A simple procedure for the isolation of high quality RNA from ripening banana fruit

Isolation of high quality RNA from ripening banana fruit tissue is difficult due to high levels of polysaccharides and other substances that interfere when using conventional procedures for RNA isolation. These substances not only decrease the yield but the quality of RNA is almost unusable. We describe here a simple RNA procedure that effectively removes these contaminating substances without affecting the yield. Following this procedure, we routinely obtained 80–150 μg of total RNA per g fresh tissue. The RNA is of good quality and suitable for northern analysis, RT-PCR and cDNA library construction. NBRI publication No. 488(NS).     

香蕉肉桂醇脱氢酶基因的克隆及表达分析

肉桂醇脱氢酶(CAD)在木质素合成过程中起关键作用。通过RACE(rapid-amplification of cDNA ends)方法从香蕉根系cDNA均一化全长文库中获得一个肉桂醇脱氢酶基因,命名为MaCAD1(GenBank登录号为KF582533)。MaCAD1是香蕉MYB基因编码框全长cDNA,包含一个1 077bp的最大开放阅读框(ORF),编码358个氨基酸。蛋白质序列同源比对发现,其含有完整的醇脱氧酶的典型保守结构域,属于典型的CAD蛋白。系统进化树比对分析表明,MaCAD1与水稻OsCAD6(CAD39907)的亲缘关系较近。组织特异性研究表明MaCAD1基因组成型表达于香蕉各个组织。在耐病和感病品种中,MaCAD1均上调表达,但在耐病品种中MaCAD1在所有时间点相对于对照增加的倍数均高于感病品种,表明MaCAD1基因在香蕉的抗病性中起着重要作用,MaCAD1可以作为一个新的响应枯萎病侵染的标记基因。     

Hormonal regulation of ripening in the strawberry,a non-climacteric fruit

Anthocyanin accumulation is one measure of ripening in the strawberry (Fragaria ananassa Duch.), a non-climacteric fruit. Neither aminoethoxyvinylglycine, an inhibitor of 1-aminocyclopropane carboxylic acid synthase, nor inhibitors of ethylene action (silver, norbornadiene) affected anthocyanin accumulation in ripening fruit. When the achenes were removed from one half of an unripe fruit there was an accelerated accumulation of anthocyanin and induction of phenylalanine ammonia lyase on the de-achened portion of the ripening fruit. These effects of achene removal could be prevented by the application of the synthetic auxins 1-naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid to the de-achened surface. The introduction of 1-naphthalene acetic acid into intact unripe strawberry fruit through the peduncle delayed their subsequent ripening, as measured by the accumulation of anthocyanin, loss of chlorophyll and decrease in firmness. These findings suggest that the decline in the concentration of auxin in the achenes as strawberry fruit mature modulates the rate of fruit ripening.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - NAA 1-naphthaleneacetic acid - PA1 phenylalanine ammonia-lyase - POA phenoxyacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Fluxes of carbohydrate metabolism in ripening bananas

The major fluxes of carbohydrate metabolism were estimated during starch breakdown by ripening bananas (Musa cavendishii Lamb ex Paxton). Hands of bananas, untreated with ethylene, were allowed to ripen in the dark at 21° C. Production of CO₂ and the contents of starch, sucrose, glucose and fructose of intact fruit were determined for a period of 10 d that included the climacteric. The detailed distribution of label was determined after supplying the following to cores of pulp from climacteric fruit: [U-¹⁴C]-, [1-¹⁴C]-, [3,4-¹⁴C]-and [6-¹⁴C]glucose, [U-¹⁴C]glycerol, ¹⁴CO₂. The data obtained were used to estimate the following fluxes, values given as mol hexose · (g FW)^–1 · h^–1 in parenthesis: starch to hexose monophosphates (5.9) and vice versa (0.4); hexose monophosphates to sucrose (7.7); sucrose to hexose (4.7); hexose to hexose monophosphate (3.8); glycolysis (0.5–1.6); triose phosphate to hexose monophosphates (0.14); oxidative pentose-phosphate pathway (0.48); CO₂ fixation in the dark (0.005). These estimates are related to our understanding of carbohydrate metabolism during ripening.We both thank Mr Richard Trethewey for his constructive criticism: S.A.H. thanks the Managers of the Broodbank Fund for a fellowship.     

