Nutrient-Specific Learning in an Omnivorous Insect: The American Cockroach Periplaneta americana L. Learns to Associate Dietary Protein with the Odors Citral and Carvone

Experiments were performed to test for nutrient-specific olfactory learning in the American cockroach, Periplaneta americana L. In a conditioning period, cockroaches were presented with two complementary foods, one of which contained protein and the other carbohydrate, this combination allowing them to select a nutritionally balanced diet. The foods were separated in space, and each was paired with one of two odors, citral or carvone. The cockroaches were then selectively deprived of one of the nutrients for 24 or 48 h. In the final (test) phase of the experiment the movement of the cockroaches toward the nutrient-associated odors was monitored. Associative learning was demonstrated with respect to protein, with protein-deprived cockroaches moving more frequently toward the protein-associated odor. No learned associations between carbohydrate and odor were demonstrated. These data are contrasted with similar experiments on an herbivorous insect, the locust Locusta migratoria.     

Taurine indirectly increases [Ca]i by inducing Ca2+ influx through the Na+-Ca2+ exchanger

Recent studies in heart cells have shown taurine to induce a sustained increase of both intracellular Ca²⁺ and Na⁺. These results led us to believe that the increase in Na⁺ by taurine could be due to Na⁺ entry through the taurine-Na⁺ cotransporter which in turn favours transarcolemmal Ca²⁺ influx through Na⁺-Ca²⁺ exchange. Therefore, we investigated the effect of -alanine, a blocker of the taurine-Na⁺ cotransporter and low concentrations of CBDMB (a pyrazine derivative, 5-(N-4chlorobenzyl)-2,4-dimethylbenzamil), a Na⁺-Ca²⁺ exchanger blocker on taurine-induced [Ca]_i increase in embryonic chick heart cells. Using Fura-2 Ca²⁺ imaging and Fluo-3 Ca²⁺ confocal microscopy techniques, taurine (20 mM) as expected, induced a sustained increase in [Ca]_i at both the cytosolic and the nuclear levels. Preexposure to 500 M of the blocker of the taurine-Na⁺ cotransporter, -alanine, prevented the amino acid-induced increase of total [Ca]_i. On the other hand, application of -alanine did not reverse the action of taurine on total [Ca]_i. However, low concentrations of the Na⁺-Ca²⁺ exchanger blocker, CBDMB, reversed the taurine-induced sustained increase of cytosolic and nuclear free calcium (in presence or absence of -alanine). Thus, the effect of taurine on [Ca]_i in heart cells appears to be due to Na⁺ entry through the taurine-Na⁺ cotransporter which in turn favours transarcolemmal Ca²⁺ influx through the Na⁺-Ca²⁺ exchanger.     

The mass culture of Dunaliella viridis (Volvocales,Chlorophyta) for oxygenated carotenoids : laboratory and pilot plant studies

The biology, and hence the mass culture, of Dunaliella viridis closely follows that of Dunaliella salina, which is successfully mass cultured for the production of -carotene. Both algae can grow at extremely high salinities and light intensities. They co-exist in the coastal salt lake, Hutt Lagoon, Western Australia. In contrast to D. salina, D. viridis does not accumulate large amounts of -carotene, producing only up to 0.7% of mixed carotenoids (lutein, zeaxathin, other oxygenated carotenoids and -carotene), compared to D. salina's ca 10% dry wt of mainly -carotene. However, in laboratory experiments, D. viridisgrew much faster and to much higher cell densities than D. salina, and attained levels of mixed carotenoids similar to those of D. salina (ca 13 mg L^–1 carotenoid). Preliminary experiments in outdoor ponds were much less promising. Harvesting by chemical flocculation was as effective as with D. salina, but extraction of carotenoids directly into vegetable oil proved inefficient. When incorporated into feed, caretonoids derived from D. viridis pigmented hen eggs acceptably. Extrapolating from laboratory results, and using costing derived from D. salina technology, the cost of production of mixed oxygenated carotenoids from D. viridis was similar to that for the production of -carotene from D. salina, at ca $A500 kg^–1.     

