Cowpea ribonuclease: properties and effect of NaCl-salinity on its activation during seed germination and seedling establishment

Pitiúba cowpea [Vigna unguiculata (L.) Walp] seeds were germinated in distilled water (control treatment) or in 100 mM NaCl solution (salt treatment), and RNase was purified from different parts of the seedlings. Seedling growth was reduced by the NaCl treatment. RNase activity was low in cotyledons of quiescent seeds, but the enzyme was activated during germination and seedling establishment. Salinity reduced cotyledon RNase activity, and this effect appeared to be due to a delay in its activation. The RNases from roots, stems, and leaves were immunologically identical to that found in cotyledons. Partially purified RNase fractions from the different parts of the seedling showed some activity with DNA as substrate. However, this DNA hydrolyzing activity was much lower than that of RNA hydrolyzing activity. The DNA hydrolyzing activity was strongly inhibited by Cu²⁺, Hg²⁺, and Zn²⁺ ions, stimulated by MgCl₂, and slowly inhibited by EDTA. This activity from the most purified fraction was inhibited by increasing concentrations of RNA in the reaction medium. It is suggested that the major biological role of this cotyledon RNase would be to hydrolyze seed storage RNA during germination and seedling establishment, and it was discussed that it might have a protective role against abiotic stress during later part of seedling establishment.     

Stored mRNA in cotyledons of Vigna unguiculata seeds: nucleotide sequence of cloned cDNA for a stored mRNA and induction of its synthesis by precocious germination

By differential hybridization screening, we previously selected a class of cDNA clones from a gt10 cDNA library that was constructed from the total poly(A)⁺ RNA of mature cowpea cotyledons (Plant Cell Physiol 31: 39–44, 1990). pSAS10, a clone of this class, hybridized with a cDNA probe complementary to poly(A)⁺ RNA from cotyledons collected 1 day after the onset of imbibition (DAI), but not with the cDNA probe from cotyledons at development stage II (13 to 15 days after flowering, DAF). pSAS10 mRNA was detectable only in cotyledons at development stage III (17 to 19 DAF) or later, and its level began to decline when seeds germinated. We have suggested that pSAS10 mRNA is likely to belong to the class of stored mRNA or the mRNA that is formed at the late stage of seed maturation, is conserved in quiescent seeds and becomes functional at the early stage of germination. We determined the nucleotide sequence of pSAS10 cDNA consisting of 459 bp and an approximately 36 bp poly(A) tract, and deduced the amino acid sequence of its product, a 10-kDa cysteine-rich polypeptide. Synthesis of pSAS10 mRNA was induced just before germination began, not only in mature seeds but also in immature seeds even at stages I (9 to 11 DAF) and II (13 to 15 DAF) if they were placed under conditions suitable for germination.     

Glutamine-synthetase isoforms appearing in sunflower cotyledons during germination

Ion-exchange chromatography has been used to separate the isoforms of glutamine synthetase (GS; EC 6.3.1.2) appearing in sunflower (Helianthus annuus L. cv. Peredovic) cotyledons during seedling growth under different light and nitrogen conditions. Both in dry and imbibed seeds, only a single form of GS (GS_s) was detected. Upon seed germination, the GS_s isoform was gradually replaced by cytosolic (GS₁) and plastidic (GS₂) isoforms. Light and nitrate decreased the levels of GS₁. In contrast, the appearance of GS₂ was greatly stimulated by light. Nitrate also had a positive effect, particularly in the light. Light and nitrate acted synergistically on the appearance of GS₂. The GS₂:GS₁ ratio in cotyledons of 9-d-old seedlings ranged from about 2, in darkness and nitrate-deprivation conditions, to 16 under light and nitrate application. The possible physiological roles of the distinct GS isoforms appearing in the epigeal cotyledons of sunflower during germination, and their differential regulation by light and nitrate, are discussed.Abbreviations GS glutamine synthetase - GS₁ cytosolic GS - GS₂ plastidic GS - GS_s GS from seeds This work was supported by a grant from Dirección General de Investigatión Científica y Técnica (PB90-0777) and Plan Andaluz de Investigación (3261), Spain. P.C. gratefully acknowledges receipt of a scholarship from Junta de Andalucía. The valuable technical assistance of Mrs. G. Alcalá is greatly appreciated. We are also grateful to Eurosemillas (Córdoba) for supplying us with sunflower seeds.     

