Kirsten Krause

Complete DNA sequences of the plastid genomes of two parasitic flowering plant species, Cuscuta reflexa and Cuscuta gronovii

BackgroundThe holoparasitic plant genus Cuscuta comprises species with photosynthetic capacity and functional chloroplasts as well as achlorophyllous and intermediate forms with restricted photosynthetic activity and degenerated chloroplasts. Previous data indicated significant differences with respect to the plastid genome coding capacity in different Cuscuta species that could correlate with their photosynthetic activity. In order to shed light on the molecular changes accompanying the parasitic lifestyle, we sequenced the plastid chromosomes of the two species Cuscuta reflexa and Cuscuta gronovii. Both species are capable of performing photosynthesis, albeit with varying efficiencies. Together with the plastid genome of Epifagus virginiana, an achlorophyllous parasitic plant whose plastid genome has been sequenced, these species represent a series of progression towards total dependency on the host plant, ranging from reduced levels of photosynthesis in C. reflexa to a restricted photosynthetic activity and degenerated chloroplasts in C. gronovii to an achlorophyllous state in E. virginiana.ResultsThe newly sequenced plastid genomes of C. reflexa and C. gronovii reveal that the chromosome structures are generally very similar to that of non-parasitic plants, although a number of species-specific insertions, deletions (indels) and sequence inversions were identified. However, we observed a gradual adaptation of the plastid genome to the different degrees of parasitism. The changes are particularly evident in C. gronovii and include (a) the parallel losses of genes for the subunits of the plastid-encoded RNA polymerase and the corresponding promoters from the plastid genome, (b) the first documented loss of the gene for a putative splicing factor, MatK, from the plastid genome and (c) a significant reduction of RNA editing.ConclusionOverall, the comparative genomic analysis of plastid DNA from parasitic plants indicates a bias towards a simplification of the plastid gene expression machinery as a consequence of an increasing dependency on the host plant. A tentative assignment of the successive events in the adaptation of the plastid genomes to parasitism can be inferred from the current data set. This includes (1) a loss of non-coding regions in photosynthetic Cuscuta species that has resulted in a condensation of the plastid genome, (2) the simplification of plastid gene expression in species with largely impaired photosynthetic capacity and (3) the deletion of a significant part of the genetic information, including the information for the photosynthetic apparatus, in non-photosynthetic parasitic plants.

Plant NBR1 is a selective autophagy substrate and a functional hybrid of the mammalian autophagic adapters NBR1 and p62/SQSTM1.

(Macro)autophagy encompasses both an unselective, bulk degradation of cytoplasmic contents as well as selective autophagy of damaged organelles, intracellular microbes, protein aggregates, cellular structures and specific soluble proteins. Selective autophagy is mediated by autophagic adapters, like p62/SQSTM1 and NBR1. p62 and NBR1 are themselves selective autophagy substrates, but they also act as cargo receptors for degradation of other substrates. Surprisingly, we found that homologs of NBR1 are distributed throughout the eukaryotic kingdom, while p62 is confined to the metazoans. As a representative of all organisms having only an NBR1 homolog we studied Arabidopsis thaliana NBR1 (AtNBR1) in more detail. AtNBR1 is more similar to mammalian NBR1 than to p62 in domain architecture and amino acid sequence. However, similar to p62, AtNBR1 homo-polymerizes via the PB1 domain. Hence, AtNBR1 has hybrid properties of mammalian NBR1 and p62. AtNBR1 has 2 UBA domains, but only the C-terminal UBA domain bound ubiquitin. AtNBR1 bound AtATG8 through a conserved LIR (LC3-interacting region) motif and required co-expression of AtATG8 or human GABARAPL2 to be recognized as an autophagic substrate in HeLa cells. To monitor the autophagic sequestration of AtNBR1 in Arabidopsis we made transgenic plants expressing AtNBR1 fused to a pH-sensitive fluorescent tag, a tandem fusion of the red, acid-insensitive mCherry and the acid-sensitive yellow fluorescent proteins. This strategy allowed us to show that AtNBR1 is an autophagy substrate degraded in the vacuole dependent on the polymerization property of the PB1 domain and of expression of AtATG7. A functional LIR was required for vacuolar import.

