Radish Cotyledons Research Articles

5-Bromodeoxyuridine inhibition of cytokinin induced radish cotyledon expansion

The mechanism of cytokinin stimulated expansion of excised radish cotyledons is unknown. We have used the thymidine (dT) analogue, 5-bromodeoxyuridine (BrdU) to examine whether DNA synthesis plays a key role in this response. Treatment with BrdU decreases cotyledon expansion. Upon the addition of thymidine together with BrdU, cotyledon expansion is rescued. [ 14C]BrdU labelling of expanding cotyledons indicates that 84% and 16% of the incorporated BrdU are located in the DNA and RNA fractions, respectively. Expanding cotyledons have tow distinct species of nuclei, containing either a 2C or a 4C complement of DNA. Cotyledons treated with BrdU accumulate nuclei in the 4C class, suggesting a slow transition of the 2C species through the S phase of the cell cycle. CsCl density gradient experiments indicate that BrdU replaces all of the available dT residues in 20% of the nuclear DNA, while BrdU was not detected in the remaining DNA fraction (80%). Cytokinin induction of radish cotyledon expansion has been proposed to result principally from cell expansion processes. Based on the results presented here, DNA synthesis is implicated as necessary for cytokinin induced radish cotyledon expansion.

Inhibitors of cytokinin metabolism III. The inhibition of cytokininN-glucosylation in radish cotyledons

Cytokinins are deactivated in radish cotyledons by conversion to 7- and 9-glucosides. In a search for inhibitors of this metabolism, the following compounds were found to be effective: (a) 6-benzylamino-2-(2-hydroxyethylamino)-9-methylpurine; (b) 3-isobutyl-1-methylxanthine; (c) papaverine; (d) theophylline; (e) caffeine; and (f) theobromine. The order of effectiveness was: (a)>(b)=(c)>(d)=(e)=(f). While the methylxanthines (b) and (d) inhibited formation of both 7- and 9-glucosides of 6-benzylaminopurine (BAP) approximately equally, compounds (a) and (c) preferentially inhibited formation of BAP 9-glucoside. Inhibition of BAP glucoside formation by (a) at 1.3 mM elevated the level of free BAP and BAP nucleotide 23- and 94-fold, respectively. While abscisic acid (ABA) suppressed conversion of zeatin riboside to zeatin 7-glucoside in radish cotyledons, it did not inhibit conversion of BAP to glucosides. Hence, ABA probably does not inhibit the glucosylating enzymes directly but rather reduces the availability of free zeatin when zeatin riboside is supplied. Auxins and nutrient supply did not affect conversion of zeatin riboside to zeatin 7-glucoside. Relative to cotyledons developed in light, those developed in darkness had a reduced capacity to convert zeatin riboside to zeatin 7-glucoside. The results presented have identified types of chemical structures which could be developed to provide more effective inhbitors of cytokininN-glucosylation.

Role of ethylene in chlorophyll degradation in radish cotyledons

The effect of age of radish seedlings on changes in chlorophyll concentration caused by ethylene was examined. Ethylene was produced at 2–4 nl g−1 h−1 following excision of cotyledons from 5-to 20-day-old seedlings. The youngest cotyledons maintained this rate, whereas ethylene synthesis declined by as much as 80% during a 24-h period in older cotyledons. The youngest cotyledons continued to accumulate chlorophyll in the dark, but after 7 days cotyledons lost chlorophyll and the proportion of chlorophyll lost increased with age. Ethylene promoted, and norbornadiene inhibited, this loss of chlorophyll; in combined treatments the effects of ethylene and norbornadiene were competitive. The maximal rate of chlorophyll loss occurred in 1μl L−1 ethylene; extrapolation of the response to concentration indicated that half-maximum loss would occur at 0.005–0.01 μl L−1 ethylene. In cotyledons from 20-day-old seedlings, chlorophyll degradation occurred mainly after 24 h from excision and transfer to the dark. Chlorophyll degradation during 48 h in the dark was affected by norbornadiene or ethylene applied from 0–24 h or from 24–48 h.

