Physcomitrella patens Reute mCherry as a tool for efficient crossing within and between ecotypes.

Physcomitrella patens is a monoecious moss that is predominantly selfing in the wild. Laboratory crossing techniques have been established and crosses between the sequenced Gransden ecotype and the genetically divergent Villersexel ecotype were used for genetic mapping. The recently introduced ecotype Reute has a high fertility rate and is genetically more closely related to the Gransden ecotype than the Villersexel ecotype. Reute sexual reproduction phenology is similar to Gransden, which should allow successful crossing. Using the Reute ecotype and an existing Gransden mutant as a test case, we applied a normalised crossing approach to demonstrate crossing potential between these ecotypes. Also, using a standard transformation approach, we generated Reute fluorescent strains expressing mCherry that allow an easy detection of crossed offspring (sporophyte). We show that Reute can be successfully crossed with a self-infertile DR5:DsRed2 mutant generated in the Gransden background. Using newly established Reute fluorescent strains, we show that they can efficiently fertilise Reute as well as Gransden wild type. The resulting progeny display Mendelian 1:1 segregation of the fluorescent marker(s), demonstrating the suitability of such strains for genetic crossing. Overall our results demonstrate that Reute is highly suitable for genetic crossing. The Reute mCherry strain can be used as a suitable background for offspring selection after crossing.

Role of ABA and ABI3 in desiccation tolerance.

We show in bryophytes that abscisic acid (ABA) pretreatment of moss (Physcomitrella patens) cells confers desiccation tolerance. In angiosperms, both ABA and the transcriptional regulator ABSCISIC ACID INSENSITIVE 3 (ABI3) are required to protect the seed during desiccation. ABA was not able to protect moss cells in stable deletion lines of ABI3 (DeltaPpabi3). Hence, moss has the same functional link between ABA, ABI3, and the desiccation tolerance phenotype that is found in angiosperms. Furthermore, we identified 22 genes that were induced during ABA pretreatment in wild-type lines. When their expression was compared with that of DeltaPpabi3 during ABA pretreatment and immediately after desiccation, a new target of ABI3 action appears to be in the recovery period.

An RNAi system in Physcomitrella patens with an internal marker for silencing allows for rapid identification of loss of function phenotypes.

RNAi is a powerful method for generating loss of function mutants, especially for targeting genes belonging to large gene families. We have recently shown that RNAi functions in the moss Physcomitrella patens. We obtained stable lines that show constitutive silencing of a nuclearly localized GFP:GUS fusion protein (NLS:GFP:GUS). However, lines that display silencing of the protein do not necessarily have reduced transcript levels. Therefore, a system has been developed that silences the NLS:GFP:GUS reporter construct at the same time as it silences a gene of interest. Gateway (Invitrogen) recombination cassettes were incorporated into these vectors to facilitate cloning of many different cDNA sequences. In addition, vectors were generated that contain genomic moss DNA sequence information to increase the production of stable moss lines. Transformation with these constructs results in strong silencing within 24 h and is stable for at least a month after transformation. FtsZ2-1, whose loss of function phenotype is known, was incorporated as a test case for analyzing phenotypes. One hundred per cent of regenerating colonies that have silenced GFP exhibit a loss of function FtsZ2-1 phenotype, validating the use of this system to assay phenotypes for plant genes of unknown function.

Molecular cloning, spatial and temporal characterization of spinach SOGA1 cDNA, encoding an alpha subunit of G protein.

Heterotrimeric G proteins are an important component of signal transduction pathway in animals. Although these proteins have been described in plants, their exact function and action mode are not clearly defined. In order to analyze the relationship between these proteins and the transduction of light signals in spinach, we have isolated by 5' and 3' RACE-PCR a 1660bp cDNA clone called SOGA1. This codes for a 383aa protein, which reveals a very strong homology with other plant Galpha subunit sequences. Genomic analysis suggested that SOGA1 belonged to a small multiple gene family. Northern blots and in-situ hybridization analyses showed that SOGA1 transcripts accumulate in all organs tested with a specific high level associated with the apex, roots and hypocotyls. Finally, a time-course analysis performed on the green tissues showed that accumulation of SOGA1 transcripts follows a circadian rhythm. However, in-situ hybridization analysis of the apex suggested the opposite behavior, while no variation was observed in the hypocotyl.