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Introduction: Individuals carrying pathogenic mutations in the BRCA1 and BRCA2 genes have a high lifetime risk of breast cancer. BRCA1 and BRCA2 are involved in DNA double-strand break repair, DNA alterations that can be caused by exposure to reactive oxygen species, a main source of which are mitochondria. Mitochondrial genome variations affect electron transport chain efficiency and reactive oxygen species production. Individuals with different mitochondrial haplogroups differ in their metabolism and sensitivity to oxidative stress. Variability in mitochondrial genetic background can alter reactive oxygen species production, leading to cancer risk. In the present study, we tested the hypothesis that mitochondrial haplogroups modify breast cancer risk in BRCA1/2 mutation carriers. Methods: We genotyped 22,214 (11,421 affected, 10,793 unaffected) mutation carriers belonging to the Consortium of Investigators of Modifiers of BRCA1/2 for 129 mitochondrial polymorphisms using the iCOGS array. Haplogroup inference and association detection were performed using a phylogenetic approach. ALTree was applied to explore the reference mitochondrial evolutionary tree and detect subclades enriched in affected or unaffected individuals. Results: We discovered that subclade T1a1 was depleted in affected BRCA2 mutation carriers compared with the rest of clade T (hazard ratio (HR) = 0.55; 95% confidence interval (CI), 0.34 to 0.88; P = 0.01). Compared with the most frequent haplogroup in the general population (that is, H and T clades), the T1a1 haplogroup has a HR of 0.62 (95% CI, 0.40 to 0.95; P = 0.03). We also identified three potential susceptibility loci, including G13708A/rs28359178, which has demonstrated an inverse association with familial breast cancer risk. Conclusions: This study illustrates how original approaches such as the phylogeny-based method we used can empower classical molecular epidemiological studies aimed at identifying association or risk modification effects.

Abstract Background Hexokinase catalyzes the phosphorylation of glucose and fructose, but it is also involved in sugar sensing in both fungi and plants. We have previously described two types of hexokinases in the moss Physcomitrella. Type A, exemplified by PpHxk1, the major hexokinase in Physcomitrella, is a soluble protein that localizes to the chloroplast stroma. Type B, exemplified by PpHxk2, has an N-terminal membrane anchor. Both types are found also in vascular plants, and localize to the chloroplast stroma and mitochondrial membranes, respectively. Results We have now characterized all 11 hexokinase encoding genes in Physcomitrella. Based on their N-terminal sequences and intracellular localizations, three of the encoded proteins are type A hexokinases and four are type B hexokinases. One of the type B hexokinases has a splice variant without a membrane anchor, that localizes to the cytosol and the nucleus. However, we also found two new types of hexokinases with no obvious orthologs in vascular plants. Type C, encoded by a single gene, has neither transit peptide nor membrane anchor, and is found in the cytosol and in the nucleus. Type D hexokinases, encoded by three genes, have membrane anchors and localize to mitochondrial membranes, but their sequences differ from those of the type B hexokinases. Interestingly, all moss hexokinases are more similar to each other in overall sequence than to hexokinases from other plants, even though characteristic sequence motifs such as the membrane anchor of the type B hexokinases are highly conserved between moss and vascular plants, indicating a common origin for hexokinases of the same type. Conclusions We conclude that the hexokinase gene family is more diverse in Physcomitrella, encoding two additional types of hexokinases that are absent in vascular plants. In particular, the presence of a cytosolic and nuclear hexokinase (type C) sets Physcomitrella apart from vascular plants, and instead resembles yeast, where all hexokinases localize to the cytosol. The fact that all moss hexokinases are more similar to each other than to hexokinases from vascular plants, even though both type A and type B hexokinases are present in all plants, further suggests that the hexokinase gene family in Physcomitrella has undergone concerted evolution.