Evidence for a Senescence-Associated Gene Induced by Darkness

A nearly full-length cDNA was isolated from a cDNA library prepared from incipiently senescent radish (Raphanus sativus L.) cotyledons using a previously isolated cDNA clone for dark-inducible mRNA as a probe (A Watanabe, N Kawakami, Y Azumi [1989] In Cell Separation in Plants, NATO ASI Series, Vol H35, pp 31-38. Springer-Verlag, Berlin). The clone detected transcripts of 800 bases which increased more than 100-fold after 24 hours of darkness. The transcripts also accumulated under light when plants were exposed to ethylene or heat stress, and (6)N-benzyladenine partially repressed its accumulation in the dark. These responses of the gene to physiological stimuli closely paralleled the effects of the stimuli on the progress of senescence of the cotyledons. We have named the gene din 1 (dark inducible gene 1). The cDNA encodes a polypeptide of 20 kilodaltons, and its nucleotide sequence shows a high (49%) similarity to a subfamily of pathogenesis-related proteins of tobacco. The predicted amino acid sequence of the product, however, shows only 20% homology to the pathogenesis-related protein.

Substituted xanthines and cytokinin analogues as inhibitors of cytokinin N-glucosylation

A series of 3-substituted xanthines, 2-(2-hydroxy-2-methylpropylamino)-9-methyl-6-benzylaminopurine and 7-benzylaminooxazolo[5,4- d]pyrimidine were synthesized as potential inhibitors of cytokinin N-glucosylation. In maize leaf segments the latter compound was found to be the most effective inhibitor tested, inhibiting the formation of the 9-glucoside of 6-benzylaminopurine (BAP) and raising the amount of free BAP. N-glucosylation of BAP in radish leaves was found to be suppressed most effectively by 1,7-dimethyl-3-(3-methylbutyl)xanthine, 1,7-dimethyl-3-(5-hexenyl)xanthine and 1,7-dimethyl-3-(3-methyl-2-butenyl)xanthine. The first two compounds were also effective inhibitors in radish cotyledons and elevated the concentrations of both free BAP and BAP nucleotide. These results indicate that the structural requirements for effective inhibitors of N-glucosylation differ between the two species.

The occurrence in plants of the growth inhibitors, the raphanusanins

The growth inhibitors, cis- and trans-raphanusanins are widespread in the genus Raphanus. However, no raphanusanins were detected in the other plant species tested. Effects of raphanusanins on the action of plant hormones, indole-3-acetic acid (IAA), gibberellic acid (GA 3) and benzyladenine (BA) were also studied using various bioassays. Raphanusanins significantly inhibited IAA activity in the Avena curvature test but had no efrect in the Avena section test. BA activity was strongly inhibited by raphanusanins in both the radish cotyledon and the Amaranthus betacyanin bioassays. In contrast, GA activity was not influenced in the range of concentrations of raphanusanins tested either in the leaf-sheath bioassay of dwarf rice or the rice endosperm bioassay. The present results suggest that raphanusanins cause growth inhibition through suppressing the action of auxin and/or cytokinin.

Calmodulin antagonists inhibit the phytochrome-induced appearance of two nuclear encoded transcripts in radish cotyledons

The effects of two calmodulin antagonists on the phytochrome-mediated appearance of two nuclear encoded transcripts in radish cotyledons have been investigated. The extent of inhibition of transcript accumulation was dependent of the time ellapsed between the administration of trifluoperazine and the light stimulus. When 1 mM trifluoperazine was administered to the seedlings 8 h before red light irradiation, the inhibition of transcript accumulation was up to 62% for the chlorophyll a b binding protein mRNA and 56% for the ribulose 1,5-bisphosphate carboxylase small subunit mRNA. Similar results were obtained with W-7 (0.1 mM).

