Lang D, Ullrich KK, Murat F, Fuchs J, Jenkins J, Haas FB, Piednoel M, Gundlach H, Van Bel M, Meyberg R, Vives C, Morata J, Symeonidi A, Hiss M, Muchero W, Kamisugi Y, Saleh O, Blanc G, Decker EL, Van Gessel N, Grimwood J, Hayes RD, Graham SW, Gunter LE, McDaniel SF, Hoernstein SNW, Larsson A, Li F-W, Perroud P-F, Phillips J, Ranjan P, Rokshar DS, Rothfels CJ, Schneider L, Shu S, Stevenson DW, Thümmler F, Tillich M, Villarreal Aguilar JC, Widiez T, Wong GK-S, Wymore A, Zhang YA, Zimmer AD, Quatrano RS, Mayer KFX, Goodstein D, Casacuberta JM, Vandepoele K, Reski R, Cuming AC, Tuskan GA, Maumus F, Salse J, Schmutz J, Rensing SA The Physcomitrella patens chromosome‐scale assembly reveals moss genome structure and evolution Plant Journal 93 515-533, 2018
DOI:10.1111/tpj.13801
View abstract
The draft genome of the moss model, Physcomitrella patens, comprised approximately 2000 unordered scaffolds. In order to enable analyses of genome structure and evolution we generated a chromosome‐scale genome assembly using genetic linkage as well as (end) sequencing of long DNA fragments. We find that 57% of the genome comprises transposable elements (TEs), some of which may be actively transposing during the life cycle. Unlike in flowering plant genomes, gene‐ and TE‐rich regions show an overall even distribution along the chromosomes. However, the chromosomes are mono‐centric with peaks of a class of Copia elements potentially coinciding with centromeres. Gene body methylation is evident in 5.7% of the protein‐coding genes, typically coinciding with low GC and low expression. Some giant virus insertions are transcriptionally active and might protect gametes from viral infection via siRNA mediated silencing. Structure‐based detection methods show that the genome evolved via two rounds of whole genome duplications (WGDs), apparently common in mosses but not in liverworts and hornworts. Several hundred genes are present in colinear regions conserved since the last common ancestor of plants. These syntenic regions are enriched for functions related to plant‐specific cell growth and tissue organization. The P. patens genome lacks the TE‐rich pericentromeric and gene‐rich distal regions typical for most flowering plant genomes. More non‐seed plant genomes are needed to unravel how plant genomes evolve, and to understand whether the P. patens genome structure is typical for mosses or bryophytes.
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Cross LL, Paudyal R, Kamisugi Y, Berry A, Cuming AC, Baker A, Warriner SL Towards designer organelles by subverting the peroxisomal import pathway Nature Communications 8, 2017
DOI:10.1038/s41467-017-00487-7
View abstract
© 2017 The Author(s). The development of 'designer' organelles could be a key strategy to enable foreign pathways to be efficiently controlled within eukaryotic biotechnology. A fundamental component of any such system will be the implementation of a bespoke protein import pathway that can selectively deliver constituent proteins to the new compartment in the presence of existing endogenous trafficking systems. Here we show that the protein-protein interactions that control the peroxisomal protein import pathway can be manipulated to create a pair of interacting partners that still support protein import in moss cells, but are orthogonal to the naturally occurring pathways. In addition to providing a valuable experimental tool to give new insights into peroxisomal protein import, the variant receptor-signal sequence pair forms the basis of a system in which normal peroxisomal function is downregulated and replaced with an alternative pathway, an essential first step in the creation of a designer organelle.
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Chater CC, Caine RS, Tomek M, Wallace S, Kamisugi Y, Cuming AC, Lang D, MacAlister CA, Casson S, Bergmann DC, Decker EL, Frank W, Gray JE, Fleming A, Reski R, Beerling DJ Origin and function of stomata in the moss Physcomitrella patens Nature Plants 2, 2016
DOI:10.1038/nplants.2016.179
View abstract
� 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. Stomata are microscopic valves on plant surfaces that originated over 400 million years (Myr) ago and facilitated the greening of Earth's continents by permitting efficient shoot-atmosphere gas exchange and planthydration 1. However, the core genetic machinery regulating stomatal development in non-vascular land plants is poorly understood 2-4 and their function has remained a matter of debate for a century 5. Here, we show that genes encoding the two basic helix-loop-helix proteins PpSMF1 (SPEECH, MUTE and FAMA-like) and PpSCREAM1 (SCRM1) in the moss Physcomitrella patens are orthologous to transcriptional regulators of stomatal development in the flowering plant Arabidopsis thaliana and essential for stomata formation in moss. Targeted P. patens knockout mutants lacking either PpSMF1 or PpSCRM1 develop gametophytes indistinguishable from wild-type plants but mutant sporophytes lack stomata. Protein-protein interaction assays reveal heterodimerization between PpSMF1 and PpSCRM1, which, together with moss-angiosperm gene complementations 6, suggests deep functional conservation of the heterodimeric SMF1 and SCRM1 unit is required to activate transcription for moss stomatal development, as in A. thaliana 7. Moreover, stomata-less sporophytes of ΔPpSMF1 and ΔPpSCRM1 mutants exhibited delayed dehiscence, implying stomata might have promoted dehiscence in the first complex land-plant sporophytes.