The Arabidopsis Plant HomeoDomain (PHD) proteins AtMS1 and AtMMD1 provide chromatin-mediated transcriptional regulation essential for tapetum-dependent pollen formation. This pollen-based male gametogenesis is a derived trait of seed plants. Male gametogenesis in the common ancestors of land plants is instead likely to have been reminiscent of that in extant bryophytes where flagellated sperms are produced by an elaborate gametophyte generation. Still, also bryophytes possess MS1/MMD1-related PHD proteins. We addressed the function of two MS1/MMD1-homologues in the bryophyte model moss Physcomitrium patens by the generation and analysis of reporter and loss-of-function lines. The two genes are together essential for both male and female fertility by providing functions in the gamete-producing inner cells of antheridia and archegonia. They are furthermore expressed in the diploid sporophyte generation suggesting a function during sporogenesis, a process proposed related by descent to pollen formation in angiosperms. We propose that the moss MS1/MMD1-related regulatory network required for completion of male and female gametogenesis, and possibly for sporogenesis, represent a heritage from ancestral land plants., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

A key aim in biology is to identify which genetic changes contributed to the evolution of form through time. Apical dominance, the inhibitory effect exerted by shoot apices on the initiation or outgrowth of distant lateral buds, is a major regulatory mechanism of plant form. 1 Nearly a century of studies in the sporophyte of flowering plants have established the phytohormone auxin as a front-runner in the search for key factors controlling apical dominance, 2 , 3 identifying critical roles for long-range polar auxin transport and local auxin biosynthesis in modulating shoot branching. 4-10 A capacity for lateral branching evolved by convergence in the gametophytic shoot of mosses and primed its diversification; 11 however, polar auxin transport is relatively unimportant in this developmental process, 12 the contribution of auxin biosynthesis genes has not been assessed, and more generally, the extent of conservation in apical dominance regulation within the land plants remains largely unknown. To fill this knowledge gap, we sought to identify genetic determinants of apical dominance in the moss Physcomitrium patens. Here, we show that leafy shoot apex decapitation releases apical dominance through massive and rapid transcriptional reprogramming of auxin-responsive genes and altering auxin biosynthesis gene activity. We pinpoint a subset of P. patens TRYPTOPHAN AMINO-TRANSFERASE (TAR) and YUCCA FLAVIN MONOOXYGENASE-LIKE (YUC) auxin biosynthesis genes expressed in the main and lateral shoot apices and show that they are essential for coordinating branch initiation and outgrowth. Our results demonstrate that local auxin biosynthesis acts as a pivotal regulator of apical dominance in moss and constitutes a shared mechanism underpinning shoot architecture control in land plants., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Clade II basic helix-loop-helix transcription factors (bHLH TFs) are essential for pollen production and tapetal nursing functions in angiosperm anthers. As pollen has been suggested to be related to bryophyte spores by descent, we characterized two Physcomitrium (Physcomitrella) patens clade II bHLH TFs (PpbHLH092 and PpbHLH098), to test if regulation of sporogenous cells and the nursing cells surrounding them is conserved between angiosperm anthers and bryophyte sporangia. We made CRISPR-Cas9 reporter and loss-of-function lines to address the function of PpbHLH092/098. We sectioned and analyzed WT and mutant sporophytes for a comprehensive stage-by-stage comparison of sporangium development. Spore precursors in the P. patens sporangium are surrounded by nursing cells showing striking similarities to tapetal cells in angiosperms. Moss clade II bHLH TFs are essential for the differentiation of these tapetal-like cells and for the production of functional spores. Clade II bHLH TFs provide a conserved role in controlling the sporophytic somatic cells surrounding and nursing the sporogenous cells in both moss sporangia and angiosperm anthers. This supports the hypothesis that such nursing functions in mosses and angiosperms, lineages separated by c. 450 million years, are related by descent., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Previous studies in the liverwort Marchantia polymorpha have shown that the putative evening complex (EC) genes LUX ARRHYTHMO (LUX) and ELF4-LIKE (EFL) have a function in the liverwort circadian clock. Here, we studied the growth phenotypes of MpLUX and MpEFL loss-of-function mutants, to establish if PHYTOCHROME-INTERACTING FACTOR (PIF) and auxin act downstream of the M. polymorpha EC in a growth-related pathway similar to the one described for the flowering plant Arabidopsis. We examined growth rates and cell properties of loss-of-function mutants, analyzed protein-protein interactions and performed gene expression studies using reporter genes. Obtained data indicate that an EC can form in M. polymorpha and that this EC regulates growth of the thallus. Altered auxin levels in Mplux mutants could explain some of the phenotypes related to an increased thallus surface area. However, because MpPIF is not regulated by the EC, and because Mppif mutants do not show reduced growth, the growth phenotype of EC-mutants is likely not mediated via MpPIF. In Arabidopsis, the circadian clock regulates elongation growth via PIF and auxin, but this is likely not an evolutionarily conserved growth mechanism in land plants. Previous inventories of orthologs to Arabidopsis clock genes in various plant lineages showed that there is high levels of structural differences between clocks of different plant lineages. Here, we conclude that there is also variation in the output pathways used by the different plant clocks to control growth and development., Competing Interests: The authors have declared that no competing interests exist.