Cloning of a cDNA encoding 1-aminocyclopropane-1-carboxylate synthase and expression of its mRNA in ripening apple fruit

1-Aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14) purified from apple (Malus sylvestris Mill.) fruit was subjected to trypsin digestion. Following separation by reversed-phase high-pressure liquid chromatography, ten tryptic peptides were sequenced. Based on the sequences of three tryptic peptides, three sets of mixed oligonucleotide probes were synthesized and used to screen a plasmid cDNA library prepared from poly(A)⁺ RNA of ripe apple fruit. A 1.5-kb (kilobase) cDNA clone which hybridized to all three probes were isolated. The clone contained an open reading frame of 1214 base pairs (bp) encoding a sequence of 404 amino acids. While the polyadenine tail at the 3-end was intact, it lacked a portion of sequence at the 5-end. Using the RNA-based polymerase chain reaction, an additional sequence of 148 bp was obtained at the 5-end. Thus, 1362 bp were sequenced and they encode 454 amino acids. The deduced amino-acid sequence contained peptide sequences corresponding to all ten tryptic fragments, confirming the identity of the cDNA clone. Comparison of the deduced amino-acid sequence between ACC synthase from apple fruit and those from tomato (Lycopersicon esculentum Mill.) and winter squash (Cucurbita maxima Duch.) fruits demonstrated the presence of seven highly conserved regions, including the previously identified region for the active site. The size of the translation product of ACC-synthase mRNA was similar to that of the mature protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), indicating that apple ACC-synthase undergoes only minor, if any, post-translational proteolytic processing. Analysis of ACC-synthase mRNA by in-vitro translation-immunoprecipitation, and by Northern blotting indicates that the ACC-synthase mRNA was undetectable in unripe fruit, but was accumulated massively during the ripening proccess. These data demonstrate that the expression of the ACC-synthase gene is developmentally regulated.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AdoMet S-adenosyl-l-methionine - HPLC high-pressure liquid chromatography - kDa kilodalton - kb kilobase - mAb monoclonal antibody - Met methionine - PCR polymerase chain reaction - poly(A)⁺ RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work was supported by grants DCB-9004129 and INT-8915155 from the National Science Foundation.     

Timing of ethylene and polygalacturonase synthesis in relation to the control of tomato fruit ripening

A critical role in the initiation of ripening has been proposed for pectolytic enzymes which are known to be involved in fruit softening. The hypothesis that tomato (Lycopersicon esculentum Mill.) ripening is controlled by the initial synthesis of the cell-wall-degrading enzyme polygalacturonase (EC 3.2.1.15), which subsequently liberates cell-wall-bound enzymes responsible for the initiation of ethylene synthesis and other ripening events, has been examined. A study of kinetics of ethylene evolution and polygalacturonase synthesis by individual fruits in a ripening series, employing an immunological method and protein purification to identify and measure polygalacturonase synthesis, showed that ethylene evolution preceded polygalacturonase synthesis by 20h. Exogenous ethylene stimulated the synthesis of polygalacturonase and other ripening events, when applied to mature green fruit, whereas the maintenance of fruits in a low ethylene environment delayed ripening and polygalacturonase synthesis. It is concluded that enhanced natural ethylene synthesis occurs prior to polygalacturonase production and that ethylene is responsible for triggering polygalacturonase synthesis indirectly. Possible mechanisms for ethylene action are discussed.     

Effects of carbon sources and auxins on in vitro propagation of banana

The effects of carbon sources (sucrose, glucose, fructose and mannitol) and auxins [indolebutyric acid (IBA) and α-naphthaleneacetic acid (NAA)] on in vitro propagation of banana (Musa spp. AAA) were studied. Over all carbon sources tested, sucrose induced highest frequency of shoot proliferation. Optimal shoot proliferation rates were achieved on the Murashige and Skoog (MS) medium supplemented with sucrose and glucose combination (1:1) at the concentration of 30 g dm⁻³. Similarly, higher frequency of root induction was obtained at IBA and NAA combination (1:1; concentration of 2 mg dm⁻³) than at other concentrations of IBA or NAA alone or their combinations.     