Disruption of plastid-encoded RNA polymerase genes in tobacco : expression of only a distinct set of genes is not based on selective transcription of the plastid chromosome

Abstract Plastids of higher plants operate with at least two distinct DNA-dependent RNA polymerases, which are encoded in the organelle (PEP) and in the nucleus (NEP), respectively. Plastid run-on assays and Northern analyses were employed to analyse gene expression in tobacco mutant plastids lacking the PEP genes rpoA, rpoB or rpoC1. Hybridisation of run-on transcripts to restriction fragments representing the entire tobacco plastid chromosome, as well as to selected plastid gene-specific probes, shows that all parts of the plastid DNA are transcribed in rpo-deficient plastids. In comparison to wild-type chloroplasts, which are characterized by preferential transcription of photosynthesis-related genes in the light, mutant plastids exhibit a different transcription pattern with less pronounced differences in the hybridisation intensities between the individual genes. The analysis of steady-state transcript patterns and transcription rates of selected genes in both types of plastids demonstrates that differences in transcription rates are not necessarily paralleled by corresponding changes in transcript levels. The accumulation of large transcripts in the mutant plastids indicates that processing of primary transcripts may be impaired in the absence of PEP. These data suggest that, contrary to the prevailing view, much of the regulation of NEP-driven plastid gene expression in the rpo-deficient mutants is not based on differential promoter usage but is exerted at post-transcriptional levels.

From chloroplasts to “cryptic” plastids: evolution of plastid genomes in parasitic plants

To date, more than 130 plastid genomes (plastomes) have been completely sequenced. Of those, 12 are strongly reduced plastid genomes from heterotrophic plants or plant-related species that exhibit a parasitic lifestyle. Half of these species are land plants while the other half consists of unicellular species that have evolved from photosynthetic algae. Due to their specialized lifestyle, parasitic lineages experienced a loss of evolutionary pressure on the plastid genome and, in particular, on the photosynthesis-related genes. This made them tolerant for the accumulation of detrimental mutations and deletions in plastid genes. That parasitic plants are naturally occurring plastome mutants makes them a rich source of information concerning plastome evolution and the mechanisms that are involved. This review reports on the progress made in recent years with parasitic plant plastomes and attempts to summarize what we can learn from analysing the genomes of functionally reduced, or cryptic, plastids. Particularly, the loss of genes for a plastid-encoded RNA polymerase as well as an intron maturase and the retention of the gene for the large subunit of the Calvin cycle enzyme Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in selected species will be discussed.

Comparative survey of plastid and mitochondrial targeting properties of transcription factors in Arabidopsis and rice

A group of nuclear transcription factors, the Whirly proteins, were recently shown to be targeted also to chloroplasts and mitochondria. In order to find out whether other proteins might share this feature, an in silico-based screening of transcription factors from Arabidopsis and rice was carried out with the aim of identifying putative N-terminal chloroplast and mitochondrial targeting sequences. For this, the individual predictions of several independent programs were combined to a consensus prediction using a naïve Bayes method. This consensus prediction shows a higher specificity at a given sensitivity value than each of the single programs. In both species, transcription factors from a variety of protein families that possess putative N-terminal plastid or mitochondrial target peptides as well as nuclear localization sequences, were found. A search for homologues within members of the AP2/EREBP protein family revealed that target peptide-containing proteins are conserved among monocotyledonous and dicotyledonous species. Fusion of one of these proteins to GFP revealed, indeed, a dual targeting activity of this protein. We propose that dually targeted transcription factors might be involved in the communication between the nucleus and the organelles in plant cells. We further discuss how recent results on the physical interaction between the organelles and the nucleus could have significance for the regulation of the localization of these proteins.

DNA-binding proteins of the Whirly family in Arabidopsis thaliana are targeted to the organelles

Arabidopsis thaliana contains three genes with high homology to potato p24 which was described as a member of the Whirly family of nuclear transcriptional activators. Computer‐based analysis revealed that all Arabidopsis Whirly (Why) proteins contain targeting sequences for either plastids or mitochondria. The functionality of these sequences was demonstrated by in vitro import assays into isolated organelles. Transient expression of GFP fusion proteins in protoplasts and onion epidermal cells confirmed the localisation of these proteins in plastids or mitochondria, respectively. The possession of organellar targeting sequences seems to be conserved among Why proteins of higher plant species, including potato p24.

