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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.

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.

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.

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.