The Metabolism of Hormones during Seed Germination and Dormancy: II. The Metabolism of 8-C-Zeatin in Bean Axes

8-¹⁴C-Zeatin is taken up rapidly and is extensively metabolized by excised bean axes during a 12-hour incubation at 26 C. Most of the radioactivity is found in the 80% ethanol soluble fraction and consists of zeatin, zeatin riboside, zeatin-5′-ribotide, as well as corresponding dihydrozeatin derivatives. The characterization of ¹⁴C-dihydrozeatin included crystallization to constant specific radioactivity. No cleavage of the zeatin side chain to adenine, hypoxanthine, their ribosides, or glycylpurine was detected. Dihydrozeatin has been previously isolated from yellow lupin seeds, and our experiments indicate that it can be derived through reduction of the side chain from preexisting cytokinin. While the total amount of zeatin metabolized is not affected by growth-inhibiting concentrations of abscisic acid or cycloheximide, the conversion to dihydrozeatin derivatives is curtailed. Although somewhat less effective than zeatin and zeatin riboside, dihydrozeatin and dihydrozeatin riboside also counteract the abscisic acid-induced growth inhibition.     

6-(gamma,gamma-Dimethylallylamino)Purine As A Precursor of Zeatin

A 6-(γ,γ-dimethylallylamino) purine-like compound was found in the culture medium of Rhizopogon roseolus, which had been shown earlier to synthesize zeatin. The role of 6-(γ,γ-dimethylallylamino) purine as a precursor of zeatin was studied. Rhizopogon was furnished with 6-(γ,γ-dimethylallylamino) purine-8-¹⁴C. Cochromatography, oxidation studies with potassium permanganate, and bromination indicated that labeled zeatin ribonucleoside was isolated from the medium. The fungus also incorporated labeled adenine, hypoxanthine, and 4-amino-5-imidazole carboxamide into zeatin ribonucleoside.     

APOLAR MOVEMENT OF ZEATIN THROUGH COLEUS PETIOLES AND PISUM ROOTS AS ESTIMATED BY BIOASSAY AND RADIOACTIVE LABELLING

The movement and polarity of zeatin, a highly active, endogenous cytokinin, through petioles and roots were tested in the classical experimental arrangement using excised 5-mm sections. Zeatin in the receiver cylinders of agar was measured by soybean callus bioassay and by liquid scintillation counting of ¹⁴C that had been added in the donor cylinders as [8-¹⁴C] zeatin. Both methods agreed in showing movement, but there was no polarity in Coleus #5 petioles. The amounts moved were about one-tenth of the GA-3 movement through petioles of the third pair of leaves of the same clone. Movement of ¹⁴C-zeatin through Pisum roots was similarly statistically significant but non-polar; the amounts moved were similar to those previously observed for polar GA-3 movement through Zea roots.     

Metabolism of C-zeatin in phaseolus embryos : occurrence of o-xylosyldihydrozeatin and its ribonucleoside

The metabolism of trans-[8-¹⁴C]zeatin was examined in embryos of Phaseolus acutifolius A. Gray P.I. 321637 and Phaseolus coccineus Lam. cvs Scarlet Runner and Desiree. In both species zeatin was converted to ribosylzeatin, ribosylzeatin 5′-monophosphate, O-glucosyl-9-ribosylzeatin and the recently discovered O-xylosyl derivatives of zeatin and ribosylzeatin (Turner, JE, DWS Mok, MC Mok, G Shaw 1987 Proc Natl Acad Sci USA. In press). Two new metabolites, identified by enzyme degradation and gas chromatography-mass spectrography analyses as O-xylosyldihydrozeatin and its ribonucleoside, were recovered from P. coccineus embryos. From this and previous studies it may be concluded that the potential to form O-xylosyl derivatives of zeatin is present only in embryos of three Phaseolus species (P. vulgaris L., P. coccineus, and P. acutifolius), but not in P. lunatus L., while the reduction of the side chain is most prominent in P. coccineus.     

