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)     

Water Content and Respiration Rate of Bean Cotyledons

The course of water uptake and respiration rate rise in cotyledonsof Phaseolus vulgaris is divided into three phases. In the first phase lasting 10–16 hrs. respiration rateis controlled by water content; desiccation and reimbibitioninfluence cotyledon water content and respiration rate alikeand the changes are reversible; low temperature prevents watercontent rising above 50 per cent. and also limits respirationrate; both processes have Q₁₀ near unity. The second phase lasting 3–8 hrs. is characterized bya pause in both water uptake and respiration rate rise. In the third phase respiration rate continues to rise untilthe fifth day, after which it falls steadily until eventuallythe cotyledons absciss. The period of rising respiration isone of metabolic activity, the rise having a high Q₁₀ and beingprevented by low temperature. Desiccation at this stage is irreversible.     

Transport and Metabolism of Dihydrozeatin Riboside in Germinating Lupin Seeds

[³H]-dihydrozeatin riboside was applied selectively to the embryonicaxes or to the cotyledons of germinating lupin (Lupinus luteusL. cv. Weiko III) seeds 6 h following the start of imbibition.There was little transport of dihydrozeatin riboside from embryoto cotyledons up to 6 h after the application, but a substantialamount of radioactivity had moved into the cotyledons at theend of the 10 h incubation period. However, there was no detectablemovement of [³H]-dihydrozeatin riboside from the cotyledonsto the embryonic axis. This indicated a highly polarized movementof cytokinins during the early stages of seed germination. Exogenouslyapplied [³H]-dihydrozeatin riboside was found to be very stable,both when applied to the embryonic axes and cotyledons of intactseed, or following excision, and there was little metabolismwith only small amounts of radioactivity found associated withdegradative metabolites. The embryonic axis of this specieshas recently been found to synthesize cytokinins within 12 hfrom the start of imbibition, and the results of this studyindicate that the embryo-derived cytokinin is probably transportedto the cotyledons where it accumulates and subsequently participatesin the control of cotyledon function. Key words: Lupinus luteus, cytokinin transport and metabolism, dihydrozeatin riboside, seed germination     

Respiratory Activity in Pea Cotyledons during Seed Development

Three phases were recognized in the course of the respirationrate of pea (Pisum sativum L.) cotyledons during seed development.(1) The respiration rate per cotyledon initially increased alongwith the mitochondrial activity. (2) During the second phase,the respiration rate increased further until a constant levelwas reached and then decreased. The mitochondria now startedto lose their capacity to oxidize malate, followed by a decreasingcapacity to oxidize succinate. (3) During the maturation phasethe respiration rate decreased further. The rate of ascorbateoxidation started to decline at this time. Ascorbate oxidationwas increasingly stimulated by cytochrome c. The changes inrespiration rate are considered in relation to changes in growthand maintenance respiration. When the water content of the seeds was maintained by storingthem at high humidity, the respiration rate of cotyledons ofearly harvested seeds decreased sharply whereas that of laterharvested seeds hardly changed. This change in response wasused to mark the transition between the second and third phase.During humid storage changes in the functional integrity ofthe mitochondria still occurred. The results are discussed in relation to the ability of peaseeds to withstand desiccation. Key words: Pisum sativum, Seed development, Respiration, Mitochondrial activity     

Studies on the Growth and Respiration of Roots: I. THE EFFECTS OF STIMULATORY AND INHIBITORY CONCENTRATIONS OF {beta}-INDOLYLACETIC ACID ON ROOT SECTIONS OF PISUM SATIVUM

