Kinetic Comparison of Cytokinin Nucleosidase Activity Isolated from Normally Ripening and Mutant Tomato Varieties

Abstract

Kinetic parameters for cytokinin nucleosidase activity which catalyzes the deribosylation of N6(A&2-isopentenyl)adenosine (I6Ado) to produce the more "active" free base N5(A&2-isopenetyl)adenine (IlAde) were compared for a normally ripening tomato (Lycopersicon esculentum L.) cultivar Rutgers, and two mutant tomato varieties (Nor and Rin). Km for nucleosidase activity in Rutgers was lower (Km = 0.1 millimolar) than that in either Nor (Km = 0.14 millimolar) or Rin (Km = 0.13 millimolar).The characterization of tomato genetic mutants has been the subject of numerous reports (6-8, 12, 13, 15, 16). Two of these genetic mutants, Nor (nonripening) and Rin (ripening inhibitor), produce fruit which develop normally but do not undergo several of the physiological changes associated with ripening in normal tomato strains (13,16 Rolle and Chism (14). Acetone powders were prepared from whole tomato fruit. These were stored at -1 5C for later use. Acetone powder (25 g) was mixed with 50 mL of 0.1 M Tris-HCl buffer (pH 7.50) and homogenized with a Potter-Elvehjem homogenizer. The temperature of the homogenates was maintained at 0 to 4°C. The resultant slurry was filtered through double layers of cheesecloth. The filtrate was maintained within the 0 to 4°C temperature range and centrifuged for 30 min at 48,200g. The resulting supernatant fraction was chromatographed on an w-aminopentyl agarose column (1.8 cm i.d. x 25 cm length) with a 0 to 1 M linear NaCl gradient in 20 mM Tris-HCl (pH 7.5) as the eluant. Active fractions which showed turbidity on elution from the column were pooled and centrifuged at 48,200g within the 0 to 4°C range. The precipitate was suspended in 0.1 M TrisHCl buffer (pH 7.5) and retained for use in assays. Protein concentrations were determined according to the method of Bradford (2) with BSA as a standard. Enzyme AssaysA stopped assay was used for monitoring enzyme activity.The incubation mixture in a final volume of 1 mL contained 0.5 mL of enzyme preparation along with the substrate dissolved in 0.1 M Tris-HCl (pH 7.5). The assay medium was incubated in a water bath maintained at 31.5°C. Aliquots (0.2 mL) of the assay medium, were withdrawn at 0 and 3 h, and mixed with equal volumes of methanol. These were frozen at -60°C, thawed, and centrifuged at 1200g in an IEC Centra 7 centrifuge. Fifty ,uL aliquots of these extracts were analyzed for activity by HPLC. HPLC

MicroRNAs and targets in senescent litchi fruit during ambient storage and post-cold storage shelf life

“…However, the effect of auxin on the delay or acceleration of fruit senescence may depend on various factors. It has been reported that auxin can inhibit the ripening process in tomato [ 41 ], while low levels of auxin are required for seed dehiscence in Arabidopsis [ 42 ].…”

A dynamic interplay between phytohormones is required for fruit development, maturation, and ripening

“…Auxin and maybe cytokinin appear to be key regulators of fruit maturation. Genetic studies have shown that the tomato ripening inhibitor ( rin ) mutant that displays a non-ripening phenotype, have higher levels of auxin and cytokinin at breaker stage compared to wild-type fruit (Davey and Van Staden, 1978; Rolle and Chism, 1989). The suppression of a rin -like MADS -box gene in apple (Ireland et al, 2013), resulted in a maintenance of high auxin concentration during fruit maturation and fruit that did not ripen (Ireland et al, 2013; Schaffer et al, 2013).…”

Molecular programme of senescence in dry and fleshy fruits

“…Auxin (response to auxin stimulus, GO:0009733) seems to play a fundamental role in fruit maturation. For example, auxin inhibits the ripening process in tomato (Davey and Van Staden, 1978;Rolle and Chism, 1989) and apple (Ireland et al, 2013), or it controls dehiscence in Arabidopsis, where low levels are required for seed dehiscence (Sorefan et al, 2009). Several of the genes involved in auxin synthesis and response are on the list of common genes.…”