Metabolism of 2-C-(+/-)-abscisic Acid in excised bean axes

Excised embryonic bean axes (Phaseolus vulgaris, var. White Marrowfat) rapidly metabolize 2-¹⁴C-(±)-abscisic acid to two compounds, M-1 and M-2, which have very low growth-inhibitory activity. Chemical tests indicate the M-1 and M-2 are not previously described abscisic acid metabolites. M-2 accumulates in the axes and evidence is presented for the hypothesis that abscisic acid → M-1 → M-2. Zeatin, which partially reverses the abscisic acid-mediated growth inhibition of axes, neither decreases abscisic acid uptake nor causes any major changes in its metabolism. It was observed that axes transferred from abscisic acid-containing solutions to buffer resume control rates of fresh weight increase while still containing considerable quantities of abscisic acid.     

8-14C-Zeatin metabolites and their transport from leaf to phloem exudate of Yucca

Transport and metabolism of 8-¹⁴ C-zeatin, applied to an attached de-tipped one-year-old mature leaf of a Yucca plant bearing a bleeding inflorescent stalk, has been studied. Radioactive zeatin ribotide was found in the exudate of the bleeding inflorescence, which was collected over a period of 5 days. Radioactive zeatin ribotide was mainly extracted from the fed leaf. Minor conversion products in this leaf were zeatin ribotide, zeatin- o -β-glucoside and zeatinriboside o -β-glucoside.
In not zeatin fed plants, zeatin- o -β-glucoside was tentatively identified as the main endogenous cytokinin in one-year-old mature leaves. In the bleeding sap of not treated plants no free bases of zeatin or zeatin ribosides were found. After alkaline phosphatase treatment zeatin-riboside was detected by combined gas chromatography-mass spectrometry, indicating the presence of zeatin ribotide in the bleeding sap. High β-glucosidase activity was found in the stern.
Results suggest that stared cytokinin glucosides from Yucca leaves are, converted by β-glucosidase in leaves and stem, transported through the inflorescent stalk as zeatin nucleotides.     

Abscisic Acid Metabolism in Salt-Stressed Cells of Dunaliella salina: Possible Interrelationship with beta-Carotene Accumulation

The interrelationship between abscisic acid (ABA) production and β-carotene accumulation was investigated in salt-stressed cells of the halotolerant green alga Dunaliella salina var bardawil. Cells were supplied with either R-[2-¹⁴C]mevalonolactone or [¹⁴C] sodium bicarbonate for 20 hours and then exposed to increased salinity (1.5 to 3.0 molar NaCl) for various lengths of time. Incorporation of label into abscisic acid and phaseic acid and the distribution of [¹⁴C]ABA between the cells and incubation media were monitored. [¹⁴C]ABA and [¹⁴C]phaseic acid were identified as products of both R-[2-¹⁴C]mevalonolactone and [¹⁴C]sodium bicarbonate metabolism. ABA metabolism was enhanced by hypersalinity stress. Actinomycin D, chloramphenicol, and cycloheximide abolished the stress-induced production of ABA, suggesting a role for gene activation in the process. Kinetic analysis of both ABA and β-carotene production demonstrated two stages of accelerated β-carotene production. In addition, ABA levels increased rapidly, and this increase occurred coincident with the early period of accelerated β-carotene production. A possible role for ABA as a regulator of carotenogenesis in cells of D. salina is therefore discussed.     