Simultaneous observations on extension growth and respirationrate (oxygen consumption) of 2-mm. sections excised from theextension zone of roots of pea (Pisum sativum) growing in distilledwater and 0·5 per cent. sucrose have yielded resultsclosely similar to those of Brown and Sutcliffe (1950). Respirationrate is not obviously correlated with growth rate either inwater or in sucrose, but it is strongly correlated with sectionlength. Respiration rate per unit section length (¬per unitfresh weight) shows a marked downward drift during extensionand is affected little by growth conditions. Tentative suggestionsare advanced to account for the small differences between driftsin o·5 per cent. sucrose and those in distilled water. Medium agitation produces an immediate and sustained stimulationof growth but no stimulation of oxygen uptake until the latergrowth stages. Thus respiration per unit section length is unaffectedby agitation at any stage. A typical growth response to ß-indolylacetic acid(IAA) was obtained, with a maximum stimulation (of about 35per cent.) at 1 part in 10¹¹ and inhibitions increasing progressivelywith concentration beyond the threshold of about i part in 10⁹.Both percentage stimulation and percentage inhibition of growthwere independent of the presence of sucrose. Respiratory responses to ß-indolylacetic acid werecomplex. In water no immediate response could be detected witheither a growth-stimulatory (10^–11) or a growth-inhibitory(10^–-8) concentration, while in 0·5 per cent. sucrosethe inhibitory concentration prevented the small immediate respiratoryrise due to the sucrose, probably by impeding sugar entry. Duringthe subsequent period of rapid growth (up to 36 hours) the smallrespiratory responses observed closely followed the small growthresponses to both concentrations of IAA, suggesting that theformer are the direct result of the differences in section lengthinduced by the auxin. When growth ceases (at 48 hours) sectionswhich have grown considerably in sucrose show respiratory ratesstill closely correlated with section length, whereas in waterboth concentrations of auxin induce marked depressions in respirationrate. It is concluded that ß-indolylacetic acid in bothgrowth-stimulatory and growth- inhibitory concentrations hasno direct effect on the activity of the respiratory enzyme systemof growing root cells. The small respiratory responses are bestexplained as resulting from differential changes in sectionsize and correlated changes in the enzyme complements of thegrowing cell.     

Cell Cycle Activation During Imbibition and Visible Germination in Embryos and Megagametophytes of Jack Pine (Pinus banksiana Lamb.)

Flow cytometric determination of cell cycle activation duringimbibition and visible germination in five families of jackpine (Pinus banksiana Lamb.) embryos and megagametophytes revealedthat in seeds that had undergone no imbibition the majorityof cells were in the 2C state. As the imbibition period increased,less of the nuclei were blocked in the G0/G1 state and morebecome active in the cell cycle. The augmentation in the nucleiactive in the 2C–4C cycle as well as those with DNA levelshigher than the 4C state occured gradually and preceeded radicleemergence. In megagametophyte tissue examined at various stagesof imbibition, cell cycle activity became apparent rapidly followingimbibition. In nuclei of green and white embryos examined separatelythe ²frequency distributions were significantly different forall three families after 144h. As imbibition period increased,fewer nuclei from the green embryos were blocked in the 2C state,and more became active in the 2C–4C cell cycle. This wasnot the case for white embryos where no significant linear relationwas noted. Cell cycle activity in the hypocotyl+cotyledons regionand the emerging radicle were examined separately. Functionalrelations found in the hypocotyl+cotyledons region were notevident in the radicle. As visible germination proceeded, cellcycle activity in the hypocotyl + cotyledons region for thisperiod of germination showed a reversal of the activity notedduring imbibition: fewer nuclei were active and once again ahigher proportion were found in the 2C state. cell cycle; C levels; DNA content; flow cytometry; germination; imbibition; jack pine; megagametophyte; Pinus banksiana Lamb     

In Vitro Synthesis of Pteroylpoly-{gamma}-glutamates by Cotyledon Extracts of Pisum sativum L.

The formation of folylpolyglutamate derivatives by germinatingpea seeds (Pisum sativum L. cv Homesteader) was examined invivo and in vitro. Differential microbiological assay of cotyledonextracts showed that total folate concentrations increased from163 ng folate equivalents per g fresh weight at day 1 to 680ng per g fresh weight at day 3 of germination. Over a 7 daygermination period, folylpolyglutamate derivatives accountedfor 46–73% of the total cotyledonary folate pool. Theconcentration of these polyglutamate forms of folate increased6.5 fold during the first four days of germination and thenremained relatively constant. Dialyzed extracts of 1–4 day old cotyledons had abilityto incorporate [³H]glutamate and [¹⁴C]tetrahydrofolate intofolylpolyglutamates. This activity was mainly associated withprotein precipitating at 35–45% of saturation with ammoniumsulphate. The folylpolyglutamate synthetase of pea cotyledonshad requirements for ATP and the monoglutamate of tetrahydrofolate.The latter folate was a more effective substrate than 5,10-methylenetetrahydrofolatebut the diglutamate of unsubstituted tetrahydrofolate was notutilized. Ion exchange chromatography of the reaction productssuggested that [³H]glutamate and [¹⁴C]tetrahydrofolate wereincorporated into di-, and tetraglutamates of tetrahydrofolate.Folates of longer glutamyl chain lengths were only detectedwhen the synthetase reaction proceeded for 12 h or longer. (Received August 23, 1985; Accepted January 22, 1986)     

