Hanayo Nakanishi

Phytochrome-interacting factor 4 and 5 (PIF4 and PIF5) activate the homeobox ATHB2 and auxin-inducible IAA29 genes in the coincidence mechanism underlying photoperiodic control of plant growth of Arabidopsis thaliana.

The plant circadian clock generates rhythms with a period close to 24 h, and it controls a wide variety of physiological and developmental events. Among clock-controlled developmental events, the best characterized is the photoperiodic control of flowering time, which is mediated through the CONSTANS (CO)-FLOWERING LOCUS T (FT) pathway in Arabidopsis thaliana. The clock also regulates the diurnal plant growth including the elongation of hypocotyls in a short day (SDs)-specific manner. In this mechanism, phytochromes (mainly phyB) and the PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5, encoding phytochrome-interacting basic helix-loop-helix (bHLH) transcription factors, play crucial roles. The time of day-specific and photoperiodic control of hypocotyl elongation is best explained by the accumulation of the PIF4 and PIF5 proteins during night-time before dawn, especially under SDs, due to coincidence between the internal (circadian rhythm) and external (photoperiod) time cues. However, the PIF4- and/or PIF5-controlled downstream factors have not yet been identified. Here, we provide evidence that ARABIDOPSIS THALIANA HOMEOBOX PROTEIN2 (ATHB2), together with auxin-inducible IAA29, is diurnally expressed with a peak at dawn under the control of PIF4 and PIF5 specifically in SDs. This coincidentally expressed transcription factor serves as a positive regulator for the elongation of hypocotyls. The expression profiles of ATHB2 were markedly altered in certain clock and phytochrome mutants, all of which show anomalous phenotypes with regard to the photoperiodic control of hypocotyl elongation. Taken together, we propose that an external coincidence model involving the clock-controlled PIF4/PIF5-ATHB2 pathway is crucial for the diurnal and photoperiodic control of plant growth in A. thaliana.

Functional roles of D2-Lys317 and the interacting chloride ion in the water oxidation reaction of photosystem II as revealed by fourier transform infrared analysis.

Photosynthetic water oxidation in plants and cyanobacteria is catalyzed by a Mn4CaO5 cluster within the photosystem II (PSII) protein complex. Two Cl– ions bound near the Mn4CaO5 cluster act as indispensable cofactors, but their functional roles remain to be clarified. We have investigated the role of the Cl– ion interacting with D2-K317 (designated Cl-1) by Fourier transform infrared spectroscopy (FTIR) analysis of the D2-K317R mutant of Synechocystis sp. PCC 6803 in combination with Cl–/NO3– replacement. The D2-K317R mutation perturbed the bands in the regions of the COO– stretching and backbone amide vibrations in the FTIR difference spectrum upon the S1 → S2 transition. In addition, this mutation altered the 15N isotope-edited NO3– bands in the spectrum of NO3–-treated PSII. These results provide the first experimental evidence that the Cl-1 site is coupled with the Mn4CaO5 cluster and its interaction is affected by the S1 → S2 transition. It was also shown that a negative band at 1748 cm–1 arising from COOH group(s) was altered to a positive intensity by the D2-K317R mutation as well as by NO3– treatment, suggesting that the Cl-1 site affects the pKa of COOH/COO– group(s) near the Mn4CaO5 cluster in a common hydrogen bond network. Together with the observation that the efficiency of the S3 → S0 transition significantly decreased in the core complexes of D2-K317R upon moderate dehydration, it is suggested that D2-K317 and Cl-1 are involved in a proton transfer pathway from the Mn4CaO5 cluster to the lumen, which functions in the S3 → S0 transition.

Classification of the Genes Involved in the Two-Component System of the Moss Physcomitrella patens

Physcomitrella patens, belonging to bryopsida, is a basal lineage of land plants. To gain insight into the diversification of the two-component system (TCS), which is widely conserved from prokaryotes to eukaryotes, we compiled TCS-associated genes by employing P. patens genome databases. The moss has a set of His-kinases (HKs), including homologs of the cytokinin- and ethylene-receptors in seed plants. In addition, it has a number of coding-sequences specifying unique HKs. We found evidence that a putative cytokinin-receptor HK in P. patans serves as a sensor for this hormone, and that the HK activity of a putative ethylene-receptor homolog is regulated by ethylene, as observed for Arabidopsis thaliana.

Genomewide characterization of the light-responsive and clock-controlled output pathways in Lotus japonicus with special emphasis of its uniqueness.

