Light-Induced Chloroplast [alpha]-Amylase in Pearl Millet (Pennisetum americanum)

In pearl millet (Pennisetum americanum) seedlings light induces the appearance of a leaf [alpha]-amylase isozyme. The leaf [alpha]-amylase isozyme was present in enriched amounts in isolated chloroplast but it could not be detected in isolated etioplasts. The chloroplast [alpha]-amylase was present in both mesophyll and bundle-sheath chloroplasts. Preliminary characterization indicated that molecular properties of chloroplast [alpha]-amylase were like those of a typical [alpha]-amylase. The plastidic [alpha]-amylase had a molecular mass of 46 kD, pH optimum of 6.2, required Ca2+ for activity and thermostability, but lost activity in the presence of ethylenediaminetetracetate. Plastidic [alpha]-amylase activity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis could be renatured in situ by Triton X-100. Western blot analysis demonstrated that this protein was antigenically similar to a maize seed [alpha]-amylase. In vivo [35S]methionine labeling of bundle-sheath strands isolated from light-grown leaves followed by immunoprecipitation revealed that bundlesheath strands synthesized plastidic [alpha]-amylase de novo.     

水稻sbe1启动子驱动的反义sbe-GUS融合基因在转基因水稻中的表达

为研究水稻基因启动子对外源基因在转基因水稻中表达的影响,构建了由sbe1启动子引导的反义sbe-GUS融合基因。经农杆菌介导,将不同的融合基因导入水稻中,定量测定转基因水稻植株不同组织中的GUS酶活力。结果表明,sbe1启动子可驱动反义sbe-GUS融合基因在转基因水稻植株的胚乳中高效表达,而在颖壳、胚和茎叶等组织中的表达活性较弱。证实sbe1启动子在驱动外源基因的表达上表现有明显的组织特异性。     

Bean [alpha]-Amylase Inhibitor Confers Resistance to the Pea Weevil (Bruchus pisorum) in Transgenic Peas (Pisum sativum L.)

Bruchid larvae cause major losses of grain legume crops through-out the world. Some bruchid species, such as the cowpea weevil and the azuki bean weevil, are pests that damage stored seeds. Others, such as the pea weevil (Bruchus pisorum), attack the crop growing in the field. We transferred the cDNA encoding the [alpha]-amylase inhibitor ([alpha]-AI) found in the seeds of the common bean (Phaseolus vulgaris) into pea (Pisum sativum) using Agrobacterium-mediated transformation. Expression was driven by the promoter of phytohemagglutinin, another bean seed protein. The [alpha]-amylase inhibitor gene was stably expressed in the transgenic pea seeds at least to the T5 seed generation, and [alpha]-AI accumulated in the seeds up to 3% of soluble protein. This level is somewhat higher than that normally found in beans, which contain 1 to 2% [alpha]-AI. In the T5 seed generation the development of pea weevil larvae was blocked at an early stage. Seed damage was minimal and seed yield was not significantly reduced in the transgenic plants. These results confirm the feasibility of protecting other grain legumes such as lentils, mungbean, groundnuts, and chickpeas against a variety of bruchids using the same approach. Although [alpha]-AI also inhibits human [alpha]-amylase, cooked peas should not have a negative impact on human energy metabolism.     

Hormonal Regulation of Expression of Two Cysteine Endopeptidase Genes in Rice Seedlings

Two cDNA probes (pRP60 and pRP80) were used to examine the hormonalregulation of expression of two cysteine endopeptidase genesin germinated whole seeds and de-embryonated seeds of rice.The pRP60 protein is a major rice cysteine endopeptidase namedREP-1 that digests rice glutelin, the main seed storage protein.The pRP80 protein has features of a cysteine endopeptidase buthas not yet been confirmed to be one. Neither mRNA was detectablein dry seeds, and the levels per seed increased sharply uponimbibition, reached peaks at d 6 to d 9, and then decreased.Both mRNAs were expressed in a seed-specific manner, but theaccumulation of pRP60 mRNA was about ten times greater thanthe other. When seeds were de-embryonated and incubated, theamounts of both mRNAs were reduced to very low levels, but inthe presence of 0.01 to 1 µM. gibberellic acid (GA₃) bothagain reached high levels. Addition of abscisic acid (ABA) oruniconazole, an inhibitor of gibberellin biosynthesis, partlyeliminated the effect of GA₃. Protein immunoblot analysis showedthat the accumulation of the REP-1 protein in seeds was regulatedby GA₃ and ABA similarly to that of pRP60 mRNA, but the changein pRP60 mRNA with time after the onset of imbibition precededthat of the REP-1 protein. (Received May 28, 1997; Accepted September 8, 1997)     

