Deducing intracellular distributions of metabolic pathways from genomic data
2014, Gruber, Ansgar, Kroth, Peter G.
In the recent years, a large number of genomes from a variety of different organisms have been sequenced. Most of the sequence data has been publicly released and can be assessed by interested users. However, this wealth of information is currently underexploited by scientists not directly involved in genome annotation. This is partially because sequencing, assembly, and automated annotation can be done much faster than the identification, classification, and prediction of the intracellular localization of the gene products. This part of the annotation process still largely relies on manual curation and addition of contextual information. Users of genome databases who are unfamiliar with the types of data available from (whole) genomes might therefore find themselves either overwhelmed by the vast amount and multiple layers of data or dissatisfied with less-than-meaningful analyses of the data.
In this chapter we present procedures and approaches to identify and characterize gene models of enzymes involved in metabolic pathways based on their similarity to known sequences. Furthermore we describe how to predict the subcellular location of the proteins using publicly available prediction servers and how to interpret the obtained results. The strategies we describe are generally applicable to organisms with primary plastids such as land plants or green algae. Additionally, we describe strategies suitable for those groups of algae with secondary plastids (for instance diatoms), which are characterized by a different cellular topology and a larger number of intracellular compartments compared to plants.
Identification and evaluation of endogenous reference genes for steady state transcript quantification by qPCR in the diatom Phaeodactylum tricornutum with constitutive expression independent from time and light
2013, Sachse, Matthias, Sturm, Sabine, Gruber, Ansgar, Kroth, Peter G.
Diatoms are unicellular algae, which due to their importance for the global primary production and their cellular and genetic complexity, became popular subjects of physiological and molecular biological research in the recent years. The increasing genomic information gathered on diatoms since the last decade promotes diverse analyses of their steady state RNA levels, which are commonly performed via quantitative real-time PCR (qPCR), a technique which excels in sensitivity and dynamic range. Up to now there are only a few studies on suitable endogenous reference genes in diatoms. Such reference genes are crucial for any relative qPCR study and must feature stable transcript levels between all the investigated experimental conditions. Therefore we expanded the data on suitable endogenous reference genes by thorough testing of ten potential genes in the model diatom Phaeodactylum tricornutum at light and time discriminate conditions. Stably expressed genes for these conditions will be of great use for any diatom study dealing with time and light dependent effects. Samples of algae grown in a 16 hours low light photoperiod and dark transitioned cells were investigated over a period of up to 33 hours. A set of three endogenous reference genes was found to be stably expressed in a light and time independent manner: the TATA box binding protein TBP, the ribosomal protein S1 RPS and the hypoxanthine-guanine phosphoribosyltransferase HPRT. Other commonly used reference genes like actin, histone H4 or 18S ribosomal ribonucleic acid did not perform well and are thus unsuited for expression analysis in light or time dependent experimental setups.
A novel type of light-harvesting antenna protein of red algal origin in algae with secondary plastids
2013, Sturm, Sabine, Engelken, Johannes, Gruber, Ansgar, Vugrinec, Sascha, Kroth, Peter G., Adamska, Iwona, Lavaud, Johann
Background: Light, the driving force of photosynthesis, can be harmful when present in excess; therefore, any light harvesting system requires photoprotection. Members of the extended light-harvesting complex (LHC) protein superfamily are involved in light harvesting as well as in photoprotection and are found in the red and green plant lineages, with a complex distribution pattern of subfamilies in the different algal lineages.
Results: Here, we demonstrate that the recently discovered "red lineage chlorophyll a/b-binding-like proteins" (RedCAPs) form a monophyletic family within this protein superfamily. The occurrence of RedCAPs was found to be restricted to the red algal lineage, including red algae (with primary plastids) as well as cryptophytes, haptophytes and heterokontophytes (with secondary plastids of red algal origin). Expression of a full-length RedCAP:GFP fusion construct in the diatom Phaeodactylum tricornutum confirmed the predicted plastid localisation of RedCAPs. Furthermore, we observed that similarly to the fucoxanthin chlorophyll a/c-binding light-harvesting antenna proteins also RedCAP transcripts in diatoms were regulated in a diurnal way at standard light conditions and strongly repressed at high light intensities.
Conclusions: The absence of RedCAPs from the green lineage implies that RedCAPs evolved in the red lineage after separation from the the green lineage. During the evolution of secondary plastids, RedCAP genes therefore must have been transferred from the nucleus of the endocytobiotic alga to the nucleus of the host cell, a process that involved complementation with pre-sequences allowing import of the gene product into the secondary plastid bound by four membranes. Based on light-dependent transcription and on localisation data, we propose that RedCAPs might participate in the light (intensity and quality)-dependent structural or functional reorganisation of the light-harvesting antennae of the photosystems upon dark to light shifts as regularly experienced by diatoms in nature. Remarkably, in plastids of the red lineage as well as in green lineage plastids, the phycobilisome based cyanobacterial light harvesting system has been replaced by light harvesting systems that are based on members of the extended LHC protein superfamily, either for one of the photosystems (PS I of red algae) or for both (diatoms). In their proposed function, the RedCAP protein family may thus have played a role in the evolutionary structural remodelling of light-harvesting antennae in the red lineage.