Putative regulatory mechanism of prothoracicotropic hormone (PTTH) secretion in the American cockroach, <Emphasis Type="Italic">Periplaneta americana</Emphasis> as inferred from co-localization of Rab8, PTTH,and protein kinase C in neurosecretory cells

Small GTPases of the Rab family act as essential regulators of vesicle transport pathways, including the exocytosis of neurohormones. These processes are not well-understood in insects. To address the physiological function of Rab proteins and their phosphorylation in insect neurosecretion, Rab8-like, prothoracicotropic hormone (PTTH)-like, and protein kinase C (PKC)-like immunohistochemical reactivities (-ir) were investigated in the brain of the American cockroach, Periplaneta americana. All the antibodies tested reacted with neurons in the pars intercerebralis, corpora cardiaca, and nervi corporis allati I. Double-labeling experiments demonstrated that all PTTH-ir were colocalized with Rab8-ir and PKC-ir in the pars intercerebralis, although exclusive reactivity was present to antisera against Rab8 or PKC. These findings support the notion that Rab8-like antigen is phosphorylated by PKC, and that this phosphorylation is involved in the axonal transport and secretion of PTTH in this species. This work was supported by a Grant-in-Aid for Scientific Research (B) 1838043, (C) 20580053, and Global Center of Education and Research Program (19GCOE01) from the Japan Society for the Promotion of Science.     

Purification of carnitine acetyltransferase from skeletal muscle of the camel (Camelus dromedarius)

The enzyme carnitine acetyltransferase (CAT) catalyzes the reversible transfer of short-chain acyl groups between coenzyme A and L-carnitine, and hence, plays an important role in the -oxidation of fatty acids. Purification and characterization of CAT from desert animal species may help in explaining the involvement of secondary pathways for energy production in these species. In this paper, we report the purification and partial characterization of CAT from the Arabian camel. CAT was purified from the skeletal muscle of the Arabian camel by ammonium sulfate and acetone fractionation, followed by chromatography on DEAF-Sepharose, agarose-Co A and Superose 12 gel filtration columns. CAT was purified by 2937-fold to a specific activity of 94 Units mg^–1. The purified CAT was a monomer of 59 kDa as judged by native and SDS-PAGE, and showed a pl of 5.2. The enzyme displayed maximum activity with propionyl-Co A. Apparent K_m for acetyl-, propionyl- and butyryl-Co A were 27.7, 17.3 and 29 M respectively, while palmitoyl-Co A was not a substrate.     

Expression of a bacterial carotene hydroxylase gene (crtZ) enhances UV tolerance in tobacco

Carotenoids are essential components of the photosynthetic apparatus involved in plant photoprotection. To investigate the protective role of zeaxanthin under high light and UV stress we have increased the capacity for its biosynthesis in tobacco plants (Nicotiana tabacum L. cv. Samsun) by transformation with a heterologous carotenoid gene encoding -carotene hydroxylase (crtZ) from Erwinia uredovora under constitutive promoter control. This enzyme is responsible for the conversion of -carotene into zeaxanthin. Although the total pigment content of the transgenics was similar to control plants, the transformants synthesized zeaxanthin more rapidly and in larger quantities than controls upon transfer to high-intensity white light. Low-light-adapted tobacco plants were shown to be susceptible to UV exposure and therefore chosen for comparative analysis of wild-type and transgenics. Overall effects of UV irradiation were studied by measuring bioproductivity and pigment content. The UV exposed transformed plants maintained a higher biomass and a greater amount of photosynthetic pigments than controls. For revelation of direct effects, photosynthesis, pigment composition and chlorophyll fluorescence were examined immediately after UV treatment. Low-light-adapted plants of the crtZ transgenics showed less reduction in photosynthetic oxygen evolution and had higher chlorophyll fluorescence levels in comparison to control plants. After 1 h of high-light pre-illumination and subsequent UV exposure a greater amount of xanthophyll cycle pigments was retained in the transformants. In addition, the transgenic plants suffered less lipid peroxidation than the wild-type after treatment with the singlet-oxygen generator rose bengal. Our results indicate that an enhancement of zeaxanthin formation in the presence of a functional xanthophyll cycle contributes to UV stress protection and prevention of UV damage.     