Inhibition of Pinus radiata Primary Needle Epicuticular Wax Biosynthesis by Trichloroacetate

High concentrations (1000 parts 10^–6) of trichloroacetate(TCA) inhibit germination of Pinus radiata seed. Seedlings growingin the presence of lower concentrations (100 parts 10^–6)take up TCA where it becomes concentrated in cotyledons anddeveloping primary needles. TCA inhibits biosynthesis of nonacosan-10-oland long chain diol constituents of the primary needle epicuticularwax concomitant with a reduction in appearance of tubular waxexcept around stomatal pores. Epicuticular wax from TCA-treatedplants consists mainly of alkyl esters, and is amorphous. Itis suggested that P. radiata stomatal subsidiary cells possesstubular epicuticular wax chemically distinct from that of epidermalcells.     

Effects of Calmodulin Antagonists on the Mitochondrial and Microsomal Ca2$ Uptake in Apple Fruit

In apple fruit, active ATP-dependent microsomal Ca^2$ uptakeand respiration-dependent mitochondrial Ca^2$ uptake were observed. The mitochondrial Ca^2$ uptake was depressed by the calmodulinantagonists chlorpromazine hydrochloride (CPZ) and N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamidehydrochloride (W-7). The Ca^2$-ATPase from apple mitochondriawas also inhibited by CPZ or W-7. The apparent K_m value forCa^2$ in mitochondrial Ca^2$ uptake (K_m=0.35 mM) was similar tothat of mitochondrial Ca^2$-ATPase (K_m=0.32 mM). The inhibitoryeffect of W-7 on the activity of the mitochondrial Ca^2$ uptakewas closely correlated with the inhibition by W-7 of mitochondrialCa^2$-ATPase (r=0.996). These findings indicate that the mitochondrialuptake of Ca^2$ in apple fruit depends on the calmodulin-mediatedactivation of Ca^2$-ATPase. The microsomal Ca^2$ uptake was depressed by CPZ, suggestingthat the microsomal Ca^2$ uptake may also be modulated by calmodulin. ¹ Contribution No. C-72, Fruit Tree Research Station. (Received June 7, 1982; Accepted October 19, 1982)     

Structural Associations Between Mitochondria and Chloroplasts in the Bundle Sheath Cells of Portulaca oleracea

Mitochondria and chloroplasts are structurally associated inthe bundle sheath cells of Portulaca oleracea L., an NAD malicenzyme type C₄ plant. These associations occur in some restrictedregions where the mitochondrial cristae extend inwards. Exposureof plants to 1.0 µ11^–1 SO₂ for 3 h induced shrinkageof mitochondria of the bundle sheath cells, which further visualizedthe structural association between the organelles. C₄ plant, chloroplast, mitochondrion, Portulaca oleracea, sulphur dioxide     

Mitochondrial Arginase Activity from Cotyledons of Developing and Germinating Seeds of Vicia faba L

Differential and sucrose density gradient centrifugation established that about 80% of the total arginase activity (EC 3.5.3.1) in cotyledons of germinating broad bean seeds (Vicia faba L.) was present in the mitochondrial fraction. The mitochondrial arginase activity was enhanced considerably by exposure to osmotic shock, by freezing and thawing, or by Triton X-100 treatment. About 10% of the total arginase activity was recovered from the 40,000g supernatant fraction. During seed maturation, arginase activity in the cotyledons decreased to about one-third of its maximal activity, while increasing over 10-fold during subsequent germination. The time courses of mitochondrial arginase, succinate oxidase, and succinate dehydrogenase activities differed considerably during germination.     

Glutamine synthesis in germinating seeds of Cucurbita moschata

During germination, an increase in glutamine and glutamine synthetase[L-glutamate: ammonia ligase (ADP), EC 6.3.1.2 [EC] ] occurred inthe cotyledons reaching a maximum at 4 to 6 days. The enzymehad a Km of 4.5 nun for L-glutamate, and 0.67 mu for hydroxylamine.Hydroxylamine exhibited substrate inhibition kinetics. The enzymewas inhibited by calcium ion, fluoride ion and p-hydroxymercuribenzoatebut not by EDTA, sodium ion or chloride ion. The sulf hydrylinhibition was reversed by dithiothreitol. In vivo synthesisof glutamine-¹⁴C from glutamate-¹⁴C was found to parallel theincrease in glutamine content and the in vitro glutamine synthetaseactivity during germination. ¹ Present address: Department of Biology, Mercyhurst College,Erie, Pennsylvania 16501, U.S.A. (Received June 12, 1971; )     

