Nobuyoshi Mochizuki

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.

The cell biology of tetrapyrroles: a life and death struggle

Tetrapyrroles such as chlorophyll and heme are co-factors for essential proteins involved in a wide variety of crucial cellular functions. Nearly 2% of the proteins encoded by the Arabidopsis thaliana genome are thought to bind tetrapyrroles, demonstrating their central role in plant metabolism. Although the enzymes required for tetrapyrrole biosynthesis are well characterized, there are still major questions about the regulation of the pathway, and the transport of tetrapyrroles within cells. These issues are important, as misregulation of tetrapyrrole metabolism can lead to severe photo-oxidative stress, and because tetrapyrroles have been implicated in signaling pathways coordinating interactions between plant organelles. In this review, we discuss the cell biology of tetrapyrrole metabolism and its implications for tetrapyrroles as signaling molecules.

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.

Functional Analysis of a 450–Amino Acid N-Terminal Fragment of Phytochrome B in Arabidopsis

Phytochrome, a major photoreceptor in plants, consists of two domains: the N-terminal photosensory domain and the C-terminal domain. Recently, the 651–amino acid photosensory domain of phytochrome B (phyB) has been shown to act as a functional photoreceptor in the nucleus. The phytochrome (PHY) domain, which is located at the C-terminal end of the photosensory domain, is required for the spectral integrity of phytochrome; however, little is known about the signal transduction activity of this domain. Here, we have established transgenic Arabidopsis thaliana lines expressing an N-terminal 450–amino acid fragment of phyB (N450) lacking the PHY domain on a phyB-deficient background. Analysis of these plants revealed that N450 can act as an active photoreceptor when attached to a short nuclear localization signal and β-glucuronidase. In vitro spectral analysis of reconstituted chromopeptides further indicated that the stability of the N450 Pfr form, an active form of phytochrome, is markedly reduced in comparison with the Pfr form of full-length phyB. Consistent with this, plants expressing N450 failed to respond to intermittent light applied at long intervals, indicating that N450 Pfr is short-lived in vivo. Taken together, our findings show that the PHY domain is dispensable for phyB signal transduction but is required for stabilizing the Pfr form of phyB.

CRYPTOCHROME2 in Vascular Bundles Regulates Flowering in Arabidopsis

Plants make full use of light signals to determine the timing of flowering. In Arabidopsis thaliana, a blue/UV-A photoreceptor, CRYPTOCHROME 2 (cry2), and a red/far-red photoreceptor, PHYTOCHROME B (phyB), are two major photoreceptors that control flowering. The light stimuli for the regulation of flowering are perceived by leaves. We have recently shown that phyB expression in mesophyll but not in vascular bundles suppresses the expression of a key flowering regulator, FLOWERING LOCUS T (FT), in vascular bundles. In this study, we asked where in the leaf cry2 perceives light stimuli to regulate flowering. To answer this question, we established transgenic Arabidopsis lines in which the cry2–green fluorescent protein (GFP) fusion was expressed under the control of organ/tissue-specific promoters in a cry2-deficient mutant background. Analysis of these lines revealed that expression of cry2-GFP in vascular bundles, but not in epidermis or mesophyll, rescued the late flowering phenotype. We further confirmed that cry2-GFP expressed in vascular bundles increased FT expression only in vascular bundles. Hence, in striking contrast with phyB, cry2 most likely regulates FT expression in a cell-autonomous manner.

Mutant Screen Distinguishes between Residues Necessary for Light-Signal Perception and Signal Transfer by Phytochrome B