Benzyladenine and derivatives-their significance and interconversion in plants

Recently benzyladenine has been isolated as a natural cytokinin from a number of plants. The natural occurrence of this cytokinin will change the attitude with which physiologists view this hormone. This review attempts to put into context what is known about this cytokinin and its derivatives and to compare and contrast its metabolism and the function and physiological action of its various metabolites. Nothing is known about the biosynthesis of benzyladenine. Its structure would suggest that its biosynthetic pathway may differ considerably from that of zeatin and iso-pentenyladenine.Abbreviations Ade adenine - Ado adenosine - BA benzyladenine - [9R]BA BA ribonucleoside - [9R-MP]BA BA nucleotide - [9R-DP]BA BA dinucleotide - [9R-TP]BA BA trinucleotide - [3G]BA BA 3 glucoside - [7G]BA BA 7 glucoside - [9G]BA BA 9 glucoside - [9R-G]BA BA 9-ribosylglucoside - [9Ala]BA BA alanine-conjugate - (2OH)BA BA ortho-OH - (2OH)[9R]BA BA ortho-Oh-riboside - KN kinetin - [9R]KN KN ribonucleoside - DHZ dihydrozeatin - Z trans-zeatin - [9R]Z zeatin ribonucleoside - [7G]Z zeatin-7-glucoside - [9G]Z zeatin-9-glucoside - [9Ala]Z zeatin alanine-conjugate - (OG)[9R]Z O-glucoside of zeatin ribonucleoside - [9R-MP]Z zeatin nucleotide - iP iso-pentenyladenine - [9R]iP iP ribonucleoside     

Effects of Thidiazuron on Cytokinin Autonomy and the Metabolism of N-(Delta-Isopentenyl)[8-C]Adenosine in Callus Tissues of Phaseolus lunatus L

The effects of a highly cytokinin-active urea derivative, N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (Thidiazuron), and zeatin on cytokinin-autonomous growth and the metabolism of N⁶-(Δ²-isopentenyl)[8-¹⁴C]adenosine ([¹⁴C]i⁶ Ado) were examined in callus tissues of two Phaseolus lunatus genotypes, cv Jackson Wonder and P.I. 260415. Tissues of cv Jackson Wonder maintained on any concentration of Thidiazuron became cytokinin autonomous, whereas only tissues exposed to suboptimal concentrations of zeatin displayed cytokinin-autonomous growth. Tissues of P.I. 260415 remained cytokinin dependent under all these conditions. The metabolism of [¹⁴C]i⁶ Ado was similar for the two genotypes, but differed with the medium used. [¹⁴C]i⁶ Ado was rapidly converted to N⁶-(Δ²-isopentenyl)[8-¹⁴C]adenosine 5′-P ([¹⁴C]i⁶ AMP) by tissues grown on zeatin-containing medium, whereas only traces of the nucleotide were formed in tissues grown on medium with Thidiazuron. Incubation with [¹⁴C] i⁶ AMP of tissues grown in the presence of Thidiazuron resulted in rapid conversion to [¹⁴C]i⁶ Ado, while [¹⁴C]i⁶ AMP persisted in tissues maintained on zeatin. Thus, Thidiazuron appears to stimulate enzyme activity converting the ribonucleotide to ribonucleoside. Although the cytokininactive phenylureas and adenine derivatives differ in their effects on cytokinin autonomy as well as nucleotide formation, the two types of effects do not seem to be related.     

The Metabolism of Hormones during Seed Germination and Dormancy: IV. The Metabolism of (S)-2-C-Abscisic Acid in Ash Seed

Embryos from dormant and stratified Fraxinus americana seed were incubated with (S)-2-¹⁴C-abscisic acid (ABA) under a variety of conditions. Both dormant and stratified embryos rapidly metabolize abscisic acid to phaseic acid, dihydrophaseic acid, and an unidentified polar metabolite apparently derived from dihydrophaseic acid. Although the stratified embryos may have an increased capacity to metabolize abscisic acid, our calculations suggest that such an increased capacity would probably not be physiologically significant.     

