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An experimental method to facilitate the identification of hybrid sporophytes in the moss Physcomitrella patens using fluorescent tagged lines.

ABSTRACT: • The sequencing of the Physcomitrella patens genome, combined with the high frequency of gene targeting in this species, makes it ideal for reverse genetic studies. For forward genetic studies, experimental crosses and genetic analysis of progeny are essential. • Since P. patens is monoicous, producing both male and female gametes on the same gametophore, and undergoing self-fertilization at a high frequency, the identification of crossed sporophytes is difficult. Usually spores from many sporophytes from a mixed culture must be tested for the production of recombinant progeny. • Here, we describe the use of transgenic lines that express a fluorescent transgene constitutively, to provide a direct visual screen for hybrid sporophytes. • We show that segregations in crosses obtained with this technique are as expected, and demonstrate its utility for the study of the rate of outcrossing between three isolates of P. patens.

SUBMITTER: Perroud PF

PROVIDER: S-EPMC3445409 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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An experimental method to facilitate the identification of hybrid sporophytes in the moss Physcomitrella patens using fluorescent tagged lines.

Perroud Pierre-François PF Cove David J DJ Quatrano Ralph S RS McDaniel Stuart F SF

The New phytologist 20110302 1

• The sequencing of the Physcomitrella patens genome, combined with the high frequency of gene targeting in this species, makes it ideal for reverse genetic studies. For forward genetic studies, experimental crosses and genetic analysis of progeny are essential. • Since P. patens is monoicous, producing both male and female gametes on the same gametophore, and undergoing self-fertilization at a high frequency, the identification of crossed sporophytes is difficult. Usually spores from many sporo ...[more]

PMID: 21366596

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Project description:Mosses are an ancient land plant lineage and are therefore important in studying the evolution of plant developmental processes. Here, we describe stomatal development in the model moss species Physcomitrium patens (previously known as Physcomitrella patens) over the duration of sporophyte development. We dissect the molecular mechanisms guiding cell division and fate and highlight how stomatal function might vary under different environmental conditions. In contrast to the asymmetric entry divisions described in Arabidopsis thaliana, moss protodermal cells can enter the stomatal lineage directly by expanding into an oval shaped guard mother cell (GMC). We observed that when two early stage P. patens GMCs form adjacently, a spacing division can occur, leading to separation of the GMCs by an intervening epidermal spacer cell. We investigated whether orthologs of Arabidopsis stomatal development regulators are required for this spacing division. Our results indicated that bHLH transcription factors PpSMF1 and PpSCRM1 are required for GMC formation. Moreover, the ligand and receptor components PpEPF1 and PpTMM are also required for orientating cell divisions and preventing single or clustered early GMCs from developing adjacent to one another. The identification of GMC spacing divisions in P. patens raises the possibility that the ability to space stomatal lineage cells could have evolved before mosses diverged from the ancestral lineage. This would have enabled plants to integrate stomatal development with sporophyte growth and could underpin the adoption of multiple bHLH transcription factors and EPF ligands to more precisely control stomatal patterning in later diverging plant lineages. We also observed that when P. patens sporophyte capsules mature in wet conditions, stomata are typically plugged whereas under drier conditions this is not the case; instead, mucilage drying leads to hollow sub-stomatal cavities. This appears to aid capsule drying and provides further evidence for early land plant stomata contributing to capsule rupture and spore release.

Project description:BackgroundHexokinase catalyzes the phosphorylation of glucose and fructose, but it is also involved in sugar sensing in both fungi and plants. We have previously described two types of hexokinases in the moss Physcomitrella. Type A, exemplified by PpHxk1, the major hexokinase in Physcomitrella, is a soluble protein that localizes to the chloroplast stroma. Type B, exemplified by PpHxk2, has an N-terminal membrane anchor. Both types are found also in vascular plants, and localize to the chloroplast stroma and mitochondrial membranes, respectively.ResultsWe have now characterized all 11 hexokinase encoding genes in Physcomitrella. Based on their N-terminal sequences and intracellular localizations, three of the encoded proteins are type A hexokinases and four are type B hexokinases. One of the type B hexokinases has a splice variant without a membrane anchor, that localizes to the cytosol and the nucleus. However, we also found two new types of hexokinases with no obvious orthologs in vascular plants. Type C, encoded by a single gene, has neither transit peptide nor membrane anchor, and is found in the cytosol and in the nucleus. Type D hexokinases, encoded by three genes, have membrane anchors and localize to mitochondrial membranes, but their sequences differ from those of the type B hexokinases. Interestingly, all moss hexokinases are more similar to each other in overall sequence than to hexokinases from other plants, even though characteristic sequence motifs such as the membrane anchor of the type B hexokinases are highly conserved between moss and vascular plants, indicating a common origin for hexokinases of the same type.ConclusionsWe conclude that the hexokinase gene family is more diverse in Physcomitrella, encoding two additional types of hexokinases that are absent in vascular plants. In particular, the presence of a cytosolic and nuclear hexokinase (type C) sets Physcomitrella apart from vascular plants, and instead resembles yeast, where all hexokinases localize to the cytosol. The fact that all moss hexokinases are more similar to each other than to hexokinases from vascular plants, even though both type A and type B hexokinases are present in all plants, further suggests that the hexokinase gene family in Physcomitrella has undergone concerted evolution.

Project description:BACKGROUND: Alternative splicing (AS) involving tandem acceptors that are separated by three nucleotides (NAGNAG) is an evolutionarily widespread class of AS, which is well studied in Homo sapiens (human) and Mus musculus (mouse). It has also been shown to be common in the model seed plants Arabidopsis thaliana and Oryza sativa (rice). In one of the first studies involving sequence-based prediction of AS in plants, we performed a genome-wide identification and characterization of NAGNAG AS in the model plant Physcomitrella patens, a moss. RESULTS: Using Sanger data, we found 295 alternatively used NAGNAG acceptors in P. patens. Using 31 features and training and test datasets of constitutive and alternative NAGNAGs, we trained a classifier to predict the splicing outcome at NAGNAG tandem splice sites (alternative splicing, constitutive at the first acceptor, or constitutive at the second acceptor). Our classifier achieved a balanced specificity and sensitivity of >or= 89%. Subsequently, a classifier trained exclusively on data well supported by transcript evidence was used to make genome-wide predictions of NAGNAG splicing outcomes. By generation of more transcript evidence from a next-generation sequencing platform (Roche 454), we found additional evidence for NAGNAG AS, with altogether 664 alternative NAGNAGs being detected in P. patens using all currently available transcript evidence. The 454 data also enabled us to validate the predictions of the classifier, with 64% (80/125) of the well-supported cases of AS being predicted correctly. CONCLUSION: NAGNAG AS is just as common in the moss P. patens as it is in the seed plants A. thaliana and O. sativa (but not conserved on the level of orthologous introns), and can be predicted with high accuracy. The most informative features are the nucleotides in the NAGNAG and in its immediate vicinity, along with the splice sites scores, as found earlier for NAGNAG AS in animals. Our results suggest that the mechanism behind NAGNAG AS in plants is similar to that in animals and is largely dependent on the splice site and its immediate neighborhood.

Dataset Information

An experimental method to facilitate the identification of hybrid sporophytes in the moss Physcomitrella patens using fluorescent tagged lines.

Publications

An experimental method to facilitate the identification of hybrid sporophytes in the moss Physcomitrella patens using fluorescent tagged lines.

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