Effects of Gibberellins and Prohexadione on the Activities of Oryzain and {alpha}-Amylase in Rice Seeds

Oryzains, cysteine proteinases of rice seeds, are induced byGA₃ in germinating rice seeds [Abe et al. (1987) Agric. Biol.Chem. 51: 1509]. The effects of GA₁, GA₃, GA₄, GA₉, and GA₂₀on the production of oryzain and -amylase were investigatedin embryoless half- and whole-seeds of rice (cv. Nipponbare).When gibberellins (GAs) were incubated with embryoless half-seeds,GA₁, GA₃ and GA₄ induced oryzain and -amylase, but GA₉, andGA₂₀ did not. GA₉ and GAM induced oryzain and -amylase productionin whole seeds, but this production was inhibited by the simultaneousapplication of prohexadione, an inhibitor of 2ß- and3ß-hydroxylation of GAs. Prohexadione did not inhibitthe activities of oryzain and -amylase induced by GA₁. Theseresults suggest that GAs possessing the 3ß-hydroxylgroup induce activities of oryzain and -amylase in rice seedsand that GA₉ and GA₂₀ have activity only after they are convertedmetabolically to active GAs, probably GA₄ and GA₁, respectively.GA₁, was more active than GA₄ in both half seeds and wholeseeds incubation. Oryzain and -amylase activities induced byGA₄ were significantly inhibited in the presence of 10^–4M prohexadione. This suggests that the conversion of GA₁, toGA₄ (13-hydroxylation) might be inhibited at a high dose ofprohexadione in whole seeds. ⁴Present address: Institute of Food Development, Kyung Hee University,Suwon 449-701, Korea     

Cyclic AMP Promotion and Abscisic Acid Inhibition of {alpha}-Amylase Activity in the Seeds of Rice (Oryza sativa L.)

Cyclic AMP and GA₃ stimulated both -amylase activity in riceendosperm and the germination of the seed. In combination theyalso induced germination of ABA-treated seeds but cyclic AMPalone failed to neutralize the inhibitory effect of ABA; withadded kinetin, however, it promoted the -amylase activity ofthe dormant seeds. The enzyme activity decreased as the storageperiod of seeds increased. Cyclic AMP and GA     

Penicillin Induction of Gibberellin and {alpha}-Amylase Biosynthesis in Rice Endosperm

Penicillin induces the synthesis of -amylase in embryoless riceendosperm and enhances the gibberellin-induced response. Penicillininduction of -amylase can be prevented by inhibitors of nucleicacid and protein synthesis, CCC and 2,4-DNP. A characteristic gibberellin-like activity in the extracts frompenicillin-treated endosperms becomes detectable after 12 hfrom the addition of penicillin. This gibberellin-like activityis located on paper chromatograms at the R_F typical for GA₃and its formation is blocked by CCC, an inhibitor of GA biogenesis.Glucose has no effect on the biosynthesis of either gibberellinor -amylase induced by penicillin. The time-course study of the levels of different constituentsshows that penicillin probably induces RNA and DNA synthesisin the first place, which results in gibberellin biosynthesis,which in turn stimulates the synthesis of -amylase. The possiblemode of action of penicillin in higher plants is discussed.     

籼稻明恢63成熟种子愈伤组织的诱导及转基因水稻的抗性检测

利用不含附加营养成分的2,4-D培养基（2mg/L）诱导明恢63的成熟种子,9天预诱导后获得了大量的愈伤组织。利用基因枪辅助的土壤农杆菌转化法将天花粉蛋白（Trichosanthin, TCS）基因转入籼稻明恢63的愈伤组织,并通过再生（含有3mg/L 6-BA, 0.5mg/L ABA和1mg/L NAA的N6培养基）获得了转基因植物。 Southern blot分析和Western blot检测证明外源基因已经插入到T0代明恢63的基因组中并获得了表达。初步研究结果表明,转基因水稻对稻瘟病菌侵染具有抗性。     

