Reassessing the role of YUCCAs in auxin biosynthesis.

It is remarkable that although auxin was the first growth-promoting plant hormone to be discovered, and although more researchers work on this hormone than on any other, we cannot be definitive about the pathways of auxin synthesis in plants. In 2001, there appeared to be a dramatic development in this field, with the announcement of a new gene, and a new intermediate, purportedly from the tryptamine pathway for converting tryptophan to the main endogenous auxin, indole-3-acetic acid (IAA). Recently, however, we presented evidence challenging the original and subsequent identifications of the intermediate concerned.

Reassessing the role of N-hydroxytryptamine in auxin biosynthesis.

The tryptamine pathway is one of five proposed pathways for the biosynthesis of indole-3-acetic acid (IAA), the primary auxin in plants. The enzymes AtYUC1 (Arabidopsis thaliana), FZY (Solanum lycopersicum), and ZmYUC (Zea mays) are reported to catalyze the conversion of tryptamine to N-hydroxytryptamine, putatively a rate-limiting step of the tryptamine pathway for IAA biosynthesis. This conclusion was based on in vitro assays followed by mass spectrometry or HPLC analyses. However, there are major inconsistencies between the mass spectra reported for the reaction products. Here, we present mass spectral data for authentic N-hydroxytryptamine, 5-hydroxytryptamine (serotonin), and tryptamine to demonstrate that at least some of the published mass spectral data for the YUC in vitro product are not consistent with N-hydroxytryptamine. We also show that tryptamine is not metabolized to IAA in pea (Pisum sativum) seeds, even though a PsYUC-like gene is strongly expressed in these organs. Combining these findings, we propose that at present there is insufficient evidence to consider N-hydroxytryptamine an intermediate for IAA biosynthesis.

Denaturation of beta-lactoglobulin in pressure-treated skim milk.

The kinetics of beta-lactoglobulin (beta-LG) denaturation in pressure-treated reconstituted skim milk samples over a wide pressurization range (100-600 MPa) and at various temperatures (10-40 degrees C) was studied. Denaturation was extremely dependent on the pressure and duration of treatment. At 100 MPa, no denaturation was observed regardless of the temperature or the holding time. At higher pressures, the level of denaturation increased with an increasing holding time at a constant pressure or with increasing pressure at a constant holding time. At 200 MPa, there was only a small effect of changing the temperature at pressurization. However, at higher pressures, increasing the temperature from 10 to 40 degrees C markedly increased the rate of denaturation. The two major genetic variants of beta-LG (A and B) behaved similarly to pressure treatment, although the B variant appeared to denature slightly faster than the A variant at low pressures (< or =400 MPa). The denaturation could be described as a second-order process for both beta-LG variants. There was a marked change in pressure dependence at about 300 MPa, which resulted in markedly different activation volumes in the two pressure ranges. Evaluation of the kinetic and thermodynamic parameters suggested that there may have been a transition from an aggregation-limited reaction to an unfolding-limited reaction as the pressure was increased.

Rheological properties of acid gels prepared from heated pH-adjusted skim milk.

Reconstituted skim milk was adjusted to pH values between 6.5 and 7.1 and heated (90 degrees C) for up to 30 min. The skim milk samples were then readjusted to pH 6.7. Acid gels prepared from heated milk had markedly higher G ' values, a reduced gelation time, and an increased gelation pH than those prepared from unheated milk. An increased pH at heating decreased the gelation time, increased the gelation pH, and increased the final G ' of acid set gels prepared from the heated milk samples. There were only small differences in the level of whey protein denaturation in the samples at different pH values, and these differences could not account for the differences in the G ' of the acid gels. The levels of denatured whey protein associated with the casein micelles decreased and the levels of soluble denatured whey proteins increased as the pH at heating was increased. The results indicated that the soluble denatured whey proteins had a greater effect on the final G ' of the acid gels than the denatured whey proteins associated with the casein micelles.

Heat-induced redistribution of disulfide bonds in milk proteins. 1. Bovine beta-lactoglobulin.

Changes in the structure and chemistry of beta-lactoglobulin (beta-LG) play an important role in the processing and functionality of milk products. In model beta-LG systems, there is evidence that the aggregates of heated beta-LG are held together by a mixture of intermolecular non-covalent association and heat-induced non-native disulfide bonds. Although a number of non-native disulfide bonds have been identified, little is known about the initial inter- and intramolecular disulfide bond rearrangements that occur as a result of heating. These interchange reactions were explored by examining the products of heat treatment to determine the novel disulfide bonds that form in the heated beta-LG aggregates. The native protein and heat-induced aggregates were hydrolyzed by trypsin, and the resulting peptides, before and after reduction with dithiothreitol, were separated by high-performance liquid chromatography and their identities confirmed by electrospray ionization mass spectrometry. Comparisons of these peptide patterns showed that some of the Cys160 was in the reduced form in heated beta-LG aggregates, indicating that the Cys160-Cys66 disulfide bond had been broken during heating. This finding suggests that disulfide bond interchange reactions between beta-LG non-native monomers, or polymers, and other proteins could occur largely via Cys160.

Heat-induced redistribution of disulfide bonds in milk proteins. 2. Disulfide bonding patterns between bovine beta-lactoglobulin and kappa-casein.

Heat treatment of milk causes the heat-denaturable whey proteins to aggregate with kappa-casein (kappa-CN) via thiol-disulfide bond interchange reactions. The particular disulfide bonds that are important in the aggregates are uncertain, although Cys(121) of beta-lactoglobulin (beta-LG) has been implicated. The reaction at 60 degrees C between beta-LG A and an activated kappa-CN formed small disulfide-bonded aggregates. The tryptic peptides from this model system included a peptide with a disulfide bond between a Cys residue in the triple-Cys peptide [beta-LG(102-124)] and kappa-CN Cys(88) and others between kappa-CN Cys(88) or kappa-CN Cys(11) and beta-LG Cys(160). Only the latter two novel disulfide bonds were identified in heated (90 degrees C/20 min) milk. Application of computational search tools, notably MS2Assign and SearchXLinks, to the mass spectrometry (MS) and collision-induced dissociation (CID)-MS data was very valuable for identifying possible disulfide-bonded peptides. In two instances, peptides with measured masses of 4275.07 and 2312.07 were tentatively assigned to beta-LG(102-135):kappa-CN(11-13) and beta-LG A(61-69):kappa-CN(87-97), respectively. However, sequencing using the CID-MS data demonstrated that they were, in fact, beta-LG(1-40) and beta-LG(41-60), respectively. This study supports the notion that reversible intramolecular disulfide-bond interchange precedes the intermolecular interchange reactions.