Cytokinins in Seedling Roots of Pea

Abstract

The natural occurrence of cytokinins existing both in a free form and as a constituent of transfer RNA was examined in serial segments of young seedling roots of pea. Purified ethanol extracts of root apices were resolved into four factors capable of inducing soybean callus tissue proliferation. The most active factor was identified as zeatin or some closely related compound; it produced polyploid divisions and tracheary element differentiation when tested on cultured pea root segments. 2Abbreviations: IPA: 6-(3-methyl-2-butenylamino)-9-,3-D-ribofuranosylpurine or isopentenyladenosine; 2iP: 6-(3-methyl-2-butenylamino)purine or isopentenyladenine; ZR: 6-(4-hydroxy-3-methyl-2-cisbutenylamino)-9-,8-Dribofuranyosylpurine or zeatin ribonucleoside; Z: 6-(4-hydroxy-3-methyl-2-transbutenylamino)purine or zeatin. Extraction of Free Cytokinins. Immediately after harvesting, the different fractions of pea root segments were immersed separately in precooled absolute ethanol at -72 C. The frozen segments were collected by filtration on Whatman No. 1 filter paper and homogenized in an all-glass homogenizer in cold 80% (v/v) ethanol. The homogenized tissue was extracted for 3 hr in two portions of 80% ethanol and centrifuged at 10,000g for 15 min at 4 C. The original absolute ethanol fraction and the two 80% ethanol extracts were combined and the alcohol evaporated in vacuo. The water phase was adjusted to pH 8.0 and extracted with three equal volumes of 1-butanol which were then combined and taken to dryness in vacuo. The residue was redissolved in about 100 ml of water and adjusted to pH 2.0 with concentrated H2SO,. Fifteen milliliters of cold saturated AgNO, solution were added, and the mixture constantly was stirred for 12 hr at 4 C and centrifuged at 7000g for 15 min. The precipitate was washed with cold 2% AgNO, solution and stirred with 20 ml of 0.2 N HCl at 50 C for 30 min before being centrifuged at 7000g. This procedure was repeated twice. The combined acidic supernatants were adjusted to pH 2.5 with concentrated NaOH and percolated through a Dowex 50W-X8, H+ (200-400 mesh) column, 1.0 cm X 25.0 cm, which was then washed with 100 ml of water and eluted with 300 ml 3 N NH,OH. The eluate was evaporated in vacuo to about 50 ml and extracted at pH 8.0 with three equal volumes of 1-butanol which were combined and taken to dryness in vacuo. The resulting residue was dissolved in 80% ethanol and applied as a streak on Whatman No. 1 paper and developed with solvent system A, 1-butanol-acetic acid-water (12: 3:5 v/v). In addition, the following solvents were employed: B: 30 mM borate buffer at pH 8.4; C: ethyl methyl ketone saturated with water; D: l-butanol-1 N ammonium hydroxide (10:7 v/v); E: 1-butanol saturated with water. Each chromatogram was thoroughly air dried and cut into 10 equal RF strips and eluted with 80% ethanol. The eluates were reduced in volume and tested for cytokinin activity using the soybean callus bioassay.

Studies on Cytokinin Production by Rhizobium

“…The increase in ,ug KE/g dry weight of bacteria between day 2 and day 3 was reproducible in different experiments and probably reflected an increase in metabolic rates during the rapid growth phase. The decrease in detectable cytokinin between day 3 and day 10 indicates that these compounds were either inactivated, metabolized, or masked by inhibitors. No cytokinin activity was detected in a 1.1 liter sample of day 0 cultures.…”

Implication of ectomycorrhizal fungi in the cytokinin relations of loblolly pine (Pinus taeda L.)*

“…Studies by Erance (1980) with Pinus proposed that nitrogen is an effector of cytokinin ponderosa Laws, inoculated with P. tinctorius also production and thus regulates the metabolic activity revealed that total biomass, lateral root numbers, of plants in response to their mineral nutrition, and short-root numbers were significantly greater in Although these studies emphasized shoot nutrition inoculated than in non-inoculated seedlings. In as an important regulator of phytohormone biocontrast, Coleman, Bledsoe & Smit (1990) observed synthesis, the mineral status of roots may be equally that inoculation of Pseudotsuga menziesii with important since root apices are an active source of Hebeloma crustuliniforme and Rhizopogon vinicolor cytokinins (Short & Torrey, 1972). decreased root dry weight, total plant biomass, and…”

“…Roots are considered the major the ectomycorrhizal association, site of cytokinin synthesis (Short & Torrey, 1972; Recent improvements in the separation and deEeldman, 1975), and therefore, stimulated lateral tection of cytokinins using high-pressure liquid short-root production, as seen with 12-month-old chromatography coupled with mass spectrometry, seedlings, and enhanced total root dry weight of 14-and cytokinin-specific immunoassays offer a tremenmonth-old seedlings, may have provided additional dous tool to plant scientists. Application of these sources of cytokinin supply in the host plant, technologies to investigate hormonal relationships in Wheeler, Henson & McLaughlin (1979) suggested symbiotic associations could rapidly expand our that increased foliar cytokinins in Alnus glutinosa knowledge of microbe-host interactions.…”

“…The resulting residue was dissolved in 200 ml distilled HgO, adjusted to pH 2 5, and percolated through a Dowex 50W-4X column (50-100 mesh, 25 ml bed volume). The column was washed with 100 ml of distilled HgO, followed by 100 ml of 70 % ethanol, and adsorbed cytokinins eluted with 500 ml of 5 N NH4OH (Short & Torrey, 1972). The eluate was reduced to dryness in vacuo and the residue redissolved in 1-5 ml of 35% ethanol.…”

Multiple transcripts of a gene for a leucine-rich repeat receptor kinase from morning glory (Ipomoea nil) originate from different TATA boxes in a tissue-specific manner

“…Additional upstream sequence information was obtained by gene-specific PCR extension from the library clone (described in Bassett et al 2000) and included approximately 1144 bp upstream of the putative translation initiation site. Total RNA was isolated according to the procedure of Short and Torrey (1972) from cotyledons, first leaves and roots obtained from plants sampled 5 and 6 days after short-day induction of flowering (induced) or continuous exposure to light (vegetative). cDNA was prepared from total morning glory RNAs using the Advantage RT for PCR kit (Clontech, Palo Alto, Calif.) and a combination of random hexamers and oligo(dT) to prime cDNA synthesis (Bassett et al 2000).…”