Abha Khandelwal

Role of ABA and ABI3 in Desiccation Tolerance

The hormone pathway that stabilizes seeds may have served more primitive seedless plants in supporting desiccation tolerance. 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 (ΔPpabi3). 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 ΔPpabi3 during ABA pretreatment and immediately after desiccation, a new target of ABI3 action appears to be in the recovery period.

Arabidopsis Transcriptome Reveals Control Circuits Regulating Redox Homeostasis and the Role of an AP2 Transcription Factor

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 (Δrrtf1). Furthermore, Δrrtf1 showed greater sensitivity to photosynthetic stress compared to the wild type.

Moonlighting activity of presenilin in plants is independent of γ-secretase and evolutionarily conserved

Presenilins (PS) provide the catalytic activity for γ-secretase, which cleaves physiologically relevant substrates including Notch, ErbB4, and APP. Recent genetic studies indicated that the contribution of PS1 to mouse development includes γ-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 γ-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.

The moss Physcomitrella patens: a novel model system for plant development and genomic studies.

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 >250,000 expressed sequence tags), 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. The methods outlined in this chapter will enhance the use of the P. patens model system in many laboratories throughout the world. David J. Cove, Pierre-François Perroud, Audra J. Charron, Stuart F. McDaniel, Abha Khandelwal, and Ralph S. Quatrano Department of Biology, Washington University, St. Louis, Missouri 63130 P R O TO CO L S 1 Culturing the Moss Physcomitrella patens, 75 2 Isolation and Regeneration of Protoplasts, 80 3 Somatic Hybridization in P. patens Using PEGinduced Protoplast Fusion, 82 4 Chemical and UV Mutagenesis of Spores and Protonemal Tissue, 84 5 Transformation Using Direct DNA Uptake by Protoplasts, 87 6 Transformation Using T-DNA Mutagenesis, 89 7 Transformation of Gametophytes Using a Biolistic Projectile Delivery System, 91 8 Isolation of DNA, RNA, and Protein from P. patens Gametophytes, 93 This chapter, with full-color images, can be found online at www.cshprotocols.org/emo.

Culturing the moss Physcomitrella patens.

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°C in a very short day. Cryopreservation may also be used. Finally, sporophytes are generated by self-fertilization and sexual crossing.

Isolation and regeneration of protoplasts of the moss Physcomitrella patens.

This method is adapted from a protocol described by Grimsley et al. (1977). For more information about P. patens as a model organism, see The Moss Physcomitrella patens: A Novel Model System for Plant Development and Genomic Studies (Cove et al. 2009a). For details about the growth of P. patens on cellophane overlay plates, see Culturing the Moss Physcomitrella patens (Cove et al. 2009b). For protocols describing the use of P. patens protoplasts after they have been isolated, see Somatic Hybridization in the Moss Physcomitrella patens Using PEG-Induced Protoplast Fusion (Cove et al. 2009c), Transformation of the Moss Physcomitrella patens Using Direct DNA Uptake by Protoplasts (Cove et al. 2009d), and Transformation of the Moss Physcomitrella patens Using T-DNA Mutagenesis (Cove et al. 2009e). For details on the use of a hemocytometer to estimate protoplast density, see Estimation of Cell Number by Hemocytometry Counting (Sambrook and Russell 2006).

Transformation of the moss Physcomitrella patens using direct DNA uptake by protoplasts.

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 and 10, respectively. (These are the frequencies of stable transformants among regenerants surviving the transformation procedure.)

Isolation of DNA, RNA, and Protein from the Moss Physcomitrella patens Gametophytes

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.

Transformation of the moss Physcomitrella patens using T-DNA mutagenesis.

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 (expressed as the frequency of stable transformants among regenerants surviving the transformation procedure).

Transformation of moss Physcomitrella patens gametophytes using a biolistic projectile delivery system.

RELATED INFORMATION For more information about P. patens as a model organism, see The Moss Physcomitrella patens: A Novel Model System for Plant Development and Genomic Studies (Cove et al. 2009a). The growth of protonemal tissue is described in Culturing the Moss Physcomitrella patens (Cove et al. 2009b), and a method for isolation of P. patens protoplasts is found in Isolation and Regeneration of Protoplasts of the Moss Physcomitrella patens (Cove et al. 2009c).