Aphidicolin and ethidium bromide as tools to verify dependence of early post-germinative growth on nuclear and organellar DNA synthesis

Embryos of Haplopappus gracilis (Nutt.) Gray were sown in aphidicolin (inhibitor of nuclear DNA synthesis) used alone or in combination with ethidium bromide (inhibitor of organellar DNA synthesis). In both cases germination and the early processes of post-germinative growth, including enlargement and greening of cotyledons, some elongation of radicle, and formation of root hairs, occurred at the same rate as in the controls. Comparable results were obtained using fluorodeoxyuridine. The effect of aphidicolin is, unlike fluorodeoxyuridine, completely reversible and the side effect on protein and RNA synthesis are negligible. Total non-dependence on nuclear and organellar DNA synthesis during early post-germinative growth may not be a generalized situation in all seeds, as was shown by some observations on lettuce and watermelon.     

Isolation and identification of a new growth inhibitor, raphanusanin, from radish seedlings and its role in light inhibition of hypocotyl growth

A neutral growth inhibitor, for which the name raphanusanin is proposed, has been isolated in crystalline form from light-exposed Sakurajima radish (Raphanus sativus var. hortensis f. gigantissimus Makino) seedlings and identified as a new compound, 3-methoxy-4-methylthio-2-piperithione by spectrometric analyses.

Applied raphanusanin inhibited the hypocotyl growth of etiolated radish and lettuce seedlings at concentrations higher than 1.5 × 10⁻⁶ molar.

The endogenous raphanusanin contents in cotyledons and hypocotyls of radish seedlings increased more under red light, but decreased or maintained the initial level in the dark. Its content in roots showed almost no change between the light and dark materials.

     

Control by Light of Hypocotyl Elongation and Levels of Endogenous Gibberellins in Seedlings of Lactuca sativa L.

Four 13-hydroxygibberellins, gibberellin A₁ (GA₁), 3-epi-GA₁,GA₁₉ and GA₂₀ were identified by full-scan GC/MS in extractsof lettuce seedlings (Lactuca sativa L. cv. Grand Rapids). Theresults suggest that the early-13-hydroxylation biosyntheticpathway to GA₁ functions in the lettuce seedlings. It was alsofound that GA₁ is active per se in the control of hypocotylelongation in lettuce seedlings. To investigate the relationshipbetween control by light of hypocotyl elongation and levelsof endogenous GAs in lettuce, endogenous levels of GAs werequantified by radioimmunoassay in seedlings that had been grownfor 5 days in the dark (5D) and in those that had been grownfor 4 days in the dark and then under white light for 1 day(4D1L). The endogenous level of GA₁ in the upper and lower partsof hypocotyls in 5D seedlings was about four times higher thanthat in 4D1L seedlings. The response of explants (hypocotylsegments with cotyledons) from dark-grown seedlings to GA₁ isknown to be similar in the dark and under white light when theexplants are treated with inhibitors of the biosynthesis ofGA. Therefore, the above information suggests that the highlevel of GA₁ in hypocotyls of dark-grown seedlings is responsiblefor the rapid elongation of hypocotyl, while irradiation bywhite light decreases the endogenous level of GA₁ in the hypocotylswith a resultant decrease in the rate of hypocotyl elongation. (Received March 13, 1992; Accepted May 21, 1992)     

Comparison of Plant Telomere Locations using a PCR-generated Synthetic Probe

We have generated a telomere-specific probe by the polymerasechain reaction and used it to localize chromosome telomeresof ten unrelated angiosperm species in in situ. Concatenationof the simple monomers, 5'-(TTTAGGG)-3', derived from the sequenceof Arabidopsis thaliana telomeres, yielded a stable, versatileand reliable probe that gave a signal of high intensity followingfluorescence in situ hybridization. Most species, includingthose with known karyotype rearrangements, showed telomere labelonly at chromosome termini. These findings are discussed inthe context of the chromosomal events responsible for generatingand stabilizing karyotype change in plants.Copyright 1993, 1999Academic Press PCR, telomere repeat sequence (TRS), in situ hybridization, comparative physical mapping, karyotype evolution, genome organization, Lathyrus sativus L., Vicia sativa L. subsp. nigra (L.) Ehrh., Vigna radiata (L.) R. Wilczek cv. Berken, Nicotiana sylvestris Speg et Comes, Haplopappus gracilis (Nutt.) A. Gray, Gibasis pulchella (Kunth) Rafin., Tradescantia commelinoides Schultes. fil., Hordeum vulgare L. cv. Sultan., Hordeum vulgare L. cv. Tuleen 346, Milium vernale Bieb., Paphiopedilum insigne (Wall.) Pfitz     