Enhanced methods for unbiased deep sequencing of Lassa and Ebola RNA viruses from clinical and biological samples

We have developed a robust RNA sequencing method for generating complete de novo assemblies with intra-host variant calls of Lassa and Ebola virus genomes in clinical and biological samples. Our method uses targeted RNase H-based digestion to remove contaminating poly(rA) carrier and ribosomal RNA. This depletion step improves both the quality of data and quantity of informative reads in unbiased total RNA sequencing libraries. We have also developed a hybrid-selection protocol to further enrich the viral content of sequencing libraries. These protocols have enabled rapid deep sequencing of both Lassa and Ebola virus and are broadly applicable to other viral genomics studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0519-7) contains supplementary material, which is available to authorized users.     

The development of photosynthetic light reactions in cultured apple embryos in relation to their embryonal dormancy

The development of photosynthetic light reactions (photoreduction and photophosphorylation) was studied in cotyledons of cultured embryos isolated from stratified and non-stratified apple seeds (Malus domestica Borb. cv. Antonówka). The results obtained show the presence of an energy transfer inhibitor in the cotyledons of the stratified embryos.Abbreviations PSII photosystem II - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - ATP adenosine triphosphate - ADP adenosine diphosphate - CCCP carbonyl cyanide m-chlorophenyl-hydrazone - EDTA ethylenediamine tetraacetic acid; P, inorganic orthophosphate - TES (N-tris [hydroxymethyl] methyl-2-aminoethane sulfonic acid) This work was partly supported by the Polish Academy of Sciences, Contract No 10.2.10     

Colloid droplets in the magnocellular secretory neurons of the reptilian hypothalamus: An immunocytochemical and lectin-histochemical study

Summary The immunocytochemical and lectin-binding properties of the magnocellular neurosecretory neurons in the hypothalamus of 2 reptilian species, the snake Natrix maura and the lizard Liolaemus cyanogaster, were investigated. Particular attention was paid to the secretory droplets present in these neurons. Antisera against bovine neurophysins I+II, arginine-vasotocin, and mesotocin were used. The following lectins were applied: concanavalin A (Con A), wheat-germ agglutinin (WGA), and Limax flavus agglutinin (LFA). Adjacent 1-m-thick methacrylate sections were used to investigate the same secretory neuron and the same colloid droplets with all three antisera and all three lectins. Several sections were treated with trypsin and urea before immunostaining or lectin binding. Con A bound to both vasotocin- and mesotocin-immunoreactive neurons, WGA exclusively to vasotocin neurons; neither of these neurons reacted with LFA. The colloid droplets were present in vasotocin neurons but absent in the mesotocin neurons. These secretory droplets showed an affinity for Con A but not for WGA, and reacted with antisera against neurophysins and vasotocin. In Natrix maura, the colloid droplets became reactive with Con A and the antisera used only after pretreatment of the sections with trypsin and urea. Within the hypothalamo-neurohypophyseal system, antiserum against vasotocin and WGA revealed the same fiber bundles. It is concluded (i) that in reptiles the vasotocin-neurophysin precursor is glycosylated, (ii) that vasotocin neurons have the exclusive capacity to form colloid droplets, and (iii) that these droplets are an intracisternal (RER) storage form of the vasotocin-neurophysin precursor.This work was partially supported by Grants BOJA 27/9/88 from the Dirección General de Universidades e Investigación de Junta de Andalucía and DGICYT PB87-0710 from the Comisión Interministerial de Ciencia y Tecnología, Madrid, to P.F.-LL.; and Grant 89-01 from the Dirección de Investigaciones, Universidad Austral de Chile, to E.M.R.     