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Cuming AC Gene tagging in Physcomitrella patens : an addition to the genetic toolbox New Phytologist 212 543-545, 2016
DOI:10.1111/nph.14201
Caine RS, Chater CC, Kamisugi Y, Cuming AC, Beerling DJ, Gray JE, Fleming AJ An ancestral stomatal patterning module revealed in the non-vascular land plant Physcomitrella patens Development 143 3306-3314, 2016
DOI:10.1242/dev.135038
View abstract
The patterning of stomata plays a vital role in plant development and has emerged as a paradigm for the role of peptide signals in the spatial control of cellular differentiation. Research in Arabidopsis has identified a series of Epidermal Patterning Factors (EPFs) which interact with an array of membrane-localised receptors and associated proteins (encoded by ERECTA and TMM genes) to control stomatal density and distribution. However, although it is well established that stomata arose very early in the evolution of land plants, until now it has been unclear whether the established angiosperm stomatal patterning system represented by the EPF/TMM/ERECTA module reflects a conserved, universal mechanism in the plant kingdom. Here, we use molecular genetics to show that the moss Physcomitrella patens has conserved homologues of angiosperm EPF, TMM and at least one ERECTA gene which function together to permit the correct patterning of stomata and that, moreover, elements of the module retain function when transferred to Arabidopsis. Our data characterise the stomatal patterning system in an evolutionary distinct branch of plants and support the hypothesis that the EPF/TMM/ERECTA module represents an ancient patterning system.
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Kamisugi Y, Whitaker JW, Cuming AC The transcriptional response to DNADouble-strand breaks in Physcomitrella patens PLoS ONE 11, 2016
DOI:10.1371/journal.pone.0161204
View abstract
� 2016 Kamisugi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The model bryophyte Physcomitrella patens is unique among plants in supporting the generation of mutant alleles by facile homologous recombination-mediated gene targeting (GT). Reasoning that targeted transgene integration occurs through the capture of transforming DNA by the homology-dependent pathway for DNA double-strand break (DNADSB) repair, we analysed the genome-wide transcriptomic response to bleomycin-induced DNA damage and generated mutants in candidate DNA repair genes. Massively parallel (Illumina) cDNA sequencing identified potential participants in gene targeting. Transcripts encoding DNA repair proteins active inmultiple repair pathways were significantly up-regulated. These included Rad51, CtIP, DNA ligase 1, Replication protein A and ATR in homology- dependent repair, Xrcc4, DNA ligase 4, Ku70 and Ku80 in non-homologous end-joining and Rad1, Tebichi/polymerase theta, PARP in microhomology-mediated end-joining. Differentially regulated cell-cycle components included up-regulated Rad9 and Hus1 DNA-damage- related checkpoint proteins and down-regulated D-type cyclins and B-type CDKs, commensurate with the imposition of a checkpoint at G 2 of the cell cycle characteristic of homology-dependent DNA-DSBrepair. Candidate genes, including ATP-dependent chromatin remodelling helicases associated with repair and recombination, were knocked out and analysed for growth defects, hypersensitivity to DNA damage and reduced GT efficiency. Targeted knockout of PpCtIP, a cell-cycle activated mediator of homology-dependent DSB resection, resulted in bleomycin-hypersensitivity and greatly reduced GT efficiency.
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Stevenson SR, Kamisugi Y, Trinh CH, Schmutz J, Jenkins JW, Grimwood J, Muchero W, Tuskan GA, Rensing SA, Lang D, Reski R, Melkonian M, Rothfels CJ, Li F-W, Larsson A, Wong GK-S, Edwards TA, Cuming AC Genetic analysis of Physcomitrella patens identifies ABSCISIC ACID NON-RESPONSIVE, a regulator of ABA responses unique to basal land plants and required for desiccation tolerance Plant Cell 28 1310-1327, 2016
DOI:10.1105/tpc.16.00091
View abstract
The anatomically simple plants that first colonised land must have acquired molecular and biochemical adaptations to drought stress. Abscisic acid (ABA) coordinates responses leading to desiccation tolerance in all land plants. We identified ABA non-responsive mutants in the model bryophyte Physcomitrella patens and genotyped a segregating population to map and identify the ABA NON-RESPONSIVE (ANR) gene encoding a modular protein kinase comprising an N-terminal PAS domain, a central EDR domain and a C-terminal MAPKKK like domain. Ppanr mutants fail to accumulate dehydration tolerance-associated gene products in response to drought, ABA or osmotic stress, and do not acquire ABA-dependent desiccation tolerance. The crystal structure of the PAS domain, determined to 1.7Å resolution, shows a conserved PAS-fold, and to dimerise with a weak dimerization interface. Targeted mutagenesis of a conserved tryptophan residue within the PAS domain generates plants with ABA non-responsive growth and strongly attenuated ABA-responsive gene expression, whereas deleting this domain retains a fully ABA responsive phenotype. ANR orthologs are found in early-diverging land plant lineages and aquatic algae, but are absent from more recently diverged vascular plants. We propose that ANR genes represent an ancestral adaptation enabling drought-stress survival of the first terrestrial colonisers, but were lost during land plant evolution.
