SummaryThe plant vacuole is a multifunctional organelle which is essential for growth and development. To visualize the dynamics of plant vacuolar membranes, g-TIP (tonoplast intrinsic protein) was fused to GFP and expressed in Arabidopsis thaliana. The marker molecule was targeted to the vacuolar membranes in most tissues, as expected. In rapidly expanding cells, some additional spherical structures were often observed within the lumen of vacuoles, which emitted strong¯uorescence. To con®rm their normal presence, we examined wild-type Arabidopsis cotyledons by transmission electron microscopy. The metal-contact rapid-freezing method revealed that the vacuolar lumen of epidermal cells contained many cytoplasmic projections, which often formed spherical structures (1±3 mm diameter) consisting of double membranes. Thus we concluded that these structures are authentic and named them`bulbs'. Threedimensional reconstruction from serial electron microscopic images demonstrates that bulbs are very intricately folded, but are continuous with the limiting vacuolar membrane. The¯uorescence intensity of bulbs is about threefold higher than that of vacuolar membrane. GFP-AtRab75c, another marker of the vacuole, did not give¯uorescent signals of bulbs in transgenic plants, but the existence of bulbs was still con®rmed by electron microscopy. These results suggest that bulbs de®ne a subregion in the continuous vacuolar membrane, where some proteins are concentrated and others segregated.

Although the physiological functions of phytochrome A (PhyA) are now known, the distribution of endogenous PhyA has not been examined. We have visualized endogenous PhyA apoprotein (PHYA) by immunolabeling cryosections of pea tissue, using PHYA-deficient mutants as negative controls. By this method, we examined the distribution of PHYA in different tissues and changes in its intracellular distribution in response to light. In apical hook cells of etiolated seedlings, PHYA immunolabeling was distributed diffusely in the cytosol. Exposure to continuous far-red (cFR) light caused a redistribution of the immunolabeling to the nucleus, first detectable after 1.5 hr and greatest at 4.5 hr. During this time, the amounts of spectrally active phytochrome and PHYA did not decline substantially. Exposure to continuous red (cR) light or to a brief pulse of red light also resulted in redistribution of immunolabeling to the nucleus, but this occurred much more rapidly and with a different pattern of intranuclear distribution than it did in response to cFR light. Exposures to cR light resulted in loss of immunolabeling, which was associated with PHYA degradation. These results indicate that the light-induced intracellular location of PHYA is wavelength dependent and imply that this is important for PhyA activity.

Although the physiological functions of phytochrome A (PhyA) are now known, the distribution of endogenous PhyA has not been examined. We have visualized endogenous PhyA apoprotein (PHYA) by immunolabeling cryosections of pea tissue, using PHYA-deficient mutants as negative controls. By this method, we examined the distribution of PHYA in different tissues and changes in its intracellular distribution in response to light. In apical hook cells of etiolated seedlings, PHYA immunolabeling was distributed diffusely in the cytosol. Exposure to continuous far-red (cFR) light caused a redistribution of the immunolabeling to the nucleus, first detectable after 1.5 hr and greatest at 4.5 hr. During this time, the amounts of spectrally active phytochrome and PHYA did not decline substantially. Exposure to continuous red (cR) light or to a brief pulse of red light also resulted in redistribution of immunolabeling to the nucleus, but this occurred much more rapidly and with a different pattern of intranuclear distribution than it did in response to cFR light. Exposures to cR light resulted in loss of immunolabeling, which was associated with PHYA degradation. These results indicate that the light-induced intracellular location of PHYA is wavelength dependent and imply that this is important for PhyA activity. INTRODUCTIONThe phytochrome family of plant photoreceptors regulates various molecular and cellular processes of plant development in response to the light environment . Phytochromes are soluble chromoproteins that convert photoreversibly between two spectrally distinct forms when sequentially absorbing red (R) and far-red (FR) light, and this interconversion occurs immediately both in vivo and in vitro (Butler et al., 1959). Phytochromes are encoded by a small gene family (phytochrome genes PHYA to PHYE in Arabidopsis; Sharrock and Quail, 1989;Clack et al., 1994). Studies with mutants deficient in specific phytochromes have shown that phytochrome A (PhyA) and phytochrome B (PhyB) have distinct action spectra for the photoinduction of seed germination (Shinomura et al., 1996) and distinct fluence and wavelength requirements for expression of the chlorophyll a / b binding protein gene ( CAB ) (Hamazato et al., 1997). The fundamental molecular basis for these differences is of great interest but has not been elucidated. Recent genetic and molecular analyses have defined differences in PhyA and PhyB activities with respect to interacting factors and signaling intermediates. Those studies suggest that PhyA and PhyB signals are transduced by overlapping signal transduction pathways (Deng and Quail, 1999).To complement such approaches, one must also consider the ways in which the concentration, photochemical activity, and localization of each phytochrome are regulated in tissues and cells (Pratt, 1994). In tissues, this regulation may affect the transmission of the light signal from the site of photoperception to the responsive organ. An analysis of the way in which photoreceptors are redistributed within the cell in respon...

The effect of scaffold shape on dentin regeneration is not well understood. In this study, porous hydroxyapatite/beta-tricalcium phosphate (HAp/beta-TCP), powdered HAp/beta-TCP, and polyglycolic acid (PGA) fiber mesh were used as scaffolds and transplanted with cultured porcine dental pulp-derived cells into the backs of nude mice. Samples were harvested after 6 weeks. Newly-formed hard tissue was observed in all transplants. When porous HAp/beta-TCP was used, dentin-like hard tissue was observed on the inner wall with minimum cell inclusions and odontoblast-like cells were aligned adjacent to the hard tissue. When HAp/beta-TCP powders or PGA were used, bone-like hard tissues showed cell inclusions and cell alignment was not observed. Hard tissue from the HAp/beta-TCP block group was positive for type I collagen, osteonectin, bone sialoprotein and dentin sialoprotein (DSP), which are markers for dentin. This result was confirmed by in situ hybridization with a dsp probe. Only the aligned cells were positive with an antisense probe. On the other hand, hard tissue from other scaffolds showed incomplete expression of both bone and dentin markers and they were negative for osteonectin and DSP. These results suggest that scaffold shape affects the type of tissue regenerated by dental pulp-derived cells.

No abstract