Autophagy is suppressed by low temperatures and is dispensable for cold acclimation in Arabidopsis.

Plants have evolved various mechanisms to adapt to the ever-changing external environment. Autophagy is one such mechanism and has been suggested to play a key role in responding to and adapting to abiotic stresses in plants. However, the role of autophagy in adaptation to cold and freezing stresses remains to be characterized in detail. Here, we investigated the role of autophagy in the low-temperature response of Arabidopsis using atg mutants. Both the atg5-1 and atg10-1 mutants exhibited normal freezing tolerance, regardless of cold acclimation. A comparison of fresh weights indicated that the difference in growth between the wild-type and atg plants under cold conditions was rather small compared with that under normal conditions. Analysis of COLD-REGULATED gene expression showed no significant differences between the atg mutants and wild type. Treatment with 3-methyladenine, an inhibitor of autophagy, did not impair the induction of COR15Apro::LUC expression upon exposure to low temperature. Evaluation of autophagic activity using transgenic plants expressing RBCS-mRFP demonstrated that autophagy was rarely induced by cold exposure, even in the dark. Taken together, these data suggest that autophagy is suppressed by low temperatures and is dispensable for cold acclimation and freezing tolerance in Arabidopsis.

Potential contribution of floral thermogenesis to cold adaptation, distribution pattern, and population structure of thermogenic and non/slightly thermogenic Symplocarpus species.

The genus Symplocarpus in basal Araceae includes both thermogenic and non/slightly thermogenic species that prefer cold environments. If floral thermogenesis of Symplocarpus contributes to cold adaptation, it would be expected that thermogenic species have a larger habitat than non/slightly thermogenic species during an ice age, leading to increased genetic diversity in the current population. To address this question, potential distribution in past environment predicted by ecological niche modeling (ENM), genetic diversity, and population structure of chloroplast and genome-wide single nucleotide polymorphisms were compared between thermogenic Symplocarpus renifolius and non/slightly thermogenic Symplocarpus nipponicus. ENM revealed that the distribution of S. nipponicus decreased, whereas that of S. renifolius expanded in the Last Glacial Maximum. Phylogeographic analyses have shown that the population structures of the two species were genetically segmented and that the genetic diversity of S. renifolius was higher than that of S. nipponicus. The phylogenetic relationship between chloroplast and nuclear DNA is topologically different in the two species, which may be due to the asymmetric gene flow ubiquitously observed in plants. The results of this study imply that floral thermogenesis of Symplocarpus contributes to expanding the distribution during an ice age, resulting in increased genetic diversity due to cold adaptation.

Molecular characterization of SrSTP14, a sugar transporter from thermogenic skunk cabbage, and its possible role in developing pollen.

In floral thermogenesis, sugars play an important role not only as energy providers but also as growth and development facilitators. Yet, the mechanisms underlying sugar translocation and transport in thermogenic plants remain to be studied. Asian skunk cabbage (Symplocarpus renifolius) is a species that can produce durable and intense heat in its reproductive organ, the spadix. Significant morphological and developmental changes in the stamen are well-characterized in this plant. In this study, we focused on the sugar transporters (STPs), SrSTP1 and SrSTP14, whose genes were identified by RNA-seq as the upregulated STPs during thermogenesis. Real-time PCR confirmed that mRNA expression of both STP genes was increased from the pre-thermogenic to the thermogenic stage in the spadix, where it is predominantly expressed in the stamen. SrSTP1 and SrSTP14 complemented the growth defects of a hexose transporter-deficient yeast strain, EBY4000, on media containing 0.02, 0.2, and 2% (w/v) glucose and galactose. Using a recently developed transient expression system in skunk cabbage leaf protoplasts, we revealed that SrSTP1 and SrSTP14-GFP fusion proteins were mainly localized to the plasma membrane. To dig further into the functional analysis of SrSTPs, tissue-specific localization of SrSTPs was investigated by in situ hybridization. Using probes for SrSTP14, mRNA expression was observed in the microspores within the developing anther at the thermogenic female stage. These results indicate that SrSTP1 and SrSTP14 transport hexoses (e.g., glucose and galactose) at the plasma membrane and suggest that SrSTP14 may play a role in pollen development through the uptake of hexoses into pollen precursor cells.

