Bacterial orthologues indicate the malarial plastid gene ycf24 is essential

ycf24 is a well conserved gene found in all major groups of bacteria, as well as on red algal plastid genomes and the vestigal plastid genome of apicomplexan pathogens like the malaria parasite Plasmodium falciparum (ORF470). Some database annotations describe Ycf24 as an ABC transporter subunit, but we find the level of significance is low. To investigate ycf24′s function we disrupted it in the cyanobacterium Synechocystis sp., strain PCC6803 which has a multi-copy genome. This showed ycf24 is essential, partial loss producing a terminal phenotype of chlorosis, reduced cell size, loss of DNA, and a striking arrest in cytokinesis. Attempts to disrupt the single copy of ycf24 in E. coli failed to give stable transformants. When Ycf24 was over-expressed in E. coli as a soluble fusion protein, it localized mostly as a band on either side of the nucleoid and nucleoid partitioning was aberrant. We propose the relict plastid organelle of apicomplexans retains its capacity for protein synthesis because Ycf24 is essential.     

Phylogenetic utility of ycf1 in orchids: a plastid gene more variable than matK

Plastid DNA sequences have been widely used by systematists for reconstructing plant phylogenies. The utility of any DNA region for phylogenetic analysis is determined by ease of amplification and sequencing, confidence of assessment in phylogenetic character alignment, and by variability across broad taxon sampling. Often, a compromise must be made between using relatively highly conserved coding regions or highly variable introns and intergenic spacers. Analyses of a combination of these types of DNA regions yield phylogenetic structure at various levels of a tree (i.e., along the spine and at the tips of the branches). Here, we demonstrate the phylogenetic utility of a heretofore unused portion of a plastid protein-coding gene, hypothetical chloroplast open reading frame 1 (ycf1), in orchids. All portions of ycf1 examined are highly variable, yet alignable across Orchidaceae, and are phylogenetically informative at the level of species. In Orchidaceae, ycf1 is more variable than matK both in total number of parsimony informative characters and in percent variability. The nrITS region is more variable than ycf1, but is more difficult to align. Although we only demonstrate the phylogenetic utility of ycf1 in orchids, it is likely to be similarly useful among other plant taxa.     

Complete plastid genome sequences of Drimys, Liriodendron, and Piper : implications for the phylogenetic relationships of magnoliids

Background  

The magnoliids with four orders, 19 families, and 8,500 species represent one of the largest clades of early diverging angiosperms. Although several recent angiosperm phylogenetic analyses supported the monophyly of magnoliids and suggested relationships among the orders, the limited number of genes examined resulted in only weak support, and these issues remain controversial. Furthermore, considerable incongruence resulted in phylogenetic reconstructions supporting three different sets of relationships among magnoliids and the two large angiosperm clades, monocots and eudicots. We sequenced the plastid genomes of three magnoliids, Drimys (Canellales), Liriodendron (Magnoliales), and Piper (Piperales), and used these data in combination with 32 other angiosperm plastid genomes to assess phylogenetic relationships among magnoliids and to examine patterns of variation of GC content.     

Arabidopsis SAMT1 defines a plastid transporter regulating plastid biogenesis and plant development

S-Adenosylmethionine (SAM) is formed exclusively in the cytosol but plays a major role in plastids; SAM can either act as a methyl donor for the biogenesis of small molecules such as prenyllipids and macromolecules or as a regulator of the synthesis of aspartate-derived amino acids. Because the biosynthesis of SAM is restricted to the cytosol, plastids require a SAM importer. However, this transporter has not yet been identified. Here, we report the molecular and functional characterization of an Arabidopsis thaliana gene designated SAM TRANSPORTER1 (SAMT1), which encodes a plastid metabolite transporter required for the import of SAM from the cytosol. Recombinant SAMT1 produced in yeast cells, when reconstituted into liposomes, mediated the counter-exchange of SAM with SAM and with S-adenosylhomocysteine, the by-product and inhibitor of transmethylation reactions using SAM. Insertional mutation in SAMT1 and virus-induced gene silencing of SAMT1 in Nicotiana benthamiana caused severe growth retardation in mutant plants. Impaired function of SAMT1 led to decreased accumulation of prenyllipids and mainly affected the chlorophyll pathway. Biochemical analysis suggests that the latter effect represents one prominent example of the multiple events triggered by undermethylation, when there is decreased SAM flux into plastids.     

