Patatin and four serine proteinase inhibitor genes are differentially expressed during potato tuber development

A highly efficient and synchronousin vitro tuberization system is described. One-node stem pieces from potato (Solanum tuberosum cv. Bintje) plants grown under short day-light conditions containing an axillary bud were cultured in the dark on a tuber-inducing medium. After 5 or 6 days all axillary buds started to develop tubers. To study gene expression during tuber development, RNA isolated from tuberizing axillary buds was used for bothin vitro translation and northern blot hybridizations. The genes encoding the proteinase inhibitors I and II (PI-I and PI-II), a Kunitz-and a Bowman-Birk-type proteinase inhibitor were already expressed in uninduced axillary buds. The length of the day-light conditions differently influenced the expression level of the individual genes. In addition, the expression of each of these genes changed specifically during the development of the axillary bud to tuber. In contrast to the expression of these proteinase inhibitor genes, patatin gene expression was only detectable from the day tuberization was manifested as a radial expansion of the axillary bud.These results are discussed with respect to the regulation of the expression of the genes studied in relation to the regulation of tuber development.     

Identification of three genes expressed primarily during development in Physarum polycephalum

During the life cycle of Physarum polycephalum, uninucleate amoebae develop into multinucleate syncytial plasmodia. These two cell types differ greatly in cellular organisation, behaviour and gene expression. Classical genetic analysis has identified the mating-type gene, matA, as the key gene controlling the initiation of plasmodium development, but nothing is known about the molecular events controlled by matA. In order to identify genes involved in regulating plasmodium formation, we constructed a subtracted cDNA library from cells undergoing development. Three genes that have their highest levels of expression during plasmodium development were identified: redA, redB (regulated in development) and mynD (myosin). Both redA and redB are single-copy genes and are not members of gene families. Although redA has no significant sequence similarities to known genes, redB has sequence similarity to invertebrate sarcoplasmic calcium-binding proteins. The mynD gene is closely related to type II myosin heavy-chain genes from many organisms and is one of a family of type II myosin genes in P. polycephalum. Our results indicate that many more red genes remain to be identified, some of which may play key roles in controlling plasmodium formation. Received: 21 June 1999 / Accepted: 17 August 1999     

Ethylene biosynthetic genes are differentially expressed during carnation (Dianthus caryophyllus L.) flower senescence

Ethylene production and expression patterns of an 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (CARAO1) and of two ACC synthase (EC 4.4.1.14) genes (CARACC3 and CARAS1) were studied in floral organs of cut carnation flowers (Dianthus caryophyllus L.) cv. White Sim. During the vase life and after treatment of fresh flowers with ethylene, production of ethylene and expression of ethylene biosynthetic genes first started in the ovary followed by the styles and the petals. ACC oxidase was expressed in all the floral organs whereas, during the vase life, tissue-specific expression of the two ACC synthase genes was observed. After treatment with a high ethylene concentration, tissue specificity of the two ACC synthase genes was lost and only a temporal difference in expression remained. In styles, poor correlation between ethylene production and ACC synthase (CARAS1) gene expression was observed suggesting that either activity is regulated at the translational level or that the CARAS1 gene product requires an additional factor for activity.Isolated petals showed no increase in ethylene production and expression of ethylene biosynthetic genes when excised from the flower before the increase in petal ethylene production (before day 7); showed rapid cessation of ethylene production and gene expression when excised during the early phase of petal ethylene production (day 7) and showed a pattern of ethylene production and gene expression similar to the pattern observed in the attached petals when isolated at day 8. The interorgan regulation of gene expression and ethylene as a signal molecule in flower senescence are discussed.     

Presenilin 1 and 2 are expressed differentially in the cerebral cortex of mice during development

Presenilin (PS) 1 and PS2 are multi-pass transmembrane proteins involved in vital brain functions. Studies using transgenic or conditional knockout models show that PS1 is implicated in crucial brain developmental processes. Conversely, PS2 knockout mice do not exhibit any abnormality in the brain morphology, suggesting that PS2 may not be involved in brain development. However, there is no holistic information available for endogenous expression of PS during brain development. Therefore, we have examined the distribution and expression profile of PS1 and PS2 mRNA and protein in the cerebral cortex of prenatal, neonatal and postnatal mice. The results revealed that the distribution and expression profile of PS1 and PS2 mRNA varied significantly in the cerebral cortex during development. In prenatal stages, both PS1 and PS2 mRNA showed high expression at embryonic day (E) 12.5 and downregulation at E18.5. Postnatally, PS1 mRNA showed upregulation from postnatal day 0 (P0) to P45 and thereafter reduction at 20weeks, but PS2 mRNA showed no significant alteration. However, they did not exhibit any significant regional variation except at E18.5, when PS2 showed reduction in temporal and medial temporal lobes as compared to frontal and parietal lobes. Furthermore, PS1 showed significant change in protein expression similar to its mRNA profile. However, PS2 protein expression did not correspond to its mRNA; it was highest at E12.5, downregulated up to P20 and then upregulated at P45 and 20weeks. Taken together, our study demonstrates for the first time that the distribution and expression profile of PS2 is different from PS1 in the mouse cerebral cortex during development.     