Recovery of plastids from photooxidative damage: Significance of a plastidic factor

Glyoxysomal citrate synthase (gCS) was purified from crude extracts of watermelon (Citrullus vulgaris Schrad.) cotyledons, yielding a homogenous protein with a subunit MW of 48 kDa. The enzyme was selectively inhibited by 5,5-dithiobis-(2-nitrobenzoic acid), allowing quantification in the presence of the mitochondrial isoenzyme (mCS). Differences were also observed with respect to inhibition by ATP (k _i=2.6 mmol · l^-1 for gCS, k _i=0.33 mmol · l^-1 for mCS). The antibodies prepared against gCS did not cross-react with mCS. The immunocytochemical localization of gCS by the indirect protein A-gold procedure was restricted to the glyoxysomal membrane or the peripheral matrix of glyoxysomes. Other compartments, e.g. the endoplasmic reticulum, were not labeled. Xenopus oocytes were used for the translation of watermelon polyadenylated RNA (poly(A)⁺RNA). A translation product with a MW of 51 kDa was immunoprecipitated by the anti-gCS antibodies. It was absent in controls without poly(A)⁺RNA or with preimmune serum. A similar translation product was also immunoprecipitated after cell-free synthesis of watermelon poly(A)⁺RNA in a reticulocyte system, in contrast to the in-vivo labeled gCS (48 kDa). It was concluded that gCS is synthesized as a higher-molecular-weight precursor.Abbreviations DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - gCS glyoxysomal citrate synthase - gMDH glyoxysomal malate dehydrogenase - k _i inhibitor constant - mCS mitochondrial citrate synthase - OAA oxaloacetate - poly(A)⁺RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Glucan-phosphorylase forms in cotyledons of Pisum sativum L.: Localization,developmental change,in-vitro translation,and processing

The occurrence, location, and biosynthesis of glucan-phosphorylase (EC 2.4.1.1) isoenzymes were studied in cotyledons of developing or germinating seeds of Pisum sativum L. Type-I and type-II isoenzymes were detected, and were also localized by indirect immunofluorescence using polyclonal anti-type-I or anti-type-II phosphorylase antibodies. Type-I isoenzyme was found in the cytosol of parenchyma cells whereas the type-II enzyme form is a plastid protein which resides either in amyloplasts (in developing seeds) or in proplastids (in germinating seeds). During seed development, type-II phosphorylase was the predominant isoenzyme and the type-I isoenzyme represented a very minor compound. During germination, the latter increased whilst type-II phosphorylase remained at a constant level. In in-vitro translation experiments, type-I isoenzyme was observed as a final-size product with an apparent molecular weight of approx. 90 kDa. In contrast, type-II phosphorylase was translated as a high-molecular-weight precursor (116 kDa) which, when incubated with a stromal fraction of isolated intact pea chloroplasts, was processed to the size of the mature protein (105 kDa).Abbreviations IgG immunoglobulin G - kDa kilodalton - poly(A)⁺ RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work has been made possible by grants from the Deutsche Forschungsgemeinschaft. The authors are endebted to Mrs. Karin Niehüser for help in the immunocytochemical studies.     

The role of cotyledons in phototropism of de-etiolated seedlings

Simulated phototropic curvatures caused by differential masking of the cotyledons of de-etiolated seedlings exposed to white light are unconnected with true phototropism. In Cucumis sativus L. and Helianthus annuus L. such curvatures result from a red-light-induced inhibition coming from the exposed cotyledon. True phototropic bending in these species under long-term exposure to fairly high irradiances (as in nature) is a response to blue light. It occurs even when cotyledons are completely covered. These results show that the cotyledons do not perceive the phototropic stimulus and need not be illuminated for phototropism to occur.     

Phylogenetic relationships among Cucumis species based on the ribosomal internal transcribed spacer sequence and microsatellite markers

