Digital Gene Expression Profiling by 5′-End Sequencing of cDNAs during Reprogramming in the Moss Physcomitrella patens

Stem cells self-renew and repeatedly produce differentiated cells during development and growth. The differentiated cells can be converted into stem cells in some metazoans and land plants with appropriate treatments. After leaves of the moss Physcomitrella patens are excised, leaf cells reenter the cell cycle and commence tip growth, which is characteristic of stem cells called chloronema apical cells. To understand the underlying molecular mechanisms, a digital gene expression profiling method using mRNA 5′-end tags (5′-DGE) was established. The 5′-DGE method produced reproducible data with a dynamic range of four orders that correlated well with qRT-PCR measurements. After the excision of leaves, the expression levels of 11% of the transcripts changed significantly within 6 h. Genes involved in stress responses and proteolysis were induced and those involved in metabolism, including photosynthesis, were reduced. The later processes of reprogramming involved photosynthesis recovery and higher macromolecule biosynthesis, including of RNA and proteins. Auxin and cytokinin signaling pathways, which are activated during stem cell formation via callus in flowering plants, are also activated during reprogramming in P. patens, although no exogenous phytohormone is applied in the moss system, suggesting that an intrinsic phytohormone regulatory system may be used in the moss

Characterization of CYCLOIDEA-like genes in controlling floral zygomorphy in the monocotyledon Alstroemeria

The CYCLOIDEA (CYC) gene controls the development of zygomorphic flowers and the determination of adaxial identity of floral organs in the model developmental system of Antirrhinum majus. However, whether CYC homologue genes also control floral zygomorphy in monocotyledon Alstroemeria plants is yet unknown. In this study, we investigated CYC-like genes in the monocotyledons Alstroemeria aurea, Alstroemeria magenta, and Alstroemeria pelegrina var. rosea, all of which have zygomorphic flowers. Since the CYC gene belongs to the T-complex protein (TCP) gene family of transcription factors, cloning of CYC-like sequences was performed using rapid amplification of cDNA ends (RACE)-polymerase chain reaction (PCR) by using degenerate primers designed for the TCP domain. We cloned 1 CYC-like sequence each from A. aurea (AaTCP1, accession number AB714967 in the GenBank/EMBL/DDBJ databases) and A. magenta (AmTCP1, AB714970), and two CYC-like sequences from A. pelegrina var. rosea (ApTCP1, AB714968; and ApTCP2, AB714969). The deduced amino acid sequences of AaTCP1, AmTCP1, ApTCP1, and ApTCP2 shared 67.7%, 67.7%, 71.0%, and 64.5% identities, respectively, with the TCP domain in CYC. Molecular phylogenetic analysis indicated that three CYC-like genes from Alstroemeria belonged to the ZinTBL1b clade in the CYC-/tb1-like subfamily. Reverse transcription (RT)-PCR and in situ hybridization analyses showed that AaTCP1 transcripts were specifically detected in flower buds and localized in the base of adaxial inner perianth of A. aurea. These results suggest that CYC-like genes are also involved in the development of floral asymmetry and the determination of adaxial identity of floral organs in the monocotyledon Alstroemeria

Photoreaction Dynamics of LOV1 and LOV2 of Phototropin from Chlamydomonas reinhardtii

Phototropin is a blue light sensor protein found in higher plants and green algae. Photochemical reactions of a variety of differently truncated constructs of a phototropin from Chlamydomonas reinhardtii (<i>Cr</i>) (LOV1, LOV1-hinge, LOV2, LOV2-linker, and hinge-LOV2) are investigated. In the dark state, LOV1 is in dynamic equilibrium between the monomer and dimer, and the main photochemical reaction is dimerization of the monomer and dissociation of the dimer. On the other hand, LOV1-hinge exists as the monomer and the photochemical reaction is the dimerization reaction associated with the unfolding of the helix of the hinge domain. LOV2 in the dark state is monomeric. The conformation changes after the photoexcitation of LOV2 and LOV2-linker are minor, which differs notably from the reaction of LOV2-Jα and LOV2-linker from Arabidopsis thaliana (<i>At</i>). The linker region, including the Jα helix, is rather stable upon photoexcitation. The helix of the hinge domain of hinge-LOV2 is slightly unfolded in the dark state, and the major photoreaction is the dimerization event. The dark recovery rate of LOV2 was found to decrease significantly in the presence of the hinge domain. These photochemical properties of <i>Cr</i> phot are considerably different from those of <i>At</i> phot regarding conformational changes and their kinetics, although <i>Cr</i> phot has been reported to rescue the phot function in <i>At</i>. The differences and the diversity of phots are discussed

Workflow for 5′-DGE library preparation.

<p>(a) 1st strand cDNA is synthesized from mRNA. (b) At the 5′ end of the mRNA, cDNA synthesis continues onto the DNA/RNA chimeric oligonucleotide. (c) Three-cycle PCR is performed to produce the double-stranded cDNA. (d) the double-strand cDNA fragments are digested by EcoP15I. (e) Digested P2-attached 5′ tag fragments are captured by streptavidin-magnet beads and ligated with P1 adapter. (f) 5′-DGE library is amplified, and 97 bp fragments are purified after PAGE.</p

Reprogramming from leaf cells to chloronema apical cells.

<p>(<b>A</b>) Upper parts of gametophores. (<b>B–D</b>, <b>F–P</b>) Dissected leaves after indicated time. (<b>E</b>) Intact leaf. Bars in <b>A</b>: 1 mm; <b>B</b>: 1 mm for <b>B–D</b> and <b>I–L</b>; <b>E</b>: 0.2 mm for <b>E–H</b> and <b>M–P</b>.</p

Expression patterns of transcription factors, epigenetic-related and reprogramming-related genes.

<p>(<b>A</b>) Heat map representation of expression patterns during reprogramming for reprogramming-related genes. Numbers in parentheses indicate JGI protein identifiers. (<b>B</b>) Expression patterns of transcription factor genes sorted according to gene expression pattern tree. Four distinct clusters are identified to the left. (<b>C</b>) Expression patterns of epigenetic-related genes.</p