As builders and major components of peatlands, Sphagnopsida (peat mosses) are very important organisms for ecosystems and world’s climate. Nowadays many Sphagnum species as well as their habitats are largely protected, while their scientific and economic relevance remains considerable. Advanced methods of in vitro cultivation provide the potential to work in a sustainable way with peat mosses and address aspects of basic research as well as biotechnological and economical topics like biomonitoring or the production of renewable substrates for horticulture (Sphagnum farming). Here, we describe the establishment of axenic in vitro cultures of the five peat moss species Sphagnum fimbriatum Wils. and Hook., Sphagnum magellanicum Brid., Sphagnum palustre L., Sphagnum rubellum Wils. and Sphagnum subnitens Russ. and Warnst. with specific focus on large-scale cultivation of S. palustre in bioreactors. Axenic, clonal cultures were established to produce high quantities of biomass under standardized laboratory conditions. For advanced production of S.palustre we tested different cultivation techniques, growth media and inocula, and analyzed the effects of tissue disruption. While cultivation on solid medium is suitable for long term storage, submerse cultivation in liquid medium yielded highest amounts of biomass. By addition of sucrose and ammonium nitrate we were able to increase the biomass by around 10- to 30-fold within 4 weeks. The morphology of in vitro-cultivated gametophores showed similar phenotypic characteristics compared to material from the field. Thus the tested culture techniques are suitable to produce S. palustre material for basic and applied research.Electronic supplementary materialThe online version of this article (doi:10.1007/s11240-014-0658-2) contains supplementary material, which is available to authorized users.

Summary Thiol‐based redox‐regulation is vital for coordinating chloroplast functions depending on illumination and has been throroughly investigated for thioredoxin‐dependent processes. In parallel, glutathione reductase (GR) maintains a highly reduced glutathione pool, enabling glutathione‐mediated redox buffering. Yet, how the redox cascades of the thioredoxin and glutathione redox machineries integrate metabolic regulation and detoxification of reactive oxygen species remains largely unresolved because null mutants of plastid/mitochondrial GR are embryo‐lethal in Arabidopsis thaliana. To investigate whether maintaining a highly reducing stromal glutathione redox potential (EGSH) via GR is necessary for functional photosynthesis and plant growth, we created knockout lines of the homologous enzyme in the model moss Physcomitrella patens. In these viable mutant lines, we found decreasing photosynthetic performance and plant growth with increasing light intensities, whereas ascorbate and zeaxanthin/antheraxanthin levels were elevated. By in vivo monitoring stromal EGSH dynamics, we show that stromal EGSH is highly reducing in wild‐type and clearly responsive to light, whereas an absence of GR leads to a partial glutathione oxidation, which is not rescued by light. By metabolic labelling, we reveal changing protein abundances in the GR knockout plants, pinpointing the adjustment of chloroplast proteostasis and the induction of plastid protein repair and degradation machineries. Our results indicate that the plastid thioredoxin system is not a functional backup for the plastid glutathione redox systems, whereas GR plays a critical role in maintaining efficient photosynthesis.

Sphagnum farming can substitute peat with renewable biomass and thus help mitigate climate change. Large volumes of the required founder material can only be supplied sustainably by axenic cultivation in bioreactors. We established axenic in vitro cultures from sporophytes of 19 Sphagnum species collected in Austria,