Nuclear regulators with a second home in organelles

In plants, increasing evidence points towards the existence of nuclear proteins that are also targeted to either mitochondria - a well-known phenomenon from yeast and mammalians - or to plastids. One such protein is Whirly1, which was the first protein to be identified in the nucleus and plastids of the same plant cell. Like Whirly1, most of the dual targeted (nucleus and organelle) proteins have functions in the maintenance of DNA, telomere structuring or gene expression. In some instances, proteins were even shown to be relocated from one compartment to another upon environmental or developmental clues. We hypothesize that one rationale of dual targeting is storage or sequestration of these proteins inside the organelles until specific conditions require their activity in the nucleus.

Molecular, functional and ultrastructural characterisation of plastids from six species of the parasitic flowering plant genus Cuscuta.

Abstract. Plastids of Cuscuta reflexa Roxb., C. subinclusa D. et H., C. gronovii Willd. and C. campestris Yunck. possess thylakoids and contain both chlorophyll a and b in a ratio similar to that of stem tissue of the systematically closely related but ‘normal’ green Ipomoea tricolor. In contrast, plastids of C. odorata R. et P. and C. grandiflora H.B.K. do not contain any chlorophyll or possess thylakoids. Light-driven electron transport, as measured by oxygen evolution and indicated by analysis of chlorophyll fluorescence, was present in all chlorophyll-containing species. The photosystem II efficiency was low and ranged from 0.511 to 0.687. The plastid rbcL gene could not be detected in C. odorata, but was present in all other tested species. Neither rbcL transcripts nor the large subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) could be detected in C. odorata and C. grandiflora. Low amounts of the large subunit of Rubisco were detected immunologically in all other Cuscuta species. Apparently, the genus Cuscuta comprises species with different degrees of plastid functionality, ranging from intact chloroplasts, via plastids with impaired protein production and gene expression to plastids with reduced plastome gene content.

Plastid transcription in the holoparasitic plant genus Cuscuta: parallel loss of the rrn16 PEP-promoter and of the rpoA and rpoB genes coding for the plastid-encoded RNA polymerase

Abstract. The holoparasitic plant genus Cuscuta comprises a range of species whose plastid genomes have different degrees of reductions in their coding capacity. In this study, four Cuscuta species, Cuscuta reflexa, C. gronovii, C. subinclusa and C. odorata, that possess substantial physiological differences, were analysed with respect to the sequence and promoter structure of the rrn16 gene coding for the ribosomal 16S rRNA. Whereas the coding region of this gene is highly conserved among all four Cuscuta species, significant differences were observed in the non-coding region 5′ of rrn16 with respect to both the length of the intergenic region between rrn16 and trnV and the promoters used to initiate transcription of the rrn16 gene. In the green species C. reflexa,rrn16 transcription starts from a functional plastid-encoded RNA polymerase (PEP) promoter that is missing in the other three species, C. gronovii, C. odorata and C. subinclusa. Instead, a 15-nucleotide-long conserved sequence immediately upstream of the mapped 5′ ends bearing the nuclear-encoded RNA polymerase (NEP) promoter motif could be identified in these three species. The lack of a PEP promoter in these species coincides with the loss of two genes that encode subunits of PEP (rpoA and rpoB).

Data‐directed top‐down Fourier‐transform mass spectrometry of a large integral membrane protein complex: Photosystem II from Galdieria sulphuraria

High‐resolution top‐down MS was used to characterize eleven integral and five peripheral subunits of the 750 kDa photosystem II complex from the eukaryotic red alga, Galdieria sulphuraria. The primary separation used LC MS with concomitant fraction collection (LC‐MS+), yielding around 40 intact mass tags at 100 ppm mass accuracy on a low‐resolution ESI mass spectrometer, whose retention and mass were used to guide subsequent high‐resolution top‐down nano‐electrospray FT ion‐cyclotron resonance MS experiments (FT‐MS). Both collisionally activated and electron capture dissociation were used to confirm the presence of eleven small subunits to mass accuracy within 5 ppm; PsbE, PsbF, PsbH, PsbI, PsbJ, PsbK, PsbL, PsbM, PsbT, PsbX and PsbZ. All subunits showed covalent modifications that fall into three classes including retention of initiating formyl‐methionine, removal of methionine at the N‐terminus with or without acetylation, and removal of a longer N‐terminal peptide. Peripheral subunits identified by top‐down analysis included oxygen‐evolving complex subunits PsbO, PsbU, PsbV, as well as Psb28 (PsbW) and Psb27 (“PsbZ‐like”). Top‐down high‐resolution MS provides the necessary precision, typically less than 5 ppm, for identification and characterization of polypeptide composition of these important membrane protein complexes.