A Model for the Growth of Individuals in Plant Populations Cultivated at Different Concentrations of Fertilizer Solution

The growth of mean individual weight is the joint outcome ofthe growth of the individuals comprising a population. Fromthe growth data of individual weight in radish (Raphanus sativusL. var. radiculus Pers.) populations cultivated at differentconcentrations of ammonium sulphate solution, a deterministicmodel was proposed for integrating individual plant weight intomean weight per plant in a population. The model constructiondepended on the relation between mean individual weight andthe amount of fertilizer supplied to a population, and uponPearson's type VII distribution. The model related the weightof any individual to the amount of fertilizer. The fitness ofthe model to observed data was satisfactory, although the modelwas simple. Using the model, properties of the growth of componentindividuals of a population were examined in relation to thegrowth of mean individual weight. fertilizer, growth, individual, mean, Pearson's type VII distribution, plant weight, population, radish, Raphanus sativus L. var. radicula Pers     

DNA Synthesis in Storage Organs of Germinating Seeds: An Autoradiographic Analysis

Storage organs of different germinating seeds were tested forDNA synthesis, which, apart from the normal replication precedingcell division, may also be involved in some regulatory processas suggested by a previous work. Both persistent cotyledons,programmed to grow into green, leaf-like organs (cotyledonsof lettuce, radish and Haplopappus gracilis), and storage organswhich senesce and die after depletion of reserves and wheregrowth and greening are only slight or non-existent (cotyledonsof garden pea, cowpea, soybean, aleurone layers of barley andendosperm of castor bean), were tested. The results showed that widely variable patterns of behaviourcan occur, making it difficult to establish a possible and generallyvalid explanation of nuclear DNA synthesis in storage organsduring germination. Possible interpretations are discussed. Key words: DNA synthesis, Storage organs, Seed germination     

Effetto del « water stress » su semi germinanti di Haplopappus gracilis (Nutt.) Gray pretrattati con gibberellina e fusicoccina

Abstract

Water stress effects on germinating seeds of Haplopappus gracilis (Nutt.) Gray pretreated with gibberellin and fusicoccin.—Achenes of Haplopappus gracilis, deprived of their coats, were germinated for various times, dried and then rehydrated. After this procedure many seeds were not able to resume regular germination and to overcome the water stress.

After having examined the morphological changes consequent to the water stress treatments we have checked if, as suggested by some Authors, there was a correlation between the resumption of DNA synthesis in the apical meristems of the rootlets and irreversible drought sensitivity.

To this purpose, embryos were pre-imbibed in water and in solutions of gibberellin (GA₃), which in Haplopappus promotes resumption of DNA synthesis, and fusicoccin (FC) which strongly stimulates germination and embryo elongation, without altering the pattern of DNA synthesis.

Our data show that embryos pre-imbibed in water or GA₃ and then dried, do not show any difference in their ability to resume germination, while embryos pre-hydrated in FC are always damaged more precociously and more severely by the dehydration treatments.

These results suggest that, since fusicoccin promotes embryo elongation through an activation of cell membranes, the physiological condition of these may be more critical for resumption of growth after the water stress than DNA replication activity.     

Requirement of sodium chloride for the action of gibberellic acid in stimulating hypocotyl elongation of a halophyte, Salicornia herbacea L.

NaCl stimulated hypocotyl elongation of the halophyte Salicorniaherbacea L. grown either in light or dark. Its optimal concentrationwas around 0.1–0.2 M and its promoting effect was muchmore prominent in the dark. Gibberellic acid at 10^–5 Mstimulated hypocotyl elongation in light but not in the dark.Indole-3-acetic acid and kinetin were ineffective in promotinghypocotyl elongation. In light, gibberellic acid and NaCl synergisticallyenhanced hypocotyl elongation when both were given simultaneously.The action of NaCl could be replaced by KCl, but not by mannitol.Osmotic pressure of the epidermis of the Salicornia hypocotylincreased in response to gibberellic acid and/or NaCl treatment.Na⁺ content in the hypocotyl increased with NaCl application.Gibberellic acid and NaCl when given alone increased the extensibilityof the hypocotyl cell wall. Synergistic interaction in increasingthe extensibility was observed between gibberellic acid andNaCl. Stress-relaxation analysis of mechanical properties ofthe hypocotyl wall revealed that gibberellic acid and NaCl actedsynergistically in decreasing minimum relaxation time. Basedon these results, a possible mechanism by which gibberellicacid and NaCl regulate hypocotyl elongation of Salicornia herbaceaL., a typical halophilic plant, is discussed. ¹ Present address: Laboratory of Biology, Tezukayama College,Gakuen Minami, Nara 631, Japan. (Received June 13, 1978; )     

