beta-carotene oxygenase activity was confined to the lipid vesicle compartment. Because an early carotenogenic enzyme in the pathway, phytoene desaturase, was found only in the chloroplast (Grünewald, K., Eckert, M., Hirschberg, J., and Hagen, C. (2000) Plant Physiol. 122, 1261-1268), a transport of intermediates from the site of early biosynthetic steps in the chloroplast to the site of oxygenation and accumulation in cytoplasmic lipid vesicles is proposed.     

Subcellular targeting of green fluorescent protein to plastids in transgenic rice plants provides a high-level expression system

In order to develop a high-level expression system in transgenic rice, we inserted a synthetic gene (sgfp) encoding a modified form of the green fluorescent protein (GFP) into two expression vectors, Act1-sgfp for an untargeted and rbcS-Tp-sgfp for a chloroplast targeted expression. Several fertile transgenic rice plants were produced by the Agrobacterium-mediated method. Confocal microscopic analyses demonstrated that, in cells expressing the Act1-sgfp, GFP fluorescence was localized within the cytoplasm and nucleoplasm whereas, in cells expressing the rbcS-Tp-sgfp fusion gene, the fluorescence was specifically targeted to chloroplasts and non-green plastids. The levels of sgfp expression were about 0.5% of the total soluble protein in mature leaf tissues of the Act1-sgfp transformed lines. In contrast, expression levels were markedly increased in mature leaf tissues of the rbcS-Tp-sgfp transformed lines, yielding about 10% of the total soluble protein. N-terminal sequencing of the localized GFPs revealed that the Tp-GFP fusion protein was correctly processed during import to non-green plastids, as well as to chloroplasts. Thus, our results demonstrate that GFP can be produced at high levels and localized in specific subcellular spaces of transgenic plants, providing a high-level expression system for general use in rice, an agronomically important cereal.     

Protein targeting into plastids: a key to understanding the symbiogenetic acquisitions of plastids

Recent progress in molecular phylogenetics has proven that photosynthetic eukaryotes acquired plastids via primary and secondary endosymbiosis and has given us information about the origin of each plastid. How a photosynthetic endosymbiont became a plastid in each group is, however, poorly understood, especially for the organisms with secondary plastids. Investigating how a nuclear-encoded plastid protein is targeted into a plastid in each photosynthetic group is one of the most important keys to understanding the evolutionary process of symbiogenetic plastid acquisition and its diversity. For organisms which originated through primary endosymbiosis, protein targeting into plastids has been well studied at the molecular level. For organisms which originated through secondary endosymbiosis, molecular-level studies have just started on the plastid-targeted protein-precursor sequences and the targeting pathways of the precursors. However, little information is available about how the proteins get across the inner two or three envelope membranes in organisms with secondary plastids. A good in vitro protein-import system for isolated plastids and a cell transformation system must be established for each group of photosynthetic eukaryotes in order to understand the mechanisms, the evolutionary processes and the diversity of symbiogenetic plastid acquisitions in photosynthetic eukaryotes.     

Taming data

A challenge in systems-level investigations of the immune response is the principled integration of disparate data sets for constructing predictive models. InnateDB (Lynn et al., 2008; http://www.innatedb.ca), a publicly available, manually curated database of experimentally verified molecular interactions and pathways involved in innate immunity, is a powerful new resource that facilitates such integrative systems-level analyses.     

Molecular evidence for the origin of plastids from a cyanobacterium-like ancestor

Summary The origin of plastids by either a single or multiple endosymbiotic event(s) and the nature of the progenitor(s) of plastids have been the subjects of much controversy. The sequence of the small subunit rRNA (Ssu rRNA) from the plastid of the chlorophyllc-containing algaCryptomonas is presented, allowing for the first time a comparison of this molecule from all of the major land plant and algal lineages. Using a distance matrix method, the phylogenetic relationships among representatives of these lineages have been inferred and the results indicate a common origin of plastids from a cyanobacterium-like ancestor. Within the plastid line of descent, there is a deep dichotomy between the chlorophyte/land plant lineage and the rhodophyte/chromophyte lineage, with the cyanelle ofCyanophora paradoxa forming the deepest branch in the latter group. Interestingly,Euglena gracilis and its colorless relativeAstasia longa are more related to the chromophytes than to the chlorophytes, raising once again the question of the origin of the euglenoid plastids.     

Proteome analysis of tobacco bright yellow-2 (BY-2) cell culture plastids as a model for undifferentiated heterotrophic plastids

We analyzed the proteome of undifferentiated plastids from a tobacco BY-2 cell culture by shotgun proteomics following multidimensional protein fractionation. The fractionation strategy initiated with the serial extraction of proteins from membranes which allowed us to distinguish soluble, peripheral, and integral membrane proteins. The majority of the identified proteins have a function in the cellular metabolism and most of them are active in amino acid synthesis pathways. A significant number of the identified proteins was not identified in chloroplast proteome analyses before. This suggests BY-2 plastid specific functions that differ from the major activities of chloroplasts. We have used the BY-2 plastid proteins reported here to assess the metabolic activities of undifferentiated heterotrophic plastids and compared the functional profile with that of differentiated heterotrophic amyloplasts. Comparative shotgun proteome analyses as reported here provide information about prevalent metabolic activities of different plastid types.     

Taming the Spindle for Containing the Chromosomes

Checkpoint controls are critical for coordination of cell cycle events, especially during exposure to perturbations or stresses. The DNA replication checkpoint is activated in S phase in response to replication stresses that impede fork progression. Mec1 and Rad53 are critical effectors of this control pathway; they maintain the integrity of stalled replication forks and prevent premature segregation of unreplicated chromosomes. It has long been thought that the checkpoint inhibits precocious segregation of chromosomes by preventing early onset of mitosis. However, recent evidence suggests that the replication checkpoint thwarts untimely chromosome separation not by inhibiting mitotic entry but by directly regulating spindle dynamics. These findings raise a number of issues which may require a revisit to the well-trodden territories of cell cycle regulation.     

Taming the symbiont for coexistence: a host PGRP neutralizes a bacterial symbiont toxin

In horizontally transmitted mutualisms between marine animals and their bacterial partners, the host environment promotes the initial colonization by specific symbionts that it harvests from the surrounding bacterioplankton. Subsequently, the host must develop long-term tolerance to immunogenic bacterial molecules, such as peptidoglycan and lipopolysaccaride derivatives. We describe the characterization of the activity of a host peptidoglycan recognition protein (EsPGRP2) during establishment of the symbiosis between the squid Euprymna scolopes and its luminous bacterial symbiont Vibrio fischeri. Using confocal immunocytochemistry, we localized EsPGRP2 to all epithelial surfaces of the animal, and determined that it is exported in association with mucus shedding. Most notably, EsPGRP2 was released by the crypt epithelia into the extracellular spaces housing the symbionts. This translocation occurred only after the symbionts had triggered host morphogenesis, a process that is induced by exposure to the peptidoglycan monomer tracheal cytotoxin (TCT), a bacterial 'toxin' that is constitutively exported by V. fischeri. Enzymatic analyses demonstrated that, like many described PGRPs, EsPGRP2 has a TCT-degrading amidase activity. The timing of EsPGRP2 export into the crypts provides evidence that the host does not export this protein until after TCT induces morphogenesis, and thereafter EsPGRP2 is constantly present in the crypts ameliorating the effects of V. fischeri TCT.