Transgenic studies of alpha(1)-adrenergic receptor subtype function

Mice with altered alpha(1)-adrenergic receptor (AR) genes have become important tools in elucidating the subtype-specific functions of the three alpha(1)-AR subtypes because of the lack of sufficiently subtype-selective pharmacological agents. Mice with a deletion (knockout, KO) or an overexpression (transgenic, TG) of the alpha(1A)-, alpha(1B)-, or alpha(1D)-AR subtypes have been generated. The alpha(1)-ARs are the principal mediators of the hypertensive response to alpha(1)-agonists in the cardiovascular system. Studies with these mice indicate that alpha(1A)-AR and alpha(1B)-AR subtypes play an important role in cardiac development and/or function as well as in blood pressure (BP) response to alpha(1)-agonists via vasoconstriction. The alpha(1B)- and alpha(1D)-subtypes also appear to be involved in central nervous system (CNS) processes such as nociceptive responses, modulation of memory consolidation and working memory. The ability to study subtype-specific functions in different mouse strains by altering the same alpha(1)-AR in different ways strengthens the conclusions drawn from these studies. Although these genetic approaches have limitations, they have significantly increased our understanding of the functions of alpha(1)-AR subtypes.     

Developmental and Hormonal Regulation of Gibberellin Biosynthesis and Catabolism in Pea Fruit

In pea (Pisum sativum), normal fruit growth requires the presence of the seeds. The coordination of growth between the seed and ovary tissues involves phytohormones; however, the specific mechanisms remain speculative. This study further explores the roles of the gibberellin (GA) biosynthesis and catabolism genes during pollination and fruit development and in seed and auxin regulation of pericarp growth. Pollination and fertilization events not only increase pericarp PsGA3ox1 message levels (codes for GA 3-oxidase that converts GA₂₀ to bioactive GA₁) but also reduce pericarp PsGA2ox1 mRNA levels (codes for GA 2-oxidase that mainly catabolizes GA₂₀ to GA₂₉), suggesting a concerted regulation to increase levels of bioactive GA₁ following these events. 4-Chloroindole-3-acetic acid (4-Cl-IAA) was found to mimic the seeds in the stimulation of PsGA3ox1 and the repression of PsGA2ox1 mRNA levels as well as the stimulation of PsGA2ox2 mRNA levels (codes for GA 2-oxidase that mainly catabolizes GA₁ to GA₈) in pericarp at 2 to 3 d after anthesis, while the other endogenous pea auxin, IAA, did not. This GA gene expression profile suggests that both seeds and 4-Cl-IAA can stimulate the production, as well as modulate the half-life, of bioactive GA₁, leading to initial fruit set and subsequent growth and development of the ovary. Consistent with these gene expression profiles, deseeded pericarps converted [¹⁴C]GA₁₂ to [¹⁴C]GA₁ only if treated with 4-Cl-IAA. These data further support the hypothesis that 4-Cl-IAA produced in the seeds is transported to the pericarp, where it differentially regulates the expression of pericarp GA biosynthesis and catabolism genes to modulate the level of bioactive GA₁ required for initial fruit set and growth.     

Developmental and Hormonal Regulation of Genes Coding for Proline-Rich Proteins in Female Inflorescences and Kernels of Maize

The pattern of expression of two genes coding for proteins rich in proline, HyPRP (hybrid proline-rich protein) and HRGP (hydroxyproline-rich glycoprotein), has been studied in maize (Zea mays) embryos by RNA analysis and in situ hybridization. mRNA accumulation is high during the first 20 d after pollination, and disappears in the maturation stages of embryogenesis. The two genes are also expressed during the development of the pistillate spikelet and during the first stages of embryo development in adjacent but different tissues. HyPRP mRNA accumulates mainly in the scutellum and HRGP mRNA mainly in the embryo axis and the suspensor. The two genes appear to be under the control of different regulatory pathways during embryogenesis. We show that HyPRP is repressed by abscisic acid and stress treatments, with the exception of cold treatment. In contrast, HRGP is affected positively by specific stress treatments.     

New pyrimido[5,4-b]indoles and [1]benzothieno[3,2-d]pyrimidines: high affinity ligands for the alpha(1)-adrenoceptor subtypes

A number of new pyrimido[5,4-b]indole and [1]benzothieno[3,2-d]pyrimidine derivatives were synthesized and evaluated for their binding and functional properties at alpha(1)-adrenergic receptor (alpha(1)-AR) subtypes. They behaved as potent alpha(1)-AR antagonists. In binding experiments, some of them (RC24 and RC23) showed very high affinity for the alpha(1D)-AR subtype.     