Light growth inhibition and growth inhibitors in Sakurajima radish seedlings

Growth of Sakurajima radish seedlings (Raphanus sativus var.hortensis f. gigantissimus Makino) was studied in relation tothe effects of light, the effectiveness of plant hormones, andthe variation in endogenous growth inhibitor content Externally applied GAs and IAA had no effect on the elongationof the hypocotyl in the light as well as in the dark. BA wasslightly promotive in the light, but was not so effective asto nullify the light-induced inhibition of elongation. ABA wasstrongly suppressive in the light as well as in the dark. Growth inhibitors in seedlings were extracted, analyzed by thin-layerchromatography and the Sakurajima radish hypocotyl elongationtest, and 3 acidic and 3 neutral inhibitors were detected. Amongthem all of the acidic and one of the neutral inhibitors increasedwith the time period of illumination, whereas the other 2 neutralsubstances remained almost unchanged in the light but decreasedin the dark. Thus the levels of all 6 inhibitors was higherin light-grown seedlings than in dark-grown ones. This suggeststhat light inhibition in Sakurajima radish hypocotyls may becontrolled by the variation in the inhibitor levels in the seedlings. (Received December 1, 1977; )     

Enhancement of Gibberellic Acid-stimulated Hypocotyl Elongation of Lettuce (Lactuca sativa L. cv. Grand Rapids) by Phlorizin and Light

The interaction of light, gibberellic acid (GA₃), and phlorizinin the growth of lettuce (Lactuca sativa L. cv. ‘GrandRapids’) hypocotyls was investigated. At all concentrationsof GA₃, phlorizin enhanced GA₃-induced growth at luminous intensitiesabove 50 ft-c (continuous light). Without GA₃, phlorizin hadno effect on hypocotyl growth in the light but it inhibitedgrowth in the dark. Both seedlings and hypocotyl sections respondedto phlorizin in the presence of GA₃. There was no iteractionbetween phlorizin and KCl. Water-growth was severly inhibitedby light. GA₃,-induced growth was slightly inhibited by light,and then only at luminous intensities above 50 ft-c. Thus, relativeto H₂O-growth, GA₃-induced growth increased with increasingluminous intensity up to 450 ft-c, where it reached saturation.It seems that a synergism may exist between light and GA₃ aswell as between phlorizin and GA₃. Lactuca sativa L, lettuce, hypocotyl elongation, gibberellic acid, phlorizin, light     

Comparative Requirement for Endogenous Ethylene during Seed Germination

Requirement for endogenous ethylene during seed germinationof the following ten species was determined: Lycopersicon esculentumMill, (tomato), Allium cepa L. (onion), Avena fatua L., dormantpure line AN-51 (wild oats), Cucumis sativus L. (cucumber),Sinapis arvensis L. (wild mustard), Tagetes erecta L. (marigold),Raphanus sativus L. (radish), Triticum aeslivum L. (wheat),Catharanthus roseus L. (periwinkle), and Phaseolus aureus L(mung bean). Experiments were done under controlled conditionssuited for the germination of each species. Criteria used todetermine the need for endogenous ethylene were: (i) temporalrelationship between ethylene production and seed germination;(ii) parallel inhibition of ethylene synthesis and seed germinationby L-     

Effect of malformin on phytochrome- and Ethrel-mediated responses

On etiolated cuttings of Phaseolus vulgaris malformin inhibitedseveral phytochromemediated responses. This included hypocotylhook-opening, leaf expansion, and inhibition of stem elongation.Malformin also inhibited anthocyanin synthesis by sorghum, buthad no effect upon lettuce seed germination in the light ordark. Malformin alleviated Ethrel-induced hook-retention, inhibitionof stem elongation, and root curvatures, but not Ethrel-inducedstimulation of lettuce seed germination in the dark. (Received February 19, 1975; )     

Requirement of cotyledons for gibberellic acid-induced hypocotyl elongation in lettuce seedlings. Isolation of the cotyledon factor active in enhancing the effect of gibberellic acid