Two modes of exocytosis from synaptosomes are differentially regulated by protein phosphatase types 2A and 2B

The inhibitors okadaic acid (OA), fostriecin (FOS) and cyclosporin A (CsA), were used to investigate the roles of protein phosphatases in regulating exocytosis in rat brain synaptosomes by measuring glutamate release and the release of the styryl dye FM 2-10. Depolarization was induced by 30 mM KCl, or 0.3 mM or 1 mM 4-aminopyridine (4AP). OA and FOS produced a similar partial inhibition of KCl- and 0.3 mM 4AP- evoked exocytosis in both assays, but had little effect upon exocytosis evoked by 1 mM 4AP. In contrast, CsA had no effect upon KCl- and 0.3 mM 4AP-evoked exocytosis, but significantly enhanced glutamate release but not FM 2-10 dye release evoked by 1 mM 4AP. None of the phosphatase inhibitors changed calcium signals from FURA-2-loaded synaptosomes either before or after depolarization. Pretreatment with 100 nM phorbol 12-myristate 13-acetate abolished the inhibitory effect of OA on exocytosis induced by 0.3 mM 4AP. Taken together, these results show that exocytosis from synaptosomes has a phosphatase-sensitive and phosphatase-insensitive component, and that there are two modes of phosphatase-sensitive exocytosis that can be elicited by different depolarization conditions. Moreover, these two modes are differentially sensitive to phosphatase 2A and 2B.     

Screening and identification of differentially expressed genes in goose hepatocytes exposed to free fatty acid

The overaccumulation of triglycerides in hepatocytes induces hepatic steatosis; however, little is known about the mechanism of goose hepatic steatosis. The aim of this study was to define an experimental model of hepatocellular steatosis with TG overaccumulation and minimal cytotoxicity, using a mixture of various proportions of oleate and palmitate free fatty acids (FFAs) to induce fat‐overloading, then using suppressive subtractive hybridization and a quantitative PCR approach to identify genes with higher or lower expression levels after the treatment of cells with FFA mixtures. Overall, 502 differentially expressed clones, representing 21 novel genes and 87 known genes, were detected by SSH. Based on functional clustering, up‐ and down‐regulated genes were mostly related to carbohydrate and lipid metabolism, enzyme activity and signal transduction. The expression of 20 selected clones involved with carbohydrate and lipid metabolism pathways was further studied by quantitative PCR. The data indicated that six clones similar to the genes ChREBP, FoxO1, apoB, IHPK2, KIF1B, and FSP27, which participate in de novo synthesis of fatty acid and secretion of very low density lipoproteins, had significantly lower expression levels in the hepatocytes treated with FFA mixtures. Meanwhile, 13 clones similar to the genes DGAT‐1, ACSL1, DHRS7, PPARα, L‐FABP, DGAT‐2, PCK, ACSL3, CPT‐1, A‐FABP, PPARβ, MAT, and ALDOB had significantly higher expression levels in the hepatocytes treated with FFA mixtures. These results suggest that several metabolic pathways are altered in goose hepatocytes, which may be useful for further research into the molecular mechanism of goose hepatic steatosis. J. Cell. Biochem. 111: 1482–1492, 2010. © 2010 Wiley‐Liss, Inc.     

Spatially regulated genes expressed during seed germination and postgerminative development are activated during embryogeny

Summary We investigated the control of genes expressed primarily during seed germination and postgerminative development in Brassica napus L. We identified cloned mRNA sequences which became prevalent within 1 day after the start of imbibition and were at low or undetectable levels in immature embryos, dry seeds, and leaves. Most postgermination-abundant mRNAs accumulated primarily, though not exclusively, in different parts of the seedling. Of the 14 cloned mRNAs, 8 were prevalent in cotyledons, 2 were abundant in seedling axes, and 4 were approximately equally distributed in both parts. We showed that although these mRNAs reached maximal levels in seedlings, the spatially regulated mRNAs were also detected at distinct embryonic stages; mRNAs prevalent in seedling axes accumulated primarily during early embryogenesis while cotyledon-abundant mRNA concentration increased during late embryogeny. We conclude that the temporal and spatial regulation of gene expression in seedlings reflects similarities and differences in the physiological functions of cotyledons and axes. Furthermore, the regulated expression of cotyledon-abundant genes during late embryogeny suggests that the mRNAs and possibly proteins may accumulate in preparation for subsequent seedling growth. Similarities in the accumulation of cotyledon-abundant mRNAs may indicate coordinate regulation of this gene set.Abbreviations DAF days after flowering - DAI days after the start of imbibition - HAI hours after the start of imbibition - kb kilobase(pairs)     

