Alcohol acyl transferase genes at a high-flavor intensity locus contribute to ester biosynthesis in kiwifruit.

Volatile esters are key compounds contributing to flavor intensity in commonly consumed fruits including apple (Malus domestica), strawberry (Fragaria spp.), and banana (Musa sapientum). In kiwifruit (Actinidia spp.), ethyl butanoate and other esters have been proposed to contribute fruity, sweet notes to commercial cultivars. Here, we investigated the genetic basis for ester production in Actinidia in an A. chinensis mapping population (AcMPO). A major quantitative trait loci for the production of multiple esters was identified at the high-flavor intensity (HiFI) locus on chromosome 20. This locus co-located with eight tandemly arrayed alcohol acyl transferase genes in the Red5 genome that were expressed in a ripening-specific fashion that corresponded with ester production. Biochemical characterization suggested two genes at the HiFI locus, alcohol acyl transferase 16-b/c (AT16-MPb/c), probably contributed most to the production of ethyl butanoate. A third gene, AT16-MPa, probably contributed more to hexyl butanoate and butyl hexanoate production, two esters that segregated in AcMPO. Sensory analysis of AcMPO indicated that fruit from segregating lines with high ester concentrations were more commonly described as being "fruity" as opposed to "beany". The downregulation of AT16-MPa-c by RNAi reduced ester production in ripe "Hort16A" fruit by >90%. Gas chromatography-olfactometry indicated the loss of the major "fruity" notes contributed by ethyl butanoate. A comparison of unimproved Actinidia germplasm with those of commercial cultivars indicated that the selection of fruit with high concentrations of alkyl esters (but not green note aldehydes) was probably an important selection trait in kiwifruit cultivation. Understanding ester production at the HiFI locus is a critical step toward maintaining and improving flavor intensity in kiwifruit.

The impact of cold storage and ethylene on volatile ester production and aroma perception in 'Hort16A' kiwifruit.

Fruit esters are regarded as key volatiles for fruit aroma. In this study, the effects of cold storage on volatile ester levels of 'Hort16A' (Actinidia chinensis Planch. var chinensis) kiwifruit were examined and the changes in aroma perception investigated. Cold storage (1.5°C) for two or four months of fruit matched for firmness and soluble solids concentration resulted in a significant reduction in aroma-related esters such as methyl/ethyl propanoate, methyl/ethyl butanoate and methyl/ethyl hexanoate. Levels of these esters, however, were restored by ethylene treatment (100ppm, 24h) before ripening. A sensory panel found that "tropical" and "fruit candy" aroma was stronger and "green" odour notes less intensively perceived in kiwifruit which were ethylene-treated after cold storage compared to untreated fruit. The key findings presented in this study may lead to further work on the ethylene pathway, and innovative storage and marketing solutions for current and novel fruit cultivars.

The AAT1 locus is critical for the biosynthesis of esters contributing to 'ripe apple' flavour in 'Royal Gala' and 'Granny Smith' apples.

The 'fruity' attributes of ripe apples (Malus × domestica) arise from our perception of a combination of volatile ester compounds. Phenotypic variability in ester production was investigated using a segregating population from a 'Royal Gala' (RG; high ester production) × 'Granny Smith' (GS; low ester production) cross, as well as in transgenic RG plants in which expression of the alcohol acyl transferase 1 (AAT1) gene was reduced. In the RG × GS population, 46 quantitative trait loci (QTLs) for the production of esters and alcohols were identified on 15 linkage groups (LGs). The major QTL for 35 individual compounds was positioned on LG2 and co-located with AAT1. Multiple AAT1 gene variants were identified in RG and GS, but only two (AAT1-RGa and AAT1-GSa) were functional. AAT1-RGa and AAT1-GSa were both highly expressed in the cortex and skin of ripe fruit, but AAT1 protein was observed mainly in the skin. Transgenic RG specifically reduced in AAT1 expression showed reduced levels of most key esters in ripe fruit. Differences in the ripe fruit aroma could be perceived by sensory analysis. The transgenic lines also showed altered ratios of biosynthetic precursor alcohols and aldehydes, and expression of a number of ester biosynthetic genes increased, presumably in response to the increased substrate pool. These results indicate that the AAT1 locus is critical for the biosynthesis of esters contributing to a 'ripe apple' flavour.

Apple SEPALLATA1/2-like genes control fruit flesh development and ripening.

Flowering plants utilize different floral structures to develop flesh tissue in fruits. Here we show that suppression of the homeologous SEPALLATA1/2-like genes MADS8 and MADS9 in the fleshy fruit apple (Malus x domestica) leads to sepaloid petals and greatly reduced fruit flesh. Immunolabelling of cell-wall epitopes and differential staining showed that the developing hypanthium (from which the apple flesh develops) of MADS8/9-suppressed apple flowers lacks a tissue layer, and the remaining flesh tissue of fully developed apples has considerably smaller cells. From these observations, it is proposed that MADS8 and MADS9 control the development of discrete zones within the hypanthium tissue, and therefore fruit flesh, and also act as foundations for development of different floral organs. At fruit maturity, the MADS8/9-suppressed apples do not ripen in terms of both developmentally controlled ripening characters, such as starch degradation, and ethylene-modulated ripening traits. Transient assays suggest that, like the RIN gene in tomato, the MADS9 gene acts as a transcriptional activator of the ethylene biosynthesis enzyme, 1-aminocyclopropane-1-carboxylate (ACC) synthase 1. The existence of a single class of genes that regulate both flesh formation and ripening provides an evolutionary tool for controlling two critical aspects of fleshy fruit development.