Effects of Chilling and ABA on [H]Gibberellin A(4) Metabolism in Somatic Embryos of Grape (Vitis vinifera L. x V. rupestris Scheele)

Previous work has indicated that changes in gibberellin (GA) metabolism may be involved in chilling-induced release from dormancy in somatic embryos of grape (Vitis vinifera L. × V. rupestris Scheele). We have chilled somatic embryos of grape for 2, 4, or 8 weeks, then incubated them with [³H]GA₄ (of high specific activity, 4.81 × 10¹⁰ becquerel per millimole) for 48 hours at 26°C. Chilling had little effect on the total amount of free [³H]GA-like metabolites formed during incubation at 26°C, but did change the relative proportions of individual metabolites. The amount of highly water-soluble [³H] metabolites formed at 26°C decreased in embryos chilled for 4 or 8 weeks. The concentration of endogenous GA precursors (e.g., GA₁₂ aldehyde-, kaurene-, and kaurenoic acid-like substances) increased in embryos chilled for 4 or 8 weeks. Treatment with abscisic acid (ABA) (known to inhibit germination in grape embryos) concurrent with [³H]GA₄ treatment at 26°C, reduced the uptake of [³H] GA₄ but had little effect on the qualitative spectrum of metabolites. However, in the embryos chilled for 8 weeks and then treated with ABA for 48 hours at 26°C, there was a higher concentration of GA precursors than in untreated control embryos. Chilled embryos thus have an enhanced potential for an increase in free GAs through synthesis from increased amounts of GA precursors, or through a reduced ability to form highly water-soluble GA metabolites (i.e., GA conjugates or polyhydroxylated free GAs).     

Estimation of osmotic parameters accompanying zeatin-induced growth of detached cucumber cotyledons

Water potential (ψ), the osmotic potential (ψ_π), and the pressure potential (ψ_p) of detached cotyledons isolated from Cucumis sativus L. cv Marketer seedlings after 0, 1.5, and 3 days growth with and without zeatin were determined. From zero time to 3 days, cotyledons incubated without exogenous zeatin exhibited a slight decrease in ψ (from −0.4 to −1.0 bars), while those grown with zeatin developed even more negative values (about −4 bars). Both groups showed rising ψ_π values (decreases in solutes per unit volume), but this rise was more dramatic in those treated with zeatin. These data indicate that the capacity of zeatin-treated cotyledons to take up water more rapidly than controls and thus expand faster must be due to wall loosening, as reflected in ψ_p values which declined during 3 days from about +11 bars to about +1.4 bars.

It was also found that freshly detached cotyledons or those grown without exogenous zeatin exhibited osmoregulation in polyethylene glycol (PEG) solutions. That is, while cotyledons initially lost H₂O into certain PEG solutions, their ψ values decreased over time and they began absorbing water after 1 to 4 hours. After 3 days growth, zeatin-treated cotyledons had lost most of this capacity of osmoregulate. It seems likely that osmoregulation in cotyledons not treated with zeatin is due to wall loosening rather than changes in ψ_π. Zeatin-treated cotyledons with already loosened walls may not have this option to deal with water stress and thus simply come to equilibrium with external PEG solutions.

     

Metabolism of delta-Aminolevulinic Acid in Red and Blue-Green Algae

δ-Aminolevulinic acid was incorporated in vivo into C-phycocyanin and B-phycoerythrin in two species of the Rhodophyta (Cyanidium caldarium, Porphyridium cruentum) and three species of the Cyanophyta (Anacystis nidulans, Plectonema boryanum, Phormidium luridum). Amino acid analysis of phycocyanin-¹⁴C from C. caldarium cells which had been incubated with δ-aminolevulinate-4-¹⁴C showed that 84% of the radioactivity incorporated was present in the phycocyanobilin chromophore and less than 16% of the radioactivity cochromatographed with amino acids. These results indicate that δ-aminolevulinate is utilized predominantly via the porphyrin pathway in C. caldarium. Conversely, analysis of phycocyanin-¹⁴C prepared from cells of A. nidulans, P. boryanum, and P. luridum which had been incubated with radiolabeled δ-aminolevulinate demonstrated that 85%, 81%, and 93%, respectively, of the radioactivity incorporated cochromatographed with amino acids. The ratio of incorporated radioactivity in amino acids and phycoerythrobilin was 40:60 in P. cruentum phycoerythrin obtained from cells which had been incubated with δ-aminolevulinate-4-¹⁴C. Succinate-2-3-¹⁴C appeared to be as good a carbon source of amino acids as did C₄ and C₅ of δ-aminolevulinate. These data demonstrate a major alternate route (other than the porphyrin pathway) of δ-aminolevulinate metabolism in red and blue-green algae. The factors responsible for the extent to which δ-aminolevulinate is utilized for synthesis of porphyrins and their derivatives and routes of δ-aminolevulinate catabolism in the organisms employed are discussed.     

