Measurement of Indole-3-Acetic Acid in Peach Fruits (Prunus persica L. Batsch cv Redhaven) during Development

Abstract

ABSTRACIThe amount of indole-3-acetic acid (IAA) was measured in peach fruits by gas chromatography-mass spectrometry-selective ion monitoring using an isotope dilution assay with I"CC6IIAA as an internal standard throughout the growing season. Ethylene evolution of the fruit was also measured. IAA levels were 25 nanograms per gram fresh weight, 18 enous IAA concentration and observed ethylene evolution were correlated, given that auxin can promote ethylene evolution (14). MATERIALS AND METHODSPlant Material. Developing fruits of Prunus persica cv Redhaven were obtained from the University of Maryland Research Farm, Silver Spring, MD. Beginning from 18 to 88 d after anthesis fruits were harvested from around the entire periphery of the tree once or twice a week for IAA and ethylene, respectively. The abscission of unpollinated flowers was completed by d 18. At 88 d after anthesis, fruits were mature, firm-ripe, and capable of softening to full-ripe within 72 h of harvest. Uniform, upright fruiting shoots of about 60 cm length at 1.8 m height were harvested at 8:00 AM and their cut bases were immediately placed in water. One shoot was cut from each of 60 trees on each sampling date. Thirty min were needed to harvest and transport the fruit to the laboratory. The fruits were removed from their shoots immediately after transport and extraction begun.To minimize fruit-to-fruit variability only basal fruits were used for analyses. Fruits displaying visible signs of senescence, disease, or insect damage were not used. On each sampling date a minimum of 10 individual peach fruits were taken for ethylene determinations. An additional 2 replicates of a minimum of 10 pooled fruits were simultaneously extracted and assayed for endogenous levels of free IAA. In addition to the above, 8 to 10 peach ovules were excised from fruits on 60, 67, 81, and 88 d postanthesis. A single replicate of pooled ovules was extracted and endogenous free IAA levels determined on each date. This sampling protocol was used to avoid potential artifacts of frozen material and allow maximum frequency of sampling time. This latter aspect was important because our primary interest was in examining trends during development.Extraction and Measurement of Free IAA. A minimum of 10 whole fruits were added to cold (-20'C) acetone; tissue:acetone ratio 3:7 (w:v). To this mixture was added 50 to 100,000 dpm of '4C-(methylene)-IAA (59 or 51.6 mCi/mmol, Amersham containing 1.2 to 23.1 ng 12C-IAA per extract) and an estimated a 10-fold greater amount of '3C6-(benzene ring)-IAA (5.05-101 sg per extract) than the amount of free IAA in the fruit (4). Thus, the '3C6-IAA served as both an internal standard and as a carrier in order to increase sample recovery. The tissues were homogenized for 2 min and allowed to equilibrate overnight at 4°C. The crude extract was filtered, and the residue washed with 70% acetone. Acetone extracts were combined and concentrated to the aqueous phase in vacuo at 45°C. The aqueous phase was chilled to 4°C, adjusted to pH 7.0, and pa...

The fading distinctions between classical patterns of ripening in climacteric and non-climacteric fruit and the ubiquity of ethylene—An overview

“…The differences between these two cultivars of Japanese plum were explained by differences in the accumulation pattern of four mRNAs of the ethylene signal transduction components including ethylene receptors Peach Peach is classified as a climacteric fruit. Miller et al 1987 reported that concomitant with the ethylene climacteric there was also a significant increase in auxin production in the fruit. Interestingly, it had been reported that auxin can stimulate the synthesis of more climacteric ethylene (Bleecker and Kende 2000) through its inductive action on the expression of the key enzyme i.e., ACCsynthase (Abel and Theologis 1996).…”

A Rapid and Simple Procedure for Purification of Indole-3-Acetic Acid Prior to GC-SIM-MS Analysis

“…The HPLC procedure was similar to that used in our prior studies (6,13,17) except that a rapid analytical column (12.5 cm x 4.6 mm) was eluted with 20% acetonitrile/ water and 1% acetic acid. The C18 column was packed with 1.2 g of S ,u Whatman ODS-3 resin using a slurry of 1:1 ethylene glycol:methanol at 8500 psi.…”

“…It was then passed through the conditioned C18 column and prepared following the separation procedure described above for free and ester IAA. Identical procedures to those described above were used for the analysis of IAA in field grown peach fruit pericarp (13), greenhouse grown tobacco leaves (Nicotiana glauca and N. langsdorffii), and caryopses obtained from imbibed wheat seeds (Triticum aestivum cv Itana).…”

“…Techniques for identification and quantitative analysis of the plant hormone IAA have been recently improved by the utilization of HPLC and GC-MS (12) and by the use of proper internal standards, for example d4-IAA (3,4,10,14,16), d5-IAA (1,2,10,15), and [13CJ]IAA (6,13,17). Solvent extraction, concentration, and solvent partitioning have been used for IAA purification although these procedures are lengthy, laborious, and result in low recoveries.…”

Untitled

“…Organic 12 fertilizers (OF) are all natural, environmentally friendly, naturally correct soil pH, and are cost 13 effective, but OFs are messy, not readily available, and take longer time to provide visible 14 benefits to crop yield and quality. BFs are typically cost effective, improve soil quality, and 15 increase crop yields by 20-30%, but BFs are may require specialized storage, provide slower 16 benefits compared CFs, and may require different equipment for application, (Aggani et al,17 2013; Banayo et al, 2012;Ishfani, 2012;Ribaudo et al, 2011 indirectly affect plant growth and/or development, (Arshad et al, 2007;DeBont and Albers, 24 1976;Miller et al, 1987;Vandeputte et al, 2005 A study conducted by Flematti et al, 2011, suggest vegetation release increased 33 concentrations of cyanohydrins during forest fires, the cyanohydrins undergo an abiotic reaction 34 in water releasing cyanide into the soil to stimulate seed germination, (Downes et al, 2013;35 Flematti et al, 2011;Nelson et al, 2012). A competing study conducted by Abeles, 2012, 36 suggests vegetation release large portions of hydrocarbons such as ethylene during forest fires as 37 a pollutant, (Abeles, 2012;Boubel et al, 1994;Brust, 2009).…”