A Nitrilase-Like Protein Interacts with GCC Box DNA-Binding Proteins Involved in Ethylene and Defense Responses

Ethylene-responsive element-binding proteins (EREBPs) of tobacco (Nicotiana tabacum L.) bind to the GCC box of many pathogenesis-related (PR) gene promoters, including osmotin (PR-5). The two GCC boxes on the osmotin promoter are known to be required, but not sufficient, for maximal ethylene responsiveness. EREBPs participate in the signal transduction pathway leading from exogenous ethylene application and pathogen infection to PR gene induction. In this study EREBP3 was used as bait in a yeast two-hybrid interaction trap with a tobacco cDNA library as prey to isolate signal transduction pathway intermediates that interact with EREBPs. One of the strongest interactors was found to encode a nitrilase-like protein (NLP). Nitrilase is an enzyme involved in auxin biosynthesis. NLP interacted with other EREBP family members, namely tobacco EREBP2 and tomato (Lycopersicon esculentum L.) Pti4/5/6. The EREBP2-EREBP3 interaction with NLP required part of the DNA-binding domain. The specificity of interaction was further confirmed by protein-binding studies in solution. We propose that the EREBP-NLP interaction serves to regulate PR gene expression by sequestration of EREBPs in the cytoplasm.     

Composition changes in retinal pigments according to habitat of chum and pink salmon

Juvenile chum Oncorhynchus keta and pink Oncorhynchus gorbuscha salmon change the composition of their visual pigments when they move from coastal waters to offshore in the Okhotsk Sea. Levels of the visual pigment rhodopsin were found to be high on the coast, while porphyropsin levels were high in offshore waters in both species. These facts suggest visual function may be similar, allowing them to coexist in the same sea area.     

8-Epixanthatin, a light-induced growth inhibitor, mediates the phototropic curvature in sunflower (Helianthus annuus) hypocotyls

The distribution of the endogenous auxin-inhibiting substance, 8-epixanthatin, was determined in the lighted and shaded sides of phototropically-stimulated, de-etiolated sunflower ( Helianthus annuus L. cv. Taiyo) hypocotyls. From 40 min after the onset of phototropic stimulation, the growth rate at the lighted side was inhibited, whereas that at the shaded side showed no change. In the lighted side, 8-epixanthatin increased by 20 min after the onset of unilateral illumination and, after 40 min, reached a 3-fold larger concentration than that in the shaded side. Unilateral application of 8-epixanthatin suppressed the growth of etiolated hypocotyls at the applied side only, causing the hypocotyls to bend at the site of application. It is concluded that phototropic curvature in sunflower hypocotyls is caused by a lateral gradient of the auxin-inhibiting substance 8-epixanthatin.     

Cytochrome c from a thermophilic bacterium has provided insights into the mechanisms of protein maturation, folding, and stability.

Cytochrome c is widely distributed in bacterial species, from mesophiles to thermophiles, and is one of the best-characterized redox proteins in terms of biogenesis, folding, structure, function, and evolution. Experimental molecular biology techniques (gene cloning and expression) have become applicable to cytochrome c, enabling its engineering and manipulation. Heterologous expression systems for cytochromes c in bacteria, for use in mutagenesis studies, have been established by extensive investigation of the biological process by which the functional structure is formed. Mutagenesis and structure analyses based on comparative studies using a thermophile Hydrogenobacter thermophilus cytochrome c-552 and its mesophilic counterpart have provided substantial clues to the mechanism underlying protein stability at the amino-acid level. The molecular mechanisms underlying protein maturation, folding, and stability in bacterial cytochromes c are beginning to be understood.     

Polypeptide synthesis using an expressed peptide as a building block for condensation with a peptide thioester: application to the synthesis of phosphorylated p21Max protein(1-101).

An expressed peptide proved to be useful as a building block for the synthesis of a polypeptide via the thioester method. A partially protected peptide segment, for use as a C-terminal building block, could be prepared from a recombinant protein; its N-terminal amino acid residue was transaminated to an alpha-oxoacyl group, the side-chain amino groups were then protected with t-butoxycarbonyl (Boc) groups, and. finally, the alpha-oxoacyl group was removed. On the other hand, an O-phosphoserine-containing peptide thioester was synthesized via a solid-phase method using Boc chemistry. These building blocks were then condensed in the presence of silver ions and an active ester component. During the condensation, epimerization at the condensation site could be suppressed by the use of N,N-dimthylformamide (DMF) as a solvent. Using this strategy, a phosphorylated partial peptide of the p21Max protein, [Ser(PO3H2)2.11]-p21Max(1-101), was successfully synthesized.     