Occurrence of Multiple Forms of {alpha}-Amylase and Absence of Starch Phosphorylase in Soya Bean Seeds

Soya bean cultivars ‘Altona’ and ‘Chestnut’have active but quite low levels of -amylase. Activity was assayedwith specific substrates, oxidized amylose and ß-limitdextrin, which were resistant to attack by ß-amylase.During seed development -amylase activity increased to a maximumin both cultivars and then declined towards maturity. Matureand germinating seeds retain low activities of -amylase. Gelelectrophoresis separated the -amylase activity into six majorbands which occurred in both cultivars. The isozyme patternwas quite similar for developing, mature and germinating seed.although the relative proportion of activity in the variousbands changed somewhat. Starch phosphorylase was not detectedin any soya bean seed samples tested, but good activity wasfound in potato tuber extracts used as a control. Mixing experimentsusing soya bean and potato extracts indicated there were noinhibiting factors in soya bean seed extracts. Soya bean seedextracts probably do not contain starch phosphorylase. Glycine max (L.), Merr, soya bean, -amylase, isozymes, phosphorylase     

Ligand modulation of [35S]GTPgammaS binding at human alpha(2A), alpha(2B) and alpha(2C) adrenoceptors

Affinities and efficacies of chemically diverse ligands--some of them used as clinical agents--were examined, employing [3H]RX821,002 and [35S]GTPgammaS binding assays, respectively, at human (h) cloned, halpha(2A), halpha(2B) and halpha(2C) adrenoceptors (AR) expressed in Chinese hamster ovary (CHO) cells. As compared to noradrenaline (NA, efficacy defined as 100%), the majority of the 13 agonists tested generally behaved as partial agonists. Amongst 18 antagonists, pK(B) and pK(i) values, which were highly correlated for each alpha(2)-AR subtype, failed to reveal any strikingly selective agents. Inverse agonist properties were not detected for any antagonist, consistent with a lack of constitutive activity suggested by the monophasic inhibition of [35S]GTPgammaS binding by GTPgammaS. These data should facilitate interpretation of experimental and clinical actions of adrenergic agonists. Moreover, they emphasize the continuing need for alpha(2)-AR subtype-selective antagonists in order to define further the roles and therapeutic relevance of halpha(2A)-, halpha(2B)-, and halpha(2C)-AR.     

Changes in {alpha} and {beta}-Amylase Activities during Germination of Seeds of Alfalfa (Medicago sativa L.)

We examined the methods available for the assay of -amylasein alfalfa (Medicago sativa) and found the Phadebas test mostsuitable. The Phadebas assay and activity staining on ampholinegels after isoelectrofocusing revealed that an amylase is presentin the dry seeds of alfalfa and that its activity decreasesrapidly after the second day of seed germination. An amylasewas purified by affinity chromatography and gel filtration.The kinds of sugar generated from soluble starch by the purifiedamylase resembled those generated by other -amylases from plants,in particular those from mung bean (Vigna radiata). These resultsindicate that the amylase in alfalfa seeds belongs to the familyof -amylases. The molecular weight and isoelectric point ofthe -amylase were determined to be 43 kDa and 4.92, respectively. The Pantrac assay and activity staining on immobiline gels afterisoelectrofocusing revealed that the activities of ß-amylasesincreased during the initial 4 to 5 days of germination. Furthermore,treatment of whole seedlings with cycloheximide or actinomycinD inhibited the increase in activity of ß-amylasesbut did not affect the reduction in activity of -amylase. During germination of alfalfa seeds, -amylase activity decreaseswhile, in contrast, ß-amylase activity increases (inthe cotyledons of germinating seeds), changes that are specificto the germinating seeds of alfalfa. (Received September 8, 1990; Accepted February 20, 1991)     

Effect of Abscisic Acid on Uptake and Metabolism of [H]Gibberellin A(1) and [H]Pseudogibberellin A(1) by Barley Half-seeds

Uptake and metabolism of 1,2-[³H]gibberellin A₁ ([³H]GA₁, I) and its 3-hydroxy epimer ([³H]pseudoGA₁, II) by barley (Hordeum vulgare L.) half-seeds were measured after 24 hours of incubation, in the presence or absence of abscisic acid in the media. Uptake of both compounds was enhanced by abscisic acid, and abscisic acid enhanced the extent of metabolism of [³H]GA₁. However, [³H]pseudoGA₁ was not metabolized, even in the presence of abscisic acid. The significance of the stereo-chemistry of the 3-hydroxyl position is discussed.     

