Lignin, one of the most abundant biopolymers on Earth, derives from the plant phenolic metabolism. It appeared upon terrestrialization and is thought critical for plant colonization of land. Early diverging land plants do not form lignin, but already have elements of its biosynthetic machinery. Here we delete in a moss the P450 oxygenase that defines the entry point in angiosperm lignin metabolism, and find that its pre-lignin pathway is essential for development. This pathway does not involve biochemical regulation via shikimate coupling, but instead is coupled with ascorbate catabolism, and controls the synthesis of the moss cuticle, which prevents desiccation and organ fusion. These cuticles share common features with lignin, cutin and suberin, and may represent the extant representative of a common ancestor. Our results demonstrate a critical role for the ancestral phenolic metabolism in moss erect growth and cuticle permeability, consistent with importance in plant adaptation to terrestrial conditions.

In seed plants, strigolactones (SLs) regulate architecture and induce mycorrhizal symbiosis in response to environmental cues. SLs are formed by combined activity of the carotenoid cleavage dioxygenases (CCDs) 7 and 8 from 9-cis-β-carotene, leading to carlactone that is converted by cytochromes P450 (clade 711; MAX1 in Arabidopsis) into various SLs. As Physcomitrella patens possesses CCD7 and CCD8 homologs but lacks MAX1, we investigated if PpCCD7 together with PpCCD8 form carlactone and how deletion of these enzymes influences growth and interactions with the environment. We investigated the enzymatic activity of PpCCD7 and PpCCD8 in vitro, identified the formed products by high performance liquid chromatography (HPLC) and LC-MS, and generated and analysed ΔCCD7 and ΔCCD8 mutants. We defined enzymatic activity of PpCCD7 as a stereospecific 9-cis-CCD and PpCCD8 as a carlactone synthase. ΔCCD7 and ΔCCD8 lines showed enhanced caulonema growth, which was revertible by adding the SL analogue GR24 or carlactone. Wild-type (WT) exudates induced seed germination in Orobanche ramosa. This activity was increased upon phosphate starvation and abolished in exudates of both mutants. Furthermore, both mutants showed increased susceptibility to phytopathogenic fungi. Our study reveals the deep evolutionary conservation of SL biosynthesis, SL function, and its regulation by biotic and abiotic cues.

SUMMARYThe moss Physcomitrella patens is an important model organism for studying plant evolution, development, physiology and biotechnology. Here we have generated microarray gene expression data covering the principal developmental stages, culture forms and some environmental/stress conditions. Example analyses of developmental stages and growth conditions as well as abiotic stress treatments demonstrate that (i) growth stage is dominant over culture conditions, (ii) liquid culture is not stressful for the plant, (iii) low pH might aid protoplastation by reduced expression of cell wall structure genes, (iv) largely the same gene pool mediates response to dehydration and rehydration, and (v) AP2/EREBP transcription factors play important roles in stress response reactions. With regard to the AP2 gene family, phylogenetic analysis and comparison with Arabidopsis thaliana shows commonalities as well as uniquely expressed family members under drought, light perturbations and protoplastation. Gene expression profiles for P. patens are available for the scientific community via the easy-to-use tool at https://www.genevestigator.com. By providing large-scale expression profiles, the usability of this model organism is further enhanced, for example by enabling selection of control genes for quantitative real-time PCR. Now, gene expression levels across a broad range of conditions can be accessed online for P. patens.

Given the vast phenotypic and genetic heterogeneity of acute and chronic myeloid malignancies, hematologists have eagerly awaited the introduction of next-generation sequencing (NGS) into the routine diagnostic armamentarium to enable a more differentiated disease classification, risk stratification, and improved therapeutic decisions. At present, an increasing number of hematologic laboratories are in the process of integrating NGS procedures into the diagnostic algorithms of patients with acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and myeloproliferative neoplasms (MPNs). Inevitably accompanying such developments, physicians and molecular biologists are facing unexpected challenges regarding the interpretation and implementation of molecular genetic results derived from NGS in myeloid malignancies. This article summarizes typical challenges that may arise in the context of NGS-based analyses at diagnosis and during follow-up of myeloid malignancies.Table 1Challenges accompanying the introduction of massive parallel sequencing in clinical routine diagnostics in hemato-oncologyChallengeBackgroundCurrent and future approachDiscrimination of leukemia-related mutations from polymorphisms or passenger mutationsDriver mutations expected to occur at higher allele frequency in patient samples than passenger mutations; driver mutations more likely to have an impact on protein function than polymorphisms or passenger mutationsOptimization of cancer-specific databases including reporting of rare physiological gene variantsImplementation of novel bioinformatic algorithms based on prediction of functional impactQuantitative and dynamic VAF monitoring (separately and together with other mutations) at follow-upDiscrimination of somatic leukemia-related mutations from CHIPCHIP is presented in ~10% of individuals aged 70 to 80 and in up to 20% in the age group > 80 yearsQuantitative and dynamic VAF monitoring (separately and together with other mutations) at follow-upClarifying the significance of CHIP in the context of myeloid malignanciesDiscrimination of leukemia-related somatic mutations from pathogenic germline alterationsChallenge to differentiate acquired somatic mutations from germline pathogenic variants at diagnosisMutation detection in germline control samples (e.g., skin fibroblasts, saliva) in mutations such as in RUNX1, CEBPAThorough medical family history followed by molecular genetic tests in relatives if necessaryHigh and stable VAF (e.g., 40–50%) at follow-up despite clinical response to treatment may be indicative for germline alterationDiscrimination of true genetic alterations from PCR, sequencing and post-sequencing artifactsMany artefacts are known to arise during NGS library preparation, sequencing and data analysisError correction using molecular identifiers that individually label original input DNA moleculesRefinement of error-correction computational methods in post-sequencing NGS data analysisConfirmation using Sanger sequencingLimited sensitivity of NGS for minimal residual diseas...

