D1-Asn-298 in photosystem II is involved in a hydrogen-bond network near the redox-active tyrosine YZ for proton exit during water oxidation.

In photosynthetic water oxidation, two water molecules are converted into one oxygen molecule and four protons at the Mn4CaO5 cluster in photosystem II (PSII) via the S-state cycle. Efficient proton exit from the catalytic site to the lumen is essential for this process. However, the exit pathways of individual protons through the PSII proteins remain to be identified. In this study, we examined the involvement of a hydrogen-bond network near the redox-active tyrosine YZ in proton transfer during the S-state cycle. We focused on spectroscopic analyses of a site-directed variant of D1-Asn-298, a residue involved in a hydrogen-bond network near YZ We found that the D1-N298A mutant of Synechocystis sp. PCC 6803 exhibits an O2 evolution activity of ∼10% of the wild-type. D1-N298A and the wild-type D1 had very similar features of thermoluminescence glow curves and of an FTIR difference spectrum upon YZ oxidation, suggesting that the hydrogen-bonded structure of YZ and electron transfer from the Mn4CaO5 cluster to YZ were little affected by substitution. In the D1-N298A mutant, however, the flash-number dependence of delayed luminescence showed a monotonic increase without oscillation, and FTIR difference spectra of the S-state cycle indicated partial and significant inhibition of the S2 → S3 and S3 → S0 transitions, respectively. These results suggest that the D1-N298A substitution inhibits the proton transfer processes in the S2 → S3 and S3 → S0 transitions. This in turn indicates that the hydrogen-bond network near YZ can be functional as a proton transfer pathway during photosynthetic water oxidation.

Genetically introduced hydrogen bond interactions reveal an asymmetric charge distribution on the radical cation of the special-pair chlorophyll P680.

The special-pair chlorophyll (Chl) P680 in photosystem II has an extremely high redox potential (Em ) to enable water oxidation in photosynthesis. Significant positive-charge localization on one of the Chl constituents, PD1 or PD2, in P680+ has been proposed to contribute to this high Em To identify the Chl molecule on which the charge is mainly localized, we genetically introduced a hydrogen bond to the 131-keto C=O group of PD1 and PD2 by changing the nearby D1-Val-157 and D2-Val-156 residues to His, respectively. Successful hydrogen bond formation at PD1 and PD2 in the obtained D1-V157H and D2-V156H mutants, respectively, was monitored by detecting 131-keto C=O vibrations in Fourier transfer infrared (FTIR) difference spectra upon oxidation of P680 and the symmetrically located redox-active tyrosines YZ and YD, and they were simulated by quantum-chemical calculations. Analysis of the P680+/P680 FTIR difference spectra of D1-V157H and D2-V156H showed that upon P680+ formation, the 131-keto C=O frequency upshifts by a much larger extent in PD1 (23 cm-1) than in PD2 (<9 cm-1). In addition, thermoluminescence measurements revealed that the D1-V157H mutation increased the Em of P680 to a larger extent than did the D2-V156H mutation. These results, together with the previous results for the mutants of the His ligands of PD1 and PD2, lead to a definite conclusion that a charge is mainly localized to PD1 in P680^.

Clock-controlled and FLOWERING LOCUS T (FT)-dependent photoperiodic pathway in Lotus japonicus II: characterization of a microRNA implicated in the control of flowering time.

Plant circadian clock generates rhythms with a period close to 24 h, and it controls a wide variety of physiological and developmental events, including the transition to reproductive growth (or flowering). During the last decade, significant research progress in Arabidopsis thaliana has been made in defining the molecular mechanism by which the circadian clock regulates flowering time in response to changes in photoperiod. In Lotus japonicus, we have found that LjFTa, which encodes a ortholog of the Arabidopsis FLOWERING LOCUS T (FT), plays an important role in the promotion of flowering, but it is not clear how the expression of LjFTa is regulated in L. japonicus. Based on current knowledge of photoperiodic control of flowering time in A. thaliana, here we examined whether a microRNA is involved in the activation of LjFTa in L. japonicus. Two putative L. japonicus genes that are responsible for the production of miR172 (designated LjmiR172a and LjmiR172b) were cloned. Overexpression of LjmiR172a/b in A. thaliana resulted in markedly accelerated flowering through enhancement of the expression of FT, concomitantly reducing the expression level of TARGET OF EARLY ACTIVATION TAGGED 1 (TOE1) transcripts, the protein product of which functions as a transcriptional repressor of FT. These results suggest that LjmiR172 genes play a positive role in the LjFTa-mediated promotion of flowering in L. japonicus.