Although land plant germ cells have received much attention, knowledge about their specification is still limited. We thus identified transcripts enriched in egg cells of the bryophyte model species Physcomitrium patens, compared the results with angiosperm egg cells, and selected important candidate genes for functional analysis. We used laser-assisted microdissection to perform a cell-type-specific transcriptome analysis on egg cells for comparison with available expression profiles of vegetative tissues and male reproductive organs. We made reporter lines and knockout mutants of the two BONOBO (PbBNB) genes and studied their role in reproduction. We observed an overlap in gene activity between bryophyte and angiosperm egg cells, but also clear differences. Strikingly, several processes that are male-germline specific in Arabidopsis are active in the P. patens egg cell. Among those were the moss PbBNB genes, which control proliferation and identity of both female and male germlines. Pathways shared between male and female germlines were most likely present in the common ancestors of land plants, besides sex-specifying factors. A set of genes may also be involved in the switches between the diploid and haploid moss generations. Nonangiosperm gene networks also contribute to the specification of the P. patens egg cell., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

The plant hormone auxin is a key factor for regulation of plant development, and this function was probably reinforced during the evolution of early land plants. We have extended the available toolbox to allow detailed studies of how auxin biosynthesis and responses are regulated in moss reproductive organs, their stem cells and gametes to better elucidate the function of auxin in the morphogenesis of early land plants. We measured auxin metabolites and identified IPyA (indole-3-pyruvic acid) as the main biosynthesis pathway in Physcomitrium (Physcomitrella) patens and established knock-out, overexpressor and reporter lines for biosynthesis genes which were analyzed alongside previously reported auxin-sensing and transport reporters. Vegetative and reproductive apical stem cells synthesize auxin. Sustained stem cell activity depends on an inability to sense the auxin produced while progeny of the stem cells respond to the auxin, aiding in the control of cell division, expansion and differentiation. Gamete precursors are dependent on a certain degree of auxin sensing, while the final differentiation is a low auxin-sensing process. Tha data presented indicate that low auxin activity may represent a conserved hallmark of land plant gametes, and that local auxin biosynthesis in apical stem cells may be part of an ancestral mechanism to control focal growth., (© 2020 The Authors New Phytologist © 2020 New Phytologist Trust.)

Efforts to reveal ancestral functions of auxin, a key regulator of plant growth and development, and its importance for evolution have been hampered by a fragmented picture of auxin response domains in early-diverging land plants. We report the mapping of auxin sensing and responses during vegetative moss development using novel reporters. We established a moss-specific ratiometric reporter (PpR2D2) for Auxin Response Element- and AUXIN RESPONSE FACTOR-independent auxin sensing in Physcomitrella patens, and its readout during vegetative development was compared with new promoter-based GmGH3::GFPGUS and DR5revV2::GFPGUS auxin response reporters. The ratiometric reporter responds rapidly to auxin in a time-, dose- and TRANSPORT INHIBITOR RESISTANT1/AUXIN F-BOX-dependent manner and marks known, anticipated and novel auxin sensing domains. It reveals proximal auxin sensing maxima in filamentous tissues and sensing minima in all five vegetative gametophytic stem cell types as well as dividing cells. PpR2D2 readout is compliant with an ancestral function of auxin as a positive regulator of differentiation vs proliferation in stem cell regions. The PpR2D2 reporter is a sensitive tool for high-resolution mapping of auxin sensing, which can increase our knowledge of auxin function in early-diverging land plants substantially, thereby advancing our understanding of its importance for plant evolution., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Auxin phytohormones control most aspects of plant development through a complex and interconnected signaling network. In the presence of auxin, AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) transcriptional repressors are targeted for degradation by the SKP1-CULLIN1-F-BOX (SCF) ubiquitin-protein ligases containing TRANSPORT INHIBITOR RESISTANT 1/AUXIN SIGNALING F-BOX (TIR1/AFB). CULLIN1-neddylation is required for SCF TIR1/AFB functionality, as exemplified by mutants deficient in the NEDD8-activating enzyme subunit AUXIN-RESISTANT 1 (AXR1). Here, we report a chemical biology screen that identifies small molecules requiring AXR1 to modulate plant development. We selected four molecules of interest, RubNeddin 1 to 4 (RN1 to -4), among which RN3 and RN4 trigger selective auxin responses at transcriptional, biochemical, and morphological levels. This selective activity is explained by their ability to consistently promote the interaction between TIR1 and a specific subset of AUX/IAA proteins, stimulating the degradation of particular AUX/IAA combinations. Finally, we performed a genetic screen using RN4, the RN with the greatest potential for dissecting auxin perception, which revealed that the chromatin remodeling ATPase BRAHMA is implicated in auxin-mediated apical hook development. These results demonstrate the power of selective auxin agonists to dissect auxin perception for plant developmental functions, as well as offering opportunities to discover new molecular players involved in auxin responses., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