The whole-genome transcriptomic cold stress response of the moss Physcomitrella patens was analyzed and correlated with phenotypic and metabolic changes. Based on time-series microarray experiments and quantitative real-time polymerase chain reaction, we characterized the transcriptomic changes related to early stress signaling and the initiation of cold acclimation. Transcription-associated protein (TAP)-encoding genes of P. patens and Arabidopsis thaliana were classified using generalized linear models. Physiological responses were monitored with pulse-amplitude-modulated fluorometry, high-performance liquid chromatography and targeted high-performance mass spectrometry. The transcript levels of 3220 genes were significantly affected by cold. Comparative classification revealed a global specialization of TAP families, a transcript accumulation of transcriptional regulators of the stimulus/stress response and a transcript decline of developmental regulators. Although transcripts of the intermediate to later response are from evolutionarily conserved genes, the early response is dominated by species-specific genes. These orphan genes may encode as yet unknown acclimation processes.

Background: Gibberellins (GA) are plant hormones that can regulate germination, elongation growth, and sex determination. They ubiquitously occur in seed plants. The discovery of gibberellin receptors, together with advances in understanding the function of key components of GA signalling in Arabidopsis and rice, reveal a fairly short GA signal transduction route. The pathway essentially consists of GID1 gibberellin receptors that interact with F-box proteins, which in turn regulate degradation of downstream DELLA proteins, suppressors of GA-controlled responses.

Summary• Sporopollenin is the main constituent of the exine layer of spore and pollen walls. Recently, several Arabidopsis genes, including polyketide synthase A (PKSA), which encodes an anther-specific chalcone synthase-like enzyme (ASCL), have been shown to be involved in sporopollenin biosynthesis. The genome of the moss Physcomitrella patens contains putative orthologs of the Arabidopsis sporopollenin biosynthesis genes.• We analyzed available P. patens expressed sequence tag (EST) data for putative moss orthologs of the Arabidopsis genes of sporopollenin biosynthesis and studied the enzymatic properties and reaction mechanism of recombinant PpASCL, the P. patens ortholog of Arabidopsis PKSA. We also generated structure models of PpASCL and Arabidopsis PKSA to study their substrate specificity.• Physcomitrella patens orthologs of Arabidopsis genes for sporopollenin biosynthesis were found to be expressed in the sporophyte generation. Similarly to Arabidopsis PKSA, PpASCL condenses hydroxy fatty acyl-CoA esters with malonyl-CoA and produces hydroxyalkyl a-pyrones that probably serve as building blocks of sporopollenin. The ASCL-specific set of Gly-Gly-Ala residues predicted by the models to be located at the floor of the putative active site is proposed to serve as the opening of an acyl-binding tunnel in ASCL.• These results suggest that ASCL functions together with other sporophytespecific enzymes to provide polyhydroxylated precursors of sporopollenin in a pathway common to land plants.

Sulfate assimilation provides reduced sulfur for synthesis of the amino acids cysteine and methionine and for a range of other metabolites. Sulfate has to be activated prior to reduction by adenylation to adenosine 5-phosphosulfate (APS). In plants, algae, and many bacteria, this compound is reduced to sulfite by APS reductase (APR); in fungi and some cyanobacteria and ␥-proteobacteria, a second activation step, phosphorylation to 3-phosphoadenosine 5-phosphosulfate (PAPS), is necessary before reduction to sulfite by PAPS reductase (PAPR). We found previously that the moss Physcomitrella patens is unique among these organisms in possessing orthologs of both APR and PAPR genes (Koprivova, A., Meyer, A. J., Schween, G., Herschbach, C., Reski, R., and Kopriva, S. (2002) J. Biol. Chem. 277, 32195-32201). To assess the function of the two enzymes, we compared their biochemical properties by analysis of purified recombinant proteins. APR from Physcomitrella is very similar to the well characterized APRs from seed plants. On the other hand, we found that the putative PAPR preferentially reduces APS. Sequence analysis, analysis of UV-visible spectra, and determination of iron revealed that this new APR, named PpAPR-B, does not contain the FeS cluster, which was previously believed to determine the substrate specificity of the otherwise relatively similar enzymes. The lack of the FeS cluster in PpAPR-B catalysis is connected with a lower turnover rate but higher stability of the protein. These findings show that APS reduction without the FeS cluster is possible and that plant sulfate assimilation is predominantly dependent on reduction of APS.Sulfur is essential for life. It is present in the amino acids cysteine and methionine as part of proteins and peptides; in many coenzymes and prosthetic groups, such as FeS centers, thiamine, lipoic acid, etc.; and in a variety of secondary metabolites, e.g. glucosinolates and alliins in plants. In these compounds, sulfur is present in the reduced form of organic thiols; however, the major form of sulfur available in nature is sulfate.