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Kamisugi Y, Mitsuya S, El-Shami M, Knight CD, Cuming AC, Baker A Giant peroxisomes in a moss (Physcomitrella patens) peroxisomal biogenesis factor 11 mutant New Phytologist 209 576-589, 2016
DOI:10.1111/nph.13739
View abstract
© 2015 New Phytologist Trust. Peroxisomal biogenesis factor 11 (PEX11) proteins are found in yeasts, mammals and plants, and play a role in peroxisome morphology and regulation of peroxisome division. The moss Physcomitrella patens has six PEX11 isoforms which fall into two subfamilies, similar tothose found in monocots and dicots. We carried out targeted gene disruption of the Phypa_PEX11-1 gene and compared the morphological and cellular phenotypes of the wild-type and mutant strains. The mutant grew more slowly and the development of gametophores was retarded. Mutant chloronemal filamentscontained large cellular structures which excluded all other cellular organelles. Expression of fluorescent reporter proteins revealed that the mutant strain had greatly enlarged peroxisomes up to 10 μm in diameter. Expression of a vacuolar membrane marker confirmed that the enlarged structures were not vacuoles, or peroxisomes sequestered within vacuoles as a result of pexophagy. Phypa_PEX11 targeted to peroxisome membranes could rescue the knock out phenotype and interacted with Fission1 on the peroxisome membrane. Moss PEX11 functions in peroxisome division similar to PEX11 in other organisms but the mutant phenotype is more extreme and environmentally determined, making P. patens a powerful system in which to address mechanisms of peroxisome proliferation and division.
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McDaniel SF, Perroud PF, Cuming AC, Szövényi P The Ceratodon purpureus Transcriptome Ushers in the Era of Moss Comparative Genomics, 2016
DOI:10.1016/bs.abr.2016.02.003
View abstract
This chapter outlines the scope of the ongoing Ceratodon purpureus genome project and provides an overview of the C. purpureus transcriptome, the evolution of the C. purpureus UV sex chromosomes, and the patterns of polymorphism in the species. Comparative analyses of the transcriptomes of a male and a female isolate showed that C. purpureus and the moss model Physcomitrella patens had highly overlapping gene sets, and that most of the genes shared between these two species evolve under strong purifying selection. However, the differences between the C. purpureus and P. patens genomes refined our understanding of the timing of gene family gain and loss across the land plants and the heterogeneity in rate of molecular evolution across the genome of these two species. Ceratodon purpureus showed a slightly greater codon usage bias compared to P. patens, which may be explained by the contrasting mating system of the two species. The C. purpureus transcr iptomes also showed evidence of a genome doubling. event ~65-76 MYA that was independent of the contemporaneous polyploidy event inferred for P. patens. These data also suggested considerable physiological and developmental divergence between the two species. Genetic mapping and molecular evolutionary analysis showed that the nonrecombining UV chromosomes of C. purpureus are actively capturing new genes, illustrating that at least this part of the genome is highly dynamic. Moreover, patterns of polymorphism were highly variable across the genome, suggesting that sexual recombination in other parts of the genome decouples even genes on the same chromosome, and they experience different patterns of natural selection. The forthcoming C. purpureus genome will build on these existing resources and enable us to answer definitively many questions regarding the evolution of land plant gene families, genome structure, and the genetic basis of adaptive variation.© 2016 Elsevier Ltd.
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Wallace S, Chater CC, Kamisugi Y, Cuming AC, Wellman CH, Beerling DJ, Fleming AJ Conservation of Male Sterility 2 function during spore and pollen wall development supports an evolutionarily early recruitment of a core component in the sporopollenin biosynthetic pathway. New Phytol 205 390-401, 2015
DOI:10.1111/nph.13012
View abstract
The early evolution of plants required the acquisition of a number of key adaptations to overcome physiological difficulties associated with survival on land. One of these was a tough sporopollenin wall that enclosed reproductive propagules and provided protection from desiccation and UV-B radiation. All land plants possess such walled spores (or their derived homologue, pollen). We took a reverse genetics approach, consisting of knock-out and complementation experiments to test the functional conservation of the sporopollenin-associated gene MALE STERILTY 2 (which is essential for pollen wall development in Arabidopsis thaliana) in the bryophyte Physcomitrella patens. Knock-outs of a putative moss homologue of the A. thaliana MS2 gene, which is highly expressed in the moss sporophyte, led to spores with highly defective walls comparable to that observed in the A. thaliana ms2 mutant, and extremely compromised germination. Conversely, the moss MS2 gene could not rescue the A. thaliana ms2 phenotype. The results presented here suggest that a core component of the biochemical and developmental pathway required for angiosperm pollen wall development was recruited early in land plant evolution but the continued increase in pollen wall complexity observed in angiosperms has been accompanied by divergence in MS2 gene function.
