Background: We describe novel plasmid vectors for transient gene expression using Agrobacterium, infiltrated into Nicotiana benthamiana leaves. We have generated a series of pGreenII cloning vectors that are ideally suited to transient gene expression, by removing elements of conventional binary vectors necessary for stable transformation such as transformation selection genes.

Ethylene is the major effector of ripening in many fleshy fruits. In apples (Malus x domestica) the addition of ethylene causes a climacteric burst of respiration, an increase in aroma, and softening of the flesh. We have generated a transgenic line of 'Royal Gala' apple that produces no detectable levels of ethylene using antisense ACC OXIDASE, resulting in apples with no ethyleneinduced ripening attributes. In response to external ethylene these antisense fruits undergo a normal climacteric burst and produced increasing concentrations of ester, polypropanoid, and terpene volatile compounds over an 8-d period. A total of 186 candidate genes that might be involved in the production of these compounds were mined from expressed sequence tags databases and full sequence obtained. Expression patterns of 179 of these were assessed using a 15,720 oligonucleotide apple microarray. Based on sequence similarity and gene expression patterns we identified 17 candidate genes that are likely to be ethylene control points for aroma production in apple. While many of the biosynthetic steps in these pathways were represented by gene families containing two or more genes, expression patterns revealed that only a single member is typically regulated by ethylene. Only certain points within the aroma biosynthesis pathways were regulated by ethylene. Often the first step, and in all pathways the last steps, contained enzymes that were ethylene regulated. This analysis suggests that the initial and final enzymatic steps with the biosynthetic pathways are important transcriptional regulation points for aroma production in apple.

Apples have long been cultivated by humans for their fruit. They produce a complex mixture of over 200 volatile compounds [1], including alcohols, aldehydes, ketones, sesquiterpenes and esters. Esters are associated with 'fruity' attributes of fruit flavor and typically increase to high levels late in the ripening process [2]. In the commercial apple cultivar, Malus pumila cv. Royal Gala, over 30 esters have been identified [3,4]. These can be broadly separated into straight chain esters and branched chain esters. In apples, straight chain esters are thought to be biosynthesized from fatty acids via the lipoxygenase pathway [5]. In contrast branched chain esters are thought to be produced from the metabolism of branched chain amino acids such as isoleucine [6]. Of the esters produced by Royal Gala, butyl acetate, hexyl acetate, and 2-methylbutyl acetate dominate the flavor of ripe fruit, with the latter two being identified by analytical sensory panels as having the greatest impact on the attractiveness of the fruit [4]. Apple flavor is characterized by combinations of ester compounds, which increase markedly during fruit ripening. The final step in ester biosynthesis is catalyzed by alcohol acyl transferases (AATs) that use coenzyme A (CoA) donors together with alcohol acceptors as substrates. The gene MpAAT1, which produces a predicted protein containing features of other plant acyl transferases, was isolated from Malus pumila (cv. Royal Gala). The MpAAT1 gene is expressed in leaves, flowers and fruit of apple. The recombinant enzyme can utilize a range of alcohol substrates from short to medium straight chain (C3-C10), branched chain, aromatic and terpene alcohols. The enzyme can also utilize a range of short to medium chain CoAs. The binding of the alcohol substrate is rate limiting compared with the binding of the CoA substrate. Among different alcohol substrates there is more variation in turnover compared with K m values. MpAAT1 is capable of producing many esters found in Royal Gala fruit, including hexyl esters, butyl acetate and 2-methylbutyl acetate. Of these, MpAAT1 prefers to produce the hexyl esters of C3, C6 and C8 CoAs. For the acetate esters, however, MpAAT1 preference depends upon substrate concentration. At low concentrations of alcohol substrate the enzyme prefers utilizing the 2-methylbutanol over hexanol and butanol, while at high concentrations of substrate hexanol can be used at a greater rate than 2-methylbutanol and butanol. Such kinetic characteristics of AATs may therefore be another important factor in understanding how the distinct flavor profiles of different fruit are produced during ripening.Abbreviations AAT, alcohol acyl transferase; coA, coenzyme A; IPTG, isopropyl thio-b-D-galactoside; MpAAT1, apple AAT1; SPME, solid phase microextraction.

Partition of fourteen volatile compounds, representing the diverse physicochemical properties of aroma compounds, was measured by static equilibrium headspace in solutions containing the components of artificial saliva, either singly or in mixtures. Comparison of a bovine salivary mucin and pig gastric mucin showed no significant difference in partition behavior of the volatiles, so gastric mucin was used. Mucin viscosity changed with pH, but binding of volatile compounds did not show a marked dependence on pH. All combinations of the salivary components were tested for their effect on partition. Three types of behavior were noted. Partition of some compounds was unaffected by mucin, and with other compounds mucin decreased partition, whereas another group showed a decrease with mucin that was affected by the presence of salivary salts and sugar. When volatiles or sugar were added to a mucin solution, the final headspace concentration depended on the order of addition, indicating some competition for binding. These solute-mucin effects are discussed in relation to mucin structure and behavior in solution.

A sequential extraction methodology, designed to measure the solid phase partitioning of metals in soils and sediments, is described. The method uses centrifugation to pass increasing concentrations of HNO 3 through the sample, followed by ICP-AES analysis of major and trace elements of the extracts. A data-processing algorithm is used to identify the number of physico-chemical components extracted, their composition and the proportion of each in each extract. The algorithm has been successfully tested on a synthetic data set and the combination of the extraction methodology and data-processing algorithm have been tested on a contaminated soil sample (NIST SRM 2710). The 14 extracts from each duplicate experiment were analysed for 19 elements and data analysis identified nine chemically distinct soil components: pore-water residual solutes; organic, easily exchangeable; a Cu–Zn dominated phase; a Pb-dominated phase; amorphous Fe oxide/oxyhydroxide; crystalline Fe oxide; Fe–Ti oxide; and Mn oxide.