Gene Expression in the Cyanobacterium <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC7120 under Desiccation

The N₂-fixing cyanobacterium Anabaena sp. PCC7120 showed an inherent capacity for desiccation tolerance. A DNA microarray covering almost the entire genome of Anabaena was used to determine the genome-wide gene expression under desiccation. RNA was extracted from cells at intervals starting from early to late desiccation. The pattern of gene expression in DNA fragments was categorized into seven types, which include four types of up-regulated and three types of down-regulated fragments. Validation of the data was carried out by RT-PCR on selected up-regulated DNA fragments and was consistent with the changes in mRNA levels. Our conclusions regarding desiccation tolerance for Anabaena sp. PCC7120 are as follows: (i) Genes for osmoprotectant metabolisms and the K⁺ transporting system are up-regulated from early to mid-desiccation; (ii) genes induced by osmotic, salt, and low-temperature stress are up-regulated under desiccation; (iii) genes for heat shock proteins are up-regulated after mid-desiccation; (iv) genes for photosynthesis and the nitrogen-transporting system are down-regulated during early desiccation; and (v) genes for RNA polymerase and ribosomal protein are down-regulated between the early and the middle phase of desiccation. Profiles of gene expression are discussed in relation to desiccation acclimation.     

Genes essential to iron transport in the cyanobacterium Synechocystis sp. strain PCC 6803

Genes encoding polypeptides of an ATP binding cassette (ABC)-type ferric iron transporter that plays a major role in iron acquisition in Synechocystis sp. strain PCC 6803 were identified. These genes are slr1295, slr0513, slr0327, and recently reported sll1878 (Katoh et al., J. Bacteriol. 182:6523-6524, 2000) and were designated futA1, futA2, futB, and futC, respectively, for their involvement in ferric iron uptake. Inactivation of these genes individually or futA1 and futA2 together greatly reduced the activity of ferric iron uptake in cells grown in complete medium or iron-deprived medium. All the fut genes are expressed in cells grown in complete medium, and expression was enhanced by iron starvation. The futA1 and futA2 genes appear to encode periplasmic proteins that play a redundant role in iron binding. The deduced products of futB and futC genes contain nucleotide-binding motifs and belong to the ABC transporter family of inner-membrane-bound and membrane-associated proteins, respectively. These results and sequence similarities among the four genes suggest that the Fut system is related to the Sfu/Fbp family of iron transporters. Inactivation of slr1392, a homologue of feoB in Escherichia coli, greatly reduced the activity of ferrous iron transport. This system is induced by intracellular low iron concentrations that are achieved in cells exposed to iron-free medium or in the fut-less mutants grown in complete medium.     

Socius is a novel Rnd GTPase-interacting protein involved in disassembly of actin stress fibers

Rho family small GTPases are key regulators of the actin cytoskeleton in various cell types. The Rnd proteins, Rnd1, Rnd2, and Rnd3/RhoE, have been recently identified as new members of the Rho family of GTPases, and expression of Rnd1 or Rnd3 in fibroblasts causes the disassembly of actin stress fibers and the retraction of the cell body to produce extensively branching cellular processes. Here we have performed a yeast two-hybrid screening by using Rnd1 as bait and identified a novel protein that specifically binds to Rnd GTPases. We named this protein Socius. Socius directly binds to Rnd GTPases through its COOH-terminal region. When transfected into COS-7 cells, Socius is translocated to the cell periphery in response to Rnd1 and Rnd3 and colocalized with the GTPases. While expression of wild-type Socius in Swiss 3T3 fibroblasts has little effect on the actin cytoskeleton, the expression of a membrane-targeted form of Socius, containing a COOH-terminal farnesylation motif (Socius-CAAX), induces a dramatic loss of stress fibers. The inhibitory effect of Socius-CAAX on stress fiber formation is enhanced by truncation of its NH(2) terminus. On the other hand, the expression of Socius-CAAX or its NH(2) terminus-truncated form suppresses the Rnd-induced retraction of the cell body and the production of extensively branching cellular processes, although the disassembly of stress fibers is observed. We propose that Socius participates in the Rnd GTPase-induced signal transduction pathways, leading to reorganization of the actin cytoskeleton.     