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Charlot F, Chelysheva L, Kamisugi Y, Vrielynck N, Guyon A, Epert A, Le Guin S, Schaefer DG, Cuming AC, Grelon M, Nogué F RAD51B plays an essential role during somatic and meiotic recombination in Physcomitrella Nucleic Acids Research 42 11965-11978, 2014
DOI:10.1093/nar/gku890
View abstract
© The Author(s) 2014. The eukaryotic RecA homologue Rad51 is a key factor in homologous recombination and recombinational repair. Rad51-like proteins have been identified in yeast (Rad55, Rad57 and Dmc1), plants and vertebrates (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3 and DMC1). RAD51 and DMC1 are thestrandexchange proteins forming a nucleofilament for strand invasion, however, the function of the paralogues in the process of homologous recombination is less clear. In yeast the two Rad51 paralogues, Rad55 and Rad57, have been shown to be involved in somatic and meiotic HR and they are essentialto the formation of the Rad51/DNA nucleofilament counterbalancing the anti-recombinase activity of the SRS2 helicase. Here, we examined the role of RAD51B in the model bryophyte Physcomitrella patens. Mutant analysis shows that RAD51B is essential for the maintenance of genome integrity, for resistance to DNA damaging agents and for gene targeting. Furthermore, we set up methods to investigate meiosis in Physcomitrella and we demonstrate that the RAD51B protein is essential for meiotic homologous recombination. Finally, we show that all these functions are independent of the SRS2 anti-recombinase protein, which is in striking contrast to what is found in budding yeast where the RAD51 paralogues are fully dependent on the SRS2 anti-recombinase function.
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Szövényi P, Perroud P-F, Symeonidi A, Stevenson S, Quatrano RS, Rensing SA, Cuming AC, Mcdaniel SF De novo assembly and comparative analysis of the Ceratodon purpureus transcriptome Molecular Ecology Resources, 2014
DOI:10.1111/1755-0998.12284
View abstract
The bryophytes are a morphologically and ecologically diverse group of plants that have recently emerged as major model systems for a variety of biological processes. In particular, the genome sequence of the moss, Physcomitrella patens, has significantly enhanced our understanding of the evolution of developmental processes in land plants. However, to fully explore the diversity within bryophytes, we need additional genomic resources. Here, we describe analyses of the transcriptomes of a male and a female isolate of the moss, Ceratodon purpureus, generated using the 454 FLX technology. Comparative analyses between C. purpureus and P. patens indicated that this strategy generated nearly complete coverage of the protonemal transcriptome. An analysis of the overlap in gene sets between C. purpureus and P. patens provides new insights into the evolution of gene family composition across the land plants. In spite of the overall transcriptomic similarity between the two species, Ka/Ks analysis of P. patens and C. purpureus suggests considerable physiological and developmental divergence. Additionally, while the codon usage was very similar between these two mosses, C. purpureus genes showed a slightly greater codon usage bias than P. patens genes potentially because of the contrasting mating system of the two species. Finally, we found evidence of a genome doubling ~65-76 MYA that likely coincided with the contemporaneous polyploidy event inferred for P. patens but postdates the divergence of P. patens and C. purpureus. The powerful laboratory tools now available for C. purpureus will enable the research community to fully exploit these genomic resources.© 2014 John Wiley&Sons Ltd.
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Cove DJ, Cuming AC Genetics and Genomics of Moss Models: Physiology Enters the Twenty-first Century PHOTOSYNTHESIS IN BRYOPHYTES AND EARLY LAND PLANTS 37 187-199, 2014
DOI:10.1007/978-94-007-6988-5_11
Kamisugi Y, Schaefer DG, Kozak J, Charlot F, Vrielynck N, Holá M, Angelis KJ, Cuming AC, Nogué F MRE11 and RAD50, but not NBS1, are essential for gene targeting in the moss Physcomitrella patens. Nucleic Acids Res 40 3496-3510, 2012
DOI:10.1093/nar/gkr1272
View abstract
The moss Physcomitrella patens is unique among plant models for the high frequency with which targeted transgene insertion occurs via homologous recombination. Transgene integration is believed to utilize existing machinery for the detection and repair of DNA double-strand breaks (DSBs). We undertook targeted knockout of the Physcomitrella genes encoding components of the principal sensor of DNA DSBs, the MRN complex. Loss of function of PpMRE11 or PpRAD50 strongly and specifically inhibited gene targeting, whilst rates of untargeted transgene integration were relatively unaffected. In contrast, disruption of the PpNBS1 gene retained the wild-type capacity to integrate transforming DNA efficiently at homologous loci. Analysis of the kinetics of DNA-DSB repair in wild-type and mutant plants by single-nucleus agarose gel electrophoresis revealed that bleomycin-induced fragmentation of genomic DNA was repaired at approximately equal rates in each genotype, although both the Ppmre11 and Pprad50 mutants exhibited severely restricted growth and development and enhanced sensitivity to UV-B and bleomycin-induced DNA damage, compared with wild-type and Ppnbs1 plants. This implies that while extensive DNA repair can occur in the absence of a functional MRN complex; this is unsupervised in nature and results in the accumulation of deleterious mutations incompatible with normal growth and development.