Chemical screening approach using single leaves identifies compounds that affect cold signaling in Arabidopsis.

The identification of chemical compounds that affect intracellular processes has greatly contributed to our understanding of plant growth and development. In most cases, these compounds have been identified in germinated seedlings. However, chemical screening using mature plants would benefit and advance our understanding of environmental responses. In this study, we developed a high-throughput screening method using single leaves of mature plants to identify small molecules that affect cold-regulated gene expression. A single excised leaf of Arabidopsis (Arabidopsis thaliana) grown in submerged cultures responded to low temperatures in terms of COLD-REGULATED (COR) gene expression. We used transgenic Arabidopsis harboring a COLD-REGULATED 15A (COR15A) promoter::luciferase (COR15Apro::LUC) construct to screen natural compounds that affect the cold induction of COR15Apro::LUC. This approach allowed us to identify derivatives of 1,4-naphthoquinone as specific inhibitors of COR gene expression. Moreover, 1,4-naphthoquinones appeared to inhibit the rapid induction of upstream C-REPEAT BINDING FACTOR (CBF) transcription factors upon exposure to low temperature, suggesting that 1,4-naphthoquinones alter upstream signaling processes. Our study offers a chemical screening scheme for identifying compounds that affect environmental responses in mature plants. This type of analysis is likely to reveal an unprecedented link between certain compounds and plant environmental responses.

Establishing an efficient protoplast transient expression system for investigation of floral thermogenesis in aroids.

KEY MESSAGE: Floral thermogenesis is an important reproductive strategy for attracting pollinators. We developed essential biological tools for studying floral thermogenesis using two species of thermogenic aroids, Symplocarpus renifolius and Alocasia odora. Aroids contain many species with intense heat-producing abilities in their inflorescences. Several genes have been proposed to be involved in thermogenesis of these species, but biological tools for gene functional analyses are lacking. In this study, we aimed to develop a protoplast-based transient expression (PTE) system for the study of thermogenic aroids. Initially, we focused on skunk cabbage (Symplocarpus renifolius) because of its ability to produce intense as well as durable heat. In this plant, leaf protoplasts were isolated from potted and shoot tip-cultured plants with high efficiency (ca. 1.0 × 105/g fresh weight), and more than half of these protoplasts were successfully transfected. Using this PTE system, we determined the protein localization of three mitochondrial energy-dissipating proteins, SrAOX, SrUCPA, and SrNDA1, fused to green fluorescent protein (GFP). These three GFP-fused proteins were localized in MitoTracker-stained mitochondria in leaf protoplasts, although the green fluorescent particles in protoplasts expressing SrUCPA-GFP were significantly enlarged. Finally, to assess whether the PTE system established in the leaves of S. renifolius is applicable for floral tissues of thermogenic aroids, inflorescences of S. renifolius and another thermogenic aroid (Alocasia odora) were used. Although protoplasts were successfully isolated from several tissues of the inflorescences, PTE systems worked well only for the protoplasts isolated from the female parts (slightly thermogenic or nonthermogenic) of A. odora inflorescences. Our developed system has a potential to be widely used in inflorescences as well as leaves in thermogenic aroids and therefore may be a useful biological tool for investigating floral thermogenesis.

Salicylic Acid Acts Antagonistically to Plastid Retrograde Signaling by Promoting the Accumulation of Photosynthesis-associated Proteins in Arabidopsis.

