Tomomichi Fujita

Comparative genomics of Physcomitrella patens gametophytic transcriptome and Arabidopsis thaliana: Implication for land plant evolution

The mosses and flowering plants diverged >400 million years ago. The mosses have haploid-dominant life cycles, whereas the flowering plants are diploid-dominant. The common ancestors of land plants have been inferred to be haploid-dominant, suggesting that genes used in the diploid body of flowering plants were recruited from the genes used in the haploid body of the ancestors during the evolution of land plants. To assess this evolutionary hypothesis, we constructed an EST library of the moss Physcomitrella patens, and compared the moss transcriptome to the genome of Arabidopsis thaliana. We constructed full-length enriched cDNA libraries from auxin-treated, cytokinin-treated, and untreated gametophytes of P. patens, and sequenced both ends of >40,000 clones. These data, together with the mRNA sequences in the public databases, were assembled into 15,883 putative transcripts. Sequence comparisons of A. thaliana and P. patens showed that at least 66% of the A. thaliana genes had homologues in P. patens. Comparison of the P. patens putative transcripts with all known proteins, revealed 9,907 putative transcripts with high levels of similarity to vascular plant genes, and 850 putative transcripts with high levels of similarity to other organisms. The haploid transcriptome of P. patens appears to be quite similar to the A. thaliana genome, supporting the evolutionary hypothesis. Our study also revealed that a number of genes are moss specific and were lost in the flowering plant lineage.

An Improved RNA Isolation Method for Plant Tissues Containing High Levels of Phenolic Compounds or Carbohydrates

Difficulties extracting high-quality RNA from recalcitrant plant tissues are often due to high levels of phenolics, carbohydrates, or other compounds that bind and/or co-precipitate with RNA. We describe here a method using soluble polyvinylpyrrolidone (PVP) and ethanol precipitation, which has been successful in several recalcitrant systems where other specialized RNA extraction methods failed to deliver suitable product. Using this method, RNA capable of reverse-transcription/PCR amplification and cDNA library construction was isolated from ripening grape berries, dry seeds of Albizia procera and radish, and leaf tissue of A. procera and Griffonia simplicifolia. This method is applicable to a variety of plant tissues.

Convergent evolution of shoots in land plants: lack of auxin polar transport in moss shoots.

SUMMARY The shoot is a repeated structure made up of stems and leaves and is the basic body plan in land plants. Vascular plants form a shoot in the diploid generation, whereas nonvascular plants such as mosses form a shoot in the haploid generation. It is not clear whether all land plants use similar molecular mechanisms in shoot development or how the genetic networks for shoot development evolved. The control of auxin distribution, especially by polar auxin transport, is essential for shoot development in flowering plants. We did not detect polar auxin transport in the gametophytic shoots of several mosses, but did detect it in the sporophytes of mosses without shoot structure. Treatment with auxin transport inhibitors resulted in abnormal embryo development, as in flowering plants, but did not cause any morphological changes in the haploid shoots. We fused the soybean auxin‐inducible promoter GH3 with a GUS reporter gene and used it to indirectly detect auxin distribution in the moss Physcomitrella patens. An auxin transport inhibitor NPA did not cause any changes in the putative distribution of auxin in the haploid shoot. These results indicate that polar auxin transport is not involved in haploid shoot development in mosses and that shoots in vascular plants and mosses are most likely regulated differently during development.

The Protein Encoded by Oncogene 6b from Agrobacterium tumefaciens Interacts with a Nuclear Protein of Tobacco

The 6b gene in the T-DNA from Agrobacterium has oncogenic activity in plant cells, inducing tumor formation, the phytohormone-independent division of cells, and alterations in leaf morphology. The product of the 6b gene appears to promote some aspects of the proliferation of plant cells, but the molecular mechanism of its action remains unknown. We report here that the 6b protein associates with a nuclear protein in tobacco that we have designated NtSIP1 (for Nicotiana tabacum 6b–interacting protein 1). NtSIP1 appears to be a transcription factor because its predicted amino acid sequence includes two regions that resemble a nuclear localization signal and a putative DNA binding motif, which is similar in terms of amino acid sequence to the triple helix motif of rice transcription factor GT-2. Expression in tobacco cells of a fusion protein composed of the DNA binding domain of the yeast GAL4 protein and the 6b protein activated the transcription of a reporter gene that was under the control of a chimeric promoter that included the GAL4 upstream activating sequence and the 35S minimal promoter of Cauliflower mosaic virus. Furthermore, nuclear localization of green fluorescent protein–fused 6b protein was enhanced by NtSIP1. A cluster of acidic residues in the 6b protein appeared to be essential for nuclear localization and for transactivation as well as for the hormone-independent growth of tobacco cells. Thus, it seems possible that the 6b protein might function in the proliferation of plant cells, at least in part, through an association with NtSIP1.

Expression and Complementation Analyses of a Chloroplast-Localized Homolog of Bacterial RecA in the Moss Physcomitrella patens

RecA protein is widespread in bacteria, and it plays a crucial role in homologous recombination. We have identified two bacterial-type recA gene homologs (PprecA1, PprecA2) in the cDNA library of the moss Physcomitrella patens. N-terminal fusion of the putative organellar targeting sequence of PpRecA2 to the green fluorescent protein (GFP) caused a targeting of PpRecA2 to the chloroplasts. Mutational analysis showed that the first AUG codon acts as initiation codon. Fusion of the full-length PpRecA2 to GFP caused the formation of foci that were colocalized with chloroplast nucleoids. The amounts of PprecA2 mRNA and protein in the cells were increased by treatment with DNA damaging agents. PprecA2 partially complemented the recA mutation in Escherichia coli. These results suggest the involvement of PpRecA2 in the repair of chloroplast DNA.