ダイコン子葉プロトプラストからの不定芽形成

Protoplast culture is prepared from cotyledons of Japanese radish (Raphanus sativus L. cv. YR-Kurama). Protoplasts formed collonies in B medium lacking Tween 80, followed by the addition of equal volume of C medium containing 1.0mg/l NAA on the 14th day in culture. Collonies were transferred for further growth onto half strength of MS medium containing 1.0mg/l NAA and 1.0mg/l BA. A shoot formation can be induced on shoot regeneration medium (1/2 MS, 0.5% Sucrose, 1.0mg/l NAA, 1.0mg/l BA).

Growth‐Promoting Activity of Chlordimeform

AbstractEarly season applications of the insecticide chlordimeform [N' ‐ (4‐chloro‐o‐tolyl)‐N,N‐dimethylformamidine] (CDF), have increased cotton (Gossypium hirsutum L.) lint yields above those expected from the pesticidal properties of the chemical. The molecular structure of CDF resembles other agricultural chemicals that have cytokinin‐like activity. The objective of this research was to determine whether CDF possesses biological activity similar to these compounds. Chlordimeform was compared with the naturally occurring cytokinin zeatin in two hormone bioassays (radish [Raphanus sativa L.] cotyledon expansion and cytokinin‐depleted soybean [Glycine max L.] callus growth). Chlordimeform induced radish cotyledon expansion when the cotyledons were incubated under continuous light. The optimal CDF concentration for cotyledon growth was 5.1 ✕ 10−4 M. Although expansion occurred under light conditions, CDF inhibited chlorophyll synthesis at the higher concentrations and did not induce growth when cotyledons were incubated in the dark. These responses were contrary to the response of cotyledons incubated with zeatin. Also, no activity was found in soybean callus culture. The data suggested that CDF has plant physiological activity, but the biological activity is not identical to that of zeatin.

Inhibitory Effect of Gabaculine on 5-Aminolevulinate Dehydratase Activity in Radish Seedlings

We have compared the activity of 5-aminolevulinate dehydratase (5-ALAD) with the amount of protein detected by specific antibodies in rocket immunoelectrophoresis. Parallel kinetic evolutions of enzymic activity and amount of antigen were observed in radish (Raphanus sativus L.) cotyledons, both in complete darkness or under standard far red light involving phytochrome. However, the treatment of seedlings with gabaculine leads to an important decrease in enzymic activity, while the specific protein content is maintained. This inhibition is not overcome by the addition of glutamic acid, but by 5-aminolevulinic acid which points to a specific control of 5-ALAD activity by its substrate. As there is no discrepancy between the enzymic activity and the amount of antigen during the time course development of seedlings, this could confirm a coordinate cellular control between 5-aminolevulinic acid formation and 5-ALAD protein synthesis, both being amplified by the action of phytochrome.

Senescence of Radish Cotyledons and Basipetal Transport of Soluble Sugars

Summary Radish seedlings with a pair of cotyledons and an intact root system were used to study induced basipetal transport of photosynthates. The maturity of the cotyledons is characterized by the level of pigment, primary photochemical reaction, absorption and fluorescence spectroscopy. Transfer of seedlings with matured cotyledons grown in continuous light to darkness induces senescence of cotyledons, which favours the movement of sugars from cotyledons to roots. Kinetin treatment of roots in darkness make them to a more effective sink and results in a rapid basipetal transport of sugars.

