ABSTRACE MATERIALS AND METHODSThe biosynthesis of cytokinins was examined in pea (Pisum sativum L.) plant organs and carrot (Daucus carota L.) root tissues. When pea roots, stems, and leaves were grown separately for three weeks on a culture medium containing 18-"4Cadenine without an exogenous supply of cytokinin and auxin, radioactive cytokinins were synthesized by each of these organs. Incubation of carrot root cambium and noncambium tissues for three days in a liquid culture medium containing 18-'Cladenine without cytokinin demonstrates that radioactive cytokinins were synthesized in the cambium but not in the noncambium tissue preparatioa. The radioactive cytokinins extracted from each of these tissues were analyzed by Sephadex LH-20 columns, reverse phase high pressure liquid chromatography, paper chromatography in various solvent systems, and paper electrophoresis. The main species of cytokinins detectable by these methods are N'4-A2-isopentyl)adenine-5'-monophosphate, 644-hydroxy-3-methyl-2-butenyl-amino)-9-,-ribofuranosylpurine-5'-monophosphate, N'-Q52-isopeatenyl)adenosine, 6-(4-hydroxy-3-methyl-2-butenylamino)-9-ft-ribofuraosylpurine, N'-(A2-isopentenyl)adenine, and 6-(4-hydroxy-3-methyl-2-butenylamino)purine. On the basis of the amounts of cytokinin synthesized per gram fresh tissues, these results indicate that the root is the major site, but not the only site, of cytokinin biosynthesis. Furthermore, cambium and possibly all actively dividing tissues are responsible for the synthesis of this group of plant hormones.There is evidence that cytokinins exist in the roots (15,16) and that cytokinins from xylem exudate may come from the roots (9,17 2Abbreviations: Ade, adenine; Ado, adenosine; i6Ade, N6-(A2-isopentenyl)adenine; i6Ado, N6-(A2-isopentenyl)adenosine; io6Ade, 6-(4-hydroxy-3-methyl-2-butenylamino) purine; io6Ado, 6-(4-hydroxy-3-methyl-2-butenylamino)-9-,-ribofuranosylpurine. imbibed for 16 h with running tap water, and surface-sterilized for 10 min with 0.5% NaOCl. The seeds were thoroughly rinsed with sterile distilled H20, and three seeds were grown on 50 ml of a basic culture medium (8)

To investigate the molecular mechanism of cytokinin regulation of nitrate reductase (NR) activity, the influence of benzyladenine (BA) on the level of NR transcript was studied in etiolated barley leaves using a barley NR cDNA as a probe. Northern blot analyses of the levels of NR poly (A)+ RNA indicate that the amount present is proportional to the concentration of BA (2 x 10(-8) to 2 x 10(-4) M) applied to the leaves. Enhancement of NR mRNA by 2 x 10(-5) M BA was clearly detected after 15 minutes of exposure of the leaves to light. The enhancement is cytokinin-specific and adenine is ineffective. Brief treatment with the protein synthesis inhibitor, cycloheximide, inhibited BA-enhanced NR activity but did not inhibit BA-enhanced NR transcript level, thus the enhancement was independent of concurrent protein synthesis. Nuclear runoff transcription studies showed that the enhancement of NR mRNA was at least partially due to increased transcription rates.

