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We identified a 23 kDa protein expressed abundantly in young barley seedlings; this protein was named P23k. P23k was shown to have evolved in barley and its allied species such as wheat, oat and rye, suggesting that P23k might be closely related to a specific biological trait of winter cereals. To determine the function of P23k, we examined the expression and localization of P23k and its mRNA in barley seedlings. P23k mRNA was markedly expressed in the scutellum of imbibed barley seeds, and was synchronously down-regulated with consumption of the starchy endosperm. In addition, ABA and sugar treatment of the scutellum showed that expression of P23k is highly dependent on the supply of glucose or sucrose, both of which are starch catabolites. Furthermore, immunocytochemical analysis showed that P23k is primarily localized around the membranes, where sugar translocation is active. These results indicate that P23k plays a role in sugar translocation in young barley seedlings.

A Lotus japonicus mutant, Ljsym75, which forms ineffective symbiotic nodules and defines a new locus involved in the process of nitrogen fixation, was characterized in detail in order to identify the stage of developmental arrest of the nodules. No nitrogen-fixing activity was detectable in Ljsym75 nodules at any stage during plant development, and plant growth was markedly retarded. Ljsym75 plants formed twice as many nodules as the wild-type Gifu, and this phenotype was not influenced by the application of low concentrations of nitrate. Although the ineffective nodules formed on Ljsym75 were anatomically similar to effective Gifu nodules, Ljsym75 nodules senesced prematurely. Microscopic examination revealed that bacteria endocytosed into Ljsym75 nodules failed to differentiate into bacteroids. Moreover, the bacteria contained no nitrogenase proteins, whereas leghemoglobin was detected in the cytosol of the nodules. These results indicate that Ljsym75 is required for bacterial differentiation into nitrogen-fixing bacteroids in nodules, and thus the Ljsym75 gene was renamed sen1 (for stationary endosymbiont nodule). Linkage analysis using DNA markers showed that Sen1 is located on chromosome 4.

All cDNAs involved in carotenoid biosynthesis leading to lycopene in yellow petals of Gentiana lutea have been cloned from a cDNA library. They encode a geranylgeranyl pyrophosphate synthase, a phytoene synthase, a phytoene desaturase and a ζ-carotene desaturase. The indicated function of all cDNAs was established by heterologous complementation in Escherichia coli. The amino acid sequences deduced from the cDNAs were between 47.5% and 78.9% identical to those reported for the corresponding enzymes from other higher plants. Southern analysis suggested that the genes for each enzyme probably represent a small multi-gene family. Tissue-specific expression of the genes and expression during flower development was investigated. The expression of the phytoene synthase gene, psy, was enhanced in flowers but transcripts were not detected in stems and leaves by northern blotting. Transcripts of the genes for geranylgeranyl pyrophosphate (ggpps), phytoene desaturase (pds) and ζ-carotene desaturase (zds) were detected in flowers and leaves but not in stems. Analysis of the expression of psy and zds in petals revealed that levels of the transcripts were lowest in young buds and highest in fully open flowers, in parallel with the formation of carotenoids. Obviously, the transcription of these genes control the accumulation of carotenoids during flower development in G. lutea. For pds only a very slight increase of mRNA was found whereas the transcripts of ggpps decreased during flower development.

For analysis of the promoter of a gene for chalcone synthase ( GTCHS1 ) from Gentiana triflora , a cDNA was cloned for chalcone synthase (CHS) from a cDNA library of petals of Gentiana . Using the sequence of GTCHS1 , the promoter region of GTCHS1 was cloned by the inverse polymerase chain reaction. The promoter was fused to a gene for β -glucuronidase (GUS) and the construct was introduced into Petunia hybrida . Measurements of the GUS activities of transformants indicated that the GTCHS1 promoter strongly directed the expression of GUS in flower limbs, while the 35S promoter of cauliflower mosaic virus (CaMV) directed expression of the reporter gene in all tissues. Histochemical staining of GUS activity revealed that the GTCHS1 promoter strongly directed the expression of GUS in the inner epidermis, at sites where most of the anthocyanin accumulated. The sequence of the GTCHS1 promoter included a consensus sequence of the MYB protein-binding site, five consensus sequences of the MYC protein-binding site, one core sequence of a G-box and three P-box-like sequences.