The appearance of polygalacturonase mRNA in tomatoes: one of a series of changes in gene expression during development and ripening

Tomato mRNA was extracted from individual fruits at different stages of development and ripening, translated in a rabbit reticulocyte lysate and the protein products analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The results indicate that there are at least two classes of mRNA under separate developmental control. One group of approximately six mRNAs is present during fruit growth and then declines at the mature-green stage. Another group of between four and eight mRNAs increases substantially in amount at the onset of ripening, after the start of enhanced ethylene synthesis by the fruit, and continues to accumulate as ripening progresses. Studies of protein synthesis in vivo show that several new proteins are synthesised by ripening fruits including the fruit-softening enzyme polygalacturonase. One of the ripening-related mRNAs is shown to code for polygalacturonase, by immunoprecipitation with serum from rabbits immunised against the purified tomato enzyme. Polygalacturonase mRNA is not detectable in green fruit but accumulates during ripening. It is proposed that the ripening-related mRNAs are the products of a group of genes that code for enzymes important in the ripening process.Abbreviation SDS sodium dodecyl sulfate     

The effect of glucose on the control of carbohydrate metabolism in ripening bananas

The effect of exogenous glucose on the major fluxes of carbohydrate metabolism in cores of climacteric fruit of banana (Musa cavendishii Lamb ex Paxton) was determined with the intention of using the effects in the application of top-down metabolic control analysis. Hands of bananas, untreated with ethylene, were allowed to ripen in the dark at 21 °C. Cores were removed from climacteric fruit and incubated in 100 or 200 mM glucose for 4 or 6 h. The rates of starch breakdown, sucrose and fructose accumulation and CO₂ production were measured. The steady-state contents of hexose monophosphates, adenylates and pyruvate were determined. In addition, the detailed distribution of label was determined after supply of the following: [U-¹⁴C]-, [1-¹⁴C]-, [3,4¹⁴C]and [6-¹⁴C]glucose, and [U-¹⁴C]glycerol. The data were used to estimate the major fluxes of carbohydrate metabolism. Supply of exogenous glucose led to increases in the size of the hexose-monophosphate pools. There was a small stimulation of the rate of sugar synthesis and a major increase in the rate of starch synthesis. Starch breakdown was inhibited. Respiration responded to the demand for ATP by sugar synthesis. The effect of glucose on fluxes and metabolite pools is discussed in relation to our understanding of the control and regulation of carbohydrate metabolism in ripening fruit.Abbreviations Glc6P glucose-6-phosphate - Glc1P glucose-1-phosphate - Fru6P fructose-6-phosphate - AEC adenylate energy charge We thank Geest Foods Group, Great Dunmow, Essex, UK for giving us the bananas. SAH thanks the managers of the Broodbank Fund for a fellowship.     

Identification of cDNA clones for tomato (Lycopersicon esculentum Mill.) mRNAs that accumulate during fruit ripening and leaf senescence in response to ethylene

Changes in gene expression during foliar senescence and fruit ripening in tomato (Lycopersicon esculentum Mill.) were examined using in-vitro translation of isolated RNA and hybridization against cDNA clones.During the period of chlorophyll loss in leaves, changes occurred in mRNA in-vitro translation products, with some being reduced in prevalence, whilst others increased. Some of the translation products which changed in abundance had similar molecular weights to those known to increase during tomato fruit ripening. By testing RNA from senescing leaves against a tomato fruit ripening-related cDNA library, seven cDNA clones were identified for mRNAs whose prevalence increased during both ripening and leaf senescence. Using dot hybridization, the pattern of expression of the mRNAs corresponding to the seven clones was examined. Maximal expression of the majority of the mRNAs coincided with the time of greatest ethylene production, in both leaves and fruit. Treatment of mature green leaves or unripe fruit with the ethylene antagonist silver thiosulphate prevented the onset of senescence or ripening, and the expression of five of the seven ripening- and senescence-related genes.The results indicate that senescence and ripening in tomato involve the expression of related genes, and that ethylene may be an important factor in controlling their expression.Abbreviations cDNA copy-DNA - MW molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate     

Expression of AODEF,a B-functional MADS-box gene,in stamens and inner tepals of the dioecious species Asparagus officinalis L.

