[Show abstract][Hide abstract] ABSTRACT: This article presents a broad overview of techniques, both analytical and technological, that are used in systems biology to analyze large-scale data sets. While focusing on the computational aspects, the associated biology of cell regulation is also discussed. Discussions are centered around two widely used high-throughput technologies, namely, DNA microarrays and MS-based proteomics. The biological principles behind these techniques are presented. Typical steps involved in generation of high-throughput data to final biological interpretations of analyzed results are discussed. Various attempts to derive systems-level biological networks utilizing microarray and proteomics data are also presented.
IEEE control systems 01/2011; · 2.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We show in bryophytes that abscisic acid (ABA) pretreatment of moss (Physcomitrella patens) cells confers desiccation tolerance. In angiosperms, both ABA and the transcriptional regulator ABSCISIC ACID INSENSITIVE 3 (ABI3) are required to protect the seed during desiccation. ABA was not able to protect moss cells in stable deletion lines of ABI3 (DeltaPpabi3). Hence, moss has the same functional link between ABA, ABI3, and the desiccation tolerance phenotype that is found in angiosperms. Furthermore, we identified 22 genes that were induced during ABA pretreatment in wild-type lines. When their expression was compared with that of DeltaPpabi3 during ABA pretreatment and immediately after desiccation, a new target of ABI3 action appears to be in the recovery period.
[Show abstract][Hide abstract] ABSTRACT: Single-repeat R3 MYB transcription factors like CPC (CAPRICE) are known to play roles in developmental processes such as root hair differentiation and trichome initiation. However, none of the six Arabidopsis single-repeat R3 MYB members has been reported to regulate flavonoid biosynthesis. We show here that CPC is a negative regulator of anthocyanin biosynthesis. In the process of using CPC to test GAL4-dependent driver lines, we observed a repression of anthocyanin synthesis upon GAL4-mediated CPC overexpression. We demonstrated that this is not due to an increase in nutrient uptake because of more root hairs. Rather, CPC expression level tightly controls anthocyanin accumulation. Microarray analysis on the whole genome showed that, of 37 000 features tested, 85 genes are repressed greater than three-fold by CPC overexpression. Of these 85, seven are late anthocyanin biosynthesis genes. Also, anthocyanin synthesis genes were shown to be down-regulated in 35S::CPC overexpression plants. Transient expression results suggest that CPC competes with the R2R3-MYB transcription factor PAP1/2, which is an activator of anthocyanin biosynthesis genes. This report adds anthocyanin biosynthesis to the set of programs that are under CPC control, indicating that this regulator is not only for developmental programs (e.g. root hairs, trichomes), but can influence anthocyanin pigment synthesis.
[Show abstract][Hide abstract] ABSTRACT: INTRODUCTION This article includes a series of methods for culturing the moss Physcomitrella patens at all stages of its life cycle. Gametophytes are axenically cultured on solid agar-based media and in shaken liquid cultures. For long-term storage of gametophytes, cultures are maintained on solid medium at 10 degrees C in a very short day. Cryopreservation may also be used. Finally, sporophytes are generated by self-fertilization and sexual crossing.
Cold Spring Harbor Protocols 02/2009; 2009(2):pdb.prot5136. · 4.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: INTRODUCTION In this protocol, the transformation of moss (Physcomitrella patens) protoplasts is performed via Agrobacterium-mediated transfer of T-DNA. Protoplasts are incubated with Agrobacterium and acetoseringone in regeneration medium. They are then washed and plated on antibiotic-containing medium to select for T-DNA insertion in stable transformants. The transformation rate for this protocol is typically 10(-4) (expressed as the frequency of stable transformants among regenerants surviving the transformation procedure).
Cold Spring Harbor Protocols 02/2009; 2009(2):pdb.prot5144. · 4.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: INTRODUCTION This method makes use of biolistic bombardment to propel gold particles coated with DNA into protonemal tissue of the moss Physcomitrella patens. It is especially suitable for transient gene expression studies, but it can be used to obtain stable transformants.