Thioredoxin h regulates calcium dependent protein kinases in plasma membranes.

Thioredoxin (Trx) is a key player in redox homeostasis in various cells, modulating the functions of target proteins by catalyzing a thiol-disulfide exchange reaction. Target proteins of cytosolic Trx-h of higher plants were studied, particularly in the plasma membrane, because plant plasma membranes include various functionally important protein molecules such as transporters and signal receptors. Plasma membrane proteins from Arabidopsis thaliana cell cultures were screened using a resin Trx-h1 mutant-immobilized, and a total of 48 candidate proteins obtained. These included two calcium-sensing proteins: a phosphoinositide-specific phospholipase 2 (AtPLC2) and a calcium-dependent protein kinase 21 (AtCPK21). A redox-dependent change in AtCPK21 kinase activity was demonstrated in vitro. Oxidation of AtCPK21 resulted in a decrease in kinase activity to 19% of that of untreated AtCPK21, but Trx-h1 effectively restored the activity to 90%. An intramolecular disulfide bond (Cys97-Cys108) that is responsible for this redox modulation was then identified. In addition, endogenous AtCPK21 was shown to be oxidized in vivo when the culture cells were treated with H2 O2 . These results suggest that redox regulation of AtCPK21 by Trx-h in response to external stimuli is important for appropriate cellular responses. The relationship between the redox regulation system and Ca(2+) signaling pathways is discussed.

Clock-controlled and FLOWERING LOCUS T (FT)-dependent photoperiodic pathway in Lotus japonicus I: verification of the flowering-associated function of an FT homolog.

During the last decade, significant research progress in the study of Arabidopsis thaliana has been made in defining the molecular mechanism by which the plant circadian clock regulates flowering time in response to changes in photoperiod. It is generally accepted that the clock-controlled CONSTANS (CO)-FLOWERING LOCUS T (FT)-mediated external coincidence mechanism underlying the photoperiodic control of flowering time is conserved in higher plants, including A. thaliana and Oryza sativa. However, it is also assumed that the mechanism differs considerably in detail among species. Here we characterized the clock-controlled CO-FT pathway in Lotus japonicus (a model legume) in comparison with that of A. thaliana. L. japonicus has at least one FT orthologous gene (named LjFTa), which is induced specifically in long-days and complements the mutational lesion of the A. thaliana FT gene. However, it was speculated that this legume might lack the upstream positive regulator CO. By employing L. japonicus phyB mutant plants, we showed that the photoreceptor mutant displays a phenotype of early flowering due to enhanced expression of LjFTa, suggesting that LjFTa is invovled in the promotion of flowering in L. japonicus. These results are discussed in the context of current knowledge of the flowering in crop legumes such as soybean and garden pea.

Exploring dynamics of molybdate in living animal cells by a genetically encoded FRET nanosensor.

Molybdenum (Mo) is an essential trace element for almost all living organisms including animals. Mo is used as a catalytic center of molybdo-enzymes for oxidation/reduction reactions of carbon, nitrogen, and sulfur metabolism. Whilst living cells are known to import inorganic molybdate oxyanion from the surrounding environment, the in vivo dynamics of cytosolic molybdate remain poorly understood as no appropriate indicator is available for this trace anion. We here describe a genetically encoded Förester-resonance-energy-transfer (FRET)-based nanosensor composed of CFP, YFP and the bacterial molybdate-sensor protein ModE. The nanosensor MolyProbe containing an optimized peptide-linker responded to nanomolar-range molybdate selectively, and increased YFP:CFP fluorescence intensity ratio by up to 109%. By introduction of the nanosensor, we have been able to successfully demonstrate the real-time dynamics of molybdate in living animal cells. Furthermore, time course analyses of the dynamics suggest that novel oxalate-sensitive- and sulfate-resistant- transporter(s) uptake molybdate in a model culture cell.

Molecular mechanisms of circadian rhythm in Lotus japonicus and Arabidopsis thaliana are sufficiently compatible to regulate heterologous core clock genes robustly.

Recent intensive studies of the model plant Arabidopsis thaliana have revealed the molecular mechanisms underlying circadian rhythms in detail. Results of phylogenetic analyses indicated that some of core clock genes are widely conserved throughout the plant kingdom. For another model plant the legume Lotus japonicus, we have reported that it has a set of putative clock genes highly homologous to A. thaliana. Taking advantage of the L. japonicus hairy root transformation system, in this study we characterized the promoter activity of A. thaliana core clock genes CCA1 and PRR5 in heterologous L. japonicus cells and found that the molecular mechanism of circadian rhythm in L. japonicus is compatible with that of A. thaliana.