The phylogenetic relationships within the genus Cucumis (a total of 25 accessions belonging to 17 species) were studied using the nuclear ribosomal DNA internal transcribed spacer (ITS) region. The analysis included commercially important species such as melon (C. melo L.) and cucumber (C. sativus). Two additional cucurbit species, watermelon and zucchini, were also included as outgroups. The data obtained reflected the clustering of Cucumis species in four main groups, comprising accessions from cucumber, melon, C. metuliferus and the wild African species. Some of the species clustered in different positions from those reported in classifications previously described by other authors. The data obtained clearly identify a division between the 2n=2x = 14 species (C. sativus) and the 2n = 2x = 24 ones (C. melo and wild species). Within the wild species we identified a subgroup that included C. sagittatus and C. globosus. Oreosyce africana, also classified as Cucumis membranifolius, was shown to be nested within Cucumis. Three accessions previously classified as independent species were shown to be genotypes of Cucumis melo. A set of melon and cucumber SSRs were also used to analyse the Cucumis species and the results were compared with the ITS data. The differential amplification of the SSRs among the accessions made it possible to distinguish three main groups: melon, cucumber and the wild species, though with less detail than applying ITS. Some SSRs were shown to be specific for melon, but other SSRs were useful for producing PCR fragments in all species of the genus.We are grateful to NCRPIS, IPK in Gatersleben, Semillas Fitó S.A., Michel Pitrat and Fernando Nuez for providing seeds. We would also like to thank Vanessa Alfaro, Trinidad Martínez and Núria Galofré for their excellent technical assistance. This work was financed by project AGL2000-0360 of Spains Ministerio de Ciencia y Tecnología (MCYT). AJMs work was supported by a postdoctoral contract from Spains MCYT.     

Preformed mRNA in Cotyledons of Ungerminated Seeds of Cicer arietinum L

Polyadenylated RNA was isolated from total RNA extracted from cotyledons of ungerminated or 18-hour-germinated chick-pea seeds by affinity chromatography on oligo(dT)-cellulose. Both poly(A)-containing RNA fractions exhibited a template activity when assayed in two cell-free translation systems, wheat germ extracts, and nuclease-treated reticulocyte lysates. Translation of preformed mRNA from cotyledons of dry seeds was completely abolished in the presence of several inhibitors of polypeptide chain initiation and also in the presence of the two “cap” analogues m⁷ GTP and m⁷ GMP. The patterns of polypeptides synthesized by translation of poly(A)-containing RNAs from cotyledons of ungerminated or 18-hour-germinated seeds, in the wheat germ system, analyzed by electrophoresis and autoradiography, were similar but not identical. It is concluded that cotyledons of dry Cicer arietinum L. seeds contain preformed mRNA.     

Purification and properties of tobacco ferredoxin-dependent glutamate synthase,and isolation of corresponding cDNA clones

Ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) was purified to electrophoretic homogeneity from leaves of tobacco (Nicotiana tabacum L.). The holoenzyme is a monomeric flavoprotein with a molecular weight of 164 kDa. Polyclonal rabbit antibodies against the purified enzyme were used to isolate a 450-bp Fd-GOGAT cDNA clone (C₁₆) from a tobacco gt11 expression library. A longer Fd-GOGAT cDNA clone (C₃₅) encoding about 70% of the amino acids of tobacco Fd-GOGAT was isolated from a tobacco gt10 cDNA library using C₁₆ as the probe. The amino-acid sequence of the protein encoded by the Fd-GOGAT cDNA clone C₃₅ was delineated. It is very likely that Fd-GOGAT is encoded by two genes in the amphidiploid genome of tobacco while only a single Fd-GOGAT gene appears to be present in the diploid genome of Nicotiana sylvestris. Two Fd-GOGAT isoenzymes could be distinguished in extracts of tobacco leaf protein. In contrast, a single Fd-GOGAT protein species was detected in leaves of Nicotiana sylvestris speg. et Comes. In tobacco leaves, the 6-kb Fd-GOGAT mRNA is about 50-fold less abundant than chloroplastic glutamine synthetase (EC 6.3.1.2) mRNA. Both Fd-GOGAT mRNA and Fd-GOGAT protein accumulated during greening of etiolated tobacco leaves, and a concomitant increase in Fd-GOGAT activity was observed. These results indicate that tobacco Fd-GOGAT gene expression is light-inducible. Levels of Fd-GOGAT mRNA in tobacco organs other than leaves were below the detection limit of our Northern-blot analysis. Polypeptides of Fd-GOGAT were present in tobacco leaves and, to a lesser extent, in pistils and anthers, but not in corollas, stems and roots. These results support organ specificity in tobacco Fd-GOGAT gene expression.Abbreviations bp base pairs - Fd-GOGAT ferredoxin-dependent glutamate synthase - GS glutamine synthetase - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate The authors wish to thank Juan Luis Gómez Pinchetti (Marine Plant Biotechnology Laboratory) for his assistance during the experiments. This study was supported by grants received from SAREC (Swedish Agency for Research Cooperation with Developing Countries), Carl Tryggers Fund for Scientific Research (K. Haglund), SJFR (Swedish Council for Forestry and Agricultural Research) (M. Björk, M. Pedersén), CITYT Spain (SAB 89-0091 and MAR 91-1237, M. Pedersén) and CICYT Spain (Z. Ramazanov, invited professor of Ministerio de Educatión y Ciencia, Spain). The planning of this cooperation was facilitated by COST-48.     

