A systems model of exocytic vesicles in Arabidopsis pollen during its germination

Abasic systems model for vesicle trafficking in Arabidopsis pollen tubes was constructed. The model was composed of transcriptome data and differential equations. The transcriptome data revealed some genes controlling vesicle trafficking in the pollen tubes, and the differential equations connected the molecular functions of the gene products. The computed pollen tube growth reasonably agreed with biological samples. Here, I expand the computational prediction into exocytic vesicles during pollen germination, which can be used to examine the accuracy of the systems model with biological samples. The computational analysis of the model predicts that the number of exocytic vesicles changes in an over 10-fold range before the vesicle trafficking system reaches the equilibrium that makes the pollen tube grow logarithmically. SYP125 (syntaxin of plants 125) is highly localized in the pollen tube tip in both the biological sample and systems model. The computational analysis predicts that SYP125 would highly localize in exocytic vesicles temporally before the pollen tube grows logarithmically. These kinetic predictions guide future research directions. © 2010 Landes Bioscience

Split luciferase complementation assay to study protein-protein interactions in Arabidopsis protoplasts

We developed a split luciferase complementation assay to study protein-protein interactions in Arabidopsis protoplasts. In this assay, the N- and C-terminal fragments of Renilla reniforms luciferase are translationally fused to bait and prey proteins, respectively. When the proteins interact, split luciferase becomes activated and emits luminescence that can be measured by a microplate luminometer. Split luciferase activity was measured by first transforming protoplasts with a DNA vector in a 96-well plate. DNA vector expressing both bait and prey genes was constructed through two independent in vitro DNA recombinant reactions, Gateway and Cre-loxP. As proof of concept, we detected the protein-protein interactions between the nuclear histones 2A and 2B, as well as between membrane proteins SYP (syntaxin of plant) 51 and SYP61, in Arabidopsis protoplasts. © 2007 The Authors

Detection of chromosomes tagged with green fluorescent protein in live Arabidopsis thaliana plants

BACKGROUND: Structural and dynamic studies of chromosomes tagged with green fluorescent protein (GFP) in yeast and cultured animal cells have revealed some surprises. Although this technology can be very powerful, only a few studies using this approach with developed multicellular systems have been reported for the study of chromatin behavior in situ. RESULTS: We established vectors and conditions to visualize tagged loci stably inserted in the Arabidopsis genome via GFP fused to a bacterial DNA-binding protein. Using this system, three-dimensional coordinates for tagged loci within nuclei from cells of a live plant can be directly determined with concomitant visualization of the position of the nucleolus. Chromosome polyploidization in epidermal cells at the elongation zone of the root in transgenic plants can be visualized in situ using this technique. CONCLUSION: We have established that GFP fusion with DNA-binding proteins can be used in conjunction with concatameric binding-site arrays to track genomic loci in living Arabidopsis plants. It should now be feasible to study the mechanisms of organization and dynamics of chromatin in specific cell types during various times of plant development, taking advantage of the well developed genetic systems and resources available for Arabidopsis

Visualizing chromosome structure/organization

With the rapid development of sequencing technologies in the past decade, many eukaryotic genomes have been resolved at the primary sequence level. However, organization of the genome within nuclei and the principles that govern such properties remain largely unclear. Optimization of fluorescence probe-based hybridization technologies combined with new advances in the instrumentation for microscopy has steadily yielded more structural information on chromosome organization in eukaryote model systems. These studies provide static snapshots of the detailed organization of chromatin. More recently, the successful application of a chromatin tagging strategy utilizing auto fluorescent fusion proteins opened a new era of chromatin studies in which the dynamic organization of the genome can be tracked in near real time. This review focuses on these new approaches to studying chromatin organization and dynamics in plants, and on future prospects in unraveling the basic principle of chromosome organization

A systems model of vesicle trafficking in Arabidopsis pollen tubes

A systems model that describes vesicle trafficking during pollen tube growth in Arabidopsis (Arabidopsis thaliana) was constructed. The model is composed of ordinary differential equations that connect the molecular functions of genes expressed in pollen. The current model requires soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) and small GTPases, Arf or Rab, to reasonably predict tube growth as a function of time. Tube growth depends on vesicle trafficking that transports phospholipid and pectin to the tube tip. The vesicle trafficking genes identified by analyzing publicly available transcriptome data comprised 328 genes. Fourteen of them are up-regulated by the gibberellin signaling pathway during pollen development, which includes the SNARE genes SYP124 and SYP125 and the Rab GTPase gene RABA4D. The model results adequately fit the pollen tube growth of both previously reported wild-type and raba4d knockout lines. Furthermore, the difference of pollen tube growth in syp124/syp125 single and double mutations was quantitatively predicted based on the model analysis. In general, a systems model approach to vesicle trafficking arguably demonstrated the importance of the functional connections in pollen tube growth and can help guide future research directions. © 2009 American Society of Plant Biologists

The Effects of Restricting Nitrogen, Phosphorus, and Potassium Fertilizers on Erianthus (\u3cem\u3eErianthus arundinaceus\u3c/em\u3e) Growth and Nutrient Contents

Low inputs and sustainability are the major concerns in bioenergy crop production (Reijnders 2006). Erianthus spp. is a relative of sugarcane and is a perennial crop with high dry matter production (Matsuo et al. 2003). It is expected to become a cellulosic bioenergy crop. However, its fertilizer requirements are still unknown because erianthus has a highly developed root system (Matsuo et al. 2003), and appears to absorb nutrients from the subsoil layer, which is hardly used by other crops. Therefore, it is necessary to experimentally restrict fertilizer application and maintain the rhizosphere to clarify the fertilizer requirements. In this study, we grew Erianthus (Erianthus arundinaceus) in pots and restricted nitrogen (N), phosphorus (P), and potassium (K) fertilizer application to evaluate the fertilizer requirements

Phosphatidic acid produced by phospholipase Dα1 and Dδ is incorporated into the internal membranes but not involved in the gene expression of RD29A in the abscisic acid signaling network in Arabidopsis thaliana

Core protein components of the abscisic acid (ABA) signaling network, pyrabactin resistance (PYR), protein phosphatases 2C (PP2C), and SNF1-related protein kinase 2 (SnRK2) are involved in the regulation of stomatal closure and gene expression downstream responses in Arabidopsis thaliana. Phosphatidic acid (PA) produced by the phospholipases Dα1 and Dδ (PLDs) in the plasma membrane has been identified as a necessary molecule in ABA-inducible stomatal closure. On the other hand, the involvement of PA in ABA-inducible gene expression has been suggested but remains a question. In this study, the involvement of PA in the ABA-inducible gene expression was examined in the model plant Arabidopsis thaliana and the canonical RD29A ABA-inducible gene that possesses a single ABA–responsive element (ABRE) in the promoter. The promoter activity and accumulation of the RD29A mRNA during ABA exposure to the plants were analyzed under conditions in which the production of PA by PLDs is abrogated through chemical and genetic modification. Changes in the subcellular localization of PA during the signal transduction were analyzed with confocal microscopy. The results obtained in this study suggest that inhibition of PA production by the PLDs does not affect the promoter activity of RD29A. PA produced by the PLDs and exogenously added PA in the plasma membrane are effectively incorporated into internal membranes to transduce the signal. However, exogenously added PA induces stomatal closure but not RD29A expression. This is because PA produced by the PLDs most likely inhibits the activity of not all but only the selected PP2C family members, the negative regulators of the RD29A promoter. This finding underscores the necessity for experimental verifications to adapt previous knowledge into a signaling network model before its construction