Changes in the Translatable RNA Population during Abscisic Acid Induce Freezing Tolerance in Bromegrass Suspension Culture

Abscisic acid (ABA) has been shown to increase freezing toleranceof bromegrass (Bromus in-ermis Leyss cv. Manchar) cell suspensioncultures from a LT₅₀ (the temperature at which 50% cells werekilled) of –7 to – 30?C in 5 days at 23?C. Our objectivewas to study the qualitative changes in the translatable RNApopulation during ABA induced frost tolernace. In vitro translationproducts of poly(A)⁺ RNA isolated from bromegrass cells withor without 75 µM ABA treatment for various periods oftime were separated by 2D-PAGE and visualized by fluorography.SDS soluble proteins from the same treatments were also separatedby 20-PAGE. After 5 days treatment, at least 22 new or increasedabundance SDS soluble polypeptides were observed. From fluorographs,29 novel or increased abundance in vitro translation productscould be detected. The pattern of changes between ABA inducedSDS-soluble proteins and translation products from the 2D gelswere similar. A time course study (0–7 days) showed that17 of the 29 translation products were detected after 1 dayABA treatment, and at least 14 were present after 1 h. Coldtreatment (+4?C) induced fewer changes in the pool of translatableRNA than with ABA treatment. Three translation products inducedby cold appear to be similar to 3 of the ABA induced translationproducts. The majority of the ABA inducible translatable RNAsappeared at 10 µM or higher which coincides with the inductionof freezing tolerance. Many of these ABA inducible RNAs persisted7 days after ABA was removed from the media and correspondinglythe LT₅₀ (–17?C) was still well above the control level(–17?C). The results suggest that ABA alters the poolof translatable RNAs during induction of freezing tolerancein bromegrass suspension culture cells. ¹Oregon Agricultural Experiment Station Technical Paper No.9256. (Received August 3, 1990; Accepted October 18, 1990)     

Characterization and Translation of Poly(A)+RNA from Wounded and Crown Gall Tissues of Potato Tubers

Poly(A)⁺ and poly(A)^–RNA from wounded potato tuber tissuesand crown gall tumors were separated from total RNA by oligodeoxythymidylicacid-cellulose affinity chromatography. The poly(A)⁺RNA wascharacterized by sucrose density gradient centrifugation, hybridizationwith ³(H)polyuridylic acid [Poly(U)] and in vitro translationin a rabbit reticulocyte lysate system. The tumor poly(A)⁺RNAwas a heterodisperse mixture from 3.5S to 35S. Upon poly(U)hybridization of the gradient fractions two major hybridizationpeaks at 7S and 21S and two peaks at 11S and 16S appeared. Inan in vitro translation system the poly(A)⁺RNA programmed thesynthesis of 23 different polypeptides of 9,000 to 79,800 daltonsmolecular weight as determined by SDS-polyacrylamide gel electrophoresis.The 21S poly(A)+RNA was about 5 times more active in in vitroprotein synthesis than the 7S poly(A)⁺RNA. The poly(A)⁺RNA from wounded tissues was also heterodisperse(from 4.5S to 31S) with a modal peak at 18S. This RNA codedfor at least 28 polypeptides, which were different from thoseof crown gall tumor tissues. On a per unit poly(A)⁺RNA basis the tumor RNA was slightly moreactive in translation than that from wounded tissues. The translationof tumor poly(A)⁺RNA was completely blocked by 0.5 mM 7-methylguanosine5'-phosphate, but not by 7-methylguanosine, suggesting the presenceof a 5'-cap structure. (Received May 15, 1982; Accepted June 30, 1982)     

Changes in mRNA of Vigna mungo Cotyledons during Seed Germination

Quantitative and qualitative changes of mRNA in Vigna mungocotyledons during seed germination have been investigated. TotalRNA is higher in dry cotyledons and declines during germination.Poly(A)⁺ RNA also is present at a relatively high level in drycotyledons, increases slightly during the first day of germination,and then decreases. Polysomal RNA is very low in dry cotyledonsbut increases rapidly during the first day of germination, andthen declines. The translational activity of the mRNA in a wheatgerm cell-free system is low on day 0 but increases rapidlyon day 1 of germination. Two-dimensional gel electrophoresisof in vitro translation products reveals that many new peptidesare synthesized on day 1 of germination. Synthesis of most ofthese polypeptides continue throughout 5 days of germination. Change in the mRNA population during germination has been investigatedusing cDNA against poly(A)⁺ RNA from 3-day-old cotyledons. Withtotal RNA of day 3 and 5, the cDNA strongly hybridized withRNA similar in size to 25 S ribosomal RNA, but no specific bandsare detected with samples of day 0 or 1. With poly(A)⁺ RNA ofday 5 or 1, the cDNA tends to hybridize with RNAs of relativelysmall molecular size. Cordycepin and -amanitin prevent the increasein poly (A)⁺ RNA content and the appearance of new mRNAs duringthe first day of germination. ¹Present address: Division of Regulation of Macromolecular Function,Institute for Protein Research, Suita City, Osaka 565, Japan. (Received January 13, 1986; Accepted June 10, 1986)     