Biosynthesis of Cytokinins in Soil

AbstractCytokinin biosynthesis in soil by rhizosphere microorganisms may increase upon the addition of physiological precursors and affect plant growth. This study was conducted to establish the effects of the purine, adenine (ADE), the isoprenoid, isopentyl alcohol (IA), and a cytokinin‐producing bacterium, on cytokinin biosynthesis in an Arlington soil (coarse‐loamy, mixed thermic Haplic Durixeralf). Various concentrations of ADE were applied, separately and in combination with 8.81 × 10−2 g IA kg−1 soil, and an inoculum of Azotobacter chroococcum (ATCC 9043), as soil treatments. Cytokinin bioactivity, isolation, and quantification of soil extracts were monitored over a period of 7 d using the radish cotyledon bioassay and HPLC‐UV spectrometry. Cytokinin production was evident as early as 2 d and reached a maximum at 5 d post‐treatment. An application of 1.35 × 10−4 g ADE kg−1 soil, 8.81 × 10−2 g IA kg−1 soil, and A. chroococcum enhanced cytokinin production of zeatin riboside and t‐zeatin in this soil up to 1.50‐ and 1.39‐fold, respectively, in comparison to controls containing no precursors. The production of zeatin riboside (78.0 µg kg−1) and t‐zeatin (22.1 µg kg−1) in the presence of ADE and IA was 1.35‐ and 2.44‐fold greater with the inoculation of A. chroococcum, when compared to the indigenous microflora alone. This study provides information on extraction and analytical detection of cytokinins in soil and reveals that microbial biosynthesis of cytokinins can be enhanced by applying physiological precursors to soil.

Temporal control of phytochrome-dependent gene expression during radish seedling development

The level of two nuclear-encoded transcripts (the small subunit of ribulose-1,5-bisphosphate carboxylase and the chlorophyll a/b-binding protein of photosystem II) and two chloroplastencoded mRNAs (the large subunit of ribulose-1,5-bisphosphate carboxylase and the chloroplast 32-kDa polypeptide) were analysed during the first 4 d of radish (Raphanus sativus L.) seedling development. A single 5-min red-light pulse increased the concentrations of all transcripts by various orders of magnitude, depending on the particular mRNA. For all transcripts the maximal effect of the red light was observed when the irradiation was applied 48 h following sowing. The red-light treatment did not lead to changes in the total RNA level or to alterations in the levels of nuclear and chloroplastic DNAs; the level of β-actin mRNA was also unaffected by the light treatment. It is concluded that during the first days of development, cells of radish cotyledons undergo light-independent molecular changes, occurring at the transcriptional level, conferring on them a transient competence towards the photoreceptor phytochrome.

Transfer of radish (Raphanus sativus L.) chloroplasts into cabbage (Brassica oleracea L.) by protoplast fusion.

Intergeneric hybrid plants were obtained through protoplast fusion between red cabbage (Brassica oleracea L.) and radish (Raphanus sativus L.). Protoplast fusion was carried out by the dextran method using protoplasts from hypocotyls and cotyledons of both cabbage and radish. The selection of somatic hybrids utilized the inactivation of cabbage protoplasts with iodoacetamide and the low cell-division ability of radish protoplasts. Ten plants were regenerated from selected colonies. Two of them developed to the flowering stage and formed male sterile flowers, which showed cytoplasmic inheritance. The plant and leaf morphology, chromosome number, acid phosphatase and peroxidase isozyme patterns, sexual compatibility, and SmaI cleavage patterns of chloroplast DNA indicated that these plants were intergeneric hybrids having the nucleus of cabbage and chloroplasts of radish.

Photosynthetic Ability of Radish Cotyledons Grown under Continuous Standard Far-Red Light: Photo-activation of NADP(H) Glyceraldehyde-3-Phosphate Dehydrogenase

An investigation of the photosynthetic competence of radish cotyledons grown under continuous standard far-red light is reported, following the study of photo-activation of NADP(H) glyceraldehyde-3-phosphate dehydrogenase. The in vitro illumination of isolated organelles using white light resulted in a 1-85-fold enhancement of enzymatic activity. This increase was dependent upon the age of the seedlings and could only be observed when organelles were extracted from far-red- or daylight-grown plants and when their structure was kept intact. Photosynthetic electron flow was involved in the modulation of the activity, as shown by the use of specific inhibitors. A dark deactivation of the enzymatic activity was also observed, leading to the conclusion that the activating regulatory process, likely to be related to ferredoxin/thioredoxin, is present in vivo under standard far-red light.