Axenic in-vitro cultivation of nineteen peat-moss (Sphagnum L.) species as a resource for basic 1 biology, biotechnology and paludiculture 2 Summary 27  The cultivation of Sphagnum mosses reduces CO2 emissions by rewetting drained peatlands 28 and by substituting peat with renewable biomass. 'Sphagnum farming' requires large volumes 29 of founder material, which can only be supplied sustainably by axenic cultivation in 30 bioreactors. 31  We established axenic in-vitro cultures from sporophytes of 19 Sphagnum species collected in 32 Austria, Germany, Latvia, Netherlands, Russia and Sweden, namely S. angustifolium, 33 S. balticum, S. capillifolium, S. centrale, S. compactum, S. cuspidatum, S. fallax, S. fimbriatum, 34 S. fuscum, S. lindbergii, S. medium/divinum, S. palustre, S. papillosum, S. rubellum, S. russowii, 35 S. squarrosum, S. subnitens, S. subfulvum, and S. warnstorfii. These species cover five of the 36 six European Sphagnum sections, namely Acutifolia, Cuspidata, Rigida, Sphagnum and 37 Squarrosa. 38  Their growth was measured in axenic suspension cultures, whereas their ploidy was 39 determined by flow cytometry and compared with the genome size of Physcomitrella patens. 40We identified haploid and diploid Sphagnum species, found that their cells are predominantly 41 arrested in the G1-phase of the cell cycle, and did not find a correlation between plant 42 productivity and ploidy. 43  With this collection, high-quality founder material for diverse large-scale applications but also 44 for basic Sphagnum research is available from the International Moss Stock Center (IMSC). 45 46

Summary The auxin efflux PIN‐FORMED (PIN) proteins are conserved in all land plants and important players in plant development. In the moss Physcomitrella (Physcomitrium patens), three canonical PINs (PpPINA–C) are expressed in the leafy shoot (gametophore). PpPINA and PpPINB show functional activity in vegetative growth and sporophyte development. Here, we examined the role of PpPINC in the life cycle of Physcomitrella. We established reporter and knockout lines for PpPINC and analysed vegetative and reproductive tissues using microscopy and transcriptomic sequencing of moss gametangia. PpPINC is expressed in immature leaves, mature gametangia and during sporophyte development. The sperm cells (spermatozoids) of pinC knockout mutants exhibit increased motility and an altered flagella phenotype. Furthermore, the pinC mutants have a higher portion of differentially expressed genes related to spermatogenesis, increased fertility and an increased abortion rate of premeiotic sporophytes. Here, we show that PpPINC is important for spermatogenesis and sporophyte retention. We propose an evolutionary conserved way of polar growth during early moss embryo development and sporophyte attachment to the gametophore while suggesting the mechanical function in sporophyte retention of a ring structure, the Lorch ring.

SummaryThe auxin efflux PIN-FORMED (PIN) proteins are conserved in all land plants and important players in plant development. In the moss Physcomitrella (Physcomitrium patens) three canonical PINs (PpPINA-C) are expressed in the gametophore. PpPINA and PpPINB show functional activity in vegetative growth and sporophyte development. Here, we examined the role of PpPINC in the life cycle of Physcomitrella.We established reporter and knockout lines for PpPINC and analysed vegetative and reproductive tissues using microscopy and transcriptomic sequencing of moss gametangia.PpPINC is expressed in immature leaves, mature gametangia and during sporophyte development. The sperm cells (spermatozoids) of knockout mutants exhibit increased motility compared to the wild type and show an altered flagella phenotype. Further, the knockout mutants have a significantly increased fertility, and an increased abortion rate of premeiotic sporophytes.Here, we show that PpPINC is an important regulator for spermatogenesis and sporophyte development. We propose an evolutionary conserved way of polar growth during early moss embryo development and sporophyte attachment, while suggesting the mechanical function in sporophyte securement of a ring structure, the Lorch ring.

Sphagnum mosses are important carbon sequesters and emerging model organisms. However, induction and long-term cultivation of thalloid protonema in several species was not achievable so far. Here, we provide protocols for a set of new tools relevant for Sphagnum molecular biology: a new way for Sphagnum protoplast isolation and regeneration, and a first protocol for transient protoplast transformation. Together, these protocols will support the emerging Sphagnum research community in basic and applied science.

Bryologie Bryologie 2019 • 40 • 20 Cryptogamie, Bryologie est une revue en fl ux continu publiée par les Publications scientifi ques du Muséum, Paris