Disruption of the plastid ycf10 open reading frame affects uptake of inorganic carbon in the chloroplast of Chlamydomonas.

The product of the chloroplast ycf10 gene has been localized in the inner chloroplast envelope membrane (Sasaki et al., 1993) and found to display sequence homology with the cyanobacterial CotA product which is altered in mutants defective in CO2 transport and proton extrusion (Katoh et al., 1996a,b). In Chlamydomonas reinhardtii, ycf10, located between the psbI and atpH genes, encodes a putative hydrophobic protein of 500 residues, which is considerably larger than its higher plant homologue because of a long insertion that separates the conserved N and C termini. Using biolistic transformation, we have disrupted ycf10 with the chloroplast aadA expression cassette and examined the phenotype of the homoplasmic transformants. These were found to grow both photoheterotrophically and photoautotrophically under low light, thereby revealing that the Ycf10 product is not essential for the photosynthetic reactions. However, under high light these transformants did not grow photoautotrophically and barely photoheterotrophically. The increased light sensitivity of the transformants appears to result from a limitation in photochemical energy utilization and/or dissipation which correlates with a greatly diminished photosynthetic response to exogenous (CO2 + HCO3-), especially under conditions where the chloroplast inorganic carbon transport system is not induced. Mass spectrometric measurements with either whole cells or isolated chloroplasts from the transformants revealed that the CO2 and HCO3- uptake systems have a reduced affinity for their substrates. The results suggest the existence of a ycf10-dependent system within the plastid envelope which promotes efficient inorganic carbon (Ci) uptake into chloroplasts.     

A mutation in Arabidopsis seedling plastid development1 affects plastid differentiation in embryo-derived tissues during seedling growth

Oilseed plants like Arabidopsis (Arabidopsis thaliana) develop green photosynthetically active embryos. Upon seed maturation, the embryonic chloroplasts degenerate into a highly reduced plastid type called the eoplast. Upon germination, eoplasts redifferentiate into chloroplasts and other plastid types. Here, we describe seedling plastid development1 (spd1), an Arabidopsis seedling albino mutant capable of producing normal green vegetative tissues. Mutant seedlings also display defects in etioplast and amyloplast development. Precocious germination of spd1 embryos showed that the albino seedling phenotype of spd1 was dependent on the passage of developing embryos through the degreening and dehydration stages of seed maturation, suggesting that SPD1 is critical during eoplast development or early stages of eoplast redifferentiation. The SPD1 gene was found to encode a protein containing a putative chloroplast-targeting sequence in its amino terminus and also domains common to P-loop ATPases. Chloroplast localization of the SPD1 protein was confirmed by targeting assays in vivo and in vitro. Although the exact function of SPD1 remains to be defined, our findings reveal aspects of plastid development unique to embryo-derived cells.     

Ancient paralogy in the cpDNA trnL-F region in Annonaceae: implications for plant molecular systematics

The plastid trnL-F region has proved useful in molecular phylogenetic studies addressing diverse evolutionary questions from biogeographic history to character evolution in a broad range of plant groups. An important assumption for phylogenetic reconstruction is that data used in combined analyses contain the same phylogenetic signal. The trnL-F region is often used in combined analyses of multiple chloroplast markers. These markers are assumed to contain congruent phylogenetic signal due to lack of recombination. Here we show that trnL-F sequences display a phylogenetic signal conflicting with that of other chloroplast markers in Annonaceae, and we demonstrate that this conflict results from ancient paralogy. TrnL-F copy 2 diverged from trnL-F copy 1 (as used in family-wide phylogenetic analyses) in a direct ancestor of the Annonaceae. Although this divergence dates back 88 million years or more, the exons of both copies appear to be intact. In this case, assuming that (putative) chloroplast markers contain the same phylogenetic signal results in an incorrect topology and an incorrect estimate of ages. Our study demonstrates that researchers should be cautious when interpreting gene phylogenies, irrespective of the genome from which they are presumed to have been sampled.     

