MicroRNAs (miRNAs) control gene expression in animals and plants. Like another class of small RNAs, siRNAs, they affect gene expression posttranscriptionally. While siRNAs in addition act in transcriptional gene silencing, a role of miRNAs in transcriptional regulation has been less clear. We show here that in moss Physcomitrella patens mutants without a DICER-LIKE1b gene, maturation of miRNAs is normal but cleavage of target RNAs is abolished and levels of these transcripts are drastically reduced. These mutants accumulate miRNA:target-RNA duplexes and show hypermethylation of the genes encoding target RNAs, leading to gene silencing. This pathway occurs also in the wild-type upon hormone treatment. We propose that initiation of epigenetic silencing by DNA methylation depends on the ratio of the miRNA and its target RNA.

SummaryIn mammalian cells, phospholipase D (PLD) and its product phosphatidic acid (PA) are involved in a number of signalling cascades, including cell proliferation, membrane traf®cking and defence responses. In plant cells a signalling role for PLD and PA is also emerging. Plants have the extra ability to phosphorylate PA to produce diacylglycerol pyrophosphate (DGPP), a newly discovered phospholipid whose formation attenuates PA levels, but which could itself be a second messenger. Here we report that increases in PA and its conversion to DGPP are common stress responses to water de®cit. Increases occur within minutes of treatment and are dependent on the level of stress. Part of the PA produced is due to PLD activity as measured by the in vivo transphosphatidylation of 1-butanol, and part is due to diacylglycerol kinase activity as monitored via 32 P-PA formation in a differential labelling protocol. Increases in PA and DGPP are found not only in the green alga Chlamydomonas moewusii and cellsuspension cultures of tomato and alfalfa when subjected to hyperosmotic stress, but also in dehydrated leaves of the resurrection plant Craterostigma plantagineum. These results provide further evidence that PLD and PA play a role in plant signalling, and provide the ®rst demonstration that DGPP is formed during physiological conditions that evoke PA synthesis.

In order to determine the degree of tolerance of the moss Physcomitrella patens to different abiotic stress conditions, we examined its tolerance against salt, osmotic and dehydration stress. Compared to other plants like Arabidopsis thaliana, P. patens exhibits a high degree of abiotic stress tolerance, making it a valuable source for the identification of genes effecting the stress adaptation. Plants that had been treated with NaCl tolerated concentrations up to 350 mM. Treatments with sorbitol revealed that plants are able to survive concentrations up to 500 mM. Furthermore, plants that had lost 92% water on a fresh-weight basis were able to recover successfully. For molecular analyses, a P. patens expressed sequence tag (EST) database was searched for cDNA sequences showing homology to stress-associated genes of seed plants and bacteria. 45 novel P. patens genes were identified and subjected to cDNA macroarray analyses to define their expression pattern in response to water deficit. Among the selected cDNAs, we were able to identify a set of genes that is specifically up-regulated upon dehydration. These genes encode proteins exerting their function in maintaining the integrity of the plant cell as well as proteins that are known to be members of signaling networks. The identified genes will serve as molecular markers and potential targets for future functional analyses.

Stomata are microscopic valves on plant surfaces that originated over 400 million years ago and facilitated the greening of Earth’s continents by permitting efficient shoot-atmosphere gas exchange and plant hydration1. However, the core genetic machinery regulating stomatal development in non-vascular land plants is poorly understood2–4 and their function has remained a matter of debate for a century5. Here, we show that genes encoding the two basic helix-loop-helix proteins PpSMF1 and PpSCRM1 in the moss Physcomitrella patens are orthologous to transcriptional regulators of stomatal development in the flowering plant Arabidopsis thaliana and essential for stomata formation in moss. Targeted knock-out P. patens mutants lacking either PpSMF1 or PpSCRM1 develop gametophytes indistinguishable from wild-type plants but mutant sporophytes lacking stomata. Protein-protein interaction assays reveal heterodimerisation between PpSMF1 and PpSCRM1 which, together with moss-angiosperm gene complementations6, suggests deep functional conservation of the heterodimeric SMF1 and SCRM1 unit required to activate transcription for moss stomatal development, as in A. thaliana7. Moreover, stomata-less sporophytes of ΔPpSMF1 and ΔPpSCRM1 mutants exhibited delayed dehiscence, implying stomata might have promoted dehiscence in the first complex land plant sporophytes.

Summary Two cDNAs encoding allene oxide cyclases (PpAOC1, PpAOC2), key enzymes in the formation of jasmonic acid (JA) and its precursor (9S,13S)‐12‐oxo‐phytodienoic acid (cis‐(+)‐OPDA), were isolated from the moss Physcomitrella patens. Recombinant PpAOC1 and PpAOC2 show substrate specificity against the allene oxide derived from 13‐hydroperoxy linolenic acid (13‐HPOTE); PpAOC2 also shows substrate specificity against the allene oxide derived from 12‐hydroperoxy arachidonic acid (12‐HPETE). In protonema and gametophores the occurrence of cis‐(+)‐OPDA, but neither JA nor the isoleucine conjugate of JA nor that of cis‐(+)‐OPDA was detected. Targeted knockout mutants for PpAOC1 and for PpAOC2 were generated, while double mutants could not be obtained. The ΔPpAOC1 and ΔPpAOC2 mutants showed reduced fertility, aberrant sporophyte morphology and interrupted sporogenesis.