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Chater C, Kamisugi Y, Movahedi M, Fleming A, Cuming AC, Gray JE, Beerling DJ Regulatory Mechanism Controlling Stomatal Behavior Conserved across 400 Million Years of Land Plant Evolution CURR BIOL 21 1025-1029, 2011
DOI:10.1016/j.cub.2011.04.032
Hooper EJ, Duckett JG, Cuming AC, Kunin WE, Pressel S Ephemerella readeri Mull. Hal. (Physcomitrella readeri (Mull. Hal.) I.G. Stone&G.A.M. Scott, Funariidae, Bryophyta): a genus and species new to Europe J BRYOL 32 256-264, 2010
DOI:10.1179/037366810X12814321877589
Caliskan M, Bashiardes S, Cuming AC A plasma membrane H(+)ATPase gene is germination-induced in wheat embryos AFR J BIOTECHNOL 9 268-272, 2010
Fraile-Escanciano A, Kamisugi Y, Cuming AC, Rodriguez-Navarro A, Benito B The SOS1 transporter of Physcomitrella patens mediates sodium efflux in planta NEW PHYTOL 188 750-761, 2010
DOI:10.1111/j.1469-8137.2010.03405.x
Cuming AC Moss as a model system for plant stress responses In Plant Stress Biology: From Genomics To Systems Biology, 2009
Cuming AC Mosses as Model Organisms for Developmental, Cellular and Molecular Biology In Bryophyte Biology, 2009
Cuming AC Plant-pathogen interactions: a view from the evolutionary basement NEW PHYTOL 183 237-239, 2009
Kamisugi Y, Cuming AC Gene targeting In The moss Physcomitrella, 2009
View abstract
Physcomitrella patens is model plant for comparative genomic analysis, and is the only plant model in which“gene targeting” – the replacement of an endogenous gene by a genetically altered variant transgene – occurs with high efficiency. We describe the parameters that determine the efficiency of gene targeting and discuss the nature of the integration events that occur following transformation of P. patens. Integration of transgenes utilizes endogenous mechanisms for the repair of DNA double-strand breaks, and the preferential integration of transforming DNA at homologous sites by Physcomitrella indicates that the predominating mechanism for DNA repair is based on homologous recombination, rather than non-homologous end-joining, as is the case in flowering plants. Physcomitrella thus provides a useful experimental model for the study of the HR-mediated DNA repair pathway in plants, and understanding how this mechanism operates in Physcomitrella may pave the way for the developmentof more efficient gene targeting strategies for the genetic modification of crop species.
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Kamisugi Y, von Stackelberg M, Lang D, Care M, Reski R, Rensing SA, Cuming AC A sequence-anchored genetic linkage map for the moss, Physcomitrella patens PLANT J 56 855-866, 2008
DOI:10.1111/j.1365-313X.2008.03637.x
Rensing SA, Lang D, Zimmer AD, Terry A, Salamov A, Shapiro H, Nishiyama T, Perroud PF, Lindquist EA, Kamisugi Y, Tanahashi T, Sakakibara K, Fujita T, Oishi K, Shin-I T, Kuroki Y, Toyoda A, Suzuki Y, Hashimoto S, Yamaguchi K, Sugano S, Kohara Y, Fujiyama A, Anterola A, Aoki S, Ashton N, Barbazuk WB, Barker E, Bennetzen JL, Blankenship R, Cho SH, Dutcher SK, Estelle M, Fawcett JA, Gundlach H, Hanada K, Heyl A, Hicks KA, Hughes J, Lohr M, Mayer K, Melkozernov A, Murata T, Nelson DR, Pils B, Prigge M, Reiss B, Renner T, Rombauts S, Rushton PJ, Sanderfoot A, Schween G, Shiu SH, Stueber K, Theodoulou FL, Tu H, Van de Peer Y, Verrier PJ, Waters E, Wood A, Yang LX, Cove D, Cuming AC, Hasebe M, Lucas S, Mishler BD, Reski R, Grigoriev IV, Quatrano RS, Boore JL The Physcomitrella genome reveals evolutionary insights into the conquest of land by plants SCIENCE 319 64-69, 2008
DOI:10.1126/science.1150646
Cuming AC, Cho SH, Kamisugi Y, Graham H, Quatrano RS Microarray analysis of transcriptional responses to abscisic acid and osmotic, salt, and drought stress in the moss, Physcomitrella patens NEW PHYTOL 176 275-287, 2007
DOI:10.1111/j.1469-8137.2007.02187.x
Kamisugi Y, Schlink K, Rensing SA, Schween G, von Stackelberg M, Cuming AC, Reski R, Cove DJ The mechanism of gene targeting in Physcomitrella patens: homologous recombination, concatenation and multiple integration NUCLEIC ACIDS RES 34 6205-6214, 2006
DOI:10.1093/nar/gkl832
Kamisugi Y, Cuming AC The evolution of the abscisic acid-response in land plants: comparative analysis of group 1 LEA gene expression in moss and cereals PLANT MOL BIOL 59 723-737, 2005