Plastids are involved in phytohormone metabolism as well as photosynthesis. However, the mechanism by which plastid retrograde signals and phytohormones cooperatively regulate plastid biogenesis remains elusive. Here, we investigated the effects of an inhibitor and a mutation that generate biogenic plastid signals on phytohormones and vice versa. Inhibition of plastid biogenesis by norflurazon (NF) treatment and the plastid protein import2 (ppi2) mutation caused a decrease in salicylic acid (SA) and jasmonic acid (JA). This effect can be attributed in part to the altered expression of genes involved in the biosynthesis and the metabolism of SA and JA. However, SA-dependent induction of the PATHOGENESIS-RELATED1 gene was virtually unaffected in NF-treated plants and the ppi2 mutant. Instead, the level of chlorophyll in these plants was partially restored by the exogenous application of SA. Consistent with this observation, the levels of some photosynthesis-associated proteins increased in the ppi2 and NF-treated plants in response to SA treatment. This regulation in true leaves seems to occur at the posttranscriptional level since SA treatment did not induce the expression of photosynthesis-associated genes. In salicylic acid induction deficient 2 and lesions simulating disease resistance 1 mutants, endogenous SA regulates the accumulation of photosynthesis-associated proteins through transcriptional and posttranscriptional mechanisms. These data indicate that SA acts antagonistically to the inhibition of plastid biogenesis by promoting the accumulation of photosynthesis-associated proteins in Arabidopsis, suggesting a possible link between SA and biogenic plastid signaling.

Induction of TOC and TIC genes during photomorphogenesis is mediated primarily by cryptochrome 1 in Arabidopsis.

The majority of genes encoding photosynthesis-associated proteins in the nucleus are induced by light during photomorphogenesis, allowing plants to establish photoautotrophic growth. Therefore, optimizing the protein import apparatus of plastids, designated as the translocon at the outer and inner envelope membranes of chloroplast (TOC-TIC) complex, upon light exposure is a prerequisite to the import of abundant nuclear-encoded photosynthesis-associated proteins. However, the mechanism that coordinates the optimization of the TOC-TIC complex with the expression of nuclear-encoded photosynthesis-associated genes remains to be characterized in detail. To address this question, we investigated the mechanism by which plastid protein import is regulated by light during photomorphogenesis in Arabidopsis. We found that the albino plastid protein import2 (ppi2) mutant lacking Toc159 protein import receptors have active photoreceptors, even though the mutant fails to induce the expression of photosynthesis-associated nuclear genes upon light illumination. In contrast, many TOC and TIC genes are rapidly induced by blue light in both WT and the ppi2 mutant. We uncovered that this regulation is mediated primarily by cryptochrome 1 (CRY1). Furthermore, deficiency of CRY1 resulted in the decrease of some TOC proteins in vivo. Our results suggest that CRY1 plays key roles in optimizing the content of the TOC-TIC apparatus to accommodate the import of abundant photosynthesis-associated proteins during photomorphogenesis.

Keratins and the plakin family cytolinker proteins control the length of epithelial microridge protrusions.

Actin filaments and microtubules create diverse cellular protrusions, but intermediate filaments, the strongest and most stable cytoskeletal elements, are not known to directly participate in the formation of protrusions. Here we show that keratin intermediate filaments directly regulate the morphogenesis of microridges, elongated protrusions arranged in elaborate maze-like patterns on the surface of mucosal epithelial cells. We found that microridges on zebrafish skin cells contained both actin and keratin filaments. Keratin filaments stabilized microridges, and overexpressing keratins lengthened them. Envoplakin and periplakin, plakin family cytolinkers that bind F-actin and keratins, localized to microridges, and were required for their morphogenesis. Strikingly, plakin protein levels directly dictate microridge length. An actin-binding domain of periplakin was required to initiate microridge morphogenesis, whereas periplakin-keratin binding was required to elongate microridges. These findings separate microridge morphogenesis into distinct steps, expand our understanding of intermediate filament functions, and identify microridges as protrusions that integrate actin and intermediate filaments.