Concanavalin A is synthesized as a glycoprotein precursor

Concanavalin A (Con A) is a tetrameric lectin which is synthesized in the cotyledons of developing jack-bean (Canavalia ensiformis (L.) D.C.) seeds and accumulates in the protein bodies of storage-parenchyma cells. The polypeptides of Con A have a molecular weight of 27000 and a relative molecular mass (M_r) of 30000 when analyzed by gel electrophoresis on denaturing polyacrylamide gels. In-vitro translation of RNA isolated from immature jack-bean cotyledons shows that Con A is synthesized as a polypeptide with M_r 34000. In-vivo pulse labeling of cotyledons with radioactive amino acids or glucosamine also resulted in the formation of a 34000-M_r polypeptide. In-vivo labeling with radioactive amino acids in the presence of tunicamycin yielded an additional polypeptide of 32000 M_r. Together these results indicate that Con A is cotranslationally processed by the removal of a signal sequence and the addition of an oligosaccharide side chain of corresponding size. Analysis of the structure of the oligogosaccharide side chain was accomplished through glycosidase digestion of glycopeptides isolated from [³H]glucosamine-labeled Con A. Incubation of the labeled glycopeptides with endoglycosidase H, -mannosidase or -N-acetylglucosaminidase, followed by gel filtration, allowed us to deduce that the oligosaccharide side chain of pro-Con A is a high-mannose oligosaccharide. Pulse-chase experiments with labeled amino acids are consistent with the interpretation that the glycosylated precursor of Con A is processed to mature Con A (M_r=30000). The 4000 decrease in M_r is interpreted to result from the removal of a small glycopeptide. The implications of the conversion of a glycoprotein pro-Con A to mature Con A are discussed in the context of the unique circular permutation of the primary structure of Con A.Abbreviations Con A concanavalin A - Glc glucose - GlcNAc N-acetylglucosamine - IgG immunoglobulin G - Man mannose - M_r relative molecular mass - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Complete nucleotide sequence of alfalfa mosaic virus RNA 4.

Alfalfa mosaic virus RNA 4, the subgenomic messenger for viral coat protein, was partially digested with RNase T1 or RNase A and the sequence of a number of fragments was deduced by in vitro labeling with polynucleotide kinase and application of RNA sequencing techniques. From overlapping fragments, the complete primary sequence of the 881 nucleotides of RNA 4 was constructed: the coding region of 660 nucleotides (not including the initiation and termination codon) is flanked by a 5' noncoding region of 39 nucleotides and a 3' noncoding region of 182 nucleotides. The RNA sequencing data completely confirm the amino acid sequence of the coat protein as deduced by Van Beynum et al. (Fur.J. Biochem. 72, 63-78, 1977).     

Cytochemical localization and ultrastructure of Aspergillus flavus in cottonseed

Cottonseeds having fluorescent fibers were harvested from fields in Arizona and examined utilizing light microscopy and transmission electron microscopy. The occurrence of fluorescent fibers indicated that seeds had been infected by Aspergillus flavus during development. Presence of A. flavus was verified by plating portions of seeds with fluorescent fibers. Hyphae, conidial heads, and conidia were identified readily in differentially-stained cotyledon tissue processed for light microscopy. Utilization of transmission electron microscopy permitted observations on lignified seed coats and cotyledons of mature cottonseeds. Hyphae were located throughout the cotyledon and in the nonlignified layers of the seed coat. The identification of hyphae in cross sections of vessel elements within the seed coat provided ultrastructural evidence supporting the hypothesis that A. flavus may enter seeds via the vascular tissue. Controls for the microscopy studies included observations on cottonseeds with no visual signs of infection and on laboratory-grown cultures of A. flavus. These observations demonstrated that the hyphae localized within fluorescent seeds had features characteristic of A. flavus and that fungal-like structures do not occur within uninfected seeds.     