Abscisic Acid Metabolism in Water-stressed Bean Leaves

Phaseic acid (PA) and dihydrophaseic acid (DPA) are the major metabolites observed when (S)-2-¹⁴C-abscisic acid (ABA) is fed to 14-day excised primary bean leaves (Phaseolus vulgaris L. cv. Red Kidney). The distribution of ¹⁴C in leaves which were wilted after feeding ABA appears to be the same as that observed in unwilted leaves. A reduction in the relative specific radioactivities of the two metabolites after wilting, compared with the specific radioactivities measured in unwilted plants, indicated that these metabolites continue to be formed endogenously after wilting. Estimates of the endogenous ABA levels showed that they rose from 0.04 μg to approximately 0.5 μg/g fresh weight within 4 hours after the beginning of a 10% wilt and remained at that level during a subsequent 20 hours of wilt. In unwilted leaves, the levels of PA and DPA were 5 times and 20 times higher than that of ABA, respectively. Both PA and DPA levels rose throughout the wilt period. PA rose from 0.20 μg to 1.0 μg and DPA from 0.8 μg to over 3 μg/g fresh weight. From these data, we calculated the rate of ABA synthesis to be at least 0.15 μg/hr.g fresh weight during this period. We have interpreted these results to mean that in wilted leaves an elevated level of ABA is maintained because the rate of synthesis and metabolism are both elevated and approximately equal.     

The influence of bud length,age of the tree and culture media on androgenesis induction inAesculus carnea Hayne anther culture

Statistical analyses of the data revealed very significant differences in androgenesis induction ofA. carnea Hayne anther culture depending on the bud length, nutrient medium composition and age of the parental tree. Significant mutual influence of all these factors was also observed. The highest number of androgenic anthers was obtained when 4 mm long buds were used. Older trees (60 and 100 yrs) gave a higher number of androgenic anthers than the younger ones (20 and 40 yrs). MS medium supplemented with 2,4-d and Kin (1 mg l^–1, each) was the most favourable for androgenesis induction. Pollen embryos (haploids and aneuploids) were formed by the division of uninuclear microspores.The highest percentage of germinated embryos and further synchronous development of the shoot and root was achieved in MS medium supplemented with IAA, GA₃ (1 mg l^–1) and activated charcoal (1%). When other germination media were used, malformations of androgenic embryos were observed.Abbreviations AC activated charcoal - H casein hydrolysate - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BAP 6-benzylaminopurine - GA₃ gibberelic acid - Kin 6-furfurylaminopurine - MS Murashige and Skoog - T thidiazurone - N phenyl-N'-1,2,3-thiadiazol-5-ylurea - Z zeatin-6-(4-hydroxy-3-methyl-trans-2-butenylamino)purine     

Metabolism of Radiolabeled β-Guaiacyl Ether-Linked Lignin Dimeric Compounds by Phanerochaete chrysosporium