The promoter of the beta-glucosidase gene from Kluyveromyces fragilis contains sequences that act as upstream repressing sequences in Saccharomyces cerevisiae

Summary The relationship between the promoter length of the Kluyveromyces fragilis -glucosidase gene and the level of its expression in Saccharomyces cerevisiae was studied by gene fusion between deleted promoter fragments of various lengths and the promoterless -galactosidase gene of Escherichia coli. The removal of a region from position-425 to-232 led to a tenfold increase in the expression of the gene. The same results were obtained for the reconstructed -glucosidase gene with the same promoter length. It is likely that the deletion of this part of the promoter removes negative regulatory elements which are functional in Saccharomyces cerevisiae. This increase in activity is the main event which may explain the high increase in gene expression (60-fold) previously observed for an upstream deletion obtained during subcloning experiments of the -glucosidase gene. It is also shown that the expression of the gene greatly depends upon the nature of the recipient strain, the growth phase of the cell and that of the vector carrying it.     

Xyloglucan (amyloid) mobilisation in the cotyledons of Tropaeolum majus L. seeds following germination

The levels of cell-wall xyloglucan (amyloid) in nasturtium (Tropaeolum majus L.) cotyledons were monitored during a 28-d period covering seed imbibition, germination and early seedling development. The activities of the following enzymes capable of hydrolysing the glycosidic linkages in the xyloglucan were assayed in cotyledon extracts over the same period: endo-(14)--glucanase (EC 3.2.1.4), -glucosidase (EC 3.2.1.21), -xylosidase and -galactosidase (EC 3.2.1.23). The endo--glucanase was assayed viscometrically using xyloglucan as substrate, and the three glycosidases using appropriate p-nitrophenylglycosides. Alpha xylosidase and -galactosidase, the enzymes which would be expected to hydrolyse the side-chains from the xyloglucan molecule, were also assyed using xyloglucan as substrate. Under our culture conditions, xyloglucan levels remained constant at 30 mg per cotyledon pair for 7 d, that is until 3 d after germination: thereafter, the amount of xyloglucan diminished to zero in a 12-d period. The most rapid period of depletion was between days 9 and 13. The mobilisation of all reserve substances from the cotyledons resulted in a weight-loss of 92 mg: xyloglucan, therefore, is an important storage substance, representing 33% by weight of the seed's substrate reserves. It is a cell-wall storage polysaccharide. Xyloglucan mobilisation was accompanied by a 17-fold increase in endo--glucanase activity, a 7-fold increase in -galactosidase and an 8-fold increase in -xylosidase activities, all determined using xyloglucan as substrate. All three activities began to increase at day 5, peaked at days 12–14 when the most rapid phase of xyloglucan breakdown was over, and had declined to zero by days 22–25. The levels of theses enzymes have been shown to be consistent with their being responsible for xyloglucan hydrolysis in vivo. Nitrophenyl--galactosidase activity increased up to day 3, remained constant and then increased again 2.5-fold from day 5, peaking at day 11. Nitrophenyl--glucosidase remained relatively constant up to day 16 and then decreased to zero by day 25. Nitrophenyl--xylosidase activity was not detected.     

Transglycosylation reactions by exoglycosidases from the termite Macrotermes subhyalinus

The ability of four exoglycosidases (-galactosidase, -glucosidase, -glucosidase and invertase) from the termite Macrotermes subhyalinus to catalyse tranglycosylation reactions was tested using lactose, cellobiose, maltose and sucrose as glycosyl donors and 2-phenylethanol as glycosyl acceptor. The experimental conditions were optimized in relation to the time course of the reaction, pH and concentrations of glycosyl donor and acceptor. Whereas the hydrolytic activity was largely predominant over the transferase activity with -galactosidase and -glucosidase, the transglycosylation activity represented 68% with -glucosidase. In addition, as demonstrated by the transglycosylation product formed, the hydrolysis of sucrose was catalysed by -glucosidase and not by invertase. On the basis of this work, -glucosidase from M. subhyalinus appears to be a valuable tool for the preparation of neoglycoconjugates.     