Photosynthetic Electron Transport Involved in PxcA-Dependent Proton Extrusion in Synechocystis sp. Strain PCC6803: Effect of pxcA Inactivation on CO2, HCO3−, and NO3− Uptake

The product of pxcA (formerly known as cotA) is involved in light-induced Na⁺-dependent proton extrusion. In the presence of 2,5-dimethyl-p-benzoquinone, net proton extrusion by Synechocystis sp. strain PCC6803 ceased after 1 min of illumination and a postillumination influx of protons was observed, suggesting that the PxcA-dependent, light-dependent proton extrusion equilibrates with a light-independent influx of protons. A photosystem I (PS I) deletion mutant extruded a large number of protons in the light. Thus, PS II-dependent electron transfer and proton translocation are major factors in light-driven proton extrusion, presumably mediated by ATP synthesis. Inhibition of CO₂ fixation by glyceraldehyde in a cytochrome c oxidase (COX) deletion mutant strongly inhibited the proton extrusion. Leakage of PS II-generated electrons to oxygen via COX appears to be required for proton extrusion when CO₂ fixation is inhibited. At pH 8.0, NO₃⁻ uptake activity was very low in the pxcA mutant at low [Na⁺] (~100 μM). At pH 6.5, the pxcA strain did not take up CO₂ or NO₃⁻ at low [Na⁺] and showed very low CO₂ uptake activity even at 15 mM Na⁺. A possible role of PxcA-dependent proton exchange in charge and pH homeostasis during uptake of CO₂, HCO₃⁻, and NO₃⁻ is discussed.     

Purification of an α-L-arabinofuranosidase from carrot cell cultures and its involvement in arabinose-rich polymer degradation

Konno, H., Yamasaki, Y. and Katoh, K. 1987. Purification of an α-L-arabinofurano-sidase from carrot cell cultures and its involvement in arabinose-rich polymer degradation.
An α-L-arabinofuranosidase (α-L-arabinofuranoside arabinofuranohydrolase, EC 3.2.1.55) was isolated from a homogenate of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The buffer-soluble enzyme was purified to homogeneity by a procedure involving ammonium sulfate fractionation, chromatography on DEAE-Sephadex A-50, Sephadex G-150, Con A-Sepharose 4B and CM-Sephadex C-50, and preparative polyacrylamide gel electrophoresis. The size of this enzyme as determined by polyacrylamide gel electrophoresis in the presence of sodium laurylsulfate and by Sephadex G-200 gel filtration was 94 and 110 kDa, respectively. The isoelectric point was at pH 4.7. The K_m and V_max values for p-nitrophenyl α-L-arabinofuranoside were 1.33 mM and 20.2 μimol (mg protein)^-1 h^-1, respectively. The optimal activity occurred at pH 4.2 with Mcllvaine buffer. The enzyme was stimulated by Ca²⁺ and Zn²⁺, whereas it was strongly inhibited by Cu²⁺, Ag²⁺, Hg²⁺, p-chloromercuri-benzoate and L-arabono-l,4-lactone. The enzyme acted on beet arabinan in an exo-fashion. Furthermore, the enzyme was partially involved in the hydrolysis of the ara-binogalactan and pectic polymer purified from carrot cell walls.     

Loss of photosystem II induced by a nitrate deficiency in photoorganotrophically grown Anabaena variabilis

