Indole-3-acetic acid (IAA) labeled with 13C in the six carbons of the benzene ring is described for use as an internal standard for quantitative mass spectral analysis of IAA by gas chromatography/selected ion mon-itoring. ['3C6jIAA was compared to the available deuterium labeled compounds and shown to offer the advantages of nonexchangeability of the isotope label, high isotopic enrichment, and chromatographic properties identical to that of the unlabeled compound. The utility of I'3C46IAA for measurement of endogenous IAA levels was demonstrated by analysis of IAA in Lemna gibba G-3.Techniques for identification and quantitative analysis of endogenous plant hormones are important for studies of the physiology and biochemistry of these regulatory messengers of higher plants. The use of combined GC-MS for both identification and quantification has proven to be the most accurate and certain method for plant hormone analysis. Even MS has its limitations and a major difficulty in the application of GC-MS to plant hormone analysis has been the limited availability of the appropriate stable isotope labeled 'heavy' internal standards.Various internal standards have been utilized for the analysis of IAA. For example, 5-methyl-IAA (26), indole-3-butyric acid (20), and indole-3-propionic acid (24) (15) synthesized two ring labeled compounds. The most effective of the two compounds described was 4,5,6,7-d4-IAA, and this internal standard has been used for a number of investigations (5, 12,13, 19,23 MATERIALS AND METHODS Apparatus. All mass spectra and GC-SIM2-MS were obtained on a Hewlett-Packard3 5992A GC-MS. The instrument was modified as follows: HPl 8740B capillary inlet system, direct capillary connection, improved ion source, Edwards E2M2 roughing pump, and a 'B' type diffusion pump. SIM analysis was done using a 4 ion program dwelling on each ion for 400 ms. For methyl esters using ['3C6JIAA as the internal standard, the ions at m/z 130, 136, 189, and 195 were monitored. The GC-MS was operated for SIM using underresolved peaks ('fat peak monitoring') and a window size of 0.30 atomic mass unit. All work was performed on WCOT fused silica columns. Two types were used: a 12 meter cross-linked methyl silicone column with a film thickness of 0.33 ,um and an internal diameter of 0.20 mm (Hewlett-Packard 19091-60312) and an 11 meter midpolarity bonded phase column of CP Sil 19 CB with a film thickness of 0.18 Am and an internal diameter of 0.31 mm (Chrompack 7732-214620). The injector was at 250°C and He at 1 ml/min was the carrier gas. With the Hewlett-Packard column the oven was at 1 60°C for 2 min followed by temperature programming at 10°C/min. The Chrompack column was held at 140°C for 2 min followed by temperature programming at 5°C/ min. Injections were made in the splitless mode with the column vented for the first 1.5 min.Fourier transform IR spectra were obtained in KBr on a Nicolet 60SX instrument.Reagents. Stable isotope labeled compounds were obtained as follows: Indole-3-acetic-2,2-d2 acid (MD-170...

Fruits of tomato, Lycopersicon esculentum Mill. cv Liberty, ripen slowly and have a prolonged keeping quality. Ethylene production and the levels of polyamines in pericarp of cv Liberty, Pik Red, and Rutgers were measured in relation to fruit development. Depending on the stage of fruit development, Liberty produced between 16 and 38% of the ethylene produced by Pik Red and Rutgers. The polyamines putrescine, spermidine, and spermine were present in all cultivars. Cadaverine was detected only in Rutgers. Levels of putrescine and spermidine declined between the immature and mature green stages of development and prior to the onset of climacteric ethylene production. In Pik Red and Rutgers, the decline persisted, whereas in Liberty, the putrescine level increased during ripening. Ripe pericarp of Liberty contained about three and six times more free (unconjugated) polyamines than Pik Red and Rutgers, respectively. No pronounced changes in spermidine or cadaverine occurred during ripening. The increase in the free polyamine level in ripe pericarp of Liberty may account for the reduction of climacteric ethylene production, and prolonged storage life.A regulatory role for polyamines in plants is suggested by their ubiquity, their abundance in actively growing tissues and their decline in senescing tissues, the regulation of their production by factors that affect plant growth and development, and their effects on plant growth and development when applied to plants (9,10,22). Little is known about the role of polyamines in fruit development. In avocado (13, 26), apple (6), pear (25), and tomato cv Rutgers (4) fruits, free polyamine levels decline during fruit development. An increase is observed in the fruits of mandarin (16), Shamouti orange (11), and tomato landrace Alcobaca containing the recessive allele alc (7) during fruit maturation and ripening. Infusing polyamines into pear fruits delayed fruit ripening (25). These findings suggest that free polyamines serve as endogenous antisenescence agents.Ethylene is a senescence-promoting hormone and accelerates fruit ripening (1, 27). Free polyamines inhibited ethylene production in a variety of tissues (2,5,8,24) opposite effects in relation to fruit ripening and senescence (3,5,9,22,26). Because of this, polyamine and ethylene physiologies may be linked during fruit development. This paper describes the changes in free polyamine levels and ethylene production during fruit development in pericarp from normal and slow ripening tomato cultivars. MATERIALS AND METHODS Plant MaterialFruits at various stages ofdevelopment were harvested from greenhouse-grown plants oftomato (Lycopersicon esculentum Mill.) cv Liberty, Pik Red, and Rutgers. Fruits were graded for maturity and ripening stages (12,17). The six fruit stages used were immature green, mature green, breaker, pink, light red, and red. Polyamine AnalysisPolyamine analyses were performed as described elsewhere (23). Because free, but not conjugated, polyamines have been implicated as endogenous antisenescenc...