Detection and Location of Seed-Borne Inoculum of Didymella bryoniae and its Transmission in Seedlings of Cucumber and Pumpkin

Of the 91 tested cucurbit seed samples from thirteen countries nine from four countries were found to be infected with Didymella bryoniae. The pathogen is located on and in the seed coat including the perisperm and in the tissue of the cotyledons. Primary seedling infection occurred on the radicle, hypoctyl and cotyledons. Infection of the radicle generally caused pre-emergence tot while infection on the hypocotyl and cotyledons developed furthere inoculum for infection of the first true leaves and the stem. Experimentally, all the isolates of D. bryoniae could infect cucumber (Cucumis sativus), oriental melon (Cucumis melo var. makuwa), pumpkin (Cucurbita pepo) and watermelon (Citrullus vulgaris) at different growth stages; the susceptibility of cucumber and pumpkin was markedly influenced by previailing humid conditions.The blotter method was found more suitable for detection of seed-borne infection than the agar plate method.     

Genotype and medium affect shoot regeneration of melon

The effects of basal media, growth regulators and gelling agents on the morphogenetic response of eleven Cucumis melo L. var. reticulatus and inodorus genotypes were examined. Regeneration was achieved from cultured cotyledons of all genotypes and the morphogenic response was affected by the genetic background. Among the combination of factors tested, MS basal medium supplemented with 2.8 M 6-benzyladenine (BA) and 1.0 M abscisic acid (ABA) solidified with agar gave the highest frequency of shoot regeneration.Abbreviations ABA abscisic acid - BA 6-benzyladenine - TDZ thidiazuron     

Acidophilic granulocytes of the marine fish gilthead seabream (<Emphasis Type="Italic">Sparus aurata</Emphasis> L.) produce interleukin-1β following infection with<Emphasis Type="Italic"> Vibrio anguillarum</Emphasis>

The fish immune response to Gram-negative bacteria is poorly understood. In this study, we use a monoclonal antibody (mAb) specific to acidophilic granulocytes from the marine fish gilthead seabream (Sparus aurata L.), together with an antiserum specific to interleukin-1 (IL-1) from this species, in order to investigate whether these cells are involved in the immune response against the pathogenic bacterium Vibrio anguillarum and, in particular, in the production of the pro-inflammatory cytokine IL-1. We found that gilthead seabream head-kidney, peritoneal exudate and peripheral blood leukocytes accumulated proIL-1 intracellularly when challenged in vitro with V. anguillarum, whereas only peritoneal exudate and blood leukocytes were able to accumulate proIL-1 following infection. Importantly, the blood leukocytes from infected animals that accumulated proIL-1 were shown to be the acidophilic granulocytes. A rapid mobilization of such cells from the head-kidney to the site of inflammation following infection with V. anguillarum was also observed. This work was supported by the Spanish Ministry of Science and Technology (grants BIO2001-2324-C02-02 and AGL2002-03529, and fellowship to J. García-Castillo), Spanish Ministry of Education, Culture and Sport (fellowship to E. Chaves-Pozo) and Fundación Séneca, Coordination Centre for Research (grant PI-51/00782/FS/01 and fellowship to P. Pelegrín).E. Chaves-Pozo and P. Pelegrín contributed equally to this work.     

Purification of glycollate oxidase from greening cucumber cotyledons

Glycollate oxidase (glycollate: oxygen oxidoreductase, EC 1.1.3.1) was purified to apparent homogeneity from crude extracts of greening cucumber cotyledons (Cucumis sat vus). Molecular sieving and chromatofocusing resulted in 700-fold purification and specific activity of 1 kat mg^-1 protein. The enzyme exhibited a M_r of 180,000, or 700,000, respectively, and is a tetramer or 16-mer made of identical subunits of M_r 43,000. Monospecific antibodies were raised against the homogeneous protein.