The tomato carotenoid cleavage dioxygenase 1 genes contribute to the formation of the flavor volatiles β‐ionone, pseudoionone, and geranylacetone

Abstract

SummaryVolatile terpenoid compounds, potentially derived from carotenoids, are important components of flavor and aroma in many fruits, vegetables and ornamentals. Despite their importance, little is known about the enzymes that generate these volatiles. The tomato genome contains two closely related genes potentially encoding carotenoid cleavage dioxygenases, LeCCD1A and LeCCD1B. A quantitative reverse transcriptasepolymerase chain reaction analysis revealed that one of these two genes, LeCCD1B, is highly expressed in ripening fruit (4 days post-breaker), where it constitutes 0.11% of total RNA. Unlike the related neoxanthin cleavage dioxygenases, import assays using pea chloroplasts showed that the LeCCD1 proteins are not plastidlocalized. The biochemical functions of the LeCCD1 proteins were determined by bacterial expression and in vitro assays, where it was shown that they symmetrically cleave multiple carotenoid substrates at the 9,10 (9¢,10¢) positions to produce a C 14 dialdehyde and two C 13 cyclohexones that vary depending on the substrate. The potential roles of the LeCCD1 genes in vivo were assessed in transgenic tomato plants constitutively expressing the LeCCD1B gene in reverse orientation. This over-expression of the antisense transcript led to 87-93% reductions in mRNA levels of both LeCCD1A and LeCCD1B in the leaves and fruits of selected lines. Transgenic plants exhibited no obvious morphological alterations. High-performance liquid chromatography analysis showed no significant modification in the carotenoid content of fruit tissue. However, volatile analysis showed a ‡50% decrease in b-ionone (a b-carotene-derived C 13 cyclohexone) and a ‡60% decrease in geranylacetone (a C 13 acyclic product likely derived from a lycopene precursor) in selected lines, implicating the LeCCD1 genes in the formation of these important flavor volatiles in vivo.

Abscisic acid levels in tomato ovaries are regulated by LeNCED1 and SlCYP707A1

“…The three other CCDs described are probably not related to ABA biosynthesis as they do not group with LeNCED1. Moreover, TC154637 and TC154638 have already been described as carotenoid cleavage dioxygenase 1 genes involved in volatile terpenoid production (Simkin et al 2004). Therefore, LeNCED1 appears to produce the most represented NCED transcript in ovary and possibly is the only NCED gene in tomato.…”

Cloning and characterisation of a maize carotenoid cleavage dioxygenase (ZmCCD1) and its involvement in the biosynthesis of apocarotenoids with various roles in mutualistic and parasitic interactions

“…3c), colonies indeed failed to develop the yellow to orange colour that did show up in the empty Fig. 2 Alignment of the deduced amino acid sequences of maize CCD1A (ZmCCD1-A) and CCD1B (ZmCCD1-B; DQ539625) with CCD1 from petunia (PhCCD1; AY576003) (Simkin et al 2004b), tomato (LeCCD1; AY576001) (Simkin et al 2004a), grape (VvCCD1; AY856353) (Mathieu et al 2005), Crocus (CsCCD1; AJ132927) (Bouvier et al 2003) and Arabidopsis (AtCCD1; AJ005813) (Neill et al 1998) vector control, showing that ZmCCD1B cleaves the carotenoids produced by the E. coli strains (Fig. 3).…”

“…CCD1 genes have been shown to be constitutively expressed in these tissues. For example, two variants of tomato CCD1 can cleave several carotenoid substrates and some of the cleavage products are present in or are emitted from tomato fruits, such as 6-methyl-5-hepten-2-one, -ionone and geranylacetone, which play a key role in tomato Xavour (Simkin et al 2004a;Vogel et al 2008). Similarly, a petunia CCD1 is leading to -ionone biosynthesis in the Xowers when -carotene is cleaved and this volatile is possibly involved in the attraction of pollinating insects (Simkin et al 2004b).…”

Biosynthesis of plant‐derived flavor compounds

“…In tomato fruit, b-ionone is present at very low concentrations (4 nl l )1 ), but due to its low odor threshold (0.007 nl l )1 ) is the second most important volatile contributing to fruit flavor (Baldwin et al, 2000). Silencing of LeCCD1A and LeCCD1B resulted in a significant decrease in the b-ionone content of ripe fruits, implying a role for these genes in C13 norisoprenoid synthesis in vivo (Simkin et al, 2004). Reduction of Petunia hybrida CCD1 transcript levels in transgenic plants led to a 58-76% decrease in b-ionone synthesis in the corollas of selected petunia lines, indicating a significant role for this enzyme in volatile synthesis (Simkin et al, 2004).…”

“…Therefore, they have been subject to extensive research in recent years with regard to their structure and flavor potential (Winterhalter and Rouseff, 2002). The synthesis of b-ionone, geranyl acetone and 6-methyl-5-hepten-2-one in tomato fruits increases 10-20-fold during fruit ripening, and these compounds were produced by the activity of the genes LeCCD1A and LeCCD1B that were isolated from tomato fruits (Simkin et al, 2004). In tomato fruit, b-ionone is present at very low concentrations (4 nl l )1 ), but due to its low odor threshold (0.007 nl l )1 ) is the second most important volatile contributing to fruit flavor (Baldwin et al, 2000).…”