Thiol modulation of the chloroplast ATP synthase is dependent on the energization of thylakoid membranes.

Thiol modulation of the chloroplast ATP synthase γ subunit has been recognized as an important regulatory system for the activation of ATP hydrolysis activity, although the physiological significance of this regulation system remains poorly characterized. Since the membrane potential required by this enzyme to initiate ATP synthesis for the reduced enzyme is lower than that needed for the oxidized form, reduction of this enzyme was interpreted as effective regulation for efficient photophosphorylation. However, no concrete evidence has been obtained to date relating to the timing and mode of chloroplast ATP synthase reduction and oxidation in green plants. In this study, thorough analysis of the redox state of regulatory cysteines of the chloroplast ATP synthase γ subunit in intact chloroplasts and leaves shows that thiol modulation of this enzyme is pivotal in prohibiting futile ATP hydrolysis activity in the dark. However, the physiological importance of efficient ATP synthesis driven by the reduced enzyme in the light could not be demonstrated. In addition, we investigated the significance of the electrochemical proton gradient in reducing the γ subunit by the reduced form of thioredoxin in chloroplasts, providing strong insights into the molecular mechanisms underlying the formation and reduction of the disulfide bond on the γ subunit in vivo.

Characterization of shade avoidance responses in Lotus japonicus.

Sessile plants must continuously adjust their growth and development to optimize photosynthetic activity under ever-fluctuating light conditions. Among such light responses in plants, one of the best-characterized events is the so-called shade avoidance, for which a low ratio of the red (R):far-red (FR) light intensities is the most prominent stimulus. Such shade avoidance responses enable plants to overtop their neighbors, thereby enhancing fitness and competitiveness in their natural habitat. Considerable progress has been achieved during the last decade in understanding the molecular mechanisms underlying the shade avoidance responses in the model rosette plant, Arabidopsis thaliana. We characterize here the fundamental aspects of the shade avoidance responses in the model legume, Lotus japonicus, based on the fact that its phyllotaxis (or morphological architecture) is quite different from that of A. thaliana. It was found that L. japonicus displays the characteristic shade avoidance syndrome (SAS) under defined laboratory conditions (a low R:FR ratio, low light intensity, and low blue light intensity) that mimic the natural canopy. In particular, the outgrowth of axillary buds (i.e., both aerial and cotyledonary shoot branching) was severely inhibited in L. japonicus grown in the shade. These results are discussed with special emphasis on the unique aspects of SAS observed with this legume.

Towards a functional dissection of thioredoxin networks in plant cells.

Thioredoxins are a ubiquitous family of redox equivalent mediators, long considered to possess a limited number of target enzymes. Recent progress in proteomic research has allowed the identification of a wide variety of candidate proteins with which this small protein may interact in vivo. Moreover, the activity of thioredoxin itself has been recently found to be subject to regulation by posttranslational modifications, adding an additional level of complexity to the function of this intriguing enzyme family. The current review charts the technical progress made in the continuing discovery of the numerous and diverse roles played by these proteins in the regulation of redox networks in plant cells.

The regulator of the F1 motor: inhibition of rotation of cyanobacterial F1-ATPase by the epsilon subunit.

The chloroplast-type F(1) ATPase is the key enzyme of energy conversion in chloroplasts, and is regulated by the endogenous inhibitor epsilon, tightly bound ADP, the membrane potential and the redox state of the gamma subunit. In order to understand the molecular mechanism of epsilon inhibition, we constructed an expression system for the alpha(3)beta(3)gamma subcomplex in thermophilic cyanobacteria allowing thorough investigation of epsilon inhibition. epsilon Inhibition was found to be ATP-independent, and different to that observed for bacterial F(1)-ATPase. The role of the additional region on the gamma subunit of chloroplast-type F(1)-ATPase in epsilon inhibition was also determined. By single molecule rotation analysis, we succeeded in assigning the pausing angular position of gamma in epsilon inhibition, which was found to be identical to that observed for ATP hydrolysis, product release and ADP inhibition, but distinctly different from the waiting position for ATP binding. These results suggest that the epsilon subunit of chloroplast-type ATP synthase plays an important regulator for the rotary motor enzyme, thus preventing wasteful ATP hydrolysis.

Inverse regulation of rotation of F1-ATPase by the mutation at the regulatory region on the gamma subunit of chloroplast ATP synthase.

