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SummaryThe Arabidopsis Plant HomeoDomain (PHD) proteins AtMS1 and AtMMD1 provide chromatin-mediated transcriptional regulation essential for tapetum-dependent pollen formation. Such 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-homologs 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 cell autonomous 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.

Shoot branching mechanisms where branches arise in association with leaves – referred to as lateral or axillary branching – evolved by convergence in the sporophyte of vascular plants and the gametophyte of bryophytes, and accompanied independent events of plant architectural diversification1. Previously, we showed that three hormonal cues, including auxin, have been recruited independently to co-ordinate branch patterning in flowering plant leafy shoots and moss gametophores (Coudert, Palubicki et al., 2015)2–4. Moreover, auxin-mediated apical dominance, which relies on local auxin production, has been proposed as a unifying molecular regulatory mechanism of branch development across land plants5. Whilst our previous work in the moss Physcomitrium patens has gathered indirect evidence supporting the notion that auxin synthesized in gametophore apices regulates branch formation at a distance2, direct genetic evidence for a role of auxin biosynthesis in gametophore branching control is still lacking. Here, we show that gametophore apex decapitation promotes branch emergence through massive and rapid transcriptional reprogramming of auxin-responsive genes and altering auxin biosynthesis gene activity. Specifically, we identify a subset of P. patens TRYPTOPHAN AMINO-TRANSFERASE (TAR) and YUCCA FLAVIN MONOOXYGENASE-LIKE (YUC) auxin biosynthesis genes expressed in apical and basal regions of the gametophore, and show that they are essential for branch initiation and outgrowth control. Our results demonstrate that local auxin biosynthesis coordinates branch patterning in moss and thus constitutes a shared and ancient feature of shoot architecture control in land plants.

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.

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 roughly 450 million years, are related by descent.

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.

Significance The plant hormone auxin coordinates almost all aspects of plant development. Throughout plant life, the expression of hundreds of genes involved in auxin regulation is orchestrated via several combinatorial and cell-specific auxin perception systems. An effective approach to dissect these complex pathways is the use of synthetic molecules that target specific processes of auxin activity. Here, we describe synthetic auxins, RubNeddins (RNs), which act as selective auxin agonists. The RN with the greatest potential for dissecting auxin perception was RN4, which we used to reveal a role for the chromatin remodeling ATPase BRAHMA in apical hook development. Therefore, the understanding of RN mode of action paves the way to dissecting specific molecular components involved in auxin-regulated developmental processes., 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 SCFTIR1/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.

Plant Biology, Department of Biology, University of Fribourg,1700 Fribourg, SwitzerlandSummaryThe emergence and radiation of multicellular land plantswas driven by crucial innovations to their body plans [1].The directional transport of the phytohormone auxin repre-sents a key, plant-specific mechanism for polarization andpatterning in complex seed plants [2–5]. Here, we showthat already in the early diverging land plant lineage, asexemplifiedbythemossPhyscomitrellapatens,auxintrans-port by PIN transporters is operational and diversified intoER-localized and plasma membrane-localized PIN proteins.Gain-of-function and loss-of-function analyses revealedthat PIN-dependent intercellular auxin transport in Phys-comitrella mediates crucial developmental transitions intip-growing filaments and waves of polarization and differ-entiation in leaf-like structures. Plasma membrane PIN pro-teinslocalizeinapolarmannertothetipsofmossfilaments,revealing an unexpected relation between polarizationmechanisms in moss tip-growing cells and multicellulartissues of seed plants. Our results trace the origins of polar-ization and auxin-mediated patterning mechanisms andhighlight the crucial role of polarized auxin transport duringthe evolution of multicellular land plants.Results and DiscussionDuring plant diversification, a spectacular evolutionary transi-tion from anatomically simple green algae to developmentallycomplex multicellular land plants took place, presumably inorder to adapt to new and challenging environments [1].The phytohormone auxin is the most versatile plant-specificsignal that governs many crucial aspects of the seed plantbody organization [2]. A unique property of auxin amongplant signaling molecules is its directional (polar) transportthrough tissues, which is essential for most auxin-regulateddevelopmental processes, such as the establishment ofthe polarity axis during embryogenesis, de novo formationof organs, and vascular tissue formation [3–5]. Auxin trans-port depends largely on specific auxin transporters, namelythe PIN-FORMED (PIN) proteins [6]. Their typically asymmet-rical (polar) localization at the plasma membrane (PM)determines the direction of auxin flow between cells [7],which in turn provides directional and positional informationfor the development of multicellular tissues by linkinginformation at the level of individual cells to a coordinateddevelopmental output [8]. On account of its universalroles in polarization and patterning processes in seedplants, it is believed that this PIN-mediated auxin transportplayed a key role in important developmental innovationsduring the diversification of land plants [9, 10]. However,in planta data on the evolution of auxin transport andpolarization machineries in early diverging land plants aremissing.In order to reconstruct the role of PIN proteins during landplant evolution, we used the moss model species Physcomi-trella patens, which is a representative of one of the earliestdiverging lineages of land plants [11]. Similar to that of theangiosperm Arabidopsis, the P. patens genome encodes twodifferent types of PIN proteins, characterized by either a shortor a long hydrophylic loop between the transmembrane re-gions (Figure 1A), the latter designated as the canonical PINprotein [12, 13]. We assessed the auxin transport capabilitiesof these different PIN proteins by several means. We per-formed auxin transport assays using radioactively labeledsubstrates in mesophyll protoplasts from transfected Nico-tiana benthamiana leaves [14] and found that P. patens PINswith a long and short loop enhanced the export of indole-3-acetic acid (IAA), but not the chemically related benzoic acid(Figure S1F available online). Using root hair growth as an es-tablished measure of auxin export capacities, we showed thatoverexpression (OE) of long moss PINs in Arabidopsis, similarto OE of a typical PM-localized long PIN from Arabidopsis(AtPIN1), inhibits root hair growth, suggesting action in stimu-latingauxinefflux.Incontrast,OEoftheshortmossPINdidnotaffect root hair growth, similar to OE of the ER-localized, shortAtPIN5 from Arabidopsis [15](Figures 1B and S1G). Finally,P. patens filaments export auxin [16, 17] that can be detectedin the cultivation medium (Figure 1C). Using this in vivo assay,we observed that OE of moss and Arabidopsis PINs stronglyenhancedauxinexportintothemedium.Also,adoublemutantlinein long mossPINs(pinapinb)slightly reduced auxinexportinto the medium (Figures 1C, S1A–S1E, and S1H). Theseresults suggest that already in one of the early divergingland plant lineages, similar to seed plants, the PIN proteinsdiversified into long PINs with auxin export function andshort PINs with presumable roles in auxin homeostasis andmetabolism.