Cells adopt a wide array of irregular and bumpy shapes, which are scaffolded by an internal structure called the cytoskeleton. This network of filaments can deform the cell membrane the way tent poles frame a canvas. Cells contain three types of cytoskeleton elements (actin filaments, intermediate filaments, and microtubules), each with unique chemical and mechanical properties. One of the main roles of the cytoskeleton is to create protrusions, a range of structures that 'stick out' of a cell to allow movement and interactions with the environment. Both actin filaments and microtubules help form protrusions, but the role of intermediate filaments remains unclear. Microridges are a type of protrusion found on cells covered by mucus, for instance on the surface of the eye, inside the mouth, or on fish skin. These small bumps are organised on the membrane of a cell in fingerprint-like arrangements. Scientists know that actin networks are necessary for microridges to form; yet, many structures supported by actin filaments are not stable over time, suggesting that another component of the cytoskeleton might be lending support. Intermediate filaments are the strongest, most stable type of cytoskeleton element, and they can connect to actin filaments via linker proteins. However, research has yet to show that this kind of cooperation happens in any membrane protrusion. Here, Inaba et al. used high-resolution microscopy to monitor microridge development in the skin of live fish. In particular, they focused on a type of intermediate filaments known as keratin filaments. This revealed that, inside microridges, the keratin and actin networks form alongside each other, with linker proteins called Envoplakin and Periplakin connecting the two structures together. Genetic experiments revealed that Envoplakin and Periplakin must attach to actin for microridges to start forming. However, the two proteins bind to keratin for protrusions to grow. This work therefore highlights how intermediate filaments and linker proteins contribute to the formation of these structures. Many tissues must be covered in mucus to remain moist and healthy. As microridges likely contribute to mucus retention, the findings by Inaba et al. may help to better understand how disorders linked to problems in mucus emerge.

Installation of authentic BicA and SbtA proteins to the chloroplast envelope membrane is achieved by the proteolytic cleavage of chimeric proteins in Arabidopsis.

To improve the photosynthetic performance of C3 plants, installing cyanobacterial bicarbonate transporters to the chloroplast inner envelope membrane (IEM) has been proposed for years. In our previous study, we successfully introduced chimeric cyanobacterial sodium-dependent bicarbonate transporters, BicA or SbtA, to the chloroplast IEM of Arabidopsis. However, the installation of authentic BicA and SbtA to the chloroplast IEM has not been achieved yet. In this study, we examined whether or not tobacco etch virus (TEV) protease targeted within chloroplasts can cleave chimeric proteins and produce authentic bicarbonate transporters. To this end, we constructed a TEV protease that carried the transit peptide and expressed it with chimeric BicA or SbtA proteins containing a TEV cleavage site in planta. Chimeric proteins were cleaved only when the TEV protease was co-expressed. The authentic forms of hemagglutinin-tagged BicA and SbtA were detected in the chloroplast IEM. In addition, cleavage of chimeric proteins at the TEV recognition site seemed to occur after the targeting of chimeric proteins to the chloroplast IEM. We conclude that the cleavage of chimeric proteins within chloroplasts is an efficient way to install authentic bicarbonate transporters to the chloroplast IEM. Furthermore, a similar approach can be applied to other bacterial plasma membrane proteins.

Alternative Oxidase Capacity of Mitochondria in Microsporophylls May Function in Cycad Thermogenesis.

