Glyoxylate Cycle Enzymes in Peroxisomes Isolated from Petals of Pumpkin (Cucurbita sp.) during Senescence

The activities of the two unique enzymes of the glyoxylate cycle,isocitrate lyase (EC 4.1.3.1 [EC] ) and malate synthase (EC 4.1.3.2 [EC] ),were undetectable in petals of pumpkin (Cucurbita sp. AmakuriNankin) until the end of blooming, but they appeared duringsenescence. The activity of catalase (EC 1.11.1.6 [EC] ) increased,glycolate oxidase (EC 1.1.3.1 [EC] ) activity did not change, whilehydroxypyruvate reductase (EC 1.1.1.81 [EC] ) activity peaked at fullblooming stage and declined thereafter. After fractionationof cellular organelles on a sucrose density gradient, we detectedisocitrate lyase and malate synthase activities in peroxisomalfractions only from petals at the senescing stage. Northernblot analysis revealed that malate synthase mRNA increased duringpetal senescence. Citrate synthase (EC 4.1.3.7 [EC] ) and malate dehydrogenase(EC 1.1.1.37 [EC] ) activities were also present, while aconitase(EC 4.2.1.3 [EC] ) was not detectable in peroxisomal fractions. Moreoverthe presence of 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35 [EC] )and urate oxidase (EC 1.7.3.3 [EC] ) in the peroxisomal fractionsfrom senescing petals indicates that peroxisomes could be involvedboth in the ß-oxidation pathway and in the purinecatabolism during petal senescence. (Received May 25, 1991; Accepted September 25, 1991)     

Nonspecific lipid transfer protein in castor bean cotyledon cells: subcellular localization and a possible role in lipid metabolism.

The subcellular localization and several biochemical activities of nonspecific lipid transfer protein (nsLTP) were investigated. A section of a castor bean cotyledon cell was labeled with anti-nsLTP serum followed by protein A-gold. Gold particles were more abundant in the glyoxysome matrix and the vessel cell wall than in other areas. Cell fractionation analysis of 6-day-old castor bean cotyledons by sucrose density gradient centrifugation demonstrated that 13% of nsLTP was distributed in the glyoxysomal fraction, identified on the basis of catalase as a marker, and 87% in the soluble fraction near the top of the gradient. The location of castor bean nsLTP in glyoxysomes was further confirmed by in vitro import experiments. The synthesized precursor of nsLTP (pro-nsLTP-C) was incorporated into intact castor bean glyoxysomes and processed to the mature form after import into the glyoxysomes, but it was not imported into canine pancreatic microsomes. Castor bean nsLTP-A was found to possess the ability to bind oleic acid and oleoyl-CoA by means of a method involving Lipidex 1000. The dissociation constants (Kd) for oleic acid and oleoyl-CoA binding to nsLTP-A were 4.8 and 5.0 microM, respectively. The saturated binding capacities (Bmax) for oleic acid and oleoyl-CoA per mol of nsLTP-A were 1.1 and 1.2 mol, respectively. When acyl-CoA oxidase activity was assayed in the glyoxysomal fraction, marked enhancement of the activity was observed in the presence of nsLTP. These results suggest the possibility that nsLTP regulates fatty acid beta-oxidation through the enhancement of acyl-CoA oxidase activity in glyoxysomes. The occurrence of castor bean nsLTP in the vessel wall was discussed.     

A transposon insertion in the Arabidopsis SSR16 gene causes an embryo-defective lethal mutation

The SSR16 gene of Arabidopsis has been identified as a gene encoding a ribosomal protein S16 homolog through analysis of a transposon insertion mutation. The insertion mutation is lethal, arresting embryonic development at approximately the transition from the globular to the heart stage of embryonic development. Co-segregation of the mutant phenotype with the transposon-borne drug-resistance marker and loss of the inserted transposon concomitant with phenotypic reversion provided evidence that the transposon had caused the mutation. Sequences flanking the insertion site were amplified from DNA of viable heterozygotes by thermal asymmetric interlaced (TAIL) PCR. The amplified fragment flanking the 3' end of the inserted element was sequenced and found to be identical to an Arabidopsis expressed sequence tag (EST). The EST, in turn, contained a coding sequence homologous to the ribosomal protein S16 (RPS16) of bacteria such as Escherichia coli, Bacillus subtilis and Salmonella typhimurium , as well as Neurospora crassa mitochondria and higher plant plastids. Thus the gene identified by the embryo-defective lethal insertion mutation encodes an RPS16 homolog and has been designated the SSR16 gene.     