The non-photosynthetic plastid in malarial parasites and other apicomplexans is derived from outside the green plastid lineage.

The discovery of a non-photosynthetic plastid genome in Plasmodium falciparum and other apicomplexans has provided a new drug target, but the evolutionary origin of the plastid has been muddled by the lack of characters, that typically define major plastid lineages. To clarify the ancestry of the plastid, we undertook a comprehensive analysis of all genomic characters shared by completely sequenced plastid genomes. Cladistic analysis of the pattern of plastid gene loss and gene rearrangements suggests that the apicomplexan plastid is derived from an ancestor outside of the green plastid lineage. Phylogenetic analysis of primary sequence data (DNA and amino acid characters) produces results that are generally independent of the analytical method, but similar genes (i.e., rpoB and rpoC) give similar topologies. The conflicting phylogenies in primary sequence data sets make it difficult to determine the the exact origin of the apicomplexan plastid and the apparent artifactual association of apicomplexan and euglenoid sequences suggests that DNA sequence data may be an inappropriate set of characters to address this phylogenetic question. At present we cannot reject our null hypothesis that the apicomplexan plastid is derived from a shared common ancestor between apicomplexans and dinoflagellates. During the analysis, we noticed that the Plasmodium tRNA-Met is probably tRNA-fMet and the tRNA-fMet is probably tRNA-Ile. We suggest that P. falciparum has lost the elongator type tRNA-Met and that similar to metazoan mitochondria there is only one species of methionine tRNA. In P. falciparum, this has been accomplished by recruiting the fMet-type tRNA to dually function in initiation and elongation. The tRNA-Ile has an unusual stem-loop in the variable region. The insertion in this region appears to have occurred after the primary origin of the plastid and further supports the monophyletic ancestory of plastids.     

The pentatricopeptide repeat gene OTP51 with two LAGLIDADG motifs is required for the cis-splicing of plastid ycf3 intron 2 in Arabidopsis thaliana

Summary The Arabidopsis thaliana chloroplast contains 20 group-II introns in its genome, and seven known splicing factors are required for the splicing of overlapping subsets of 19 of them. We describe an additional protein (OTP51) that specifically promotes the splicing of the only group-II intron for which no splicing factor has been described previously. This protein is a pentatricopeptide repeat (PPR) protein containing two LAGLIDADG motifs found in group-I intron maturases in other organisms. Amino acids thought to be important for the homing endonuclease activity of other LAGLIDADG proteins are missing in this protein, but the amino acids described to be important for maturase activity are conserved. OTP51 is absolutely required for the splicing of ycf3 intron 2, and also influences the splicing of several other group-IIa introns. Loss of OTP51 has far-reaching consequences for photosystem-I and photosystem-II assembly, and for the photosynthetic fluorescence characteristics of mutant plants.     

Floral development and floral phyllotaxis in Anaxagorea (Annonaceae)

Background and Aims Anaxagorea is the phylogenetically basalmost genus in the large tropical Annonaceae (custard apple family) of Magnoliales, but its floral structure is unknown in many respects. The aim of this study is to analyse evolutionarily interesting floral features in comparison with other genera of the Annonaceae and the sister family Eupomatiaceae. Methods Live flowers of Anaxagorea crassipetala were examined in the field with vital staining, liquid-fixed material was studied with scanning electron microscopy, and microtome section series were studied with light microscopy. In addition, herbarium material of two other Anaxagorea species was cursorily studied with the dissecting microscope. Key Results Floral phyllotaxis in Anaxagorea is regularly whorled (with complex whorls) as in all other Annonaceae with a low or medium number of floral organs studied so far (in those with numerous stamens and carpels, phyllotaxis becoming irregular in the androecium and gynoecium). The carpels are completely plicate as in almost all other Annonaceae. In these features Anaxagorea differs sharply from the sister family Eupomatiaceae, which has spiral floral phyllotaxis and ascidiate carpels. Flat stamens and the presence of inner staminodes differ from most other Annonaceae and may be plesiomorphic in Anaxagorea. However, the inner staminodes appear to be non-secretory in most Anaxagorea species, which differs from inner staminodes in other families of Magnoliales (Eupomatiaceae, Degeneriacae, Himantandraceae), which are secretory. Conclusions Floral phyllotaxis in Anaxagorea shows that there is no signature of a basal spiral pattern in Annonaceae and that complex whorls are an apomorphy not just for a part of the family but for the family in its entirety, and irregular phyllotaxis is derived. This and the presence of completely plicate carpels in Anaxagorea makes the family homogeneous and distinguishes it from the closest relatives in Magnoliales.     