Senescence-Specific Increase in Cytosolic Glutamine Synthetase and Its mRNA in Radish Cotyledons

Changes in the levels of cytosolic and chloroplastic isoforms of glutamine synthetase were examined in senescing radish (Raphanus sativus L. cv Comet) cotyledons by immunoblotting analysis using antibodies raised separately against maize glutamine synthetase isoforms. Translatable mRNAs for these isoforms were also examined by analyzing translation products from poly(A)(+) RNA in a wheat germ system with the antibodies. The relative content of cytosolic isoform (GS(1)) increased twofold in the cotyledons that were placed in the dark for 72 hours to accelerate senescence, while that of chloroplastic isoform (GS(2)) declined to half of its initial level. The dark-treatment also increased the relative level of translatable mRNA for GS(1) sevenfold after 72 hours, and decreased rapidly that for GS(2) and for other nuclear-coded chloroplast proteins as well. Cotyledons also accumulated GS(1) mRNA when they became senescent after a lengthy growth period under continuous light. These observations suggested that GS(1) genes were activated, while those for GS(2) were repressed, and eventually the population of the enzyme was altered in senescent cotyledonary cells. The role of increased cytosolic enzyme is discussed in relation to the nitrogen metabolism in senescent leaves.

Cytokinin-like activities of nucleocyclitols

Although the 9-substituted adenines are commonly inactive as cytokinins, the nucleocyclitol 3-(-adenin-9-yl)-3-deoxy-1,5,6-tri-0-(methylsulfonyl)-muco-inositol (NI) proved to be active in the following bioassays: cell proliferation in soybean cotyledon callus tissue, cell expansion in excised radish cotyledons, and delay of senescence in detached leaves. In these assays, the effect of the compound, applied at the same molar concentration as benzyl adenine, was lower or less uniform than BA. NI completely failed to promote germination of lettuce seeds in conditions of secondary dormancy or thermodormancy, whre BA is effective. NI can substitute for BA in some though not all of the numerous responses evoked by cytokinins.

Cadmium‐induced changes in the composition and structure of the light‐harvesting chlorophyll a/b protein complex II in radish cotyledons

Five‐day‐old etiolated radish (Raphanux salivux L. cv. Saxa) seedlings exposed to white continuous light in the presence of Cd2+ (0.2 mM) showed characteristic changes in their light‐harvesting chlorophyll a/b protein complex II after 48 h of greening. The content of its oligomeric supramolecular form was greatly diminished with a concomitant increase in the level of the monomer. The isolation of highly purified light‐harvesting chlorophyll a/b protein complex II from control and Cd2+ treated radish cotyledons and a detailed analysis of its structure and composition revealed that first of all, Cd2+ altered the content of the specific phosphatidylglycerol fatty acid ‐trans‐Δ3‐hexadecenoic acid, widely accepted as a component responsible for the oligomerization of this chlorophyll‐protein complex. This fatty acid in the thylakoid membrane phosphatidylglycerol pool seems to be very sensitive to different environmental stresses lowering its content, which indicates the vital significance of this component for the supramolecular organization and proper functioning of the light‐harvesting chlorophyll a/b protein complex II.

Change in Gene Expression in Radish Cotyledons during Dark-Induced Senescence

Change in Gene Expression in Radish Cotyledons during Dark-Induced Senescence

5-aminolevulinate dehydratase activity in thylakoid-related structures of etiochloroplasts from radish cotyledons

5-Aminolevulinate dehydratase activity was studied in etiochloroplasts purified from radish cotyledons grown for 120 hr under continuous far-red light. The activity was mainly recovered from the stroma of the organelles, as previously observed, but also from the membrane fraction. The bound activity was not altered after incubation in the presence of NaCl or detergents indicating that the enzyme was neither associated with the pelletable fraction by electrostatic forces nor entrapped in lipid vesicles. After separation of prothylakoids and prolamellar bodies on a sucrose gradient, 5-aminolevulinate dehydratase activity and two-thirds of the total protein were observed in prothylakoids. Carotenoids were mainly present in the prolamellar bodies fraction. A few chlorophylls were observed in prothylakoids and prolamellar bodies, indicating that impure fractions were obtained, but that 5-aminolevulinate dehydratase activity was actually bound to thylakoid-related structures.