When photoorganotrophically trained cells of Anabaena variabiliswere grown in nitrate-free medium, they lost the activity ofphotosynthetic oxygen evolution and became devoid of phycobilinpigments. These cells (H cells) lacked the fluorescence emissioncharacteristic of photosystem II chlorophyll, and their lamellarfragments failed to photoreduce DPIP even in the presence ofdiphenylcarbazide as the electron donor, suggesting that theloss of photosynthetic oxygen evolution in H cells is primarilydue to degeneration of an integral part of photosystem II. These characteristics of H cells closely resembled those ofheterocysts differentiated from normal, vegetative cells in[i] the deficiency of phycobilin pigments, [ii] the loss ofphotosystem II activity, [iii] the photoorganotrophic mode ofcell growth, depending upon the organic substances furnishedexternally or provided from neighboring vegetative cells, and[iv] the manner of transformation from normal cells, both typesof cells being induced in the absence of nitrate. In spite ofsuch similarity, light and electron microscopic observationsrevealed that H cells differed significantly from heterocysts.Furthermore, the readiness with which H cells resumed photosystemII activity and the independence of this resumption from cellgrowth exclude the possibility that the nutritional enrichmentof heterocysts was responsible for the loss of photosyntheticactivity. ¹Present address: Ocean Research Institute, University of Tokyo,Nakano, Tokyo 164. (Received May 14, 1975; )     

Loss of the Photosynthetic Capacity and Proteins in Senescing Leaves at Top Positions of Two Cultivars of Rice in Relation to the Source Capacities of the Leaves for Carbon and Nitrogen

Senescing leaves are a source organ of both carbon and nitrogenbut, because degradation of chloroplast proteins and exportof their degradation products to sink organs give rise to lossof the photosynthetic capacity, the leaves serve as the sourceof nitrogen only at the cost of their source capacity for carbon.Changes in the photosynthetic capacity and levels of proteinsin leaves at the top four positions of two cultivars of rice,Nipponbare and Akenohoshi, during the ripening stage were investigatedtaking the trade-off relationship between the two source capacitiesinto consideration. Rate of light-saturated photosynthesis (Pmax)in leaves decreased more rapidly in Nipponbare than in Akenohoshiafter heading. Various proteins were also degraded during senescence,with Nipponbare leaves showing faster loss of proteins thanthe corresponding leaves of Akenohoshi. Decline in Pmax wascorrelated, similarly in the two cultivars, with losses of ribulose-l,5-bisphosphatecarboxylase/oxygenase, soluble proteins, chlorophyll a thatbound to the reaction center complexes of the two photosystems,the activity of whole-chain electron transport, and a majorpart of insoluble proteins during senescence. The results suggestthat degradation of proteins during senescence of rice leavesis coordinated so as to enable the leaves to perform photosynthesiswith a high use efficiency of protein and export nitrogen ata low or nearly minimum cost of the source capacity for carbon. ⁵ Present address: Section of Biology, University of California,Davis, California 95616, U.S.A. ⁶ Present address: Faculty of Agriculture, Utsunomiya University,350 Mine-machi, Utsunomiya, 321 Japan.     

Effect of plant growth substances on the formation of carpelloid structures from tobacco ovules

Excised ovules with placentae developed into carpelloids consisting of stigma-like, style-like and ovule-like structures. Kinetin was necessary for the formation of carpelloids in immature ovules which were excised from the flower buds containing pollen grains at bicellular stage. However, plant growth substances were not necessary for these formations in younger ovules which were excised from the flower buds containing pollen grains at unicellular stage. The morphological characteristics of the stigma-like and style-like structures were similar to those of normal tissues, and their functions were the same.     

Production and immunohistochemical characterization of a monoclonal antibody raised to proteoglycan purified from a human yolk sac tumour

Summary A large proteoglycan with chondroitin sulphate and dermatan sulphate side chains has been isolated and purified from a yolk sac tumour of the left ovary from a 23-year-old female. A monoclonal antibody, designated 2B1, was produced which reacted specifically with the intact molecule of the large proteoglycan and the chondroitinase ABC-treated core molecule. The localization of substances showing cross-reactivity to this antibody was studied in a variety of human tissues by means of indirect immunohistochemistry. The interstitial elements of nearly all tissues of a 5-month-old foetus were intensely reactive with the antibody, but in adult tissues structures that gave positive reactions were limited; only the perivascular and perimuscular fibrous elements were reactive, except for the aorta, which reacted extensively. In contrast, the interstitial elements of the carcinoma tissues tested were intensely reactive. Thus antibody 2B1 can be regarded as a useful tool for studies on the immunohistochemical localization of large proteoglycan in various human tissues.