Microtubules regulate dynamic organization of vacuoles in Physcomitrella patens.

Eukaryotic cells have developed several essential membrane components. In flowering plants, appropriate structures and distributions of the major membrane components are predominantly regulated by actin microfilaments. In this study, we have focused on the regulatory mechanism of vacuolar structures in the moss, Physcomitrella patens. The high ability of P. patens to undergo homologous recombination enabled us stably to express green fluorescent protein (GFP) or red fluorescent protein (RFP) fusion proteins, and the simple body structure of P. patens enabled us to perform detailed visualization of the intracellular vacuolar and cytoskeletal structures. Three-dimensional analysis and high-speed time-lapse observations revealed surprisingly complex structures and dynamics of the vacuole, with inner sheets and tubular protrusions, and frequent rearrangements by separation and fusion of the membranes. Depolymerization of microtubules dramatically affected these structures and movements. Dual observation of microtubules and vacuolar membranes revealed that microtubules induced tubular protrusions and cytoplasmic strands of the vacuoles, indicative of interactions between microtubules and vacuolar membranes. These results demonstrate a novel function of microtubules in maintaining the distribution of the vacuole and suggest a functional divergence of cytoskeletal functions in land plant evolution.

Convergences and divergences in polar auxin transport and shoot development in land plant evolution

A shoot is a reiterated structure consisting of stems and leaves and is the prevailing body plan in most land plant lineages. Vascular plants form shoots in the diploid generation, whereas mosses do so in the haploid generation.1 However, whether these plants use similar molecular mechanisms in shoot development and how the genetic networks for shoot development evolved is not clear. In our recent paper,2 we examined polar auxin transport in several mosses, which is essential for shoot development in angiosperms. Surprisingly, we did not detect polar auxin transport in the gametophytic shoots of mosses, but did detect it in the sporophytes, which have no shoot structure, indicating that shoots in vascular plants and mosses are most likely regulated differently. Here we discuss the convergent evolution of shoots and diverged auxin regulation in land plants.

Quantitative imaging of directional transport through plasmodesmata in moss protonemata via single-cell photoconversion of Dendra2

Cell-to-cell transport of molecules in plants must be properly regulated for plant growth and development. One specialized mechanism that plants have evolved involves transport through plasmodesmata (PD), but when and how transport of molecules via PD is regulated among individual cells remains largely unknown, particularly at the single-cell level. Here, we developed a tool for quantitatively analyzing cell-to-cell transport via PD at a single-cell level using protonemata of Physcomitrella patens and a photoconvertible fluorescent protein, Dendra2. In the filamentous protonemal tissues, one-dimensional intercellular communication can be observed easily. Using this system, we found that Dendra2 was directionally transported toward the apex of the growing protonemata. However, this directional transport could be eliminated by incubation in the dark or treatment with a metabolic inhibitor. Thus, we propose that directional transport of macromolecules can occur via PD in moss protonemata, and may be affected by the photosynthetic and metabolic activity of cells.

Habituation as a Tumorous State That Is Interchangeable with a Normal State in Plant Cells

Publisher Summary Plant cells cultured in vitro normally need an exogenous supply of plant hormones, namely an auxin and/or a cytokinin, for continued growth. Sometimes the cells lose this requirement during subculturing and become able to grow on hormone-free medium or on medium that lacks one or other of the hormones. This phenomenon is called as auxin habituation. The chapter presents the phemomena of habituation and genetic tumors as systems that allow the interconversion of two different states (normal and tumorous) without any apparent genetic modification. With respect to reversion from a tumorous to a normal state, there are a few reports in the case of crown gall and hairy root induced by Agrobacterium infection. In these cases, evidence for methylation and subsequent inactivation of genes in the T-DNA have been presented. Similar modifications of genes may occur in genetic tumors, but the modification reactions must be much more rapidly reversible than methylation. In the case of habituation, reversibility to a normal, hormone-requiring state is reduced during subcultures. This is the case in decreases of organforming capacity and also in increases in the variability of regenerated plants.

Genome of the pitcher plant Cephalotus reveals genetic changes associated with carnivory

Carnivorous plants exploit animals as a nutritional source and have inspired long-standing questions about the origin and evolution of carnivory-related traits. To investigate the molecular bases of carnivory, we sequenced the genome of the heterophyllous pitcher plant Cephalotus follicularis, in which we succeeded in regulating the developmental switch between carnivorous and non-carnivorous leaves. Transcriptome comparison of the two leaf types and gene repertoire analysis identified genetic changes associated with prey attraction, capture, digestion and nutrient absorption. Analysis of digestive fluid proteins from C. follicularis and three other carnivorous plants with independent carnivorous origins revealed repeated co-options of stress-responsive protein lineages coupled with convergent amino acid substitutions to acquire digestive physiology. These results imply constraints on the available routes to evolve plant carnivory.