Akira Nagatani

Isolation and Initial Characterization of Arabidopsis Mutants That Are Deficient in Phytochrome A

Phytochrome, a red/far-red-light photoreceptor protein of plants, is encoded by a small gene family. Phytochrome A (PHYA), the product of the PHYA gene, is the predominant molecular species of phytochrome in etiolated tissue and has been best characterized biochemically. To define a role for PHYA, we isolated new mutants, designated fre1 (far-red elongated), in Arabidopsis thaliana that were specifically deficient in PHYA spectral activity and protein accumulation. These mutants were identified on the basis of their long hypocotyl phenotype under continuous far-red light. Although the fre1 mutants lacked the hypocotyl response to continuous far-red light, their responses to continuous white light and to end-of-day far-red-light treatments were normal. Thus, PHYA appears to play only a minor role in the regulation of hypocotyl elongation under natural conditions. In contrast, the fre1 mutation affected greening a fre1 mutant was less able than the wild type to deetiolate after growth in the dark. However, the potentiation effect of a red-light pulse on accumulation of chlorophyll was not changed significantly in the fre1 mutants. Thus, the function of PHYA might be highly specialized and restricted to certain phases of Arabidopsis development.

The Phototropin Family of Photoreceptors

The past decade has seen dramatic advances in our knowledge of plant photoreceptors and in our understanding of the signal transduction pathways that they activate ([Briggs and Olney, 2001][1]). A major part of these advances has been the identification and characterization of photoreceptors that

Dimers of the N-terminal domain of phytochrome B are functional in the nucleus

A plant modulates its developmental processes in response to light by several informational photoreceptors such as phytochrome. Phytochrome is a dimeric chromoprotein which regulates various aspects of plant development from seed germination to flowering. Upon absorption of red light, phytochrome translocates from the cytoplasm to the nucleus, and regulates gene expression through interaction with transcription factors such as PIF3 (refs 5–7). The phytochrome polypeptide has two domains: the amino-terminal photosensory domain with a chromophore and the carboxy-terminal domain which contains signalling motifs such as a kinase domain. The latter is widely believed to transduce the signal to downstream components. Here we show that the C-terminal domain of Arabidopsis phytochrome B (phyB), which is known as the most important member of the phytochrome family, is not directly involved in signal transduction. The N-terminal domain isolated from phyB, when dimerized and localized in the nucleus, triggered full phyB responses with much higher photosensitivity than the full-length phyB. These findings indicate that the C-terminal domain attenuates the activity of phyB rather than positively transducing the signal.

Photochemical Properties of the Flavin Mononucleotide-Binding Domains of the Phototropins from Arabidopsis, Rice, and Chlamydomonas reinhardtii

Phototropins (phot1 and phot2, formerly designated nph1 and npl1) are blue-light receptors that mediate phototropism, blue light-induced chloroplast relocation, and blue light-induced stomatal opening in Arabidopsis. Phototropins contain two light, oxygen, or voltage (LOV) domains at their N termini (LOV1 and LOV2), each a binding site for the chromophore flavin mononucleotide (FMN). Their C termini contain a serine/threonine protein kinase domain. Here, we examine the kinetic properties of the LOV domains of Arabidopsis phot1 and phot2, rice (Oryza sativa) phot1 and phot2, andChlamydomonas reinhardtii phot. When expressed inEscherichia coli, purified LOV domains from all phototropins examined bind FMN tightly and undergo a self-contained photocycle, characterized by fluorescence and absorption changes induced by blue light (T. Sakai, T. Kagawa, M. Kasahara, T.E. Swartz, J.M. Christie, W.R. Briggs, M. Wada, K. Okada [2001] Proc Natl Acad Sci USA 98: 6969–6974; M. Salomon, J.M. Christie, E. Knieb, U. Lempert, W.R. Briggs [2000] Biochemistry 39: 9401–9410). The photocycle involves the light-induced formation of a cysteinyl adduct to the C(4a) carbon of the FMN chromophore, which subsequently breaks down in darkness. In each case, the relative quantum efficiencies for the photoreaction and the rate constants for dark recovery of LOV1, LOV2, and peptides containing both LOV domains are presented. Moreover, the data obtained from full-length Arabidopsis phot1 and phot2 expressed in insect cells closely resemble those obtained for the tandem LOV-domain fusion proteins expressed in E. coli. For both Arabidopsis and rice phototropins, the LOV domains of phot1 differ from those of phot2 in their reaction kinetic properties and relative quantum efficiencies. Thus, in addition to differing in amino acid sequence, the phototropins can be distinguished on the basis of the photochemical cycles of their LOV domains. The LOV domains ofC. reinhardtii phot also undergo light-activated spectral changes consistent with cysteinyl adduct formation. Thus, the phototropin family extends over a wide evolutionary range from unicellular algae to higher plants.