Cone thermogenesis is a widespread phenomenon in cycads and may function to promote volatile emissions that affect pollinator behavior. Given their large population size and intense and durable heat-producing effects, cycads are important organisms for comprehensive studies of plant thermogenesis. However, knowledge of mitochondrial morphology and function in cone thermogenesis is limited. Therefore, we investigated these mitochondrial properties in the thermogenic cycad species Cycas revoluta Male cones generated heat even in cool weather conditions. Female cones produced heat, but to a lesser extent than male cones. Ultrastructural analyses of the two major tissues of male cones, microsporophylls and microsporangia, revealed the existence of a population of mitochondria with a distinct morphology in the microsporophylls. In these cells, we observed large mitochondria (cross-sectional area of 2 µm2 or more) with a uniform matrix density that occupied >10% of the total mitochondrial volume. Despite the size difference, many nonlarge mitochondria (cross-sectional area <2 µm2) also exhibited a shape and a matrix density similar to those of large mitochondria. Alternative oxidase (AOX) capacity and expression levels in microsporophylls were much higher than those in microsporangia. The AOX genes expressed in male cones revealed two different AOX complementary DNA sequences: CrAOX1 and CrAOX2 The expression level of CrAOX1 mRNA in the microsporophylls was 100 times greater than that of CrAOX2 mRNA. Collectively, these results suggest that distinctive mitochondrial morphology and CrAOX1-mediated respiration in microsporophylls might play a role in cycad cone thermogenesis.

Investigating Localization of Chimeric Transporter Proteins within Chloroplasts of Arabidopsis thaliana.

In this protocol, we describe a method to design chimeric proteins for specific targeting to the inner envelope membrane (IEM) of Arabidopsis chloroplasts and the confirmation of their localization by biochemical analysis. Specific targeting to the chloroplast IEM can be achieved by fusing the protein of interest with a transit peptide and an IEM targeting signal. This protocol makes it possible to investigate the localization of chimeric proteins in chloroplasts using a small number of transgenic plants by using a modified method of chloroplast isolation and fractionation. IEM localization of chimeric proteins can be further assessed by trypsin digestion and alkaline extraction. Here, the localization of the chimeric bicarbonate transporter, designated as SbtAII, is detected by Western blotting using antibodies against Staphylococcal protein A. This protocol is adapted from Uehara et al., 2016 .

Ubiquitin-Proteasome-Dependent Regulation of Bidirectional Communication between Plastids and the Nucleus.

Plastids are DNA-containing organelles and can have unique differentiation states depending on age, tissue, and environment. Plastid biogenesis is optimized by bidirectional communication between plastids and the nucleus. Import of nuclear-encoded proteins into plastids serves as anterograde signals and vice versa, plastids themselves send retrograde signals to the nucleus, thereby controlling de novo synthesis of nuclear-encoded plastid proteins. Recently, it has become increasingly evident that the ubiquitin-proteasome system regulates both the import of anterograde plastid proteins and retrograde signaling from plastids to the nucleus. Targets of ubiquitin-proteasome regulation include unimported chloroplast precursor proteins in the cytosol, protein translocation machinery at the chloroplast surface, and transcription factors in the nucleus. This review will focus on the mechanism through which the ubiquitin-proteasome system optimizes plastid biogenesis and plant development through the regulation of nuclear-plastid interactions.

Ubiquitin-Proteasome Dependent Regulation of the GOLDEN2-LIKE 1 Transcription Factor in Response to Plastid Signals.

Arabidopsis (Arabidopsis thaliana) GOLDEN2-LIKE (GLK) transcription factors promote chloroplast biogenesis by regulating the expression of photosynthesis-related genes. Arabidopsis GLK1 is also known to participate in retrograde signaling from chloroplasts to the nucleus. To elucidate the mechanism by which GLK1 is regulated in response to plastid signals, we biochemically characterized Arabidopsis GLK1 protein. Expression analysis of GLK1 protein indicated that GLK1 accumulates in aerial tissues. Both tissue-specific and Suc-dependent accumulation of GLK1 were regulated primarily at the transcriptional level. In contrast, norflurazon- or lincomycin-treated gun1-101 mutant expressing normal levels of GLK1 mRNA failed to accumulate GLK1 protein, suggesting that plastid signals directly regulate the accumulation of GLK1 protein in a GUN1-independent manner. Treatment of the glk1glk2 mutant expressing functional GFP-GLK1 with a proteasome inhibitor, MG-132, induced the accumulation of polyubiquitinated GFP-GLK1. Furthermore, the level of endogenous GLK1 in plants with damaged plastids was partially restored when those plants were treated with MG-132. Collectively, these data indicate that the ubiquitin-proteasome system participates in the degradation of Arabidopsis GLK1 in response to plastid signals.