Response of Glutamine Synthetase and Glutamate Synthase Isoforms to Nitrogen Sources in Rice Cell Cultures

As a model system with no photorespiration and no long distancetransport, rice cell cultures (Oryza saliva L. cv Sasanishiki)were used to investigate the effect of nitrogen sources on thelevels of isoforms of glutamine synthetase (GS) and glutamatesynthase (GOGAT). Isoforms of GS and GOGAT were analyzed byimmunoblotting methods and their activities in early growthphase of the cells. Cytosolic type GS (41 kDa subunit) and NADH-GOGATwere the major isoforms in the rice cells grown in normal R-2medium. However, contents of plastid type GS (44 kDa subunit)and Fd-GOGAT increased in response to NO_–³ supply. NADH-GOGATactivity also increased following the supply of NO_–³.In vitro translated products from poly(A)⁺RNA prepared fromthe cells showed that the precursor of plastid type GS (49 kDa)was detected at 48 h after the inoculation. Supply of NH₊⁴ resultedin an increase in NADH-GOGAT activity but had no effect on thelevels of Fd-GOGAT, of polypeptides of the plastid type GS orof the corresponding mRNAs. (Received May 30, 1990; Accepted August 23, 1990)     

Solute Leakage from Solanum nigrum L. Seeds Exposed to High Temperatures during Imbibition

Exposure of Solanum nigrum L. seeds to high temperatures duringimbibition affected their leakage pattern: (1) The rate of leakageof total electrolytes was markedly increased with elevationof temperature. The increase was highest during the first 3h of imbibition but with a reduced rate thereafter. (2) Leakageof Na⁺ was almost complete after 6 h of imbibition at both temperatures,but much more Na⁺ leaked out at 50?C than at 25?C. (3) A markedincrease in leakage of K⁺ occurred after 24 h of exposure to50?C so that after 96 h three times more K⁺ leaked out at 50?Cthan at 25?C. (4) After 6 h of imbibition Ca11 and Mg⁺⁺ continuedto leak out at 25?C and at 50?C at a similar rate. (5) Imbibitionat an elevated temperature induced a marked increase in theleakage of both nucleic acids and proteins. (6) Malate dehydrogenasewas not detected in the leachate at 25?C, but was found after48 h at 50?C. It is assumed that this enzyme was of cytoplasmicorigin, indicating heat damage to membranes. The possible roleof the above phenomena in the loss of viability of the seedsdue to exposure to high temperature during imbibition is discussed. Key words: Leakage, Germination, S. nigrum     

Size and Levels of mRNA for Acid Invertase in Ripe Tomato Fruit

Poly(A)⁺RNA was isolated from ripe tomato fruit and translatedin a wheat germ cell-free translation system. A 74-kDa polypeptidewas detected as a putative precursor of acid invertase by immunoprecipitationwith antiserum raised against SDS-treated acid invertase (denaturedform) from tomato fruit. The molecular mass of the mRNA foracid invertase was estimated to be about 8 ? 10⁵ Da (2.4 k nucleotides)by sucrose density gradient centrifugation. Mature, green tomatofruit contained very low levels of invertase mRNA. When mature,green tomato fruit were stored at 22?C, levels of invertasemRNA per gram fresh weight increased to a maximum after fourdays and then declined. (Received May 24, 1989; Accepted May 2, 1990)     

Amelioration of Salinity Effect in Salt Tolerant Rice (Oryza sativa L.) by Foliar Application of Putrescine

Amelioration of NaCl (electric conductivity 15 mmho cm^–1)toxicity in the salt tolerant rice cultivar Co 43 was investigatedon 25th and 45th day old plants by foliar application of diamineputrescine (10 µM) in a pot culture experiment. Exogenoussupply of putrescine on salt stressed plants considerably increasedthe shoot growth (fresh weight and dry weight) and grain yield.Foliar application of putrescine inhibited the Na⁺ and Cl^–uptake, and accelerated the accumulation of K⁺, Ca²⁺, Mg²⁺,proline and endogenous putrescine in the leaves of salt-stressedplants. Furthermore, putrescine application prevented degradationof chlorophyll and inhibited the reductions of soluble protein,total protein, RNA and DNA contents, and elevated their concentrationsin the leaves of plants exposed to salinization. The physiologicalrole of putrescine in several stress-related processes is discussedwith salt tolerance of plants. (Received March 15, 1991; Accepted May 10, 1991)     