Phanerochaete chrysosporium metabolized the radiolabeled lignin model compounds [γ-¹⁴C]guaiacylglycerol-β-guaiacyl ether and [4-methoxy-¹⁴C]veratrylglycerol-β-guaiacyl ether (VI) to ¹⁴CO₂ in stationary and in shaking cultures. ¹⁴CO₂ evolution was greater in stationary culture. ¹⁴CO₂ evolution from [γ-¹⁴C]guaiacyl-glycerol-β-guaiacyl ether and [4-methoxy-¹⁴C]veratrylglycerol-β-guaiacyl ether in stationary cultures was two- to threefold greater when 100% O₂ rather than air (21% O₂) was the gas phase above the cultures. ¹⁴CO₂ evolution from the metabolism of the substrates occurred only as the culture entered the stationary phase of growth. The presence of substrate levels of nitrogen in the medium suppressed ¹⁴CO₂ evolution from both substrates in stationary cultures. [¹⁴C]veratryl alcohol and 4-ethoxy-3-methoxybenzyl alcohol were formed as products of the metabolism of VI and 4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether, respectively.     

Embryogenesis and plant regeneration from maize zygotes by in vitro culture of fertilized embryo sacs

Summary Fertilized embryo sacs of Zea mays were isolated and cultured In vitro. Each explant contained one zygote and 2–4 endosperm nuclei which formed, respectively, embryo and cellular endosperm during the culture. In our double-layer/two-phase culture system, NBM medium (Mòl et al. 1993) supplemented with 0.1–1.0 mg·l^–1 zeatin and 12 % sucrose showed the best results. On this medium, embryos were isolated from 37–54 % of two-week-old explants. They were similar to maize embryos developing in vivo. We have shown that development of stage-2 embryos (according to Abbe and Stein 1954) with two leaf primordia and normally differentiated provascular tissue is possible from the maize zygote in an in vitro culture system. Some embryos with enlarged and deformed scutellum or whole apical parts were also found. Up to 62 % of the embryos germinating on a simple medium regenerated into mature and fertile plants; i.e. 23 % of explants yielded plants. This unproved culture method results in better embryo differentiation and 14-fold increase of regeneration frequency than previous protocol.Abbreviations BAP benzylaminopurin - ZT Zeatin     

Cytokinin Biosynthesis in Germinating Maize Embryos

Three-day-old germinating embryos of Zea mays were incubatedwith [8-¹⁴C]adenine for 8 and 24 h. Use of high performanceliquid and thin layer chromatography provided evidence for theincorporation of adenine by isolated germinating embryos intotrans-zeatin. These results are at variance with similar labellingstudies and possible reasons for this discrepancy are discussed. Key words: Adenine, germination, maize kernels, zeatin     

Phytochemical Studies on the Tobacco Alkaloids. XII. Identification of gamma-Methylaminobutyraldehyde and its Precursor Role in Nicotine Biosynthesis

γ-Methylaminobutyraldehyde (N-methylpyrroline) labeled with ¹⁴C was isolated from tobacco roots which had metabolized ornithine-2-¹⁴C. It was labeled most strongly 4 hours after adding ornithine-2-¹⁴C to the root, also labeled by putrescine-1,4-¹⁴C and methionine-¹⁴CH₃, and observed in the root but not in the aerial portions of tobacco plants. γ-Methyl-aminobutyraldehyde when added back to the root was an efficient precursor of nicotine. Identity of γ-methylaminobutyraldehyde from tobacco roots was confirmed by comparison with the authentic compound.     

Rapid Increase in Adenosine 5'-Triphosphate during Early Wheat Embryo Germination

The ATP content of isolated wheat (Triticum aestivum L. var. Polk) embryos increases 5-fold during the first 30 minutes and 10-fold during the first hour of germination to 80% of maximum. The ATP level remains at approximately 800 nanomoles per gram of tissue during the next 15 hours. ADP, AMP, and total adenosine phosphates decrease between 1 and 6.5 hours, while adenylate energy charge increases from 0.6 to 0.8 and remains constant. The rapid increase in ATP during imbibition is consistent with the energy requirement for polyribosome formation and protein synthesis during the first hours of germination. A method for determining nanomole quantities of ATP in tissue extracts by isotopic dilution of γ-³²P-ATP in the hexokinase reaction is outlined.