The role of cotyledons in hypocotyl elongation caused by gibberellicacid was studied using young seedlings of lettuce, Lactuca saliva,var. ‘Grand Rapids’. Removal of cotyledons fromintact seedlings resulted in a depression of hypocotyl elongationcaused by gibberellic acid. Gibberellic acid-induced hypocotylelongation in decotylized seedlings, was however, substantiallyenhanced by incubating the seedlings together with excised cotyledons.The exudate from excised cotyledons also enhanced the effectof gibberellic acid on hypocotyl elongation in decotylized seedlings.This active principle (named the cotyledon factor) in the cotyledonexudate was stable against heating at 100?C for 15 min, permeatedthe dialysis membrane, and was extractable with ethyl acetate.Biological activity of the cotyledon factor was not replacedby indole-3-acetic acid, kinetin, cyclic AMP, vitamins, sucroseor inorganic nutrients. The biological significance of the cotyledonfactor is discussed in relation to the action of gibberellicacid. (Received February 14, 1973; )     

Action spectra for the inhibition of growth in radish hypocotyls

In etiolated seedlings of Raphanus sativus L. the inhibition of hypocotyl elongation by continuous light showed a major bimodal peak of action in the red and far-red, and two minor peaks in the blue regions of the spectrum. It is argued that, under conditions of prolonged irradiation, phytochrome is the pigment controlling the inhibition of hypocotyl elongation by red and far-red light, but that its mode of action in far-red is different from that in red. A distinct pigment is postulated for blue light.Abbreviations B blue - FR far red - G green - R red - HIR high irradiance reaction - P_r and P_fr red and far red absorbing forms of phytochrome - R red     

Growth and Development of Radish (Raphanus sativus, L.) Under Selected Light Environments

Plants of radish (Raphanus sativus L.) were grown under selectedlight conditions in controlled environmental chambers in orderto monitor the role of photoperiod, irradiance level and inputlight energy in plant development. Results indicated that thedaily input of light energy was the most important light factoraffecting leaf development while photoperiod and irradiancelevel had the major influences on storage organ development.Distribution of assimilates to leaves and storage organs variedunder different light regimes with long photoperiods and highirradiances producing the largest storage organs. Once initiated,the rate of storage organ growth was similar under all testedlight environments. Raphanus sativus L., radish, growth, development, light, photoperiod, assimilate distribution, storage organ     

Opening of the hypocotyl hook in seedlings as influenced by light and adjacent tissues

The influence of the cotyledons and apical bud and the root system on the light-induced opening of the hypocotyl hook of etiolated seedlings of Gossypium hirsutum L., Phaseolus vulgaris L., Helianthus annuus L., Ipomoea alla L., Ipomoea sp., Cucumis sativus L., Linum usitatissimum L., Hibiscus esculentus L., and Raphanus sativus L. was studied. Light stimulated the opening of hypocotyl hook in all cases, but the cotyledons and roots had different effects in different plants. Generally, the presence of cotyledons and the remainder of the shoot apical to the hook inhibited light-mediated opening, but in Gossypium the organs stimulated light-mediated opening. Presence of roots either promoted opening, had no effect, or had an effect only when the cotyledons were present. In the dark the adjacent organs had a reduced effect over that shown in the light, but one cultivar of cotton, Acala SJ1, opened the hook in the dark without cotyledons as much as under any condition in the light. The variation between species in hook opening may related to the need of that process for a proper hormonal balance, as affected by light, which must be obtained from adjacent tissues.     

Flow Cytometric Determination of Nuclear Replication Stage in Seed Tissues

Flow cytometric determination of DNA levels in embryos of fullymatured seeds of various plant species revealed large amountsof 2C DNA signals, indicating that most cells had arrested thecell cycle at the presynthetic G₁ phase of nuclear division.The accumulation of cells at G₁ was found both in orthodox andin recalcitrant (i.e. Castanea sativa) seed species. As recalcitrantseeds are characterized by the absence of maturation drying,the arrest of the cell cycle in the presynthetic phase may notbe linked to the seed water status. Apart from the 2C signal, 4C values were found in the embryoof some seed species (e.g. Raphanus sativus) indicating thatcells were arrested in G₂ Cells arrested in G₂ were primarilylocated in the root-tip region of the embryo. In addition, combinationsof higher C values (i.e. 8C, 12C, 16C and 64C) were observedin the endosperm of Solanum melongena and Lycopersicon esculentum,and in the root-tip cells of Phaseolus vulgaris and Spinaciaoleracea. These mixtures of polyploid nuclei (also called 'polysomaty')may arise from a developmentally controlled cellular endoreduplicationand indicates that in each cell type of the seed the amountof DNA is regulated both spatially and temporally.Copyright1993, 1999 Academic Press Endive, Cichorium endiva, lettuce, Lactuca sativa, egg-plant, Solanum melongena, pepper, Capsicum annuum, tomato, Lycopersicon esculentum, radish, Raphanus sativus, bean Phaseolus vulgaris, spinach, Spinacia oleracea, chestnut, Castanea sativa, beech, Fagus sylvatica, pine, Pinus nigra, DNA content, flow cytometry, seed, nuclear replication stage, C levels, storage     