Cold Spring Harbor Protocols 02/2009; 2009(2):pdb.prot5145. · 4.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: INTRODUCTION This protocol describes how to isolate individual protoplasts from young gametophyte tissue of the moss Physcomitrella patens and how to regenerate them into plants if desired. Protonemal tissue is grown on cellophane-overlay plates, harvested, and treated with 0.5% Driselase to produce protoplasts. After filtration and washing, the protoplasts are regenerated on solid medium.
Cold Spring Harbor Protocols 02/2009; 2009(2):pdb.prot5140. · 4.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: INTRODUCTION As an alternative to sexual crossing, protoplasts from two strains of moss (Physcomitrella patens) can be hybridized using polyethylene glycol (PEG). Although the efficiency is low, it requires no sophisticated apparatus. Hybrids are readily obtained using complementary auxotrophic mutants or strains with transgenic antibiotic resistance markers. It is now routine to obtain hybrids using transgenic strains that are hygromycin- or G418-resistant by selecting hybrids on medium containing both antibiotics.
Cold Spring Harbor Protocols 02/2009; 2009(2):pdb.prot5141. · 4.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: INTRODUCTION This protocol describes how to mutagenize spores and protonemal tissue from moss (Physcomitrella patens) using chemicals or ultraviolet (UV) light. Spores are mutagenized using the alkylating agents N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and ethyl methanesulfonate (EMS), and protonemal tissue is mutagenized with NTG and UV light. Compared to alkylating agents, UV is less effective as a mutagen, but it may be advantageous because it is less hazardous and may not lead to clustered lesions.
Cold Spring Harbor Protocols 02/2009; 2009(2):pdb.prot5142. · 4.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: INTRODUCTION The moss Physcomitrella patens has been used as an experimental organism for more than 80 years. Within the last 15 years, its use as a model to explore plant functions has increased enormously. The ability to use gene targeting and RNA interference methods to study gene function, the availability of many tools for comparative and functional genomics (including a sequenced and assembled genome, physical and genetic maps, and more than 250,000 expressed sequence tags [ESTs]), and a dominant haploid phase that allows direct forward genetic analysis have all led to a surge of new activity. P. patens can be easily cultured and spends the majority of its life cycle in the haploid state, allowing the application of experimental techniques similar to those used in microbes and yeast. Its development is relatively simple, and it generates only a few tissues that contain a limited number of cell types. Although mosses lack vascular tissue, true roots/stems/leaves, and flowers and seeds, many signaling pathways found in angiosperms are intact in moss. For example, the phytohormones auxin, cytokinin, and abscisic acid, as well as the photomorphogenic pigments phytochrome and cryptochrome, are all interwoven into distinct but overlapping pathways and linked to clear developmental phenotypes. In addition, about one-quarter of the moss genome contains genes with no known function based on sequence motifs, raising the likelihood of successful discovery efforts to identify new and novel gene functions.
Cold Spring Harbor Protocols 02/2009; 2009(2):pdb.emo115. · 4.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: INTRODUCTION This protocol describes a series of procedures for isolating nucleic acids (DNA and RNA) and proteins from moss (Physcomitrella patens) tissue. This series includes a rapid, small-scale procedure for isolating DNA, which results in genomic DNA that is only suitable for polymerase chain reaction (PCR), as well as a method for obtaining much larger amounts of genomic DNA suitable for Southern analysis. The latter method makes use of the Nucleon PhytoPure Genomic DNA Extraction Kit (GE Healthcare). The RNA extraction procedure uses the Plant RNA Isolation Mini Kit (Agilent) with some modifications. A method for extracting proteins from moss gametophytes is also presented.
Cold Spring Harbor Protocols 02/2009; 2009(2):pdb.prot5146. · 4.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: INTRODUCTION This protocol describes how to transform moss (Physcomitrella patens) protoplasts using polyethylene glycol (PEG)-mediated DNA uptake. The transformation rates for direct uptake by protoplasts of DNA with and without genomic sequence (a targeting construct) are typically 10(-5) and 10(-3), respectively. (These are the frequencies of stable transformants among regenerants surviving the transformation procedure.).