The signalling molecule auxin regulates many fundamental aspects of growth and development in plants. We review and discuss what is known about auxin-regulated development in mosses, with special emphasis on the model species Physcomitrella patens. It is well established that mosses and other early diverging plants produce and respond to auxin. By sequencing the P. patens genome, it became clear that it encodes many core proteins important for auxin homeostasis, perception, and signalling, which have also been identified in flowering plants. This suggests that the auxin molecular network was present in the last common ancestor of flowering plants and mosses. Despite fundamental differences in their life cycles, key processes such as organ initiation and outgrowth, branching, tropic responses, as well as cell differentiation, division, and expansion appear to be regulated by auxin in the two lineages. This knowledge paves the way for studies aimed at a better understanding of the origin and evolution of auxin function and how auxin may have contributed to the evolution of land plants., (© The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Autophagy, a major catabolic process in eukaryotes, was initially related to cell tolerance to nutrient depletion. In plants autophagy has also been widely related to tolerance to biotic and abiotic stresses (through the induction or repression of programmed cell death, PCD) as well as to promotion of developmentally regulated PCD, starch degradation or caloric restriction important for life span. Much less is known regarding its role in plant cell differentiation. Here we show that macroautophagy, the autophagy pathway driven by engulfment of cytoplasmic components by autophagosomes and its subsequent degradation in vacuoles, is highly active during germ cell differentiation in the early diverging land plant Physcomitrella patens. Our data provide evidence that suppression of ATG5-mediated autophagy results in reduced density of the egg cell-mediated mucilage that surrounds the mature egg, pointing toward a potential role of autophagy in extracellular mucilage formation. In addition, we found that ATG5- and ATG7-mediated autophagy is essential for the differentiation and cytoplasmic reduction of the flagellated motile sperm and hence for sperm fertility. The similarities between the need of macroautophagy for sperm differentiation in moss and mouse are striking, strongly pointing toward an ancestral function of autophagy not only as a protector against nutrient stress, but also in gamete differentiation.

Catabacter hongkongensis was isolated and cultured from human blood for the first time in Scandinavia. The patient, an 83-year-old man from Dalarna, Sweden, recovered without antibiotic treatment, although a high mortality rate associated with C. hongkongensis infection had been reported from China, Canada and France. The genome of the strain ABBA15k was sequenced, assembled and analysed. In contrast to the type strain of the species HKU16 T , no antibiotic resistance was observed in Scandinavian strain ABBA15k. The strain was deposited as CCUG 68271, and the draft genome sequence is available from the DNA Data Bank of Japan (DDBJ), the European Molecular Biology Laboratory (EMBL), and GenBank under the accession number LLYX00000000.