Garden asparagus (Asparagus officinalis L.) is a dioecious species with male and female flowers on separate unisexual individuals. Since B- and C-functional MADS-box genes specify male and female reproductive organs, it is important to characterize these genes to clarify the mechanism of sex determination in monoecious and dioecious species. In this study, we isolated and characterized AODEF gene, a B-functional gene in the development of male and female flowers of A. officinalis. Southern hybridization identified a single copy of AODEF gene in asparagus genome. Northern blot analysis showed that this gene was specifically expressed in flower buds and not in vegetative tissues. In situ hybridization showed that during early hermaphrodite stages, AODEFgene was expressed in the inner tepal and stamen whorls (whorls 2 and 3, respectively), but not in the outer tepals (whorl 1), in both male and female flowers. In late unisexual developmental stages, the expression of AODEF gene was still detected in the inner tepals and stamens of male flowers, but the expression was reduced in whorls 2 and 3 of female flowers. Our results suggest that AODEF gene is probably not involved in tepal development in asparagus and that the expression of AODEF gene is probably controlled directly or indirectly by sex determination gene in the late developmental stages.     

Expression of phytoene synthase gene (Psy) is enhanced during fruit ripening of Cara Cara navel orange (Citrus sinensis Osbeck)

Citrus is an important fruit crop as regards accumulation of carotenoids. In plant carotenoid biosynthesis, phytoene synthase gene (Psy) plays a key role in catalyzing the head-to-head condensation of geranylgeranyl diphosphate molecules to produce colorless phytoene. In the present paper, we reported the phytoene contents determination and characterization of Psy during fruit ripening of “Washington” navel orange and its red-fleshed mutant “Cara Cara”. Results showed that phytoene was exclusively accumulated in peel and pulp of “Cara Cara”. Although phytoene was observed accumulating with fruit ripening of “Cara Cara”, the contents in pulp were 10 times higher than those in peel. The isolated two Psy cDNAs were both 1520 bp in full length, containing 436 deduced amino acid residues, with a different amino acid at 412th. Genomic hybridization results showed that one or two copies might be present in “Cara Cara” and “Washington” genomes. During “Cara Cara” and “Washington” fruit coloration, expression of Psy was observed to be up-regulated, as revealed by tissue specific profiles in the flavedo, albedo, segment membrane and juice sacs. However, Psy expression in albedo of “Cara Cara” was higher than that in “Washington”, as evidenced by phytoene accumulation in the peel.     

The effect of hypoxia on the control of carbohydrate metabolism in ripening bananas

The aim of this work was to determine the effects of hypoxia on the major fluxes of carbohydrate metabolism in climacteric fruit of banana (Musa cavendishii Lamb ex Paxton). Hands of bananas, untreated with ethylene, were allowed to ripen in air at 21°C in the dark. When the climacteric began, fruit were transferred to 15 or 10% oxygen and were analysed once the climacteric peak had been reached 8–12 h later. The rates of starch breakdown, sucrose, glucose and fructose accumulation, and CO₂ production were determined, as were the contents of hexose monophosphates, adenylates and pyruvate. In addition, the detailed distribution of label was determined after supplying [U-¹⁴C]-, [1-¹⁴C]-, [3,4-¹⁴C]- and [6-¹⁴C]glucose, and [U-¹⁴C]glycerol to cores of tissue under hypoxia. The data were used to estimate the major fluxes of carbohydrate metabolism. There was a reduction in the rate of respiration. The ATP/ADP ratio was unaffected but there was a significant increase in the content of AMP. In 15% oxygen only minor changes in fluxes were observed. In 10% oxygen starch breakdown was reduced and starch synthesis was not detected. The rate of sucrose synthesis decreased, as did the rate of re-entry of hexose sugars into the hexose monophosphate pool. There was a large increase in both the glycolytic flux and in the flux from triose phosphates to hexose monophosphates. It is argued that the increase in these fluxes is due to activation of pyrophosphate: fructose-6-phosphate 1-phosphotransferase, and that this enzyme has an important role in hypoxia. The results are discussed in relation to our understanding of the control of carbohydrate metabolism in hypoxia.Abbreviations Glc6P glucose-6-phosphate - Glc1P glucose-1-phosphate - Fru6P fructose-6-phosphate - PP_i inorganic pyro-phosphate We thank Geest Foods Group, Great Dunmow, Essex, UK for giving us the bananas. S.A.H. thanks the managers of the Brood bank Fund for a fellowship.     