Variation in the olfactory response of 13 populations of the predatory mite Amblyseius womersleyi to Tetranychus urticae-infested plant volatiles (Acari: Phytoseiidae, Tetranychidae)

We studied the response of the predatory mite Amblyseius womersleyi collected in 13 different sites in Japan toward Tetranychus urticae-infested kidney bean leaf volatiles in a Y-tube olfactometer. The predatory mites were collected from eight plant species infested by one of three tetranychid mite species. The predators' responses to the infested-leaf volatiles varied from 33% to 97% among the populations. The predators collected at 10 sites showed a significant preference for infested-leaf volatiles, whereas those collected at three tea plantations did not distinguish between the infested- and uninfested-leaf volatiles. We discussed the possible factors that affected the olfactory response of A. womersleyi towards the infested leaf volatiles.     

Cajaflavanone and cajanone released from Cajanus cajan (L. Millsp.) roots induce nod genes of Bradyrhizobium sp.

A broad-host-range plasmid (pEA2-21) containing a Bradyrhizobium sp (F-4) nod DABC-lacZ translation fusion was constructed and used to monitor nod gene expression in response to pigeonpea root exudate. Two nod-inducing compounds were isolated and identified. Spectral analysis using ultraviolet absorption, infrared spectra, proton nuclear magnetic resonance, and mass spectrometry showed that the two inducers were 5,4-dihydroxy-6-(3-methyl-2-butenyl)-2, 2-dimethyl pyrano-[5, 6:7, 8]-flavanone (cajaflavanone) and 2,4,5-trihydroxy-5-isopentenyl-6, 7-dimethylchromene iso-flavanone (cajanone). When pEA2-21 was introduced into Rhizobium trifolii and R. meliloti cajanone and cajaflavanone did not induce nod gene indicating that specificity of induction appears to be influenced by the host-strain genome.     

Role of the N-terminal region of the A chain in beta 1-bungarotoxin from the venom of Bungarus multicinctus (Taiwan-banded krait)

The N-terminal -amino groups of ₁-bungarotoxin (₁-Bgt) fromBungarus multicinctus venom were modified with trinitrobenzene sulfonic acid and the modified derivative was separated by high performance liquid chromatography. The trinitrophenylated (TNP) derivative contained two TNP groups at the -amino groups of A chain and B chain and showed a marked decrease in enzymatic activity. Methionine residues at positions 6 and 8 of the A chain were oxidized with chloramine T or cleaved with cyanogen bromide to remove the N-terminal octapeptide. Oxidation of methionine residues and removal of the N-terminal octapeptide caused a precipitous decrease in enzymatic activity, whereas antigenicity remained unchanged. The presence of dihexanoyllecithin influenced the interaction between ₁-Bgt and 8-antilinonaphthalene sulfonate (ANS) and revealed that ₁-Bgt consists of two types of ANS-binding sites, one at the substrate binding site of the A chain and the other might be at the B chain. The modified derivatives still retained their affinity for Ca²⁺ and ANS, indicating that the N-terminal region is not involved in Ca²⁺ and substrate binding. A fluorescence study revealed that the -amino group of the A chain was in the vicinity of substrate binding site and that the TNP -amino groups were in proximity to Trp-19 of the A chain. In addition, the study showed that the N-terminal region is important for stabilizing the architectural environment of Trp-19. The results, together with the proposal that Trp-19 of the A chain is involved in substrate binding, suggest that the N-terminal region of the A chain plays a crucial role in maintaining a functional active site for ₁-Bgt.     