DOI:10.1007/s11103-005-0909-z
Kamisugi Y, Cuming AC, Cove DJ Parameters determining the efficiency of gene targeting in the moss Physcomitrella patens NUCLEIC ACIDS RES 33, 2005
DOI:10.1093/nar/gnil172
Caliskan M, Ozcan B, Turan C, Cuming AC Localization of germin genes and their products in developing wheat coleoptiles J BIOCHEM MOL BIOL 37 339-342, 2004
Caliskan M, Turet M, Cuming AC Formation of wheat (Triticum aestivum L.) embryogenic callus involves peroxide-generating germin-like oxalate oxidase PLANTA 219 132-140, 2004
DOI:10.1007/s00425-003-1199-9
Caliskan M, Turet-Sayin M, Turan C, Cuming AC Identification of germin isoforms in wheat callus CEREAL RES COMMUN 32 355-361, 2004
Caliskan M, Bashiardes S, Ozcan B, Cuming AC Isolation and localization of new germination-related sequences from wheat embryos J BIOCHEM MOL BIOL 36 580-585, 2003
Kamisugi Y, Cuming AC Genomes and databases In New frontiers in bryology– physiology, molecular biology and functional genomics, 2003
Knight CD, Cove DJ, Cuming AC, Quatrano RS Moss Gene Technology In Molecular PlantBiology - a practical approach, 2002
Machuka JS, Bashiardes S, Ruben E, Spooner K, Cuming AC, Knight CD, Cove DJ Sequence analysis of Expressed sequence tags from an ABA-treated cDNA library identifies stress response genes in the moss Physcomitrella patens. Plant and Cell Physiology 40 378-387, 1999
Caliskan M, Cuming AC Spatial specificity of H2O2-generating oxalate oxidase gene expression during wheat embryo germination PLANT J 15 165-171, 1998
ROBERTSON M, CUMING AC, CHANDLER PM SEQUENCE-ANALYSIS AND HORMONAL-REGULATION OF A DEHYDRIN PROMOTER FROM BARLEY, HORDEUM-VULGARE PHYSIOL PLANTARUM 94 470-478, 1995
KNIGHT CD, SEHGAL A, ATWAL K, WALLACE JC, COVE DJ, COATES D, QUATRANO RS, BAHADUR S, STOCKLEY PG, CUMING AC MOLECULAR RESPONSES TO ABSCISIC-ACID AND STRESS ARE CONSERVED BETWEEN MOSS AND CEREALS PLANT CELL 7 499-506, 1995
Robertson M, Cuming AC, Chandler PM Sequence analysis and hormonal regulation of a dehydrin promoter from barley, Hordeum vulgare Physiologia Plantarum 94 470-478, 1995
DOI:10.1111/j.1399-3054.1995.tb00956.x
View abstract
A dehydrin gene (dhn7) was isolated from a barley (Hordeum vulgare L. cv. Himalaya) genomic library. A 2138‐bp fragment of the gene was sequenced and the deduced amino acid sequence showed 99% identity with that of a previously identified cDNA, Dhn 1, which encodes one of a group of closely related proteins induced by dehydration stress. Transient expression assays in barley aleurone protoplasts defined regions of the dhn7 promoter that mediate abscisic acid (ABA) responsive transcription. A fragment (‐935 to +42) of the gene was found to confer concentration‐dependent ABA‐responsiveness measured by expression of a β‐glucuronidase (GUS) reporter gene. Analysis of a 5’deletion series ofthe promoter defined a region (‐285 to ‐233) that modulated the amplitude of the ABA‐response, whilst ABA‐responsiveness, per se, required a region (‐233 to ‐211) that included a conserved motif containing the G‐box‐like sequence TACGTGG. Gibberellic acid (GA 3 ) antagonised ABA‐induced accumulation of dehydrin mRNA in aleurone cells, but did not antagonise ABA‐induced transcriptional activation as revealed by GUS activity. The antagonistic effect of GA 3 on ABA‐induced dehydrin expression might therefore operate post‐transcriptionally. Copyright © 1995, Wiley Blackwell. All rights reserved
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ONDE S, FUTERS TS, CUMING AC RAPID ANALYSIS OF AN OSMOTIC-STRESS RESPONSIVE PROMOTER BY TRANSIENT EXPRESSION IN INTACT WHEAT EMBRYOS J EXP BOT 45 561-566, 1994
Futers TS, Onde S, Turet M, Cuming AC Sequence analysis of two tandemly linked Em genes from wheat. Plant Mol Biol 23 1067-1072, 1993
View abstract
DNA sequences are presented for two members of the wheat Em gene family. The sequences correspond to the two linked genes at the Xem-1AL locus. Comparisons of these sequences with that of another wheat Em gene and two Em cDNA clones reveals substantial homology within the protein-coding regions, and the presence in the 5'-flanking regions of the genomic sequences of motifs characteristic of ABA-responsive cis-acting elements.