The emergence and radiation of multicellular land plants was driven by crucial innovations to their body plans. The directional transport of the phytohormone auxin represents a key, plant-specific mechanism for polarization and patterning in complex seed plants. Here, we show that already in the early diverging land plant lineage, as exemplified by the moss Physcomitrella patens, auxin transport by PIN transporters is operational and diversified into ER-localized and plasma membrane-localized PIN proteins. Gain-of-function and loss-of-function analyses revealed that PIN-dependent intercellular auxin transport in Physcomitrella mediates crucial developmental transitions in tip-growing filaments and waves of polarization and differentiation in leaf-like structures. Plasma membrane PIN proteins localize in a polar manner to the tips of moss filaments, revealing an unexpected relation between polarization mechanisms in moss tip-growing cells and multicellular tissues of seed plants. Our results trace the origins of polarization and auxin-mediated patterning mechanisms and highlight the crucial role of polarized auxin transport during the evolution of multicellular land plants., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In order to establish a reference for analysis of the function of auxin and the auxin biosynthesis regulators SHORT INTERNODE/STYLISH (SHI/STY) during Physcomitrella patens reproductive development, we have described male (antheridial) and female(archegonial) development in detail, including temporal and positional information of organ initiation. This has allowed us to define discrete stages of organ morphogenesis and to show that reproductive organ development in P. patens is highly organized and that organ phyllotaxis differs between vegetative and reproductive development. Using the PpSHI1 and PpSHI2 reporter and knockout lines, the auxin reporters GmGH3(pro):GUS and PpPINA(pro):GFP-GUS, and the auxin-conjugating transgene PpSHI2(pro):IAAL, we could show that the PpSHI genes, and by inference also auxin, play important roles for reproductive organ development in moss. The PpSHI genes are required for the apical opening of the reproductive organs, the final differentiation of the egg cell, and the progression of canal cells into a cell death program. The apical cells of the archegonium, the canal cells, and the egg cell are also sites of auxin responsiveness and are affected by reduced levels of active auxin, suggesting that auxin mediates PpSHI function in the reproductive organs.

Investigation of the production of secondary metabolites of Heterobasidion occidentale led to the isolation and identification of six sesquiterpenes (illudolone A and B, illudolactone A and B, deoxyfomannosin A and B) along with the well-known sesquiterpene fomannosin and the previously described benzohydrofuran fomannoxin. The structures and relative configurations of the compounds were determined by 1D and 2D NMR spectroscopic analysis as well as by HRMS. Their absolute configuration and biosynthesis were suggested and discussed in relation to fomannosin. Four compounds showed growth inhibiting activity against several basidiomycetes, Phlebiopsis gigantea, Phanerochaete chrysosporium and H. occidentale, and toxicity towards the moss Physcomitrella patens. In addition, one compound displayed activity against the bacterium Variovorax paradoxus as well as against the ascomycete Fusarium oxysporum., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Hexokinase catalyzes the phosphorylation of glucose and fructose, but it is also involved in sugar sensing in both fungi and plants. We have previously described two types of hexokinases in the moss Physcomitrella. Type A, exemplified by PpHxk1, the major hexokinase in Physcomitrella, is a soluble protein that localizes to the chloroplast stroma. Type B, exemplified by PpHxk2, has an N-terminal membrane anchor. Both types are found also in vascular plants, and localize to the chloroplast stroma and mitochondrial membranes, respectively., Results: We have now characterized all 11 hexokinase encoding genes in Physcomitrella. Based on their N-terminal sequences and intracellular localizations, three of the encoded proteins are type A hexokinases and four are type B hexokinases. One of the type B hexokinases has a splice variant without a membrane anchor, that localizes to the cytosol and the nucleus. However, we also found two new types of hexokinases with no obvious orthologs in vascular plants. Type C, encoded by a single gene, has neither transit peptide nor membrane anchor, and is found in the cytosol and in the nucleus. Type D hexokinases, encoded by three genes, have membrane anchors and localize to mitochondrial membranes, but their sequences differ from those of the type B hexokinases. Interestingly, all moss hexokinases are more similar to each other in overall sequence than to hexokinases from other plants, even though characteristic sequence motifs such as the membrane anchor of the type B hexokinases are highly conserved between moss and vascular plants, indicating a common origin for hexokinases of the same type., Conclusions: We conclude that the hexokinase gene family is more diverse in Physcomitrella, encoding two additional types of hexokinases that are absent in vascular plants. In particular, the presence of a cytosolic and nuclear hexokinase (type C) sets Physcomitrella apart from vascular plants, and instead resembles yeast, where all hexokinases localize to the cytosol. The fact that all moss hexokinases are more similar to each other than to hexokinases from vascular plants, even though both type A and type B hexokinases are present in all plants, further suggests that the hexokinase gene family in Physcomitrella has undergone concerted evolution.

