SummaryMicro-RNAs (miRNAs) are one class of endogenous tiny RNAs that play important regulatory roles in plant development and responses to external stimuli. To date, miRNAs have been cloned from higher plants such as Arabidopsis, rice and pumpkin, and there is limited information on their identity in lower plants including Bryophytes. Bryophytes are among the oldest groups of land plants among the earth's flora, and are important for our understanding of the transition to life on land. To identify miRNAs that might have played a role early in land plant evolution, we constructed a library of small RNAs from the juvenile gametophyte (protonema) of the moss Physcomitrella patens. Sequence analysis revealed five higher plant miRNA homologues, including three members of the miR319 family, previously shown to be involved in the regulation of leaf morphogenesis, and miR156, which has been suggested to regulate several members of the SQUAMOSA PROMOTER BINDING-LIKE (SPL) family in Arabidopsis. We have cloned PpSBP3, a moss SPL homologue that contains an miR156 complementary site, and demonstrated that its mRNA is cleaved within that site suggesting that it is an miR156 target in moss. Six additional candidate moss miRNAs were identified and shown to be expressed in the gametophyte, some of which were developmentally regulated or upregulated by auxin. Our observations suggest that miRNAs play important regulatory roles in mosses.

To come to a better understanding of the evolution and function of the SBP-box transcription factor family in plants, we identified, isolated and characterized 13 of its members from the moss Physcomitrella patens. For the majority of the moss SBP-box genes, clear orthologous relationships with family members of flowering plants could be established by phylogenetic analysis based on the conserved DNA-binding SBPdomain, as well as additional synapomorphic molecular characters. The P. patens SBP-box genes cluster in four separable groups. One of these consists exclusively of moss genes; the three others are shared with family members of Arabidopsis and rice. Besides the family defining DNAbinding SBP-domain, other features can be found conserved between moss and other plant SBP-domain proteins. An AHA-like motif conserved from the unicellular alga Chlamydomonas reinhardtii to flowering plants, was found able to promote transcription in a heterologous yeast system. The conservation of a functional microRNA response element in the mRNA of three of the moss SBP-box genes supports the idea of an ancient origin of microRNA dependent regulation of SBP-box gene family members.As our current knowledge concerning the roles of SBP-box genes in plant development is scarce and the model system P. patens allows targeted mutation, the material we isolated and characterized will be helpful to generate the mutant phenotypes necessary to further elucidate these roles.

Cryptochromes are blue light absorbing photoreceptors found in many organisms and involved in numerous developmental processes. At least two highly similar cryptochromes are known to aVect branching during gametophytic development in the moss Physcomitrella patens. We uncovered a relationship between these cryptochromes and the expression of particular members of the SBP-box genes, a plant speciWc transcription factor family. Transcript levels of the respective moss SBP-box genes, all belonging to the LG1-subfamily, were found to be dependent, albeit not exclusively, on blue light. Moreover, disruptant lines generated for two moss representatives of this SBP-box gene subfamily, both showed enhanced caulonema side branch formation, a phenotype opposite to that of the ppcry1a/1b double disruptant line. In this report we show that PpCRY1a and PpCRY1b act negatively on the transcript levels of several related moss SBP-box genes and that at least PpSBP1 and PpSBP4 act as negative regulators of side branch formation.

MADS-box genes encode for a large family of transcription-regulating proteins, which were isolated from all groups of eukaryotic organisms. The plant-specific MIKC-type MADS-box genes have been intensively analyzed for their roles in controlling developmental processes. Well-known are the MADS-box genes acting as homeotic selector genes in the differentiation of whorls of floral organs in seed plants. The MADS-box gene family has also been studied in non-flowering plants, such as lycophytes, pteridophytes, and bryophytes. The analysis of MADS-box genes in the moss Physcomitrella patens led to the identification of a new class of MIKC-type genes, designated as MIKC*-type genes. The MIKC*-type genes possess a number of structural features which clearly distinguish them from the already known MIKC-type genes. Recently, orthologues of the Physcomitrella MIKC*-type genes were found in Arabidopsis thaliana, demonstrating the conservation of these genes in tracheophytes. Here, we report the isolation of two new MIKC*-type MADS-box genes from Physcomitrella. Structural features and expression patterns of these genes were analyzed. The contribution of our findings to a better understanding of the evolution of MIKC*-type genes in land plants is discussed.