The steady-state level of Mg-protoporphyrin IX is not a determinant of plastid-to-nucleus signaling in Arabidopsis

The plastid plays a vital role in various cellular activities within plant cells including photosynthesis and other metabolic pathways. It is believed that the functional status of the plastid is somehow monitored by the nucleus to optimize the expression of genes encoding plastid proteins. The currently dominant model for plastid-derived signaling (“plastid signaling”) proposes that Mg-protoporphyrin IX (MgProto) is a negative signal that represses the expression of a wide range of nuclear genes encoding plastid-localized proteins when plastid development is inhibited. In this study, we have re-evaluated this hypothesis by quantifying the steady-state levels of MgProto (as well as its neighboring intermediates protoporphyrin IX and Mg-Proto monomethyl ester [MgProtoMe]) in Arabidopsis plants with altered plastid signaling responses as monitored by expression of the Lhcb1, RBCS, HEMA1, BAM3 and CA1 genes. In addition, we have examined the correlation between gene expression and MgProto (MgProtoMe) in a range of mutants and conditions in which the steady-state levels of MgProto (MgProtoMe) have been modified. Overall we found that there was no correlation between the steady-state levels of MgProto (MgProtoMe) and Lhcb1 expression or with any of the other genes tested. Taking these results together, we propose that the current model on plastid signaling must be revised.

Arabidopsis PHYTOCHROME INTERACTING FACTOR Proteins Promote Phytochrome B Polyubiquitination by COP1 E3 Ligase in the Nucleus

This work identifies COP1 as the ubiquitin E3 ligase for not only phytochrome B but also other members of the stable phytochrome family and shows that PIF transcription factors enhance phyB ubiquitination by COP1 in vitro. It provides a molecular mechanism for the termination of red light signal transduction. Many plant photoresponses from germination to shade avoidance are mediated by phytochrome B (phyB). In darkness, phyB exists as the inactive Pr in the cytosol but upon red (R) light treatment, the active Pfr translocates into nuclei to initiate signaling. Degradation of phyB Pfr likely regulates signal termination, but the mechanism is not understood. Here, we show that phyB is stable in darkness, but in R, a fraction of phyB translocates into nuclei and becomes degraded by 26S proteasomes. Nuclear phyB degradation is mediated by COP1 E3 ligase, which preferentially interacts with the PhyB N-terminal region (PhyB-N). PhyB-N polyubiquitination by CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) in vitro can be enhanced by different PHYTOCHROME INTERACTING FACTOR (PIF) proteins that promote COP1/PhyB interaction. Consistent with these results, nuclear phyB accumulates to higher levels in pif single and double mutants and in cop1-4. Our results identify COP1 as an E3 ligase for phyB and other stable phytochromes and uncover the mechanism by which PIFs negatively regulate phyB levels.