During the last decade, tremendous progress has been made in understanding the molecular mechanisms underlying the plant circadian clock in Arabidopsis thaliana, mainly taking advantage of the availability of its entire genomic sequence. It is also well understood how the clock controls the photomorphogenesis of seedlings, including the shade avoidance response, and how the clock controls the photoperiodic flowering time in the spring annual long-days herb A. thaliana. Based on this, here we attempt to shed light on these clock-controlled fundamental and physiological events in Lotus japonicus, which is a perennial temperate legume with a morphological nature quite different from Arabidopsis. In the Lotus database, we first compiled as many clock-, light-, and flowering-associated coding sequences as possible, which appear to be orthologous or homologous to the Arabidopsis counterparts. Then we focused on the PHYTOCHROME INTERACTING FACTOR4 (PIF4)-mediated photomorphogenic pathway and the FLOWERING LOCUS T (FT)-mediated photoperiodic flowering pathway. It was shown in L. japonicus that the putative LjPIF4 homologue is expressed in a manner dependent on the circadian clock, and the putative LjFT orthologue is expressed coincidentally and especially in the long-days conditions, as in the case of A. thaliana. LjFT is capable of promoting flowering in A. thaliana, whereas the function of LjPIF4 seems to be divergent to a certain extent from that of AtPIF4. These results are discussed with emphasis on the intriguing differences between these model plant species.

Functional Characterization of HY5 Homolog Genes Involved in Early Light-Signaling in Physcomitrella patens

The developmental programs of Physcomitrella patens, a basal lineage of land plants, are regulated by phytohormones and light-signaling responses. In this study, our attention was focused on the HY5-family of transcription factors, which are known to play important roles immediately downstream of photoreceptors during the early photomorphogenesis of Arabidopsis thaliana. We retrieved two HY5-homologs, named PpHY5a and PpHY5b, from the whole genome sequence database of P. patens. Arabidopsis transgenic plants overproducing the basic leucine zipper (bZIP) domain of PpHY5a exhibited a phenotype of short hypocotyls, suggesting a functional relationship between PpHY5 and Arabidopsis HY5. A loss-of-function Δhy5a Δhy5b double mutant was defective in the vigorous protrusion of caulonema cells from the protonema networks of P. patens under light and dark conditions. These results suggest that the function of HY5-homologs in P. patens is evolutionarily conserved, and is implicated in a process of caulonema development.

Development of a novel cryogenic microscope with numerical aperture of 0.9 and its application to photosynthesis research

A novel cryogenic optical-microscope system was developed in which the objective lens is set inside of the cryostat adiabatic vacuum space. Being isolated from the sample when it was cooled, the objective lens was maintained at room temperature during the cryogenic measurement. Therefore, the authors were able to use a color-aberration corrected objective lens with a numerical aperture of 0.9. The lens is equipped with an air vent for compatibility to the vacuum. The theoretically expected spatial resolutions of 0.39μm along the lateral direction and 1.3μm along the axial direction were achieved by the developed system. The system was applied to the observations of non-uniform distributions of the photosystems in the cells of a green alga, Chlamydomonas reinhardtii, at 94K. Gaussian decomposition analysis of the fluorescence spectra at all the pixels clearly demonstrated a non-uniform distribution of the two photosystems, as reflected in the variable ratios of the fluorescence intensities assigned to photosystem II and to those assigned to photosystem I. The system was also applied to the fluorescence spectroscopy of single isolated photosystem I complexes at 90K. The fluorescence, assigned to be emitted from a single photosystem I trimer, showed an intermittent fluctuation called blinking, which is typical for a fluorescence signal from a single molecule. The vibronic fluorescence bands at around 790nm were observed for single photosystem I trimers, suggesting that the color aberration is not serious up to the 800nm spectral region.

Light-Responsive Double B-Box Containing Transcription Factors Are Conserved in Physcomitrella patens

In the model seed plant Arabidopsis thaliana, a sub-family of B-box containing transcriptional factors (BBXs), which is classified in the BBX-IV group based on the domain structure, contains two tandem B-box domains and plays crucial roles in early photomorphogenesis under the control of blue light receptors, cry1 and cry2. The results of an examination of light responsiveness of representative Physcomitrella BBX-IV genes and their heterologous expression in Arabidopsis suggested that the light signaling-related characteristics of the BBX-IV subfamily are evolutionarily conserved in a moss, which is a basal lineage of land plants.

Crystal structures of the gastric proton pump

The gastric proton pump—the H+, K+-ATPase—is a P-type ATPase responsible for acidifying the gastric juice down to pH 1. This corresponds to a million-fold proton gradient across the membrane of the parietal cell, the steepest known cation gradient of any mammalian tissue. The H+, K+-ATPase is an important target for drugs that treat gastric acid-related diseases. Here we present crystal structures of the H+, K+-ATPase in complex with two blockers, vonoprazan and SCH28080, in the luminal-open state, at 2.8 Å resolution. The drugs have partially overlapping but clearly distinct binding modes in the middle of a conduit running from the gastric lumen to the cation-binding site. The crystal structures suggest that the tight configuration at the cation-binding site lowers the pKa value of Glu820 sufficiently to enable the release of a proton even into the pH 1 environment of the stomach.Crystal structures of the gastric proton pump in complex with two inhibitory drugs reveal the mechanism that generates the steep acidic gradient across the membranes of parietal cells.