The biosynthetic pathway of the S-alk(en)yl-L-cysteine sulphoxides (flavour precursors) in species of Allium

Pulse labelling experiments with ³⁵SO₄ ^2- fed for 24h to intact plants (shooted onion sets)of Allium cepa (onion) showed that >70% of the label appeared in the S-alkenyl-L-cysteine sulphoxides within 18h, reached a maximum at 48h and thereafter decreased. The amount of label detected in the -glutamyl peptide fractions was below 20% of the total label at any time. It is concluded that in intact plants (at the growth stage used) the -glutamyl peptides are not the immediate precursors of the S-alkenyl-L-cysteine sulphoxides. The major S-alkenyl-L-cysteine sulphoxide in onion was found to be compartmentalized mainly within the endoplasmatic reticulum.Abbreviations AllCysSO (+)-S-2-propenyl-L-cysteine sulphoxide - MeCysSO (+)-S-methyl-L-cysteine sulphoxide - PrenCysSO trans-(+)-S-1-propenyl-L-cysteine sulphoxide - ProCysSO (+)-S-propyl-L-cysteine sulphoxide     

Activities of growth inhibitors isolated from light-grown dwarf pea shoots

The growth inhibitory activities of 6 endogenous growth inhibitors isolated from light-grown dwarf peas (Pisum sativum cv. Progress No. 9) were examined in the epicotyl of dark-grown seedlings of the same cultivar in the dark in order to examine the possible contribution of these compounds to the growth inhibition brought about by red light. The activities of these natural inhibitors, including two A-2 and A-2 of as yet undetermined structure, were compared with those of synthetic growth retardants and benzyladenine. Samples were applied directly into the epicotyls via a glass capillary tube. In 24-h tests doses for a 25% inhibition (I₂₅) were: A-2, 4.3 × 10^-2: cis-xanthoxin, 1.2 × 10^-1 ; A-2, 1.6 × 10^-1; trans-xanthoxin, 1.2; R,S-dihydromaleimide, 3.5 × 10² and pisatin, 4.0 × 10² nmol plant^-1 . In 72-h tests, I₂₅'s were: benzyladenine, 1.5; AMO-1618 (ammonium-(5-hydroxycarvacryl)-trimethylchloride piperidine carboxylate), 2.4; R,S-dihydromaleimide, 4.0 × 10² and CCC (chlorocholine chloride), 1.1 × 10³ nmol plant^-1. -D-Glucosyl-R-dihydromaleimide had no activity at all. Benzyladenine caused the thickening as well as elongation inhibition of the epicotyls of intact plants. The possible involvement of A-2 and in the red light growth inhibition of dwarf peas is discussed.Abbreviations AMO-1618 ammonium-(5-hydroxycarvacryl)-trimethylchloride piperidine carboxylate - CCC chlorocholine chloride - G-DHMD -D-glucosyl-R-dihydromaleimide - I₂₅ dose required for a 25% growth inhibition - R red light author for correspondence     

Variation in the inheritance of expression among subclones for unselected (uidA) and selected (bar) transgenes in maize (Zea mays L.)

Variation in the inheritance of expression among subclones for an unselected (uidA) and a selected (bar) transgene was analyzed in two individual transformation events in maize. The unselectable gene (uidA) and the selectable gene (bar), on two separate plasmids, were transferred to maize (Hi-II derivative) by particle bombardment of embryogenic calli or suspension cells. A total of 188 fertile T1 plants were obtained from one transformant (transformation event BG which integrated uidA and bar). A total of 98 fertile T1 plants were obtained from a second transformant (transformation event B which integrated bar). Through self-pollination and/or cross-pollination in the greenhouse, approximately 10 000 T2 progeny were obtained from event BG, and more than 1000 T2 progeny were obtained from event B. Segregation of transgene expression was analyzed statistically in a total of 2350 T2 progeny from 40 T1 subclones of event BG and in 217 T2 progeny from six T1 subclones from event B. Variation in the inheritance of expression among subclones for the two transgenes (uidA and bar) was observed in the two transformants. A significant difference was observed between the use of the female or male as the transgenic parent in the inheritance of expression for the two transgenes in event BG. No inheritance through the pollen was observed in two of four T1 subclones analyzed in event B. Co-expression analysis of event BG showed that both transgenes were co-expressed in 67.7% of the T2 plants which expressed at least one of the two transgenes. Of the T2 expressing plants, 30.4% expressed only bar, and 1.9% expressed only uidA. Inactivation of the unselected (uidA) and the selected (bar) transgenes was observed in individual T2 plants.     