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Taylor RM, Cuming AC Selective proteolysis of the wheat Em polypeptide. Identification of an endopeptidase activity in germinating wheat embryos. FEBS Lett 331 71-75, 1993
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The 'Em' polypeptide is the most abundant cytosolic polypeptide in mature wheat embryos. It is selectively and completely degraded within the first 24 h of germination. Extracts from germinated embryos contain endopeptidase activities which degrade the Em polypeptide. These are separable into a major and minor component by ion-exchange chromatography and the use of inhibitors shows the major component to be a cysteine proteinase. This activity shows a strong preference for the Em polypeptide as a substrate, being inactive against polypeptides which are not developmentally regulated and showing only low activity towards developmentally related, but otherwise nonhomologous 'dehydrin' polypeptides.
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Taylor RM, Cuming AC Purification of an endoproteinase that digests the wheat 'Em' protein in vitro, and determination of its cleavage sites. FEBS Lett 331 76-80, 1993
View abstract
Germinating wheat embryos contain two endoproteolytic activities which digest the prominent 'Em' polypeptide. These are easily assayed in clarified embryonic homogenates and are distinguishable by the pattern of their peptide products and by their different pH optima. One activity has a pH optimum of 4.0; the second activity is a cysteine endoproteinase with a preference for the 'Em' protein as its substrate. It is maximally active between pH 5.5 and 6 at 25 degrees C. Analysis of the early cleavage products of the cysteine proteinase indicates scissile bonds between residues Glu32-Ala33 and Asn36-Leu37 in the 'Em' polypeptide. This endoproteinase has been purified and identified as a single polypeptide species of ca. 38,000 kDa.
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LANE BG, DUNWELL JM, RAY JA, SCHMITT MR, CUMING AC GERMIN, A PROTEIN MARKER OF EARLY PLANT DEVELOPMENT, IS AN OXALATE OXIDASE J BIOL CHEM 268 12239-12242, 1993
BUTLER WM, CUMING AC DIFFERENTIAL MOLECULAR RESPONSES TO ABSCISIC-ACID AND OSMOTIC-STRESS IN VIVIPAROUS MAIZE EMBRYOS PLANTA 189 47-54, 1993
Lane BG, Cuming AC, Frégeau J, Carpita NC, Hurkman WJ, Bernier F, Dratewka-Kos E, Kennedy TD Germin isoforms are discrete temporal markers of wheat development. Pseudogermin is a uniquely thermostable water-soluble oligomeric protein in ungerminated embryos and like germin in germinated embryos, it is incorporated into cell walls. Eur J Biochem 209 961-969, 1992
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Nascent synthesis and accumulation of germin and its mRNA mark the onset of renewed growth when wheat embryos are germinated in water. Germin is a water-soluble, pepsin-resistant protein that is not found in immature embryos, or in mature embryos before their germination. An antiserum was raised by injecting rabbits with germin that was freed of other proteins by pepsinization and gel filtration. The antiserum has been used to detect, in extracts of mature embryos from dry, ungerminated wheat grains, a protein that is antigenically related to germin. The antigenically related protein has been named pseudogermin. Pseudogermin accumulates, maximally, between 20-25-days postanthesis, then declines appreciably in amount by 30-days postanthesis, in soluble extracts of immature embryos from several wheat varieties. The antiserum was also used to identify germin and pseudogermin among the proteins extracted from cell walls and to bind immunogold to cell walls preparatory to visualizing freeze-cleaved embryos by scanning electron microscopy. Wall-associated germin accounts for about 40% of the total germin in germinating wheat embryos. Appearance of germin in the apoplast is the most conspicuous germination-related change in the distribution of cell-wall proteins. It seems that germin may act at the level of the apoplast and that pseudogermin may subsume the role of germin at low water potentials during embryogenesis. The N-terminal eicosapeptide sequences in germin and pseudogermin are very similar but SDS/PAGE analysis detects discrete differences between the mobilities of their constituent monomers as well as gross differences between the stabilities of the parent oligomers. Like germin, pseudogermin is a water-soluble, pepsin-resistant protein, but pseudogermin has unprecedented disulphide-independent thermostability properties that have never been previously reported for a water-soluble oligomeric protein. Polysaccharides that co-purify with otherwise pure specimens of germin (and pseudogermin) have been isolated for analysis and shown to be highly substituted glucuronogalactoarabinoxylans. The possible biological significance of selective and tenacious association between germin and glucuronogalactoarabinoxylans is discussed in relation to cell expansion during embryogenic and germinative development of wheat, as are some peculiarities of amino-acid sequence that suggest a possible relation between germin and a proton-specific ion pump: gastric ATPase.