Cold Spring Harbor Protocols 02/2009; 2009(2):pdb.prot5143. · 4.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sensors and regulatory circuits that maintain redox homeostasis play a central role in adjusting plant metabolism and development to changing environmental conditions. We report here control networks in Arabidopsis (Arabidopsis thaliana) that respond to photosynthetic stress. We independently subjected Arabidopsis leaves to two commonly used photosystem II inhibitors: high light (HL) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Microarray analysis of expression patterns during the period of redox adjustment to these inhibitors reveals that 20% and 8% of the transcriptome are under HL and DCMU regulation, respectively. Approximately 6% comprise a subset of genes common to both perturbations, the redox responsive genes (RRGs). A redox network was generated in an attempt to identify genes whose expression is tightly coordinated during adjustment to homeostasis, using expression of these RRGs under HL conditions. Ten subnetworks were identified from the network. Hierarchal subclustering of subnetworks responding to the DCMU stress identified novel groups of genes that were tightly controlled while adjusting to homeostasis. Upstream analysis of the promoters of the genes in these clusters revealed different motifs for each subnetwork, including motifs that were previously identified with responses to other stresses, such as light, dehydration, or abscisic acid. Functional categorization of RRGs demonstrated involvement of genes in many metabolic pathways, including several families of transcription factors, especially those in the AP2 family. Using a T-DNA insertion in one AP2 transcription factor (redox-responsive transcription factor 1 [RRTF1]) from the RRGs, we showed that the genes predicted to be within the subnetwork containing RRTF1 were changed in this insertion line (Deltarrtf1). Furthermore, Deltarrtf1 showed greater sensitivity to photosynthetic stress compared to the wild type.
[Show abstract][Hide abstract] ABSTRACT: Presenilins (PS) provide the catalytic activity for gamma-secretase, which cleaves physiologically relevant substrates including Notch, ErbB4, and APP. Recent genetic studies indicated that the contribution of PS1 to mouse development includes gamma-secretase-independent functions that cannot be easily explained by any of the demonstrated or hypothesized functions of this protein. To begin a nonbiased analysis of PS1 activity unencumbered by the dominant effect stemming from loss of Notch function, we characterized PS functions in the early land plant Physcomitrella patens, which lacks Notch, ErbB4, and APP. Removal of P. patens PS resulted in phenotypic abnormalities. Further assays performed to delineate the defective pathways in PS-deficient P. patens implicated improper function of the cytoskeletal network. Importantly, this characterization of a nonmetazoan PS uncovered a previously undescribed, evolutionarily conserved function (human PS1 can rescue the growth and light responses) that is gamma-secretase-independent (mutants with substitutions of the catalytic aspartyl residues retain the activity). Introduction of PpPS into PS-deficient mouse embryonic fibroblasts rescues normal growth rates, demonstrating that at least some metazoan functions of PS are evolutionarily conserved.
Proceedings of the National Academy of Sciences 09/2007; 104(33):13337-42. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The sequenced genome of the moss Physcomitrella patens provides a powerful tool for comparative analyses of land plant genomes. In parallel, several tools for studying gene function have been developed in P. patens, including RNA interference, inducible promoters and gene targeting, a unique attribute of this plant system. The results of these initiatives are now being realized. For example, transcriptomic analyses illustrate commonalities among plant lineages in gene content, structure, and regulation. Transgenic studies show that the regulatory factors ABSCISIC ACID INSENSITIVE3 (ABI3) and LEAFY (LFY) have molecular functions that are conserved between moss and angiosperms, in spite of the fact that they function in non-homologous tissues. Future work in P. patens will contribute to our understanding of the molecular basis of plant development and evolution.
Current Opinion in Plant Biology 05/2007; 10(2):182-9. · 8.46 Impact Factor