Cotyledonary mRNA Species and Germinability of Immature Vigna unguiculata Seeds

We have defined four stages in the development of cowpea seeds:I(9–11 days after flowering), II (13–15), HI (17–19)and IV (22–24). Poly A⁺ RNA fractions were prepared fromcotyledons of developing (stages I–IV) and germinating(0, 12, 24 and 48 h after imbibition) seeds. Poly A⁺ RNAs fromstages I–III exhibited high translation activities witha maximum at stage II, and the activity was markedly reducedat stage IV. In cotyledons of germinating seeds, the translationactivity was low until 12 h after the onset of imbibition butrose thereafter. Analysis of in vitro translation products withSDS-polyacrylamide gel electrophoresis and fluorography showedthat the abundant mRNA population underwent a distinct changebetween stages II and III of seed development. Since the mRNApopulation at stage III was very similar to that of stage IV(mature seeds), it appears that, as far as mRNA species areconcerned, the prerequisites for germination are fully availablein the developing seeds by stage III. This assumption was supportedby the fact that immature seeds at stage III exhibited highgermination rates and normal axial growth and produced -amylaseat levels similar to those produced by mature seeds. Severalpolypeptides which have been regarded as translation productsof stored mRNA (poly A⁺ RNA from dry seeds) were detected atearlier stages of germination. (Received September 29, 1988; Accepted January 25, 1989)     

Preformed mRNA and Light-induced Germination of Pine (Pinus thunbergii) Seed

The mRNAs were isolated from dry, dark-imbibed and light-imbibedpine (Pinus thunbergii) seeds, in which germination is inducedupon exposure to light, and their translational activities ina wheat embryo cell-free system were examined. A portion ofthe mRNA extracted from each type of seed appeared to be poly(A)⁺RNA.A significant translational activity was already present inthe RNA fraction isolated from the dry pine seeds. The preformedmRNA seems not to be translated in vivo during the dark-imbibition,since most of the in vivo protein synthesis did not occur untilthe seeds were exposed to light. The SDS polyacrylamide gelelectrophoregrams of the polypeptides synthesized in vitro inresponse to either the preformed mRNA or the mRNAs from thedark-imbibed or light-imbibed seeds were qualitatively identical;thus it seems that the preformed mRNA is conserved during darkimbibition, and that its translation is initiated after theexposure of the imbibed seeds to light. (Received August 10, 1981; Accepted May 15, 1982)     

Gene Activity during Germination of Spores of the Fern, Onoclea sensibilis: RNA and Protein Synthesis and the Role of Stored mRNA

Pattern of ³H-uridine incorporation into RNA of spores of Onocleasensibilis imbibed in complete darkness (non-germinating conditions)and induced to germinate in red light was followed by oligo-dTcellulose chromatography, gel electrophoresis coupled with fluorographyand autoradiography. In dark-imbibed spores, RNA synthesis wasinitiated about 24 h after sowing, with most of the label accumulatingin the high mol. wt. poly(A)^–RNA fraction. There was noincorporation of the label into poly(A) ⁺ RNA until 48 h aftersowing. In contrast, photo-induced spores began to synthesizeall fractions of RNA within 12 h after sowing and by 24 h, incorporationof ³H-uridine into RNA of irradiated spores was nearly 70-foldhigher than that into dark-imbibed spores. Protein synthesis,as monitored by ³H-arginine incorporation into the acid-insolublefraction and by autoradiography, was initiated in spores within1–2 h after sowing under both conditions. Autoradiographicexperiments also showed that the onset of protein synthesisin the cytoplasm of the germinating spore is independent ofthe transport of newly synthesized nuclear RNA. One-dimensionalsodium dodecyl sulphate-polyacrylamide gel electrophoresis of³⁵S-methionine-labelled proteins revealed a good correspondencebetween proteins synthesized in a cell-free translation systemdirected by poly(A) ⁺RNA of dormant spores and those synthesizedin vivo by dark-imbibed and photo-induced spores. These resultsindicate that stored mRNAs of O. sensibilis spores are functionallycompetent and provide templates for the synthesis of proteinsduring dark-imbibition and germination. Key words: Onoclea sensibilis, fern spore germination, gene expression, protein synthesis, sensitive fern, stored mRNA     

Characteristic Changes in Relaxation Times of Water Protons in Vigna radiata Seedlings Exposed to Temperature Stress