The plant hormone auxin plays fundamental roles in vascular plants. Although exogenous auxin also stimulates developmental transitions and growth in non-vascular plants, the effects of manipulating endogenous auxin levels have thus far not been reported. Here, we have altered the levels and sites of auxin production and accumulation in the moss Physcomitrella patens by changing the expression level of homologues of the Arabidopsis SHI/STY family proteins, which are positive regulators of auxin biosynthesis genes. Constitutive expression of PpSHI1 resulted in elevated auxin levels, increased and ectopic expression of the auxin response reporter GmGH3pro:GUS, and in an increased caulonema/chloronema ratio, an effect also induced by exogenous auxin application. In addition, we observed premature ageing and necrosis in cells ectopically expressing PpSHI1. Knockout of either of the two PpSHI genes resulted in reduced auxin levels and auxin biosynthesis rates in leafy shoots, reduced internode elongation, delayed ageing, a decreased caulonema/chloronema ratio and an increased number of axillary hairs, which constitute potential auxin biosynthesis sites. Some of the identified auxin functions appear to be analogous in vascular and non-vascular plants. Furthermore, the spatiotemporal expression of the PpSHI genes and GmGH3pro:GUS strongly overlap, suggesting that local auxin biosynthesis is important for the regulation of auxin peak formation in non-vascular plants.

The establishment and maintenance of auxin maxima in vascular plants is regulated by auxin biosynthesis and polar intercellular auxin flow. The disruption of normal auxin biosynthesis in mouse-ear cress (Arabidopsis thaliana) leads to severe abnormalities, suggesting that spatiotemporal regulation of auxin biosynthesis is fundamental for normal growth and development. We have shown previously that the induction of the SHORT-INTERNODES/STYLISH (SHI/STY) family member STY1 results in increased transcript levels of the YUCCA (YUC) family member YUC4 and also higher auxin levels and auxin biosynthesis rates in Arabidopsis seedlings. We have also shown previously that SHI/STY family members redundantly affect development of flowers and leaves. Here, we further examine the function of STY1 by analyzing its DNA and protein binding properties. Our results suggest that STY1, and most likely other SHI/STY members, are DNA binding transcriptional activators that target genes encoding proteins mediating auxin biosynthesis. This suggests that the SHI/STY family members are essential regulators of auxin-mediated leaf and flower development. Furthermore, the lack of a shoot apical meristem in seedlings carrying a fusion construct between STY1 and a repressor domain, SRDX, suggests that STY1, and other SHI/STY members, has a role in the formation and/or maintenance of the shoot apical meristem, possibly by regulating auxin levels in the embryo.

Mosses (Bryophyta) are nonvascular plants that constitute a large part of the photosynthesizing biomass and carbon storage on Earth. Little is known about how this important portion of flora maintains its health status. This study assessed whether the moss, Physcomitrella patens, responds to treatment with chitosan, a fungal cell wall-derived compound inducing defense against fungal pathogens in vascular plants. Application of chitosan to liquid culture of P. patens caused a rapid increase in peroxidase activity in the medium. For identification of the peroxidase(s), matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF)/MS, other methods and the whole-genome sequence of P. patens were utilized. Peroxidase gene knock-out mutants were made and inoculated with fungi. The peroxidase activity resulted from a single secreted class III peroxidase (Prx34) which belonged to a P. patens specific phylogenetic cluster in analysis of the 45 putative class III peroxidases of P. patens and those of Arabidopsis and rice. Saprophytic and pathogenic fungi isolated from another moss killed the Prx34 knockout mutants but did not damage wild-type P. patens. The data point out the first specific host factor that is pivotal for pathogen defense in a nonvascular plant. Furthermore, results provide conclusive evidence that class III peroxidases in plants are needed in defense against hostile invasion by fungi.