Induction of a Citrus gene highly homologous to plant and yeast thi genes involved in thiamine biosynthesis during natural and ethylene-induced fruit maturation

Maturing citrus fruit undergo pigment changes which can be enhanced by exogenous ethylene. In order to identify genes induced by ethylene in citrus fruit peel, we cloned the gene c-thi1. mRNA corresponding to c-thi1 increased gradually in the peel during natural fruit maturation and in response to ethylene. GA₃ pretreatment reduced the inductive effect of ethylene. Levels of c-thi1 increased also in juice sacs but the effect of ethylene was much less prominent. c-thi1 is homologous to yeast and plant genes encoding for an enzyme belonging to the pathway of thiamine biosynthesis. The data suggest that thiamine is involved in citrus fruit maturation.     

A comparison of 1-aminocyclopropane-1-carboxylate synthase in vitro translation product and in-vivo-labeled protein in ripening tomatoes

We determined the time course of increases in 1-aminocyclopropane-1-carboxylate (ACC) synthase activity in ripening tomato (Lycopersicon esculentum (L.) Mill.) pericarp discs following wounding and treatment with 75 mM LiCl. Over the course of 24 h, we detected oscillations in the amount of enzyme activity from an initial peak at 6 h to a subsequent, even higher level at 18 h. In-vitro translation products derived from poly(A)⁺ RNAs isolated at various times of treatment and in-vivo-labeled proteins were immunoprecipitated using antibodies specific for ACC synthase. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography showed that wounding and treatment with LiCl induced an accumulation of translatable ACC-synthase-specific mRNAs. In addition, single, prominent bands were apparent for both in-vivo and in-vitro samples but their molecular masses differed. It appears that the in-vitro translation product is a polypeptide of 56 kDa while the in-vivo-labeled enzyme has a molecular mass of 47 kDa. The authors greatly appreciate the skilled technical assistance of Renate deZacks and Gail Robinson. This research was supported by the National Science Foundation through Grant No. DCB-8718873 and by the Department of Energy through Contract No. DE-AC02-76ER-01338.     

Clues about the ancestral roles of plant MADS-box genes from a functional analysis of moss homologues

Classic MIKC-type MADS-box genes (MIKC ^c genes) are indispensable elements in the genetic programming of pattern formation, including the segmental organisation of angiosperm flowers, in seed plants. Since little is known about the functions of MIKC ^c genes in non-seed plants, a functional analysis of moss MIKC ^c homologues was performed using the genetically amenable, simple model plant, Physcomitrella patens. Expression of moss homologues was knocked down using an antisense RNA approach or abolished by generating transformants with gene knockouts. The knocked down (“antisense”) transformants displayed a multifaceted mutant phenotype comprising delayed gametangia formation, diminished sporophyte yield and, in the most extremely affected cases, abnormal sporophyte development and altered leaf morphogenesis. Knocked out transformants were phenotypically normal. Analysis of in situ MIKC ^c gene expression using transgenic strains containing MIKC ^c promoter–GUS fusions showed that these genes are generally expressed ubiquitously in vegetative and reproductive tissues. We conclude that MIKC ^c genes play significant roles in morphogenetic programming of the moss. Functional redundancy characterises some members of the gene group. Our findings provide clues concerning the ancestral roles of some MIKC ^c genes that may be represented in the genomes of diverse extant plant taxa. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.