Seed development and function inArtabotrys hexapetalus (Annonaceae)

Until now seeds ofAnnonaceae were characterized as mesotestal only. The seed ofArtabotrys hexapetalus, however, is meso- and endotestal. An outer mechanical layer which surrounds the seed as a lignified fibrous tissue is derived from the mesotesta. A complex inner mechanical layer develops from a partially multi-layered endotesta built up by crystal-containing stone cells. The multi-layered endotesta forms a prominent seed plug in the micropylar region which is prolonged along both sides of the perichalaza as inner walls. The endotesta is one-layered on the sides of the seed and participates in rumination. In addition the endotesta may serve for deposition of end-products of metabolism. The complex growing process of the perichalaza and its surrounding tissues is described in detail. The perichalazal pad of tanniferous cells forms an U-shaped septum, in conjunction with the endotesta, dividing the seed into two chambers. During seed development it functions as transmitter of nutrients from the outer chamber filled with starch grains to the nucellus, endosperm and embryo contained in the inner one. During germination this pad probably serves for the uptake of water. — At the initial phases of germination the seed dehisces into two valves along the raphal and antiraphal side. Later on an additional parenchymatous operculum covering the seed apex disintegrates and the endotestal plug fixed by its two prolongations splits along a preformed fracture line into two parts to release the seedling. Rudiments of an aril are recognizable in young seeds only. — The data obtained fromArtabotrys hexapetalus are discussed and compared with published information on seeds in other annonaceous taxa. For systematic considerations the necessity to define the origin of the annonaceous seed plug from one or the other integument is emphasized as it may prove to be an important differential character withinAnnonaceae.     

Comparative development of aseptate and septate anthers of Annonaceae

We compared anther development in 13 genera and 15 species of Annonaceae to document the nature and development of anther septa. In aseptate anthers, all sporogenous initials proceed to sporogenesis and meiosis. In septate anthers, a small number of sporogenous initials, in a discontinuous distribution pattern, differentiate into sporogenous cells; the remaining initials become sterile and form cellular septa that partition each anther lobe into multiple sporangial chambers. In species where the septum is 1-2 cell layers thick, the entire septum becomes tapetal (T-type septa) and breaks down before anther dehiscence. In species in which the septum is three or more cell layers thick, only the layer in direct contact with the sporogenous cells becomes tapetal, and the remaining cells become parenchymatous (P-type septa). These thicker P-type septa are sometimes visible in dehisced anthers. Both types are homologous in ontogeny and are highly associated with the production of compound pollen. We propose that the evolution of anther septation in Annonaceae was mainly driven by the requirement for highly efficient nutrient and physical support to the development of large, compound pollen units.     

Changes in the plastid ultrastructure duringSedum rotundifolium leaf development

The ultrastructure of plastids was investigated in succulent leaves ofSedum rotundifolium to examine their changes during development. Leaves were categorized as etiolated, immature, young, and mature, based on their developmental stage and size. Of particular interest were the features of the tubular inclusion bodies (TIBs) and starch grains. These, along with vacuole size, showed remarkable changes over time. Etioplasts of unexposed leaves had prolamellar bodies, abundant starch grains, large TIB, few plastoglobuli, and thylakoid systems. Membranes of the thylakoids were still continuous with those of the prolamellar body. The plastids were often influenced by the presence and profile of inclusion bodies and starch grains throughout the early stages. Morphology was highly variable in the etioplasts but consistently hemispherical or ovoid in mature chloroplasts. TIB was most abundant in the etiolated leaves, but disappeared completely with development. Starch grains also became significantly reduced in size. Both young and mature mesophyll cells exhibited a normal chloroplast ultra-structure and huge central vacuoles, with an extremely thin peripheral cytoplasm. Grana were extensive and comprised a large portion of the chloroplasts. Traces of peripheral reticulum were also discovered in the chloroplasts of expanded leaves. The implications of these ultrastructural changes in the tubular inclusions and starch grains are discussed with relevance to Crassulacean acid metabolism (CAM).     