Characterization of two PEBP genes, SrFT and SrMFT, in thermogenic skunk cabbage (Symplocarpus renifolius).

Floral thermogenesis has been found in dozens of primitive seed plants and the reproductive organs in these plants produce heat during anthesis. Thus, characterization of the molecular mechanisms underlying flowering is required to fully understand the role of thermogenesis, but this aspect of thermogenic plant development is largely unknown. In this study, extensive database searches and cloning experiments suggest that thermogenic skunk cabbage (Symplocarpus renifolius), which is a member of the family Araceae, possesses two genes encoding phosphatidyl ethanolamine-binding proteins (PEBP), FLOWERING LOCUS T (SrFT) and MOTHER OF FT AND TFL1 (SrMFT). Functional analyses of SrFT and SrMFT in Arabidopsis indicate that SrFT promotes flowering, whereas SrMFT does not. In S. renifolius, the stage- and tissue-specific expression of SrFT was more evident than that of SrMFT. SrFT was highly expressed in flowers and leaves and was mainly localized in fibrovascular tissues. In addition, microarray analysis revealed that, within floral tissues, SrFT was co-regulated with the genes associated with cellular respiration and mitochondrial function, including ALTERNATIVE OXIDASE gene proposed to play a major role in floral thermogenesis. Taken together, these data suggest that, among the PEBP genes, SrFT plays a role in flowering and floral development in the thermogenic skunk cabbage.

Transport of the outer dynein arm complex to cilia requires a cytoplasmic protein Lrrc6.

Lrrc6 encodes a cytoplasmic protein that is expressed specifically in cells with motile cilia including the node, trachea and testes of the mice. A mutation of Lrrc6 has been identified in human patients with primary ciliary dyskinesia (PCD). Mutant mice lacking Lrrc6 show typical PCD defects such as hydrocephalus and laterality defects. We found that in the absence of Lrrc6, the morphology of motile cilia remained normal, but their motility was completely lost. The 9 + 2 arrangement of microtubules remained normal in Lrrc6(-/-) mice, but the outer dynein arms (ODAs), the structures essential for the ciliary beating, were absent from the cilia. In the absence of Lrrc6, ODA proteins such as DNAH5, DNAH9 and IC2, which are assembled in the cytoplasm and transported to the ciliary axoneme, remained in the cytoplasm and were not transported to the ciliary axoneme. The IC2-IC1 interaction, which is the first step of ODA assembly, was normal in Lrrc6(-/-) mice testes. Our results suggest that ODA proteins may be transported from the cytoplasm to the cilia by an Lrrc6-dependent mechanism.

Specific and Efficient Targeting of Cyanobacterial Bicarbonate Transporters to the Inner Envelope Membrane of Chloroplasts in Arabidopsis.

Installation of cyanobacterial bicarbonate transporters to the inner envelope membrane (IEM) of chloroplasts in C3 plants has been thought to improve photosynthetic performance. However, the method to deliver cyanobacterial bicarbonate transporters to the chloroplast IEM remains to be established. In this study, we provide evidence that the cyanobacterial bicarbonate transporters, BicA and SbtA, can be specifically installed into the chloroplast IEM using the chloroplast IEM targeting signal in conjunction with the transit peptide. We fused the transit peptide and the mature portion of Cor413im1, whose targeting mechanism to the IEM has been characterized in detail, to either BicA or SbtA isolated from Synechocystis sp. PCC6803. Among the seven chimeric constructs tested, we confirmed that four chimeric bicarbonate transporters, designated as BicAI, BicAII, SbtAII, and SbtAIII, were expressed in Arabidopsis. Furthermore, these chimeric transporters were specifically targeted to the chloroplast IEM. They were also resistant to alkaline extraction but can be solubilized by Triton X-100, indicating that they are integral membrane proteins in the chloroplast IEM. One of the transporters, BicA, could reside in the chloroplast IEM even after removal of the IEM targeting signal. Taken together, our results indicate that the addition of IEM targeting signal, as well as the transit peptide, to bicarbonate transporters allows us to efficiently target nuclear-encoded chimeric bicarbonate transporters to the chloroplast IEM.

