Light-Induced Nuclear Translocation of Endogenous Pea Phytochrome A Visualized by Immunocytochemical Procedures

Abstract

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...

Intracellular localization of phytochrome in Robinia pseudoacacia pulvini

“…Bars = 1 lm (a), 0.5 lm (b) of pulvinar motor cells. This cytoplasmic phytochrome localization agrees with the general acceptance that phy A is uniformly distributed in the cytosol in dark-grown seedlings (McCurdy and Pratt 1986;Hisada et al 2000). However, in contrast to observations made with etiolated Glycine max seedlings (Cope and Pratt 1992), Pisum sativum epicotyls (Saunders et al 1983) and A. sativa coleoptiles (McCurdy and Pratt 1986;Speth et al 1986), R light did not induce a sequestered distribution of phytochrome in green pulvini.…”

“…Alternatively, phytochrome redistribution in Robinia pulvini might require a dark period of more than 30 min to become detectable. It is known that the kinetics of the intracellular redistribution of phytochrome diers between plant species and experimental conditions (Speth et al 1987;Cope and Pratt 1992;Hisada et al 2000).…”

“…MoÈ singer et al (1987) have reported data that clearly relate phy A with barley nuclei and, more recently, Sakamoto and Nagatani (1996) reported immunodetection of phy B in isolated Arabidopsis nuclei. Finally, there is evidence that phy A and phy B can be translocated from cytosol to nuclei in an R/FR light-dependent manner (Kircher et al 1999;Yamaguchi et al 1999;Hisada et al 2000).…”

Light‐Activated Intracellular Movement of Phytochrome

“…The nuclear import of PHYA:GFP could also be almost completely abolished by 24-h pre-treatment with red light (Kim et al, 2000). We note that similar results were obtained by Hisada et al (2000), who analysed continuous far-red light-and light pulse-dependent intracellular localization of phyA in pea seedlings, using cytochemical methods.…”

Phytochrome A requires jasmonate for photodestruction

“…Coleoptile tips were cryosectioned, and transverse sections of 8 m thickness were subjected to immunoXuorescence staining of phyA as described in detail in Hisada et al (2000). The phytochrome signal was visualized using the monoclonal antibodies mAR07 and mAR08 (Nagatani et al 1987) as undiluted hybridoma supernatant, and an anti-mouse IgG antibody conjugated to Xuorescein-isothiocyanate (Amersham Japan, Tokyo, Japan) in a dilution of 1:20.…”