The cyanide-resistant alternative oxidase (AOX) is a homodimeric protein whose activity can be regulated by the oxidation/reduction state and by α-keto acids. To further clarify the role of AOX in the skunk cabbage, Symplocarpus renifolius, we have performed expression and functional analyses of the encoding gene. Among the various tissues in the skunk cabbage, SrAOX transcripts were found to be specifically expressed in the thermogenic spadix. Moreover, our data demonstrate that the SrAOX protein exists as a non-covalently associated dimer in the thermogenic spadix, and is more sensitive to pyruvate than to other carboxylic acids. Our results suggest that the pyruvate-mediated modification of SrAOX activity plays a significant role in thermoregulation in the skunk cabbage.

Actin filaments are among the major components of the cytoskeleton, and participate in various cellular dynamic processes. However, conflicting results had been obtained on the localization of actin filaments on the mitotic apparatus and their participation in the process of chromosome segregation. We demonstrated by using rhodamine-phalloidin staining, the localization of actin filaments on the mitotic spindles of tobacco BY-2 cells when the cells were treated with cytochalasin D. At prophase, several clear spots were observed at or near the kinetochores of the chromosomes. At anaphase, the actin filaments that appeared to be pulling chromosomes toward the division poles were demonstrated. However, as there was a slight possibility that these results might have been the artifacts of cytochalasin D treatment or the phalloidin staining, we analyzed the localization of actin filaments at the mitotic apparatus immunologically. We cloned a novel BY-2 alpha-type actin cDNA and prepared a BY-2 actin antibody. The fluorescence of the anti-BY-2 actin antibody was clearly observed at the mitotic apparatus in both non-treated and cytochalasin D-treated BY-2 cells during mitosis. The facts that similar results were obtained in both actin staining with rhodamine-phalloidin and immunostaining with actin antibody strongly indicate the participation of actin in the organization of the spindle body or in the process of chromosome segregation. Furthermore, both filamentous actin and spindle bodies disappeared in the cells treated with propyzamide, which depolymerizes microtubules, supporting the notion that actin filaments are associated with microtubules organizing the spindle body.

cDNAs encoding lycopene epsilon -cyclase, lycopene beta-cyclase, beta-carotene hydroxylase and zeaxanthin epoxidase were isolated from a Gentiana lutea petal cDNA library. The function of all cDNAs was analyzed by complementation in Escherichia coli. Transcript levels during different stages of flower development of G. lutea were determined and compared to the carotenoid composition. Expression of all genes increased by a factor of up to 2, with the exception of the lycopene epsilon -cyclase gene. The transcript amount of the latter was strongly decreased. These results indicate that during flower development, carotenoid formation is enhanced. Moreover, metabolites are shifted away from the biosynthetic branch to lutein and are channeled into beta-carotene and derivatives.

All cDNAs involved in carotenoid biosynthesis leading to lycopene in yellow petals of Gentiana lutea have been cloned from a cDNA library. They encode a geranylgeranyl pyrophosphate synthase, a phytoene synthase, a phytoene desaturase and a zeta-carotene desaturase. The indicated function of all cDNAs was established by heterologous complementation in Escherichia coli. The amino acid sequences deduced from the cDNAs were between 47.5% and 78.9% identical to those reported for the corresponding enzymes from other higher plants. Southern analysis suggested that the genes for each enzyme probably represent a small multi-gene family. Tissue-specific expression of the genes and expression during flower development was investigated. The expression of the phytoene synthase gene, psy, was enhanced in flowers but transcripts were not detected in stems and leaves by northern blotting. Transcripts of the genes for geranylgeranyl pyrophosphate (ggpps), phytoene desaturase (pds) and zeta-carotene desaturase (zds) were detected in flowers and leaves but not in stems. Analysis of the expression of psy and zds in petals revealed that levels of the transcripts were lowest in young buds and highest in fully open flowers, in parallel with the formation of carotenoids. Obviously, the transcription of these genes control the accumulation of carotenoids during flower development in G. lutea. For pds only a very slight increase of mRNA was found whereas the transcripts of ggpps decreased during flower development.