The phytochromes (phyA to phyE) are a major plant photoreceptor family that regulate a diversity of developmental processes in response to light. The N-terminal 651–amino acid domain of phyB (N651), which binds an open tetrapyrrole chromophore, acts to perceive and transduce regulatory light signals in the cell nucleus. The N651 domain comprises several subdomains: the N-terminal extension, the Per/Arnt/Sim (PAS)-like subdomain (PLD), the cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF) subdomain, and the phytochrome (PHY) subdomain. To define functional roles for these subdomains, we mutagenized an Arabidopsis thaliana line expressing N651 fused in tandem to green fluorescent protein, β-glucuronidase, and a nuclear localization signal. A large-scale screen for long hypocotyl mutants identified 14 novel intragenic missense mutations in the N651 moiety. These new mutations, along with eight previously identified mutations, were distributed throughout N651, indicating that each subdomain has an important function. In vitro analysis of the spectral properties of these mutants enabled them to be classified into two principal classes: light-signal perception mutants (those with defective spectral activity), and signaling mutants (those normal in light perception but defective in intracellular signal transfer). Most spectral mutants were found in the GAF and PHY subdomains. On the other hand, the signaling mutants tend to be located in the N-terminal extension and PLD. These observations indicate that the N-terminal extension and PLD are mainly involved in signal transfer, but that the C-terminal GAF and PHY subdomains are responsible for light perception. Among the signaling mutants, R110Q, G111D, G112D, and R325K were particularly interesting. Alignment with the recently described three-dimensional structure of the PAS-GAF domain of a bacterial phytochrome suggests that these four mutations reside in the vicinity of the phytochrome light-sensing knot.

Functional analysis of Arabidopsis thaliana isoforms of the Mg-chelatase CHLI subunit

The first step of chlorophyll biosynthesis is catalyzed by a Mg-chelatase composed of the subunits CHLI, CHLD and CHLH. Mg-chelatase requires ATP hydrolysis that can be attributed to CHLI. Arabidopsis has two CHLI isoforms, CHLI1 and CHLI2, that have similar expression profiles, but it has been suggested that CHLI2 has limited function in the Mg-chelatase complex. Recently, we showed that Arabidopsis CHLI1 is an ATPase and a target of chloroplast thioredoxin. Here, we demonstrate that CHLI2 also has ATPase activity but with a lower Vmax and higher Km ATP than CHLI1. We confirmed the thioredoxin-dependent reduction of a disulfide bond in CHLI2 and thiol-modulation of its ATPase activity. We then examined the physiological contribution of CHLI2 using a chli2 T-DNA knockout line. Although visible phenotype of homozygous chli2 mutants was almost comparable to wild type, the mutant accumulated significantly less chlorophyll. Furthermore, cs/cs; chli2/chli2 double mutants were almost albino. There were three phenotypes among progenies segregated from the cs/cs; CHLI2/chli2 parent: cs-like pale green, yellow, and almost albino were obtained in the approximate ratio of 1:2:0.7. PCR analysis confirmed that the chli2 mutation is semidominant on a homozygous cs background. These results reveal that although CHLI2 plays a limited role in chlorophyll biosynthesis, this subunit certainly contributes to the assembly of the Mg-chelatase complex.

Subcellular Sites of the Signal Transduction and Degradation of Phytochrome A

Phytochrome regulates various physiological and developmental processes throughout the life cycle of plants. Among the members of the phytochrome family, phytochrome A (phyA) exclusively mediates the far-red light high irradiance response (FR-HIR), which is elicited by continuous far-red light. In FR-HIR, nuclear accumulation of phyA, which precedes physiological responses, is proposed to be required for the response. In contrast to FR, red light induces rapid degradation of phyA to suppress undesirable long-term photomorphogenic responses of phyA. In the present study, we compared biological activities between phyA derivatives to which either a nuclear localization (NLS) or export (NES) signal sequence was attached. Those derivatives were expressed under the control of the PHYA promoter in the Arabidopsis phyA mutant. Detailed microscopic observation revealed that the phyA-green fluorescent protein (GFP) without a signal sequence is localized exclusively in the cytoplasm in darkness. Rapid nuclear entry was observed after exposure to both red and far-red light. Interestingly, both phyA-GFP-NLS and phyA-GFP-NES were rapidly degraded under continuous red light. Furthermore, a proteasome inhibitor delayed degradation equally under these two conditions. Therefore, similar mechanisms for phyA degradation may exist in the cytoplasm and nucleus. As expected from previous reports, phyA-GFP-NLS, but not phyA-GFP-NES, mediated different aspects of FR-HIR, such as inhibition of hypocotyl elongation and rapid induction of gene expression, confirming that phyA nuclear localization is required for FR-HIR. In addition, a detailed time course analysis of phyA-GFP and phyA-GFP-NLS responses revealed that they were almost indistinguishable, raising the question of the physiological relevance of phyA cytoplasmic retention in darkness.