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Morris PC, Kumar A, Bowles DJ, Cuming AC Osmotic stress and abscisic acid induce expression of the wheat Em genes. Eur J Biochem 190 625-630, 1990
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The early-methionine-labelled (Em) polypeptide is the single most abundant cytosolic protein of dry wheat embryos. It is encoded by messenger RNA which accumulates during the later (maturation) stages of embryogenesis. The accumulation of Em mRNA can be induced in isolated developing embryos, in culture, by the application of the plant growth regulator, abscisic acid, which prevents precocious germination. Precocious germination is also inhibited by the culture of embryos under conditions of osmotic stress when accumulation of Em mRNA is induced. This induction occurs in the absence of any significant increase in the endogenous levels of embryonic abscisic acid although there is a requirement for the continued presence of the growth regulator. Additionally, expression of Em genes can be repeated during early germination, if imbibing embryos are subjected to osmotic stress. Induction of Em-gene expression by osmotic stress is consistent with the proposed role of the Em polypeptide in mediating the remarkable tolerance of cereal embryos to the programmed desiccation undergone during their maturation.
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FUTERS TS, VAUGHAN TJ, SHARP PJ, CUMING AC MOLECULAR-CLONING AND CHROMOSOMAL LOCATION OF GENES ENCODING THE EARLY-METHIONINE-LABELED (EM) POLYPEPTIDE OF TRITICUM-AESTIVUM L VAR CHINESE SPRING THEOR APPL GENET 80 43-48, 1990
WILLIAMSON JD, QUATRANO RS, CUMING AC EM POLYPEPTIDE AND ITS MESSENGER-RNA LEVELS ARE MODULATED BY ABSCISIC-ACID DURING EMBRYOGENESIS IN WHEAT EUR J BIOCHEM 152 501-507, 1985
CUMING AC DEVELOPMENTAL REGULATION OF GENE-EXPRESSION IN WHEAT EMBRYOS - MOLECULAR-CLONING OF A DNA-SEQUENCE ENCODING THE EARLY METHIONINE-LABELED (EM) POLYPEPTIDE EUR J BIOCHEM 145 351-357, 1984
CUMING AC CONTROL OF RIBOSOMAL-PROTEIN SYNTHESIS DURING WHEAT EMBRYO INBIBITION BIOCHIM BIOPHYS ACTA 783 42-52, 1984
CUMING AC, BENNETT J BIOSYNTHESIS OF THE LIGHT-HARVESTING CHLOROPHYLL A-B PROTEIN - CONTROL OF MESSENGER-RNA ACTIVITY BY LIGHT EUR J BIOCHEM 118 71-80, 1981
CUMING AC, KENNEDY TD, LANE BG WHEAT EMBRYO RIBONUCLEATES .14. MASS ISOLATION OF MESSENGER-RNA FROM WHEAT-GERM AND COMPARISON OF ITS TRANSLATIONAL CAPACITY WITH THAT OF MESSENGER-RNA FROM IMBIBING WHEAT EMBRYOS CAN J BIOCHEM CELL B 57 1170-1175, 1979
CUMING AC, LANE BG PROTEIN-SYNTHESIS IN IMBIBING WHEAT EMBRYOS EUR J BIOCHEM 99 217-224, 1979
CUMING AC, LANE BG WHEAT EMBRYO RIBONUCLEATES .11. CONSERVED MESSENGER-RNA IN DRY WHEAT EMBRYOS AND ITS RELATION TO PROTEIN-SYNTHESIS DURING EARLY IMBIBITION CAN J BIOCHEM CELL B 56 365-&, 1978
CUMING AC, OSBORNE DJ MEMBRANE TURNOVER IN IMBIBED AND DORMANT EMBRYOS OF WILD OAT (AVENA-FATUA L) .1. PROTEIN TURNOVER AND MEMBRANE REPLACEMENT PLANTA 139 209-217, 1978
CUMING AC, OSBORNE DJ MEMBRANE TURNOVER IN IMBIBED DORMANT EMBRYOS OF WILD OAT (AVENA-FATUA L) .2. PHOSPHOLIPID TURNOVER AND MEMBRANE REPLACEMENT PLANTA 139 219-226, 1978