The yeast Snf1, animal AMPK, and plant SnRK1 protein kinases constitute a family of related proteins that have been proposed to serve as metabolic sensors of the eukaryotic cell. We have previously reported the characterization of two redundant SnRK1 encoding genes (PpSNF1a and PpSNF1b) in the moss Physcomitrella patens. Phenotypic analysis of the snf1a snf1b double knockout mutant suggested that SnRK1 is important for the plant's ability to recognize and adapt to conditions of limited energy supply, and also suggested a possible role of SnRK1 in the control of plant development. We have now used a yeast two-hybrid system to screen for PpSnf1a interacting proteins. Two new moss genes were found, PpSKI1 and PpSKI2, which encode highly similar proteins with homologues in vascular plants. Fusions of the two encoded proteins to the green fluorescent protein localize to the nucleus. Knockout mutants for either gene have an excess of gametophores under low light conditions, and exhibit reduced gametophore stem lengths. Possible functions of the new proteins and their connection to the SnRK1 kinase are discussed.

The yeast Snf1 protein kinase and its animal homologue, the AMP-activated protein kinase, play important roles in metabolic regulation, by serving as energy gauges that turn off energy-consuming processes and mobilize energy reserves during low-energy conditions. The closest homologue of these kinases in plants is Snf1-related protein kinase 1 (SnRK1). We have cloned two SnRK1-encoding genes, PpSNF1a and PpSNF1b, in the moss Physcomitrella patens, where gene function can be studied directly by gene targeting in the haploid gametophyte. A snf1a snf1b double knockout mutant is viable, but lacks all Snf1-like protein kinase activity. The mutant has a complex phenotype that includes developmental abnormalities, premature senescence and altered sensitivities to plant hormones. Remarkably, the double knockout mutant also requires continuous light, and is unable to grow in a normal day-night light cycle. This suggests that SnRK1 is needed for metabolic changes that help the plant cope with the dark hours of the night.

Hexokinase catalyzes the first step in the metabolism of glucose but has also been proposed to be involved in sugar sensing and signaling both in yeast and in plants. We have cloned a hexokinase gene, PpHXK1, in the moss Physcomitrella patens where gene function can be studied directly by gene targeting. PpHxk1 is a novel type of chloroplast stromal hexokinase that differs from previously studied membrane-bound plant hexokinases. Enzyme assays on a knock-out mutant revealed that PpHxk1 is the major glucose-phosphorylating enzyme in Physcomitrella, accounting for 80% of the total activity in protonemal tissue. The mutant is deficient in the response to glucose, which in wild type moss induces the formation of caulonemal filaments that protrude from the edge of the colony. Growth on glucose in the dark is strongly reduced in the mutant. Sequence data suggest that most plants including Physcomitrella and Arabidopsis have both chloroplast-imported hexokinases similar to PpHxk1 and traditional membrane-bound hexokinases. We propose that the two types of plant hexokinases have distinct physiological roles.

We describe a method for identifying signal transducing proteins from other organisms by their ability to turn on a signalling pathway when they are expressed at high level in yeast. The method was tested on a cDNA library from Arabidopsis thaliana, which was screened for clones that can activate glucose repression in the absence of glucose. Six clones were characterized. One of them codes for AtGRH1, a new F-box protein that shows similarity to GRR1, a yeast protein involved in glucose repression. The ability of AtGRHI to activate glucose repression is dependent on the MIG1 repressor. Two-hybrid experiments revealed that AtGRH1 can interact with AtSKP1a and AtSKP1b, two recently identified SKP1 homologues in Arabidopsis. Other clones identified in the screen encode the transcription factor AtEBP, the 14-3-3 protein AtGF14 and two new proteins: AtMYR1 and AtPOZ1. None of these proteins turn on glucose repression. Instead, they illustrate various other ways by which foreign proteins can interfere with expression of a yeast gene. We conclude that our method worked as expected in at least one case, and that it could be applied to other signalling pathways that are conserved between yeast and higher eukaryotes.