Cytokinins in Germinating Seeds of Phaseolus vulgaris L. II. Transport and Metabolism of 8[14C]t-Zeatin Applied to the Radicle

The application of 8[¹⁴C]t-zeatin to the radicle tips of germinatingPhaseolus vulgaris seeds revealed that cytokinins are transportedrapidly from the embryonic axis to the cotyledons, and are utilizedand metabolized extensively in these organs. The informationobtained on the transportation between the different parts ofthe embryo is consistent with the view that the mobilizationof food reserves from the cotyledons is controlled by cytokininswhich originate in the embryonic axis. Tentative identificationof the radioactive metabolites formed indicate that the appliedzeatin was altered by side-chain cleavage and by substitutionto the adenine ring. Phaseolus vulgaris, bean, germination, cytokinins, transport, radicle     

Mitochondrial folates and methionyl-tRNA transformylase activity during germination and early growth of seeds

Mitochondria were isolated from the cotyledons of pea (Pisum sativum cv Homesteader) and peanut (Arachis hypogaea cv Early Spanish) seeds over a 7-day growth period. The rate of mitochondrial oxygen uptake increased 3-4-fold during the first 4 days of growth and parallel changes were observed in the respiratory control and ADP/O ratios. In both species, the total cotyledonary pool of folate derivatives increased 3-4-fold during this period of germination whereas that associated with isolated mitochondria increased 5-10-fold. Until day 3 of growth, the mitochondrial folates were principally polyglutamates of 10-formyltetrahydrofolate but between day 4 and day 7 increasing levels of 5-methyltetrahydrofolate polyglutamates were detected. Pea and peanut mitochondria contained methionyl-tRNA transformylase (EC 2.1.2.9) activity that displayed an absolute requirement for 10-formyl-tetrahydrofolate. The specific activity of this enzyme rose during germination, reaching maximal levels between days 3 and 4. Isolated pea mitochondria had the ability to incorporate [³H]leucine and [³⁵S]methionine into protein in a reaction that required ADP and malate but was strongly inhibited by chloramphenicol. Organelles isolated after 4 days of germination incorporated leucine at rates ca 5-fold greater than shown by mitochondria of 16-hour-old seedlings. The inter-relationships between respiratory activity, mitochondrial formyltetrahydrofolates and methionyl-tRNA transformylase activity suggest a role for organelle protein synthesis during germination of these legume species.     

CO2-Fixation, Glycolate Formation, and Enzyme Activities of Photorespiration and Photosynthesis during Greening and Senescence of Sunflower Cotyledons

Time-courses of ¹⁴CO₂-fixation and of enzyme activities involvedin photorespiration and photosynthesis were determined duringthe life span of cotyledons from sunflower seedlings (Helianthusannuus L.). Glycolate formation in vivo was estimated from theresults of combined labelling and inhibitor experiments. NADPH-glyceraldehyde-3-phosphatedehydrogenase, NADPH-glyoxylate reductase and chlorophyll werewell correlated with the time-course of ¹⁴CO₂-fixation (photosynthesis).There was, however, a considerable discrepancy between the developmentalsequence of photosynthesis and that of both ribulose-l,5-bisphosphatecarboxylase and glycolate oxidase. Furthermore, time-coursesof glycolate oxidase activity in vitro and of glycolate formationin vivo differed significantly. Therefore, the use of glycolateoxidase as a marker for the activity of photorespiration ingreening sunflower cotyledons may be questionable. Results from¹⁴CO₂-labelling experiments with cotyledons treated with theglycolate oxidase inhibitor 2-hydroxy butynoic acid suggestthat glycolate formation relative to CO₂-fixation is reducedin senescent cotyledons. Key words: Development, glycolate oxidase, photorespiration, ribulose-l,5-bisphosphate carboxylase, oxygenase     

Functional aspects of Abscisic Acid Metabolism in Cotyledons of Phaseolus vulgaris L. Seedlings