An endogenous factor from soybean (Glycine max L.) cell cultures activates phosphorylation of a protein which is dephosphorylated in vivo in elicitor-challenged cells

The existence of specific binding sites for a -glucan elicitor of phytoalexin synthesis derived from the fungus Phytophthora megasperma f.sp. glycinea at the plasma membrane of soybean (Glycine max L.) tissues (W.E. Schmidt, J. Ebel (1987) Proc. Natl. Acad. Sci. USA 84, 4117–4121) might imply that stimulation of phytoalexin formation by the elicitor is a membrane-mediated process. Addition of the -glucan elicitor to soybean cellsuspension cultures, which has previously been shown to induce phytoalexin accumulation, also results in rapid changes in the phosphate turnover of several phosphoproteins. The effect of the elicitor on protein phosphorylation was tested after labeling of the cells with [³²P]orthophosphate. As shown by analysis using one-and two-dimensional gel electrophoresis, decreases as well as increases in the labeling of several phosphoroteins occurred rapidly, being detectable within 5 min after elicitor application, and persisted for at least 15 min. As judged by their relative molecular masses (M_r) and isoelectric points (pI), a number of proteins which were radioactively labeled in vivo were also phosphorylated in vitro by endogenous protein-kinase activity in the presence of Ca²⁺. The most pronounced effect was observed with a protein substrate with M_r=69000 and pI=5.7 (pp69) whose phosphate labeling markedly decreased in response to elicitor treatment in vivo. Phosphorylation of pp69 in vitro in the presence of -[³²P]ATP was strongly enhanced by a phosphorylation-stimulating factor (effector) derived from soybean cell cultures and occurred predominantly at serine residues. The effector possessed a low apparent M_r (1000), was negatively charged at pH 7.3, and was relatively heat stable. The effector was inactivated by treatment with alkaline phosphatase from calf intestine. Phosphorylation of pp69 was only slightly stimulated by Ca²⁺, and was insensitive to cAMP, cGMP, calmodulin, a lipid mixture, a ganglioside mixture, or spermine under the assay conditions used. A 10 mM concentration of 3-phosphoglycerate increased pp69 phosphorylation to the extent of about 50% of that induced by the soybean effector. There was no evidence, however, that such concentrations of 3-phosphoglycerate occurred in effector preparations. The results are discussed in relation to hypothetical signal transduction during elicitor action on soybean cells.Abbreviations M_r relative molecular mass - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TPCK L-1-tosylamide-2-phenylethyl chloromethyl ketone     

Side reactions in the synthesis of phosphotyrosine-containing peptides

Summary A series of phosphopeptides Tyr(PO₃H₂)-Val-Pro-Xxx-Leu (Xxx=Met, Met(O), Nle, Dab or Cys), derived from the native platelet-derived growth factor- receptor (PDGF-) sequence, has been prepared to study their interaction with the src-homology 2 (SH2) domains of the p85 subunit of PI3 kinase. The phosphopeptides were synthesized using Fmoc methodology incorporating N-Boc dibenzyl-protected phosphotyrosine (Boc-Tyr[PO₃(Bzl)₂]) as the N-terminal amino acid, since the benzyl groups can be removed during resin cleavage with TFA. Only peptides containing methionine were found to exist partially as S-benzyl sulfonium salts after TFA cleavage from the resin. The desired peptide could be obtained from the S-benzyl sulfonium salt by hydrogenolysis.     