Tryptophan decarboxylase is encoded by two autonomously regulated genes in Camptotheca acuminata which are differentially expressed during development and stress

Camptothecin (CPT) is a valuable anti-cancer monoterpene alkaloid produced by the Chinese tree Camptotheca acuminata . Tryptophan decarboxylase (TDC) supplies tryptamine for the indole moiety of CPT and its derivatives, and is considered a key step in monoterpene indole alkaloid biosynthesis as it links primary and secondary metabolism. This report describes the isolation and characterization of tdc1 and tdc2 , two autonomously regulated TDC genes from Camptotheca . When expressed in Escherichia coli , the products of each gene could decarboxylate tryptophan, but were inactive against tyrosine, phenylalanine and 3,4-dihydroxyphenylalanine (dopa). tdc1 was developmentally regulated, having its highest expression level in the apex, young stem and bark, tissues which also contain the highest levels of CPT. Expression of tdc1 also increased during seedling development and was correlated with alkaloid accumulation during germination. tdc2 expression was induced in Camptotheca leaf discs and cell suspension cultures treated with fungal elicitor or methyl jasmonate, treatments which did not affect tdc1 expression. Unlike tdc1 , tdc2 expression was not detected in any unstressed Camptotheca tissues nor in developing seedlings. These data suggest that tdc1 may be part of a developmentally regulated chemical defense system in Camptotheca , while tdc2 serves as part of a defense system induced during pathogen challenge.     

Chalcone synthase-like genes active during corolla development are differentially expressed and encode enzymes with different catalytic properties in Gerbera hybrida (Asteraceae)

Recent studies on chalcone synthase (CHS) and the related stilbene synthase (STS) suggest that the structure of chs-like genes in plants has evolved into different forms, whose members have both different regulation and capacity to code for different but related enzymatic activities. We have studied the diversity of chs-like genes by analysing the structure, expression patterns and catalytic properties of the corresponding enzymes of three genes that are active during corolla development in Gerbera hybrida. The expression patterns demonstrate that chs-like genes are representatives of three distinct genetic programmes that are active during organ differentiation in gerbera. Gchs1 and gchs3 code for typical CHS enzymes, and their gene expression pattern temporally correlates with flavonol (gchs1, gchs3) and anthocyanin (gchs1) synthesis during corolla development. Gchs2 is different. The expression pattern does not correlate with the pigmentation pattern, the amino acid sequence deviates considerably from the consensus of typical CHSs, and the catalytic properties are different. The data indicate that it represents a new member in the large superfamily of chs and chs-related genes.     

Isolation and characterization of two tightly linked catalase genes from castor bean that are differentially regulated

Two catalase genes,cat1 andcat2, have been isolated from the castor bean genome. They were located in the same direction on a chromosome at a distance of 2.4 kb,cat1 being on the downstream side ofcat2. The two genes contained introns at the same positions except that one of the 7 introns incat1 is missing incat2 and the corresponding introns differed in size and sequence between the two genes. The translated regions of the two genes had the same number of nucleotides and exhibited 81.3% nucleotide sequence identity. In addition to introns, the nucleotide sequences of the 5-and 3-flanking regions are highly divergent between the two genes. In etiolated seedlings,cat1 mRNA was present abundantly in endosperms and cotyledons and only in a small amount in roots. Thecat1 mRNA could not be detected in hypocotyls. By contrast,cat2 mRNA is most abundant in hypocotyls and roots, while endosperms and cotyledons contained only low levels ofcat2 mRNA. Although neithercat1 norcat2 mRNA could be detected in dry seeds, both mRNAs showed temporal accumulation in the endosperm in response to germination. These results suggest that expression of two tightly linked catalase genes of castor bean,cat1 andcat2, are differentially regulated during development.     

Two classes of differentially regulated glutamine synthetase genes are expressed in the soybean nodule: a nodule-specific class and a constitutively expressed class

We have characterized two sets of cDNA clones representing the glutamine synthetase (GS) mRNA in soybean nodules. Using the 3-untranslated regions of a representative member of each set, as gene member(s) specific probes, we have shown that one set of the GS genes are expressed in a nodule-specific manner, while the other set is expressed in other tissues, besides the nodules. The nodule-specific GS genes are expressed in a developmentally regulated manner in the nodules, independent of the onset of nitrogen fixation. The other class of GS genes is expressed constitutively in all tissues tested, but its expression level is dramatically enhanced in nodules following onset of N2 fixation. The latter set of genes is also expressed in cotyledons of germinating seedlings in a developmentally regulated manner. Analysis of hybrid select translation products and genomic Southern blots suggests that multiple gene members in each class are expressed in the nodules.