Chromoplasts--the last stages in plastid development.

The results of investigations on the development of chromoplast fine structures in various plants are reviewed. Emphasis is placed on the specific pigment-containing structures and their development during chromoplast formation. There is a large variety of these structures, although four fundamental types can be discerned. These are plastoglobules, membranes, crystals, and tubules. During chromoplast development, various types of structure follow one after the other, or they may even be present simultaneously in the same chromoplast. Depending on the structures present in chromoplasts their pigment content also varies. It is still not clear whether the type of structure defines the pigment content of the chromoplast or vice-versa. Various possible ways of chromoplast development and dedifferentiation are discussed.     

Higher substitution rates and lower dN/dS for the plastid genes in Gnetales than other gymnosperms

Previous studies have shown that Gnetales exhibit an increase in the rates of substitution in plastid genes. However, the relative roles of selection and drift in driving the observed increase in substitution rate remain unclear. Here we estimated the nonsynonymous (dN) and synonymous (dS) substitution rates and the dN/dS ratios for 59 plastid protein-coding genes conserved across Gnetales and other gymnosperms with maximum likelihood methods. Our results show that: (1) values of dN and dS for 48 and 50 genes, respectively, are significantly higher in Gnetales relative to the other sampled gymnosperms; (2) the acceleration of dN is of lower magnitude than that of dS; (3) dN/dS is significantly reduced for 22 of the 59 plastid genes in Gnetales; and (4) dN/dS is extremely heterogeneous among the Gnetalean genes, varying by up to a factor of 40. We propose that while biological characteristics such as generation time and plant height may contribute to the rate increase in Gnetales, the special habitats that they occupy are linked to the gene-specific reduction of dN/dS.     

Phytochrome-mediated plastid development in etiolated pea stem apices

The effects of red and far-red light on growth and plastid development in the stem apices of etiolated pea seedlings have been examined. Changes were determined in various growth parameters (DNA, soluble protein and fresh weight) and also in the activities of the plastid-localized enzymes ribulose-1,5-bisphosphate carboxylase, NADP-glyceraldehyde-3-phosphate dehydrogenase and alkaline-1,6-bisphosphatase and the non-photosynthetic (cytoplasmic) enzymes NADP-isocitrate dehydrogenase, enolase and NAD-malate dehydrogenase. Changes in the amounts of Fraction I protein were also measured. Brief daily irradiation with low intensity red light increased growth 5·1–7·6-fold which was correlated with increases of about 3·5-fold in activities of the non-photosynthetic enzymes. The chloroplast enzymes, however, showed much greater increases in activity ranging from 15- to 91-fold. Fraction I protein increased 11·7-fold. These increases approached the levels attained in fully green leaves. All these responses were largely prevented by far-red light indicating that they were mediated by phytochrome. In experiments with red light given at daily intervals there was a lag of 24 hr before the initially very low activity of ribulose-1,5-bisphosphate carboxylase increased. Fraction I protein which was initially present in significant amounts showed a similar lag in its synthesis. However, for 3 days after the initial irradiation, the rate of increase of the enzymic activity was much greater than the rate of net synthesis of Fraction I protein. A single initial red irradiation was as effective as 3 daily irradiations in increasing the activity of ribulose-1,5-bisphosphate carboxylase. A fourth irradiation, however, gave an additional response which exceeded that of the single initial irradiation. It was shown that there was a rapid activation of ribulose-1,5-bisphosphate carboxylase by either continuous white or 3 min of red light. The red light response was slowly reversed in the dark. These results are discussed with particular emphasis on the relation between growth and plastid development in a phytochrome-mediated system.