During the early stages of germination and vegetative development,cotyledons of intact bean (Phaseolus vulgaris L.) seedlingsshowed active ABA catabolism causing a low endogenous ABA content.At the end of the substrate mobilizing phase, when the cotyledonsbecame senescent, a drastic increase of the endogenous ABA contentlinked with a decrease of the ABA catabolic activity was observed.This indicates that a close connection exists between senescenceand endogenous ABA content and metabolism in bean cotyledons. Removal of the apical growth region induced in the cotyledonsactivation of the ABA catabolism and the endogenous ABA concentrationdecreased below the detection limit (0.1 ng/g fr wt) at theonset of the outgrowth of the axillary buds. From then, apicaldominance was restored and the cotyledons returned to the senescentstate, which was correlated with a drastic increase of theirendogenous ABA content. ¹ Bevoegd verklaard navorser N. F. W. O. ² Wetenschappelijk medewerker F. K. F. O. (Received November 25, 1980; Accepted February 13, 1981)     

Immunocytochemical Localization of Uricase in Peroxisomes of Soybean Cotyledons

Antibodies specific for nodule uricase were used for immunocytochemistryto demonstrate the presence of uricase in cotyledons of soybean(Glycine max) during germination and early seedling growth.The enzyme was localized exclusively in peroxisomes. ¹Permanent address: Department of Plant Cytology and Cytochemistry,University of Lodz, Lodz, Poland ²Current address: Department of Plant Science, University ofArizona, Tucson, AZ 85721, U.S.A.     

Mitochondrial Interaction with Helminthosporium maydis Race T Toxin: Blocking by Dicyclohexylcarbodiimide

Treatment of mitochondria isolated from Texas male sterile cytoplasmcorn (T mitochondria) with high concentrations of dicyclohexylcarbodiimide(DCCD) (140 nmol DCCD mg^–1 mitochondrial protein) completelyand immediately inhibited T mitochondrial swelling by Helminthosporiummaydis Race T toxin (HmT toxin). In order to obtain a specificinteraction between DCCD and the ATPase complex T mitochondriawere incubated with lower DCCD concentrations (1–5 nmolDCCD mg^–1 mitochondrial protein) for up to 8 h at 4 °C.After 8 h incubation in the presence of 3.75 nmol DCCD mg^–1mitochondrial protein, toxin-induced swelling was decreasedby 69%. Specificity of DCCD action upon the ATPase complex wasconfirmed by (1) SDS gel electrophoresis and fluorographic analysesof proteins from [¹⁴C]-DCCD-treated T mitochondria and immunoprecipitatesand (2) physiological experiments showing that DCCD exertednone of its other documented effects. These data suggest thatHmT toxin interacts with the ATPase complex of T mitochondriaeither at or near the DCCD-binding protein within the membranesector of the complex. Key words: Zea mays L., Helminthosporium maydis, Mitochondria     

Increases in activities of membrane-bound hydrolases in pea cotyledons during germination

Membrane-bound proteinase and acid phosphatase activities, butnot cytosol proteinase activity, in pea cotyledons increasedafter lag phases during germination. The activity hydrolyzingN--benzoyl-D,L-arginine P-nitroanilide in the membrane fractionincreased rapidly in the imbibition stage. Whether the increasesare due to de novo synthesis of the enzyme proteins was studied. ¹ Present address: Department of Pathology, Aichi Medical University,Nagakute, Aichi, Japan. (Received May 28, 1973; )     

Development of Mitochondrial Activity in Pea Cotyledons Following Imbibition; Influence of the Embryonic Axis

The development of mitochondria in cotyledons during the initialstages following imbibition and the subsequent degenerationwere faster when the embryonic axis was attached. This was moreclearly demonstrated when mitochondrial activity was determinedusing malate or -oxoglutarate as substrate rather than NADHor succinate. Cycloheximide did not inhibit the initial developmentof mitochondria in attached cotyledons, although it inhibitedthe incorporation of ¹⁴C-amino acid into protein. Abscisic acidinhibited almost completely the initial increase in mitochondrialactivities of attached cotyledons. The activities of severalenzymes in mitochondrial fractions were almost the same in attachedand detached cotyledons during 4 d after, imbibition. The degenerationof mitochondria was not accompanied by the loss of enzymes.It was inferred that the initial development and the subsequentdegeneration of mitochondria in cotyledons during 4 d afterimbibition was brought about by the structural improvement anddisorganization of mitochondria, respectively. The initial differencesin the development of mitochondrial activities between attachedand detached cotyledons were suggested to be attributable todifferences in the development of the activities of electrontransfer pathway from endogenous NADH to ubiquinone.