Phytochrome B in the Mesophyll Delays Flowering by Suppressing FLOWERING LOCUS T Expression in Arabidopsis Vascular Bundles

Light is one of the most important environmental factors that determine the timing of a plants transition from the vegetative to reproductive, or flowering, phase. Not only daylength but also the spectrum of light greatly affect flowering. The shade of nearby vegetation reduces the ratio of red to far-red light and can trigger shade avoidance responses, including stem elongation and the acceleration of flowering. Phytochrome B (phyB) acts as a photoreceptor for this response. Physiological studies have suggested that leaves can perceive and respond to shade. However, little is known about the mechanisms involved in the processing of light signals within leaves. In this study, we used an enhancer-trap system to establish Arabidopsis thaliana transgenic lines that express phyB–green fluorescent protein (GFP) fusion protein in tissue-specific manners. The analysis of these lines demonstrated that phyB-GFP in mesophyll cells affected flowering, whereas phyB-GFP in vascular bundles did not. Furthermore, mesophyll phyB-GFP suppressed the expression of a key flowering regulator, FLOWERING LOCUS T, in the vascular bundles of cotyledons. Hence, a novel intertissue signaling from mesophyll to vascular bundles is revealed as a critical step for the regulation of flowering by phyB.

Involvement of auxin and brassinosteroid in the regulation of petiole elongation under the shade.

Plants grown under a canopy recognize changes in light quality and modify their growth patterns; this modification is known as shade avoidance syndrome. In leaves, leaf blade expansion is suppressed, whereas petiole elongation is promoted under the shade. However, the mechanisms that control these responses are largely unclear. Here, we demonstrate that both auxin and brassinosteroid (BR) are required for the normal leaf responses to shade in Arabidopsis (Arabidopsis thaliana). The microarray analysis of leaf blades and petioles treated with end-of-day far-red light (EODFR) revealed that almost half of the genes induced by the treatment in both parts were previously identified as auxin-responsive genes. Likewise, BR-responsive genes were overrepresented in the EODFR-induced genes. Hence, the auxin and BR responses were elevated by EODFR treatment in both leaf blades and petioles, although opposing growth responses were observed in these two parts. The analysis of the auxin-deficient doc1/big mutant and the BR-deficient rot3/cyp90c1 mutant further indicates that auxin and BR were equally required for the normal petiole elongation response to the shade stimulus. In addition, the spotlight irradiation experiment revealed that phytochrome in leaf blades but not that in petioles regulated petiole elongation, which was probably mediated through regulation of the auxin/BR responses in petioles. On the basis of these findings, we conclude that auxin and BR cooperatively promote petiole elongation in response to the shade stimulus under the control of phytochrome in the leaf blade.

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

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.

Type-B monogalactosyldiacylglycerol synthases are involved in phosphate starvation-induced lipid remodeling, and are crucial for low-phosphate adaptation

Mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively) constitute the bulk of membrane lipids in plant chloroplasts. Mutant analyses in Arabidopsis have shown that these galactolipids are essential for chloroplast biogenesis and photoautotrophic growth. Moreover, these non-phosphorous lipids are proposed to participate in low-phosphate (Pi) adaptations. Under Pi-limited conditions, a drastic accumulation of DGDG occurs concomitantly with a large reduction in membrane phospholipids, suggesting that plants substitute DGDG for phospholipids during Pi starvation. Previously, we reported that among the three MGDG synthase genes (MGD1, MGD2 and MGD3), the type-B MGD2 and MGD3 are upregulated in parallel with DGDG synthase genes during Pi starvation. Here, we describe the identification and characterization of T-DNA insertional mutants of Arabidopsis type-B MGD genes. Under Pi-starved conditions, the mgd3-1 mutant showed a drastic reduction in DGDG accumulation, particularly in the root, indicating that MGD3 is the main isoform responsible for DGDG biosynthesis in Pi-starved roots. Moreover, in the roots of mgd2 mgd3 plants, Pi stress-induced accumulation of DGDG was almost fully abolished, showing that type-B MGD enzymes are essential for membrane lipid remodeling in Pi-starved roots. Reductions in fresh weight, root growth and photosynthetic performance were also observed in these mutants under Pi-starved conditions. These results demonstrate that Pi stress-induced membrane lipid remodeling is important in plant growth during Pi starvation. The widespread distribution of type-B MGD genes in land plants suggests that membrane lipid remodeling mediated by type-B MGD enzymes is a potent adaptation to Pi deficiency for land plants.