Changes in the bound aroma profiles of 'Hayward' and 'Hort16A' kiwifruit (Actinidia spp.) during ripening and GC-olfactometry analysis.

Bound volatiles are recognised as a potential source of aroma compounds in fruits. In this study, the bound volatiles of Actinidia deliciosa 'Hayward' and A. chinensis 'Hort16A' were studied at three different ripening stages. The bound volatile content tended to increase as the fruit ripened from under-ripe to ripe, and then decreased in over-ripe fruit. Glycosides of (Z)-3-hexen-1-ol and hexanol (green-note volatiles) were present in considerable amounts. ß-Glucosidase activity in 'Hayward' and 'Hort16A' remained fairly constant throughout ripening. GC-olfactometry analysis of the hydrolysates of ripe 'Hayward' and 'Hort16A' revealed the presence of 2-phenylethanol, ß-damascenone, vanillin and 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF). This is the first report of DMHF in 'Hayward' kiwifruit. For both 'Hayward' and 'Hort16A', the odour-active compounds found in the bound volatile extracts were different from those reported as contributors to the aroma of the ripe fruit, suggesting that bound volatiles are probably not significant contributors to the aroma of ripe kiwifruit.

Characterisation of bound volatile compounds of a low flavour kiwifruit species: Actinidia eriantha.

Aroma compounds in fruit are known to occur in free and glycosidically bound forms. The bound volatile fraction of a low flavour kiwifruit species, Actinidia eriantha, was studied. The fruit have a bland and grassy flavour. Glycosidic precursors were isolated from juice by adsorption onto an Amberlite XAD-2 column. After enzymatic hydrolysis with Rapidase AR2000, the released aglycones were analysed by GC-MS. Alcohols, terpenoids and phenolics were the most numerously represented compound classes. Alcohols, benzenoids and phenolics showed the highest concentrations. Major compounds were 2-phenylethanol, furfuryl alcohol, (Z)-3-hexen-1-ol, coniferyl alcohol, isoamyl alcohol and linolenic acid. Several of the bound compounds found, including linoleic, linolenic and benzoic acids and coniferyl alcohol, are precursors of odorous volatiles. Many compounds detected as bound volatiles have not been previously reported as free volatiles in A. eriantha. The bound volatile composition of A. eriantha also showed differences with those of other kiwifruit species.

Characterization of the bound volatile extract from baby kiwi (Actinidia arguta).

The glycosidically bound volatile fraction of baby kiwi ( Actinidia arguta ) was studied. Glycosidic precursors were isolated from juice by adsorption onto an Amberlite XAD-2 column. After enzymatic hydrolysis with Rapidase AR2000, the released aglycones were analyzed by GC-MS. Alcohols, terpenoids, and benzenoids were the most abundant compound classes. Aromatic compounds and norisoprenoids showed the highest concentrations. Major compounds were 2,5-dimethyl-4-hydroxy-3(2H)-furanone (Furaneol), benzyl alcohol, 3-hydroxy-ß-damascone, hexanal, and (Z)-3-hexen-1-ol. Precursors of aroma compounds including benzoic acid, cinnamic acid, and coniferyl alcohol were also found. Eugenol, raspberry ketone, and 4-vinylguaiacol were identified for the first time in the fruit of an Actinidia species. The high concentration of 2,5-dimethyl-4-hydroxy-3(2H)-furanone in bound form (95.36 µg/kg) is particularly interesting and justifies further investigation.

Tuning the herbivore-induced ethylene burst: the role of transcript accumulation and ethylene perception in Nicotiana attenuata.

Caterpillar-induced ethylene emissions play an important role in plant-herbivore interactions. The ethylene burst that ensues after attack exceeds wound-induced ethylene emissions, but the mechanisms responsible remain unknown. Adding larval oral secretions (OS) to wounds mimics this ethylene burst. We demonstrate that fatty acid-amino acid conjugates are the responsible elicitors in Manduca sexta OS, and identify genes that are important in OS-elicited ethylene biosynthesis and perception in the larvae's host, Nicotiana attenuata, by examining the consequences of gene silencing on OS-elicited ethylene emissions, as quantified by photo-acoustic spectroscopy. OS elicitation increased transcript accumulation of ACC synthase (ACS), virus-induced gene silencing of ACS halved the OS-elicited ethylene release, and ACC supplementation to ACS-silenced plants restored ethylene emissions, demonstrating that ACS activity limits the rate of release. Silencing three wound- or OS-elicited ACC oxidase (ACO) genes with an ACO consensus fragment abolished the OS-elicited ethylene release. Virus-induced gene silencing of each ACO individually revealed that only NaACO2a and NaACO3 regulate the OS-elicited ethylene release. Transforming plants with various etr1-1 constructs rendered them differentially 'deaf' to ethylene, and dramatically increased the OS-elicited ethylene burst, largely without regulating the transcripts of biosynthetic genes. The volume of the OS-elicited ethylene 'scream' was proportional to the plant's deafness, as determined by 1-MCP treatments. We conclude that the OS-elicited ethylene burst is tuned by a tag-team of transcriptional responses and ethylene perception. Ethylene signaling is shown to be essential in regulating two traits that are important in the N. attenuata-M. sexta interaction: OS-induced nicotine levels and floral longevity.