To gain an overview of plant factors controlling nodule number and organogenesis, an extensive screening using model legume Lotus japonicus was carried out. This screening involved 40,000 M2 seeds, and 32 stable mutant lines were isolated. From these, 16 mutant lines maintaining the phenotypic variation were selected and genetically analyzed. With respect to nodule number, four loci were identified, Ljsym77, Ljsym78, slippery root (slp), and radial organization1 (rdo1). The former two mutants have an increased number of nodules, while the latter two have a decreased number. Ljsym78-1 and Ljsym78-2 are hypernodulating mutants with a branched root system and were found to be allelic to Ljsym16. The phenotype of the Ljsym77 mutant was highly pleiotropic, being deficient in light and gravity responses. The slp mutant was isolated as a low-nodulating mutant lacking root hairs. Concerning nodule organogenesis, nine symbiotic loci were identified, including the two loci alb1 and fen1. Mutants affecting the developmental process of nodule organogenesis were placed in three phenotypic categories: Nod¯ (Ljsym70 to Ljsym73), Hist¯ (alb1-1, alb1-2, and Ljsym79), and Fix¯ (fen1, Ljsym75, and Ljsym81).

Two distinct mitochondrial energy dissipating systems, alternative oxidase (AOX) and uncoupling protein (UCP), have been implicated as crucial components of thermogenesis in plants and animals, respectively. To further clarify the physiological roles of AOX and UCP during homeothermic heat production in the thermogenic skunk cabbage (Symplocarpus renifolius), we identified the thermogenic cells and performed expression and functional analyses of these genes in this organism. Thermographic analysis combined with in situ hybridization revealed that the putative thermogenic cells surround the stamens in the florets of skunk cabbage and coexpress transcripts for SrAOX, encoding Symplocarpus AOX, and SrUCPb, encoding a novel UCP that lacks a fifth transmembrane segment. Mitochondria isolated from the thermogenic florets exhibited substantial linoleic acid (LA)-inducible uncoupling activities. Moreover, our results demonstrate that LA is capable of inhibiting the mitochondrial AOX pathway, whereas the proportion of pyruvate-stimulated AOX capacity was not significantly affected by LA. Intriguingly, the protein expression levels for SrAOX and SrUCPb were unaffected even when the ambient air temperatures increased from 10.3°C to 23.1°C or from 8.3°C to 24.9°C. Thus, our results suggest that functional coexpression of AOX and UCP underlies the molecular basis of heat production, and that posttranslational modifications of these proteins play a crucial role in regulating homeothermic heat production under conditions of natural ambient temperature fluctuations in skunk cabbage.

The ultrastructural changes in plastids ofBuxus sempervirens L. leaves were observed during their seasonal yellowing and regreening. The disintegration of chloroplasts into globular type chromoplasts in yellowing leaves and their direct restoration to functional chloroplasts again in regreening leaves were followed. The results presented an example of recent information indicating the essential sense of the reversible reciprocation of plastid transformation.

Direction and degree of plastid development in the tissue differentiation of sporangiogenesis inThelypteris palustris were studied, and are discussed through aspects of the plastome continuity and cell differentiation.

Leaves of tomato plants (Lycopersicon esculentum cv. Fukuju no.2) inoculated with TMV-L11A (attenuated) or TMV-L (virulent) were compared by electron microscopy during the course of virus infection. Estimates were also made of the numbers of virions, the chlorophyll (a+b) content and the chlorophyll (a/b) ratio. Five days after inoculation, no differences were found in chloroplast structure, chlorophyll content or chlorophyll (a/b) ratio between healthy and infected plants, although a large number of virions were observed in these plants with both strains. From 10 to 30 days post-inoculation, changes in chlorophyll content and chlorophyll (a/b) ratios became apparent, the amount being in the order healthy>L11A>L. Virion accumulation was also much greater in cells infected with L than that with L11A. In both cases, chloroplasts were swollen and finally often ruptured. Their lamellar systems, however, remained essentially undamaged. Even though the L11A never induced mosaic symptoms, this attenuated strain also caused some ultrastructural changes in chloroplasts.