We present evidence that the role of tryptophan and other potential intermediates in the pathways that could lead to indole derivatives needs to be reexamined. Two lines of Lemna gibba were tested for uptake of [15N-indole]-labeled tryptophan isomers and incorporation of that label into free indole-3-acetic acid (IAA). Both lines required levels of L-['5Njtryptophan 2 to 3 orders of magnitude over endogenous levels in order to obtain measurable incorporation of label into IAA. Labeled L-tryptophan was extract-able from plant tissue after feeding and showed no measurable isomerization into D-tryptophan. D-['5N]tryptophan supplied to Lemna at rates of approximately 400 times excess of endogenous D-tryptophan levels (to yield an isotopic enrichment equal to that which allowed detection of the incorporation of L-tryptophan into IAA), did not result in measurable incorporation of label into free IAA. These results demonstrate that L-tryptophan is a more direct precursor to IAA than the D isomer and suggest (a) that the availability of tryptophan in vivo is not a limiting factor in the biosynthesis of IAA, thus implying that other regulatory mechanisms are in operation and (b) that L-tryptophan also may not be a primary precursor to IAA in plants. Many studies of IAA biosynthesis have shown that TRP4 is the primary precursor of IAA by one of several different pathways (for reviews, see refs. 1, 24). However, studies of in vivo biosynthesis ofIAA in maize seedlings have demonstrated that conversion of TRP to IAA does not occur at significant rates (9, 17, 21). The simple model for TRP involvement in IAA biosynthesis is further complicated by the natural occurrence of the 12 carbon indole, IBA, in some plant tissues (8, ' 3Present address: USDA/ARS Plant Hormone Laboratory, Belts-ville, MD 20705. 4Abbreviations: TRP, tryptophan; IBA, indole-3-butyric acid; PL, parental line; GC-MS-SIM, selected ion monitoring GC-MS; DIP-MS, direct insertion probe-MS; El, electron impact. 1203 23). The metabolic conversion of an indolic precursor to IBA has not been demonstrated; however, the side chain reduction of IBA to IAA has been shown to occur in plants (10, 12). These results present an interesting problem with respect to the hypothesis that TRP is the primary precursor to indoleal-kanoic acids in plants. The in vivo role of TRP and other potential intermediates in pathways that could lead to indole derivatives clearly needs to be reexamined. Recently, D-TRP, rather than the much more abundant L isomer, has been proposed to be a direct biosynthetic precursor of IAA based on evidence of gibberellin enhanced race-mization of L-TRP to D-TRP, which could then be transa-minated to indolepyruvic acid (19, 20). The evidence for the in vivo operation of this pathway is difficult to interpret because correction of the specific activity of the applied labeled compounds, necessitated by the differences in uptake and size of internal pools, was not done. Our studies of IAA biosynthesis in vivo have been facilitated by the use of an IAA overp...

Resonance Raman and electronic absorption spectra obtained at various pH values for the Fe3+ form of distal F54 mutants of Coprinus cinereus peroxidase are reported, together with the Fe2+ form and fluoride and imidazole adducts at pH 6.0, 5.0, and 10.5, respectively. The distal phenylalanine residue has been replaced by the small aliphatic residues glycine and valine and the hydrogen-bonding aromatic residues tyrosine and tryptophan (F54G, -V, -Y, and -W, respectively). These mutations resulted in transitions between ferric high-spin five-coordinate and six-coordinate forms, and caused a decrease of the pKa of the alkaline transition together with a higher tendency for unfolding. The mutations also alter the ability of the proteins to bind fluoride in such a way that those that are six-coordinate at pH 5.0 bind more strongly than both wild-type CIP and F54Y which are five-coordinate at this pH value. The data provide evidence that the architecture of the distal pocket of CIP is altered by the mutations. Direct evidence is provided that the distal phenylalanine plays an important role in controlling the conjugation between the vinyl double bonds and the porphyrin macrocycle, as indicated by the reorientation of the vinyl groups upon mutation of phenylalanine with the small aliphatic side chains of glycine and valine residues. Furthermore, it appears that the presence of the hydrogen-bonding tyrosine or tryptophan in the cavity increases the pKa of the distal histidine for protonation compared with that of wild-type CIP.