SUMMARYWe have developed an assay based on rice embryogenic callus for rapid functional characterization of metabolic genes. We validated the assay using a selection of well-characterized genes with known functions in the carotenoid biosynthesis pathway, allowing rapid visual screening of callus phenotypes based on tissue color. We then used the system to identify the functions of two uncharacterized genes: a chemically synthesized b-carotene ketolase gene optimized for maize codon usage, and a wild-type Arabidopsis thaliana ortholog of the cauliflower , we found that the wild-type Orange allele was sufficient to induce chromoplast differentiation. We also found that chromoplast differentiation was induced by increasing the availability of precursors and thus driving flux through the pathway, even in the absence of Orange. Remarkably, we found that diverse endosperm-specific promoters were highly active in rice callus despite their restricted activity in mature plants. Our callus system provides a unique opportunity to predict the effect of metabolic engineering in complex pathways, and provides a starting point for quantitative modeling and the rational design of engineering strategies using synthetic biology. We discuss the impact of our data on analysis and engineering of the carotenoid biosynthesis pathway.

Various carotenoids were analyzed by ultra-high-pressure liquid chromatography with tandem mass spectrometry detection (UHPLC-MS/MS). Three different techniques to ionize the carotenoids were compared: electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI). For all of the carotenoids tested, it was possible to obtain characteristic transitions for their unequivocal identification using each ionization technique. APCI was shown to be a more powerful technique to ionize the carotenoids than ESI or APPI. Transitions to differentiate carotenoids that coelute were determined to distinguish antheraxanthin from astaxanthin and lutein from zeaxanthin. In addition, four dopants were evaluated to improve ionization and enhance the carotenoid signal strength in APPI. These dopants were acetone, toluene, anisole, and chlorobenzene. Carotenoids improved their response in almost all cases when a dopant was used. The use of dopants allowed the enhancement of the carotenoid signals strength up to 178-fold.

The effect of different water activities (aw, 0.968, 0.956, 0.944, 0.925) and temperature (25 degrees C and 30 degrees C) on colonization and production of fumonisin B1 (FB1) and B2 (FB2) on sterile layers of maize by Fusarium proliferatum and F. moniliforme isolates was determined over periods of 6 weeks. Generally, both F. moniliforme and F. proliferatum grew faster with increasing aw and best at 30 degrees C. All three isolates produced more FB1 than FB2 regardless of aw or temperature. Very little FB1 and FB2 were produced at 0.925 aw, with maximum produced at 0.956 and 0.968 aw at both temperatures tested. Most FB1 and FB2 were produced by F. moniliforme (25N), followed by F. proliferatum isolates (73N and 131N). At all aw levels and both temperatures there was an increase in FB1 and FB2 concentration with time. Statistical analyses of aw, temperature, time, two- and three-way interactions showed some significant differences between isolates and FB1 and FB2 production.

We performed a number of tests with the aim to develop an effective extraction method for the analysis of carotenoid content in maize seed. Mixtures of methanol–ethyl acetate (6:4, v/v) and methanol–tetrahydrofuran (1:1, v/v) were the most effective solvent systems for carotenoid extraction from maize endosperm under the conditions assayed. In addition, we also addressed sample preparation prior to the analysis of carotenoids by liquid chromatography (LC). The LC response of extracted carotenoids and standards in several solvents was evaluated and results were related to the degree of solubility of these pigments. Three key factors were found to be important when selecting a suitable injection solvent: compatibility between the mobile phase and injection solvent, carotenoid polarity and content in the matrix.

Two isolates each of Fusarium moniliforme and Fusarium proliferatum isolated from maize were compared for growth and fumonisin B 1 production on maize, wheat, and barley extract agars and on irradiated maize, wheat, and barley grain in relation to water availability and temperature. Growth rates of both Fusarium spp. were similar or higher on wheat and barley than on maize extract agar. Regardless of temperature, all isolates produced fumonisin B 1 only on irradiated maize grain, but not on wheat or barley. This might be a reason for the low natural occurrence of fumonisins on those grains.