In F1-ATPase, the rotation of the central axis subunit gamma relative to the surrounding alpha3beta3 subunits is coupled to ATP hydrolysis. We previously reported that the introduced regulatory region of the gamma subunit of chloroplast F1-ATPase can modulate rotation of the gamma subunit of the thermophilic bacterial F1-ATPase (Bald, D., Noji, H., Yoshida, M., Hirono-Hara, Y., and Hisabori, T. (2001) J. Biol. Chem. 276, 39505-39507). The attenuated enzyme activity of this chimeric enzyme under oxidizing conditions was characterized by frequent and long pauses of rotation of gamma. In this study, we report an inverse regulation of the gamma subunit rotation in the newly engineered F1-chimeric complex whose three negatively charged residues Glu210-Asp211-Glu212 adjacent to two cysteine residues of the regulatory region derived from chloroplast F1-ATPase gamma were deleted. ATP hydrolysis activity of the mutant complex was stimulated up to 2-fold by the formation of the disulfide bond at the regulatory region by oxidation. We successfully observed inverse redox switching of rotation of gamma using this mutant complex. The complex exhibited long and frequent pauses in its gamma rotation when reduced, but the rotation rates between pauses remained unaltered. Hence, the suppression or activation of the redox-sensitive F1-ATPase can be explained in terms of the change in the rotation behavior at a single molecule level. These results obtained by the single molecule analysis of the redox regulation provide further insights into the regulation mechanism of the rotary enzyme.

Chloroplast cyclophilin is a target protein of thioredoxin. Thiol modulation of the peptidyl-prolyl cis-trans isomerase activity.

Chloroplast cyclophilin has been identified as a potential candidate of enzymes in chloroplasts that are regulated by thioredoxin (Motohashi, K., Kondoh, A., Stumpp, M. T., and Hisabori, T. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 11224-11229). In the present study we found that the peptidyl-prolyl cis-trans isomerase activity of cyclophilin is fully inactivated in the oxidized form. Reduction of cyclophilin by thioredoxin-m recovered the isomerase activity. Two crucial disulfide bonds were determined by disulfide-linked peptide mapping. The relevance of these cysteines for isomerase activity was confirmed by the mutagenesis studies. Because four cysteine residues in Arabidopsis thaliana cyclophilin were conserved in the isoforms from several organisms, it appears that this redox regulation must be one of the common regulation systems of cyclophilin.

Molecular evolution of the modulator of chloroplast ATP synthase: origin of the conformational change dependent regulation.

Chloroplast ATP synthase synthesizes ATP by utilizing a proton gradient as an energy supply, which is generated by photosynthetic electron transport. The activity of the chloroplast ATP synthase is regulated in several specific ways to avoid futile hydrolysis of ATP under various physiological conditions. Several regulatory signals such as Delta mu H(+), tight binding of ADP and its release, thiol modulation, and inhibition by the intrinsic inhibitory subunit epsilon are sensed by this complex. In this review, we describe the function of two regulatory subunits, gamma and epsilon, of ATP synthase based on their possible conformational changes and discuss the evolutionary origin of these regulation systems.

Distinct N-terminal regulatory domains of Ca(2+)/H(+) antiporters.

The regulation of intracellular Ca(2+) levels is achieved in part by high-capacity vacuolar Ca(2+)/H(+) antiporters. An N-terminal regulatory region (NRR) on the Arabidopsis Ca(2+)/H(+) antiporter CAX1 (cation exchanger 1) has been shown previously to regulate Ca(2+) transport by a mechanism of N-terminal auto-inhibition. Here, we examine the regulation of other CAX transporters, both within Arabidopsis and from another plant, mung bean (Vigna radiata), to ascertain if this mechanism is commonly used among Ca(2+)/H(+) antiporters. Biochemical analysis of mung bean VCAX1 expressed in yeast (Saccharomyces cerevisiae) showed that N-terminal truncated VCAX1 had approximately 70% greater antiport activity compared with full-length VCAX1. A synthetic peptide corresponding to the NRR of CAX1, which can strongly inhibit Ca(2+) transport by CAX1, could not dramatically inhibit Ca(2+) transport by truncated VCAX1. The N terminus of Arabidopsis CAX3 was also shown to contain an NRR. Additions of either the CAX3 or VCAX1 regulatory regions to the N terminus of an N-terminal truncated CAX1 failed to inhibit CAX1 activity. When fused to N-terminal truncated CAX1, both the CAX3 and VCAX1 regulatory regions could only auto-inhibit CAX1 after mutagenesis of specific amino acids within this NRR region. These findings demonstrate that N-terminal regulation is present in other plant CAX transporters, and suggest distinct regulatory features among these transporters.