Purification, cDNA cloning and Northern-blot analysis of mitochondrial chaperonin 60 from pumpkin cotyledons.

Two different cDNA clones, pMCPN60-1 and pMCPN60-2, encoding the mitochondrial homologues of chaperonin 60 (Cpn60) were isolated from a cDNA library of germinating pumpkin cotyledons by use of mixtures of synthetic oligonucleotides based on the N-terminal amino acid sequence of the protein. Determination of the complete nucleotide sequences of the two cDNA revealed that pMCPN60-1 and pMCPN60-2 each contain one open reading frame that encodes a protein of 575 amino acids with molecular masses of 61052 Da and 61127 Da, respectively. The deduced amino acid sequences of the two polypeptides include a 32-residue N-terminal putative mitochondrial presequence attached to the mature polypeptides, and they are 95.3% identical. From a comparison of deduced amino acid sequences with other Cpn60, it appears that the mature polypeptides of pumpkin mitochondrial Cpn60 are 44-59% identical to the other Cpn60, namely, GroEL of Escherichia coli, the 60-kDa heat-shock protein (Hsp60) of mitochondria in the yeast Saccharomyces cerevisiae, P1 protein of mammalian mitochondria and the Ribulose-1,5-bisphosphate carboxylase/oxygenase subunit-binding proteins alpha and beta of plastids in higher plants. Genomic Southern-blot analysis identified at least two copies of the gene for mitochondrial Cpn60 in the pumpkin genome. The levels of mRNA for mitochondrial Cpn60 in cotyledons, hooks and hypocotyls of pumpkin seedlings increased in response to heat stress, as deduced from Northern-blot analysis, indicating that pumpkin mitochondrial Cpn60 is a heat-induced stress protein.     

Historical Changes in the Ecosystem Condition of a Small Mountain Lake over the Past 60 Years as Revealed by Plankton Remains and Daphnia Ephippial Carapaces Stored in Lake Sediments

To examine if changes in species composition of a plankton community in the past due to anthropogenic activities can be clarified in lakes without any monitoring data, we analyzed genetically ephippial carapaces of Daphnia with plankton remains stored in the bottom sediments of Lake Hataya Ohunma in Japan. In the lake, abundance of most plankton remains in the sediments was limited and TP flux was at low levels (2–4 mg/m²/y) before 1970. However TP flux increased two-fold during the period from 1980s to 1990s. In parallel with this increase, abundance of most plankton remains increased although abundance of benthic testate amoebae’s remains decreased, indicating that the lake trophic condition had changed from oligo- to mesotrophic for the past 60 years. According to cluster analysis, the stratigraphic sediments were divided into two periods with different features of the phytoplankton composition. Chronological comparison with events in the watershed suggested that eutrophication occurred because of an increase in visitors to the watershed and deposition of atmospheric dust. In this lake more than 50% of resting eggs produced by Daphnia over the past 60 years hatched. However, genetic analysis of the ephippial carapaces (remains) showed that the Daphnia population was originally composed of D. dentifera but that D. galeata, or its hybrid with D. dentifera, invaded and increased the population density when the lake was eutrophied. Subsequently, large D. pulex established populations in the 1980s when largemouth bass were anonymously introduced. These results indicated that the Lake Hataya Ohunma plankton community underwent significant changes despite the fact that there were no notable changes in land cover or land use in the watershed. Since increases in atmospheric deposition and release of fish have occurred in many Japanese lakes, the changes in the plankton community described here may be widespread in these lakes.     

Eutrophication of mountain lakes in Japan due to increasing deposition of anthropogenically produced dust

Atmospheric dust has wide-reaching effects, not only influencing climate conditions, but also ecosystems. The eastern region of the Asian continent is one of the largest emitters of dust in the world, and recent economic growth in the region has been accompanied by an increase in anthropogenic emissions. However, the effects of increased Asian dusts on aquatic ecosystems are not well understood. We examined fossil pigments and zooplankton remains from ²¹⁰Pb-dated sediments taken from high mountain lakes of Hourai-Numa and Hachiman-Numa, located in the Towada-Hachimantai National Park of Japan Islands, to uncover historical changes in the phyto- and zooplankton community over the past 100?years. Simultaneously, we measured the geochemical variables of TOC, TN, TP, ??¹³C, ??¹⁵N, and lead isotopes (²⁰⁷Pb/²⁰⁶Pb, ²⁰⁸Pb/²⁰⁶Pb) in the sediments to identify environmental factors causing such changes. As a result, despite few anthropogenic activities in the watersheds, alpine lakes in Japan had increased algal and herbivore plankton biomasses by 3?C6 fold for recent years depending on the surrounding terrestrial vegetation and landscape conditions. Biological and biogeochemical proxies recorded from the lake sediments showed that this eutrophication occurred after the 1990s when P deposition increased as a result of atmospheric loading of dust transported from the Asian continent. The continued increase of anthropogenically produced dust may therefore impart damaging impacts on mountain ecosystems even if they are protected from direct anthropogenic disturbances.     