Water protons in hypocotyl tissues from etiolated seedlingsof Vigna radiata that were exposed to temperature stress showedcharacteristic relaxation behaviors for ¹H-NMR. Cold stresstreatment (0C) caused gradual prolongation of NMR relaxationtimes (T₁). After exposure of tissues to cold stress for 24h, T₁ returned to the initial value as a result of subsequentincubation at normal temperature (20C). By contrast, heat stresstreatment (40C) induced a time-dependent decrease in T₁, whichdid not return completely to the initial value upon subsequentincubation at 20C after exposure to heat stress for 4 h. Weexamined changes in various physical factors that influencethe response of T₁ to temperature stress, namely, water contentand the concentrations of protein, diamagnetic (K⁺, Na⁺, Ca²⁺and Mg²⁺) and paramagnetic (Mn²⁺ and Fe²⁺) ions in the tissues.From the relationships between T₁ and these factors in vitro,we could not interpret the responses of T₁ to the temperaturestress only in terms of a change in water content. A synergisticeffect of an Mn²⁺ -protein complex and pH might be essentialfor the mechanism of changes in T₁ that are due to cold stress.The influence of heat stress on structural water in tissuesis discussed in terms of water-protein interactions. (Received December 28, 1992; Accepted May 6, 1993)     

Stimulation of synthesis and translational activity of polyadenylated messenger RNA in wounded potato tubers by 2, 4-dichlorophenoxyacetic acid

Mechanical wounding of potato tubers induced a rapid synthesis of RNA in the wounded tissues. Both total and polyadenylated RNA increased with time after wounding. Treatment of wounded tissues with the synthetic hormone 2,4-dichlorophenoxyacetic acid (2,4-D, 10^?4 M) further stimulated their syntheses. The poly (A) ⁺RNA from hormone treated tissues was more active in in vitro protein synthesis. The in vitro translation of poly (A) ⁺RNA from both hormone treated and untreated tissues was inhibited by 7-methylguanosin-5′ phosphate, while 7-methylguanosine had no effect, suggesting that both poly (A) ⁺RNAs contained a blocked 5′-cap structure and that the cap structure was important for in vitro protein synthesis.     

The Effect of Root Temperature on Growth and Uptake of Ammonium and Nitrate by Brassica napus L. cv. Bien venu in Flowing Solution Culture: II. UPTAKE FROM SOLUTIONS CONTAINING NH4NO3

Macduff, J. H., Hopper, M. J. and Wild, A. 1987. The effectof root temperature on growth and uptake of ammonium and nitrateby Brassica napus L. CV. Bien venu in flowing solution culture.II. Uptake from solutions containing NH₄NO₃.—J. exp. Bot.38: 53–66 The effects of root temperature on uptake and assimilation ofNH₄⁺ and NO₃^– by oilseed rape (Brassica napus L. CV. Bienvenu) were examined. Plants were grown for 49 d in flowing nutrientsolution at pH 6?0 with root temperature decrementally reducedfrom 20?C to 5?C; and then exposed to different root temperatures(3, 5, 7, 9, 11, 13, 17 or 25?C) held constant for 14 d. Theair temperature was 20/15?C day/night and nitrogen was suppliedautomatically to maintain 10 mmol m^–3 NH₄NO₃ in solution.Total uptake of nitrogen over 14 d increased threefold between3–13?C but was constant above 13?C. Net uptake of NH₄⁺exceeded that of NO₃^– at all temperatures except 17?C,and represented 47–65% of the total uptake of nitrogen.Unit absorption rates of NH₄⁺ and of 1?5–2?7 for NO₃^–suggested that NO₃^– absorption was more sensitive thanNH₄⁺ absorption to temperature. Rates of absorption were relativelystable at 3?C and 5?C compared with those at 17?C and 25?C whichincreased sharply after 10 d. Tissue concentration of N in theshoot, expressed on a fresh weight basis, was independent ofroot temperature throughout, but doubled between 3–25?Cwhen expressed on a dry weight basis. The apparent proportionof net uptake of NO₃^– that was assimilated was inverselyrelated to root temperature. The results are used to examinethe relation between unit absorption rate adn shoot:root ratioin the context of short and long term responses to change ofroot temperature Key words: Brassica napus, oilseed rape, root temperature, nitrogen uptake     