A recessive heterochronic mutation, plastochron1, shortens the plastochron and elongates the vegetative phase in rice

We describe two recessive alleles of a rice heterochronic gene, plastochron1-1 (pla1-1) and pla1-2, that reduce the length of the plastochron to approximately half that of the wild type. Because the onset of the reproductive phase in pla1 was not temporally affected, the number of leaves produced in the vegetative phase was nearly twice that produced in the wild type. Panicle development was severely disturbed in pla1 mutants. In pla1-1, many primordia of primary rachis branches were converted into vegetative shoots. These ectopic shoots repeated the initiation of panicle development and the conversion of primary rachis branches into shoots. In the weak allele pla1-2, however, only the basal one or two primordia developed as vegetative shoots, and the remaining primordia developed to produce a truncated panicle. These results indicate that both vegetative and reproductive programs are expressed simultaneously during the reproductive phase of pla1; however, the degree varied depending on the strength of the allele. Accordingly, pla1 is a heterochronic mutation that extends the vegetative period. The shoot apical meristem of pla1 was larger than that of the wild type, although the shape was not modified. An in situ hybridization experiment using the histone H4 gene as a probe revealed that cell divisions are accelerated in the pla1 meristem. The PLA1 gene is considered to regulate the duration of the vegetative phase by controlling the rate of leaf production in the meristem.     

Transgenic peppermint (Mentha×piperita L.) plants obtained by cocultivation with Agrobacterium tumefaciens

The first transgenic peppermint (Mentha×piperita L. cultivar Black Mitcham) plants have been obtained by Agrobacterium-mediated transformation by cocultivation with morphogenically responsive leaf explants. Basal leaf explants with petioles, from leaves closest to the apex of in-vitro-culture-maintained shoots (5 cm), exhibited optimal shoot organogenetic responsiveness on medium supplemented with thidiazuron (8.4 μm). Shoot formation occurred at sites of excision on the leaf blade and petiole either directly from cells of the explant or via a primary callus. Analyses of transient GUS activity data indicated that DNA delivery by microprojectile bombardment was more effective than Agrobacterium infection. However, no transgenic plants were obtained from over 22,000 leaf explants after particle bombardment. Cocultivation of leaf explants with Agrobacterium strain EHA 105 and kanamycin selection produced transgenic plants. Greater transient and stable -glucuronidase (GUS) activities were detected in explants or propagules transformed with the construct where gusA was driven by the pBISN1 promoter rather than a CaMV 35S promoter. Eight plants were subsequently regenerated and verified as transgenic based on detection of the nptII transgene by PCR and Southern blot analyses. The Southern analyses indicated that the plants were derived from eight unique transformation events. All transgenic plants appeared morphologically normal. Analyses of GUS activities in leaves sampled from different portions of these transgenic plants, 10 months after transfer to the greenhouse, indicated that six out of the eight original regenerants were uniformly transformed, i.e., did not exhibit chimeric sectors. Received: 12 December 1997 / Revision received: 3 June 1997 / Accepted: 18 July 1997     

Immunocytochemical and chemical analyses of Golgi vesicles isolated from the germinated pollen ofCamellia japonica

As a first step towards studying the biochemical relationship between Golgi vesicles (GVs) and tube wall components, isolation of GVs from the growing pollen tubes ofCamellia japonica was attempted using a centrifugation method with mannitol. The isolated GV was identified ultrastructurally and immunocytochemically. The main components of the GV were proteins and carbohydrates. The main monosaccharides of GV polysaccharides were galactose, arabinose and uronic acid, and pectins and arabinogalactan proteins also were detected immunochemically. An antiserum against the isolated GVs reacted with the outer layer of the pollen tube wall and the intine layers of the grain wall as well as thein situ GVs in the pollen tube and the grain cytoplasm. We have thus successfully isolated GVs and shown that they contain pectic substances and arabinogalactan proteins which contribute to formation of the pollen tube primary wall.     

Phage display selection can differentiate insecticidal activity of soybean cystatins

Plant cysteine proteinase inhibitors (phytocystatins) have been implicated as defensive molecules against Coleopteran and Hemipteran insect pests. Two soybean cystatins, soyacystatin N (scN) and soyacystatin L (scL), have 70% sequence identity but scN is a much more potent inhibitor of papain, vicilin peptidohydrolase and insect gut proteinases. When these cystatins were displayed on phage particles, papain-binding affinity and CPI activity of scN were substantially greater than those of scL, in direct correlation with their relative CPI activity as soluble recombinant proteins. Furthermore, scN substantially delayed cowpea weevil (Callosobruchus maculatus (F.)) growth and development in insect feeding bioassays, whereas scL was essentially inactive as an insecticide. Papain biopanning selection of phage-displayed soyacystatins resulted in a 200–1000-fold greater enrichment for scN relative to scL. These results establish that binding affinity of cystatins can be used in phage display biopanning procedures to select variants with greater insecticidal activity, illustrating the potential of phage display and biopanning selection for directed molecular evolution of biological activity of these plant defensive proteins.