Isolation and characterization of a cDNA encoding mitochondrial chaperonin 10 from Arabidopsis thaliana by functional complementation of an Escherichia coli groES mutant

Chaperonin (Cpn) is one of the molecular chaperones. Cpn10 is a co-factor of Cpn60, which regulates Cpn60-mediated protein folding. It is known that Cpn10 is located in mitochondria and chloroplasts in plant cells. The Escherichia coli homologue of Cpn10 is called GroES. A cDNA for the Cpn10 homologue was isolated from Arabidopsis thaliana by functional complementation of the E. coli groES mutant. The cDNA was 647 bp long and encoded a polypeptide of 98 amino acids. The deduced amino acid sequence showed approximately 50% identity to mammalian mitochondrial Cpn10s and 30% identity to GroES. A Northern blot analysis revealed that the mRNA for the Cpn10 homologue was expressed uniformly in various organs and was markedly induced by heat-shock treatment. The Cpn10 homologue was constitutively expressed in transgenic tobaccos. Immunogold and immunoblot analyses following the subcellular fractionation of leaves from transgenic tobaccos revealed that the Cpn10 homologue was localized in mitochondria and accumulated at a high level in transgenic tobaccos.     

cDNA cloning and differential gene expression of three catalases in pumpkin

Three cDNA clones (cat1, cat2, cat3) for catalase (EC 1.11.1.6) were isolated from a cDNA library of pumpkin (Cucurbita sp.) cotyledons. In northern blotting using the cDNA-specific probe, the cat1 mRNA levels were high in seeds and early seedlings of pumpkin. The expression pattern of cat1 was similar to that of malate synthase, a characteristic enzyme of glyoxysomes. These data suggest that cat1 might encode a catalase associated with glyoxysomal functions. Furthermore, immunocytochemical analysis using cat1-specific anti-peptide antibody directly showed that cat1 encoding catalase is located in glyoxysomes. The cat2 mRNA was present at high levels in green cotyledons, mature leaf, stem and green hypocotyl of light-grown pumpkin plant, and correlated with chlorophyll content in the tissues. The tissue-specific expression of cat2 had a strong resemblance to that of glycolate oxidase, a characteristic enzyme of leaf peroxisomes. During germination of pumpkin seeds, cat2 mRNA levels increased in response to light, although the increase in cat2 mRNA by light was less than that of glycolate oxidase. cat3 mRNA was abundant in green cotyledons, etiolated cotyledons, green hypocotyl and root, but not in young leaf. cat3 mRNA expression was not dependent on light, but was constitutive in mature tissues. Interestingly, cat1 mRNA levels increased during senescence of pumpkin cotyledons, whereas cat2 and cat3 mRNAs disappeared during senescence, suggesting that cat1 encoding catalase may be involved in the senescence process. Thus, in pumpkin, three catalase genes are differentially regulated and may exhibit different functions.     

The Arabidopsis ortholog of the DEAH-box ATPase Prp16 influences auxin-mediated development

In animals and yeasts, the DEAH-box RNA-dependent ATPase Prp16 facilitates pre-mRNA splicing. However, in Chlamydomonas reinhardtii and Caenorhabditis elegans, Prp16 orthologs are not important for general pre-mRNA splicing, but are required for gene silencing and sex determination, respectively. The CLUMSY VEIN (CUV) gene, which encodes a unique Prp16 ortholog in Arabidopsis thaliana, influences auxin-mediated development. A loss-of-function cuv-1 mutation tells us that CUV does not facilitate splicing of pre-mRNA substrates indiscriminately, but differentially effects splicing and expression of genes. Here we show that CUV influences root-meristem maintenance and planar polarity of root-hair positioning, both of which are processes regulated by auxin. We propose that Arabidopsis PRP16/CUV differentially facilitates the expression of genes, including genes involved in auxin biosynthesis, transport, perception and signaling, and that in this way it influences auxin-mediated development.