Targeting of a polytopic membrane protein to the inner envelope membrane of chloroplasts in vivo involves multiple transmembrane segments.

The inner envelope membrane (IEM) of the chloroplast plays crucial roles in forming an osmotic barrier and controlling metabolite exchange between the organelle and the cytosol. The IEM therefore harbours a number of membrane proteins and requires the import and integration of these nuclear-encoded proteins for its biogenesis. Recent studies have demonstrated that the transmembrane segment of single-spanning IEM proteins plays key roles in determining their IEM localization. However, few studies have focused on the molecular mechanisms by which polytopic membrane proteins are targeted to the IEM. In this study, we investigated the targeting mechanism of polytopic IEM proteins using the protein Cor413im1 as a model substrate. Cor413im1 does not utilize a soluble intermediate for its targeting to the IEM. Furthermore, we show that the putative fifth transmembrane segment of Cor413im1 is necessary for its targeting to the IEM. The C-terminal portion containing this transmembrane segment is also able to deliver Cor413im1 protein to the IEM. However, the fifth transmembrane segment of Cor413im1 itself is insufficient to target a fusion protein to the IEM. These data suggest that the targeting of polytopic membrane proteins to the chloroplast IEM in vivo involves multiple transmembrane segments and that chloroplasts have evolved a unique mechanism for the integration of polytopic proteins to the IEM.

[Effects of exercise intervention on exercise behavior in community-dwelling elderly subjects: a randomized controlled trial].

AIM: The objective of the present study was to evaluate the long-term effectiveness of an exercise program in modifying the exercise behavior of the community-dwelling elderly subjects. METHODS: This study was a single-blinded randomized controlled trial. The subjects included 52 males and 65 females 65 years of age or over who were randomly assigned to an exercise-intervention group or a health-education group. The stages of change in exercise behavior were evaluated before and one-year after the intervention period. The subjects' physical function (muscle strength, balance, walking speed) and self-efficacy in each domain of the physical function were measured during the intervention period. RESULTS: There were no significant differences in the stages of change before the intervention between the two groups. Significant differences in the stages of change were observed in "relapse" of stages at two points in time between the two groups (p<.01). A logistic regression analysis showed that "progression" of stages was associated with improvements in the timed up and go test (AOR 2.7; 95% CI 1.3-5.8) and sit and reach (AOR 1.14; 95%CI 1.0-1.3), while "relapse" of stages was associated with the group allocation (AOR 4.6; 95%CI 1.1-18.8), self-efficacy in "Walking" (AOR 1.54; 95%CI 1.0-2.3) and "Stair climbing" (AOR 0.68; 95%CI 0.5-0.9) with respect to physical activity during the intervention period. CONCLUSIONS: The results suggest that exercise intervention in community-dwelling elderly subjects is effective in preventing "relapse" of exercise behavior over long periods.

Isolation and gene expression analysis of a papain-type cysteine protease in thermogenic skunk cabbage (Symplocarpus renifolius).

Skunk cabbage (Symplocarpus renifolius) spadices contain abundant transcripts for cysteine protease (CP). From thermogenic spadices, we isolated SrCPA, a highly expressed CP gene that encoded a papain-type CP. SrCPA is structurally similar to other plant CPs, including the senescence-associated CPs found in aroids. The expression of SrCPA increased during floral development, and was observed in all floral tissues except for the stamens.