Acetylcholine promotes the emergence and elongation of lateral roots of Raphanus sativus

Radish (Raphanus sativus L.) was grown on four layers of paper towel moistened with distilled water with and without acetylcholine (ACh) for five days in the dark after sowing. ACh at 1 nM promoted the growth (emergence and elongation) of lateral roots of radish plants, but had no effect on the stems and main roots. Moreover, ACh enhanced the dry weight of roots [main (primary) + lateral roots]. Neostigmine, an inhibitor of acetylcholinesterase (AChE) also promoted the emergence and elongation of lateral roots, and atropine, a competitive inhibitor of ACh receptor, suppressed the emergence and elongation. ACh promoted the activities of glyceraldehyde-3-phosephate dehydrogenase (G-3-PD), nicotinamide adenine dinucleotide-specific isocitrate dehydrogenase (NAD-ICDH), succinate dehydrogenase (SDH) and cytochrome-c oxidase (Cyt-c OD) in seedlings. Moreover, ACh suppressed the activity of AChE and increased the amount of proteins and pyridine nucleotides (NAD and NADH) in the roots of the seedlings. It also increased the activities of NAD-forming enzymes [NAD synthetase and ATP-nicotinamide mononucleotide (ATP-NMN) adenyltransferase], and enhanced the amount of DNA in the roots of the seedlings. The relationship between ACh and the emergence and growth of lateral roots was discussed from a biochemical viewpoint.Key words: acetylcholine, acetylcholine esterase, DNA, lateral root, NAD, NAD-forming enzyme, protein, Raphanus sativus     

Occurrence of beet western yellows virus in sugar beet in Czechoslovakia

The beet western yellows virus (BWYV) was identified in sugar beet plants with leaf yellowing symptoms. When transmitted toSinapis alba L. the virus isolate caused severe symptoms of yellowing and violetting of the interveinal leaf tissue of this plant. By aphidsMyzus persicae (Sulz.) the virus isolate was transmitted toLactuca sativa L.,Raphanus sativus L. var.radicula Pers.,Baphanus sativus L. ssp.sativus L. ap., and toBrassica oleracea L. var.gemmifera DC. InLactuca sativa plants the virus induces a yellowing along with thickenning and brittleness of leaves and with mild dwarfing of the plants. InBaphanus sativus var.radicula andBaphanus sativus ssp.sativus plants it brings about a yellowing of the leaf margins with a change in consistency as was the case in lettuce, and inBrassica oleracea var.gemmifera it causes violet spots on the lower leaf sides. The transmission was proved in repeated experiments by a backtransmission to beet andSinapis alba and further transmission from beet toSinapis alba. The transmission of the virus isolate toVicia faba L.,Datura stramonium L., andPetunia hybrida hort. was unsuccessful. In the course of transmissions the isolate properties did not change. In its host range the virus resembles the Duffus’ strain 3 BWYV, isolated from beet in the U.S.A. This is the first characteristic of an Europian BWYV isolate, as obtained from naturally infected beet plants.     

Ferredoxin Isolated from Plant Non-Photosynthetic Tissues. Purficaition and Characterization

A ferredoxin was isolated from non-photosynthetic tissues ofthe lower storage root of radish (Raphanus sativus L. var. acantiformiscultivar Miyashige) in a pure form by conventional means. Itshowed the characteristic features in its absorption spectrumof chloroplast-type ferredoxin. However, amino acid compositionand amino (N)- terminal sequence were different from those ofradish leaf ferredoxin. Root ferredoxin was able to transferelectrons from dithionite to nitrite reductase [EC 1.7.7.1 [EC] ]isolated from mung bean seedling roots and also to mediate NADP⁺photoreduction in spinach broken chloroplasts. It therefore is suggested that a set of distinctive molecularspecies of ferredoxin is present in non-photosynthetic tissuesand functions as a redox mediator in ferredox-independent enzymesystems. (Received October 18, 1985; Accepted January 16, 1986)