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.

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 GmGH3pro:GUS and PpPINApro:GFP-GUS, and the auxin-conjugating transgene PpSHI2pro: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.

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.

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.

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 AtGRH1 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 γ 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 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.

Since a proper balance between anabolic and catabolic reactions is essential for all eukaryotes, the basic mechanisms for regulation of the energy and carbon metabolism have been conserved throughout evolution. The moss Physcomitrella patens, which belongs to one of the basal clades among land plants, has many unique properties that make it an excellent plant model system. We have used a yeast two-hybrid system to identify novel possible regulators or targets of the moss Snf1-related kinases, previously shown to regulate energy homeostasis. The function of the identified interactors PpSki1 and PpSki2 was analyzed in order to better understand the biological role of plant Snf1-related kinases. The recently completed genome sequence of Physcomitrella was used in a comparative approach to study to what extent key enzyme and gene families involved in transport and metabolism of sugars and in regulation of the energy and carbon metabolism are conserved between mosses and vascular plants. It has long been known that transformed DNA can replicate episomally in Physcomitrella. We have now shown that such DNA can be rescued back into E. coli. Surprisingly, we found that the original plasmid can be recovered from moss transformants obtained with circular DNA. Plasmids rescued from transformants obtained with linearized DNA had been repaired either by homologous recombination or by cohesive end re-ligation. These findings suggest that methods using shuttle plasmids are feasible in Physcomitrella. Hexokinase, a key enzyme in the carbon metabolism, catalyzes the first step in hexose metabolism, but is also involved in sugar sensing and signaling. We have now made an initial characterization of the complete hexokinase family in Physcomitrella which is encoded by 11 genes. Two new types of plant hexokinases, types C and D, were found in addition to the previously described types A and B.

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 filamentous gametophyte of the moss Physcomitrella patens consists of two filament types called chloronemata and caulonemata. Chloronemal cells are photosynthetically active with numerous chloroplasts, while caulonemata help to spread the colony by radial growth. The balance between the two filament types is affected by external factors such as light and plant hormones. In the present study, caulonema formation and chloronemal branching have been monitored during high and low light conditions and in the presence of glucose, auxin, or cytokinin. These experiments were performed both in a wild-type strain and in a hxk1 knockout mutant which lacks the major hexokinase of Physcomitrella. It was found that caulonema formation is induced by high energy conditions such as high light and external glucose, while chloronemal branching is stimulated by low energy conditions such as reduced light, and in the hxk1 mutant. The hxk1 mutation also causes buds to appear on chloronemal filaments, which is rarely seen in the wild type, and shows increased sensitivity to cytokinin and abscisic acid. Based on these findings a model is proposed in which the energy supply of the moss colony regulates the balance between chloronemal and caulonemal growth.

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.

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.

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.