Two S-adenosylmethionine synthetase (SAMS) cDNAs, PcSAMS1 and PcSAMS2, have been identified in Pinus contorta. We found that the two genes are differentially expressed during root development. Thus, PcSAMS1 is preferentially expressed in roots and exhibits a specific expression pattern in the meristem at the onset of adventitious root development, whereas PcSAMS2 is expressed in roots as well as in shoots and is down-regulated during adventitious root formation. The expression of the two SAMS genes is different from the SAMS activity levels during adventitious root formation. We conclude that other SAMS genes that remain to be characterized may contribute to the observed SAMS activity, or that the activities of PcSAMS1 and PcSAMS2 are affected by post-transcriptional regulation. The deduced amino acid sequences of PcSAMS1 and PcSAMS2 are highly divergent, suggesting different functional roles. However, both carry the two perfectly conserved motifs that are common to all plant SAMS. At the protein level, PcSAMS2 shares about 90% identity to other isolated eukaryotic SAMS, while PcSAMS1 shares less than 50% identity with other plant SAMS. In a phylogenetic comparison, PcSAMS1 seems to have diverged significantly from all other SAMS genes. Nevertheless, PcSAMS1 was able to complement a Saccharomyces cerevisiae sam1 sam2 double mutant, indicating that it encodes a functional SAMS enzyme.

A high proportion of purebred Hampshire pigs carries the dominant RN- mutation, which causes high glycogen content in skeletal muscle. The mutation has beneficial effects on meat content but detrimental effects on processing yield. Here, it is shown that the mutation is a nonconservative substitution (R200Q) in the PRKAG3 gene, which encodes a muscle-specific isoform of the regulatory gamma subunit of adenosine monophosphate-activated protein kinase (AMPK). Loss-of-function mutations in the homologous gene in yeast (SNF4) cause defects in glucose metabolism, including glycogen storage. Further analysis of the PRKAG3 signaling pathway may provide insights into muscle physiology as well as the pathogenesis of noninsulin-dependent diabetes mellitus in humans, a metabolic disorder associated with impaired glycogen synthesis.

The three-dimensional structure of mouse ribonucleotide reductase R2 has been determined at 2.3 A resolution using molecular replacement and refined to an R-value of 19.1% (Rfree = 25%) with good stereo-chemistry. The overall tertiary structure architecture of mouse R2 is similar to that from Escherichia coli R2. However, several important structural differences are observed. Unlike the E. coli protein, the mouse dimer is completely devoid of beta-strands. The sequences differ significantly between the mouse and E. coli R2s, but there is high sequence identity among the eukaryotic R2 proteins, and the identities are localized over the whole sequence. Therefore, the three-dimensional structures of other mammalian ribonucleotide reductase R2 proteins are expected to be very similar to that of the mouse enzyme. In mouse R2 a narrow hydrophobic channel leads to the proposed binding site for molecular oxygen near to the iron-radical site in the interior of the protein. In E. coli R2 this channel is blocked by the phenyl ring of a tyrosine residue, which in mouse R2 is a serine. These structural variations may explain the observed differences in sensitivity to radical scavengers. The structure determination is based on diffraction data from crystals grown at pH 4.7. Unexpectedly, the protein is not iron-free, but contains one iron ion bound at one of the dinuclear iron sites. This ferric ion is bound with partial occupancy and is coordinated by three glutamic acids (one bidentate) and one histidine in a bipyramidal coordination that has a free apical coordination position. Soaking of crystals in a solution of ferrous salt at pH 4.7 increased the occupancy on the already occupied site, but without any detectable binding at the second site.

The R2 protein component of mouse ribonucleotide reductase has been obtained from overproducing Escherichia coli bacteria. It has been crystallized using NaCl as precipitant. The crystals are orthorhombic, space group C222(1), with cell dimensions a = 76.9 A, b = 108.9 A, c = 92.7 A and diffract to at least 2.5 A. The asymmetric unit of the crystals contains one monomer. Rotation and translation function searches using a model based on the weakly homologous E. coli R2 gave one significant peak. Rotation about a crystallographic 2-fold axis parallel to the a-axis produces an R2 dimer with dimer interactions very similar to those found for E. coli R2.

A radical-free preparation of a highly purified ribonucleotide reductase from calf thymus was shown to generate an M2-specific tyrosine free radical on incubation with iron and dithiothreitol in the presence of air. The radical is essential for activity but once formed has a half-life of about 10 min. Using the calf thymus enzyme, there is a continual requirement of oxygen and iron for ribonucleotide reduction indicating a continual regeneration of the radical during enzyme catalysis. We therefore propose that one way a cell may regulate ribonucleotide reductase activity is by controlling the generation of M2-specific tyrosine free radicals within existing M2 molecules.