To investigate the mechanisms by which the expression of a specific gene may be modulated by multiple hormones, the regulation of nitrate reductase (NR) mRNA levels by cytokinin and abscisic acid (ABA) was studied in etiolated barley (Hordeum vulgare L. cv Robust) leaves using a barley NR cDNA as a probe. Northem blot analyses of the levels of NR mRNA indicate that accumulation of the mRNA was enhanced by the cytokinin, benzyladenine (BA), and suppressed by ABA and that the hormonal effects on NR gene expression were responsive to dose concentrations (10-' to 10-4 molar). The NR mRNA levels were influenced by the BA/ABA concentration ratios, and the inhibition of the NR transcript levels by ABA was not totally reversed by addition of equal concentrations (10-to 10-4 molar) of BA. Nuclear runoff transcription studies showed that the level of transcription was suppressed by ABA, and addition of an equal concentration of BA partially reversed the ABA action. Thus, the interaction of cytokinin and ABA on NR gene expression is, at least in part, at the level of transcription.is, at least in part, at the level of transcription ( 13,21). Genes having related 5'-regulatory region DNA sequences are similarly regulated by either ABA (13, 29), GA3 ( 15), ethylene (2), or auxin (20), but the regulatory elements of genes responsive to these hormones have not been fully identified.The genetics and molecular biology of NR3 have been studied extensively (3, 5, 8,17,23), and the NR activity has been shown to be enhanced by cytokinins (16,18,19) (27). However, the mechanisms by which specific genes are regulated by multiple hormones are not well understood.The primary actions of cytokinins and ABA in the plant cells have not been fully elucidated at the molecular level. Cytokinins enhance and/or suppress the levels of specific mRNAs (4, 7, 9,19,26), and the enhancement has been shown to be mainly at the level of transcription (19) or posttranscription (9). ABA, like other phytohormones, induces, promotes, or suppresses the levels of specific mRNAs (1 1, 12, 21, 24), and the influence of ABA on gene expression Growth of PlantsBarley (Hordeum vulgare L. cv Robust ) seeds were soaked in water for 8 h and grown on sterile vermiculite at 25°C in the dark for 4 d. The seedlings (7-9 cm in length) were gently removed from the vermiculite and rinsed with water under green light. About 60 seedlings were placed into each 400-mL beaker containing 100 mL of 50 mm KNO3. The leaves were sprayed with 8 mL of the treatment solution (water or hormones dissolved in water) and placed at 25°C in darkness or cool-white fluorescent light (about 16 klx) for various time intervals. Excised leaves were frozen immediately in liquid nitrogen and stored at -700C. NR AssayThe following protocol was used to isolate and assay for NR in vitro. Barley leaves (3 g) were crushed in a mortar at 0 to 4°C in 3 mL of 25 mm potassium phosphate buffer (pH 7.5) containing 5 mm cysteine and 5 mM EDTA. The extract was strained through two layers of cheesecloth and...

Adenosine nucleosidase (adenosine ribohydrolase, EC 3.2.2.7) which catalyzes the deribosylation of N'-(A2-isopentenyl)adenosine and adenosine to form the corresponding bases was partially purified from wheat gern. This enzyme (molecular weight 59,000 ± 3,000) deribosylates the ribonucleosides at an optimum pH of 4.7. Km values for the cytokinin nucleoside and esine are 238 and 1.43 micromolar, respectively, in 50 milHolar Tris-citrate buffer (pH 4.7) at 30 C. The presence of adenosine and other cytokinn nucleosides inlhbited the hydrolysis of N64A'.isopentenyl)adenosine but this reaction wa insensitive to guanosine, uridine, or 3'deoxy oine. It is hypothesized that an adequate level of "active cytokinin" in plant cells may be provided through the deribosylation of cytokinin riboside in concert with other cytokinin metabolic enzymes.Cytokinin bases and cytokinin ribonucleosides have been found in various plant cells, and these cytokinins are metabolized in the plant cell to form different metabolites (3,4,9,11,16,18). The relative amount of a specific metabolite formed may differ not only from one plant to another, but also in one particular plant or tissue under different physiological conditions. One of the major metabolites formed from a cytokinin ribonucleoside has been reported to be its corresponding base (9, 11, 13). In the de novo biosynthesis of cytokinins using a crude enzyme system prepared from plant cells, a cytokinin nucleotide, nucleoside, and base were formed from 5'-AMP and A2-isopentenylpyrophosphate (5). These observations indicate that in plant cells there are enzyme systems catalyzing the formation of cytokinin base from its nucleoside, which in turn can be formed from the corresponding nucleotide (7). Although hydrolytic conversion of Ado2 to Ade by adenosine nucleosidase (adenosine ribohydrolase, EC 3.2.2.7) has been shown to occur in plant (10,14,17) and microbial (20) cells, the role of this enzyme in cytokinin metabolism has not been defined.We describe here the partial purification of adenosine nucleosidase from wheat germ, the properties of the enzyme, and the kinetics of the deribosylation of cytokinin ribonucleoside by this enzyme system. ' This work was supported by the National Science Foundation Re-search Grant PCM 79 03832. 2Abbreviations: Ado, adenosine; Ade, adenine; i6Ado, N6-(A2-isopentenyl)adenosine; i?Ade, N6-(A2-isopentenyl)adenine; t-io6Ado, trans-6-(4-hydroxy-3-methyl-2-butenylamino)-9-B-D-ribofuranosylpurine. Extraction and Fractionation of Enzyme. Wheat germ (135 g) frozen with liquid N2 was homogenized in a Waring Blendor in 10 mM Tris-HCI buffer (pH 7.0) (4 volumes/weight). The homogenate was filtered through double layers of cheesecloth. The filtrate was centrifuged for 10 min at 10,000g and the resulting supernatant was centrifuged again for 25 min at 20,000g. The supernatant is referred to as crude extract. The following steps were employed to further purify the extract:Step 1: Low pH Fractionation. The extract was brought to 30 C and its pH was adjust...