Cell-free synthesis of opiate binding sites

A procedure is described for the detection of opiate binding sites synthesized during in vitro translation of various mRNA preparations. RNA were isolated from membrane bound polysomes which were prepared from NG 108-15 hybridoma, C6BU1 glioma cells, as well as from N18TG2, NB2aAg and NB41A3 neuroblastoma cells. Polyadenylated [poly(A)⁺] RNA were purified, translated in vitro in a rabbit reticulocyte lysate and the translation products assayed for their ability to bind [³H] bremazocine. Bound and free ligands were separated by column chromatography. After translation of poly(A)⁺ RNA obtained from NG 108-15 cells we demonstrated a stereospecific, saturable binding of [³H]bremazocine (displaced by levorphanol and not by dextrorphan) with a K_d of 2.4 ± 1.0 nM. The total amount of opiate binding sites synthesized was 6.2 ± 0.5 fmol per μg of poly(A)⁺ RNA. Opiate binding sites were undetectable at zero time and a plateau was reached after translation had proceeded for 20 min. Five time less opiate binding sites were synthesized when the poly(A)⁺ RNA purified from N18TG2 neuroblastoma cells were used under the same experimental conditions. There was no detectable binding of opiate ligands with poly(A)⁺ RNA obtained from C6BU1 glioma cells, NB2aAg or NB41A3 neuroblastoma cells.     

The genes encoding light-harvesting subunits of Cyclotella cryptica (Bacillariophyceae) constitute a complex and heterogeneous family

Total RNA was isolated from the diatom Cyclotella cryptica and separated into poly(A)⁺ and poly(A)^? fractions. These fractions were subjected to in vitro translation/immunoprecipitation experiments using an antiserum directed against the predominant light-harvesting complex of Cy. cryptica (ccry antiserum) and a heterologous antiserum raised against the light-harvesting complex of the cryptophyte Cryptomonas maculata (cmac antiserum). From translation reactions programmed with poly(A)⁺ RNA the ccry-antiserum immunoprecipitated polypeptides with relative molecular weights (Mr) of 27?000, 25?000, 23?000 and 21?000, while the cmac-antiserum precipitated proteins with Mrs of 32?500 and 27?000, respectively. Subsequent cDNA synthesis and immunological screening of the cDNA library with both antisera resulted in the isolation of six cDNA clones encoding light-harvesting subunits. Full-length precursors were 199-210 amino acids in length and had Mrs of 20?000–23?000. The lengths of the putative signal peptides were 29 or 30 amino acids. Pairwise comparison revealed that the similarity between the clones ranged from 54–99% on the nucleotide level and from 36–99% at the amino acid level. In agreement with the data from the screens with the two antisera, the genes clustered into two groups. The data provide evidence that the genes constitute a heterogeneous multigene family and that the light-harvesting system of Cy. cryptica might be as complex as that of higher plants and green algae.     

Natural Ageing: Poly(A) Polymerase in Germinating Embryos of Triticum durum Wheat

The viability of seeds is associated with ageing and storageconditions. A loss of viability is accompanied by slow germination,reduced growth, and a decline in protein and poly(A)⁺RNA synthesis.This paper reports on the activity of poly(A) polymerase indry and germinating embryos of Triticum durum Desf. cv. Cappellicaryopses of different ages and viability. The enzyme was presentas a single form during ageing and germination. The poly(A)polymerase was active at decreasing levels in all aged dry embryos,in parallel with loss of viability. Its activity strongly increasedduring the germination only in viable embryos. The observedincrease was due to de novo synthesis of the enzyme. Poly(A)polymerase synthesis was low during germination of less viableembryos and absent in older ones. Reduced poly(A) polymeraseactivity in dry or germinated wheat embryos may cause a shorteningof poly(A) chains in vitro and a decline in poly(A)⁺RNA synthesis.Copyright1995, 1999 Academic Press Triticum durum Desf. cv. Cappelli, wheat, embryo, natural ageing, poly(A) polymerase     

Detection and Characterization of Protein Kinases in Rice Phloem Sap

Calcium-dependent protein kinases were detected and characterizedin the phloem sap of rice plants. Protein phosphorylation wasactivated in the presence of micromolar levels of free Ca²⁺ions but was not activated by a polyamine in vitro. Mg²⁺ ionswere essential for protein phosphorylation and K⁺ ions inhibitedthe protein phosphorylation. Analysis by two-dimensional polyacrylamideelectrophoresis revealed that 17-kDa protein with a pI of 5.0was the most highly phosphorylated protein in the phloem sapof rice plants. A protein of 65 kDa, which was autophosphorylated,had a Ca²⁺-dependent protein kinase activity and the mobilityof this band in SDS gel was changed in the presence of calcium.These results suggest that a signal-transport system may existin the sieve tubes of rice plants that operates via the phosphorylationof proteins by calcium dependent protein kinases. (Received December 20, 1993; Accepted October 8, 1994)