Cloning,characterization and functional expression of a new beta-D-fructofuranosidase (Os beta fruct2) cDNA from Oryza sativa

A new cDNA (Osfruct2) encoding an acid –d-fructofuranosidase from rice has been cloned, sequenced and expressed in Pichia pastoris. The full-length cDNA is 2453 base pairs long and encodes a pre-pro-protein of 682 amino acids. The cDNA fragment coding for mature enzyme was sub-cloned into vector pPICZA for extracellular expression in the methylotrophic yeast. The recombinant product was purified by Ni²⁺-nitrilotriacetic acid agarose column and biochemically characterized. The enzyme could hydrolyze sucrose and raffinose. Molecular mass is 66 kDa. The activity optimum was at pH 4.8 and 40 °C.     

Calpain processing of brain microtubules from the Atlantic Cod,Gadus morhua

Viral infection of cultured cells with transforming viruses causes an increase in cell-surface N-linked 1-6 (GlcNAc1-6Man) branching of complex-type oligosaccharides. Similar observations have been made after transfection of cells with activated oncogenes, which is associated with an induction of tumorigenic and metastatic properties. In this study, the effects of transfection of both activated and proto-Ha-ras oncogenes into NIH3T3 cells were analyzed. The results showed that, in comparison with NIH3T3 cells, bothras transfectants have increased sensitivity to the cytotoxic action of L-PHA. An increase in 1-6 branching and an increased level of N-acetylglucosaminyltransferase V (GlcNAc-T V), the enzyme which initiates the 1-6 branching were also observed. The levels of GlcNAc-T I and 1-4 Gal-T remained unchanged in activated Ha-ras transfected NIH3T3 cells. These data suggest that a specific induction of GlcNAc-T V occurs after transfection with either the proto- or activated Ha-ras oncogenes, which is responsible for the increased 1-6 branching previously observed. (Mol Cell Biochem122: 85–92, 1993)Abbreviations Asn Asparagine - BHK Baby Hamster Kidney (cells) - 1-4 Gal-T or Gal-T 1-4 Galactosyltransferase - BSL-II Banderia Simplifolia Lectin II - CHO Chinese Hamster Ovary (cells) - ConA Concanavalin A - D-MEM Dulbecco's Modified Eagle's Medium - E-PHA Erythroagglutinin of Phytohemagglutinin - Fuc Fucose - Gn or GlcNAc N-acetylglucosamine - GlcNAc-T I N-acetylglucosaminyltransferase I - LCA Lens Culinaris Agglutinin - L-PHA Leukoagglutinin of Phytohemagglutinin - M or Man Mannose - PBS Phosphate-Buffered Saline - PL Pea Lectin - RIC Ricin - SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis - UDP-Gal Uridine-Diphosphate-Galactose - UDP-Gn Uridine-Diphosphate-N-acetylglucosamine - WGA Wheat Germ Agglutinin     

Analysis of a dehiscence zone endo-polygalacturonase in oilseed rape (Brassica napus) and Arabidopsis thaliana: evidence for roles in cell separation in dehiscence and abscission zones, and in stylar tissues during pollen tube growth

The oilseed rape (Brassica napus) endo-polygalacturonase (endo-PG) RDPG1 is involved in middle lamella breakdown during silique opening. We investigated tissue-specific expression of RDPG1 in transgenic Arabidopsis thaliana. Cellular localization of endo-PG protein in Arabidopsis siliques was determined by immuno-electron microscopy. An Arabidopsis orthologue, ADPG1, was isolated and aligned with the sequence of RDPG1. The proximal 5 sequences as well as introns are largely conserved. Analysis of the histological GUS-staining pattern of two RDPG1 promoter-GUS (-glucuronidase) constructs in transgenic Arabidopsis revealed that the conserved proximal part of the 5-flanking region directs expression in dehiscence zones of siliques and anthers, floral abscission zones and stylar tissues during pollen tube growth, branch points between stems and pedicel and expression associated with the apical meristem of seedlings, while the distal part of theRDPG1 5-flanking region contains elements involved in vascular-associated expression in petals, cotyledons and roots. Subsequent RT-PCR analysis, on RNA from the corresponding rape tissues, confirms the staining pattern revealed in transgenic Arabidopsis, thereby justifying the use of Arabidopsis as a reliable model system for analysis of oilseed rape regulatory sequences.