种子贮藏蛋白的运输、积累和基因表达调控

种子中贮藏蛋白的运输和积累途径主要有：(1)蛋白质合成后经内膜系统转移到蛋白质贮藏液泡(PSV)中积累;(2)合成的蛋白质直接在粗糙内质网的膜囊中积累形成蛋白质体;(3)贮藏蛋白不经高尔基体的加工由粗糙内质网上合成后直接运输到PSV中积累。贮藏蛋白基因的表达受该基因的顺式作用元件和反式作用因子的共同调控,此外染色体的结构也影响贮藏蛋白基因的表达。     

Hormonal regulation of serum alpha 1-antitrypsin and hepatic alpha 1-antitrypsin mRNA in rats

We evaluated the effects of pituitary dependent hormones on alpha 1-antitrypsin in male rats. Hepatic alpha 1-antitrypsin mRNA was measured by in vitro translation and by specific hybridization with a mouse cDNA alpha 1-antitrypsin probe. Hypophysectomy caused a 50-75% decrease in serum elastase inhibitory capacity (measuring functional alpha 1-antitrypsin) and hepatic alpha 1-antitrypsin mRNA content. In hypophysectomized animals, no increase in elastase inhibitory capacity or alpha 1-antitrypsin mRNA levels by translation was found when met-human growth hormone alone or corticosterone, dihydrotestosterone and thyroxine were given together. Growth hormone increased alpha 1-antitrypsin mRNA by hybridization to a small extent. Addition of growth hormone to the combination of corticosterone, dihydrotestosterone, and thyroxine increased serum elastase inhibitory capacity and alpha 1-antitrypsin mRNA. We conclude that growth hormone acts synergistically with the other pituitary dependent hormones to regulate serum and hepatic mRNA levels of alpha 1-antitrypsin.     

Metabolism of [H]Gibberellin A(5) by Immature Seeds of Apricot (Prunus armeniaca L.)

Immature seeds of apricot (Prunus armeniaca L.) were fed the native gibberellin A₅ (GA₅) as 1- and 1,2-[³H]GA₅ (5.3 Curies per millimole to 16 milliCuries per millimole) at doses (42 nanograms to 10.6 micrograms per seed) 2 to 530 times the expected endogenous level. After 4 days of incubation, seeds were extracted and free [³H]GA-like metabolites were separated from the highly H₂O-soluble [³H]metabolites. For high specific activity feeds the retention times (Rts) of radioactive peaks were compared with Rts of authentic GAs on sequential gradient-eluted → isocratic eluted reversed-phase C₁₈ high performance liquid chromatography (HPLC) -radiocounting (RC). From high substrate feeds (530 and 230 × expected endogenous levels) HPLC-RC peak groupings were subjected to capillary gas chromatography-selected ion monitoring (GC-SIM), usually six characteristic ions. The major free GA metabolites of [³H] GA₅ were identified as GA₁, GA₃, and GA₆ by GC-SIM. The major highly water soluble metabolite of [³H]GA₅ at all levels of substrate GA₅ had chromatographic characteristics similar to authentic GA₁-glucosyl ester. Expressed as a percentage of recovered radioactivity, low substrate [³H]GA₅ feeds (2 × expected endogenous level) yielded a broad spectrum of metabolites eluting at the Rts where GA₁, GA₃, GA₅ methyl ester, GA₆, GA₂₂, GA₂₉ (17, 14, 1.6, 7, 1.1, 0.5%, respectively) and GA glucosyl conjugates of GA₁, GA₃, GA₅, and GA₈ (33, 11, 1, 0.1%, respectively) elute. Metabolites were also present at Rts where GA glucosyl conjugates of GA₆ and GA₂₉ would be expected to elute (8 and 0.1%, respectively). Only 5% of the radioactivity remained as GA₅. Increasing substrate GA₅ levels increased the proportion of metabolites with HPLC Rts similar to GA₁, GA₆, and especially GA₁ glucosyl ester, primarily at the expense of metabolites with HPLC Rts similar to GA₃, GA₃-glucosyl ester, and a postulated conjugate of GA₆. There was evidence that high doses of substrate GA₅ induced new metabolites which often, but not always, differed from GA₁, GA₃, and GA₆ in HPLC Rt. These same metabolites, when analyzed by GC-SIM yielded m/e ions the same as the M⁺ and other characteristic m/e ions of the above GAs, albeit at differing GC Rt and relative intensities.