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Transgenic approach is an excellent way for the clarification of gene function, but it is generally difficult to create transgenic plants for most of the fruit trees including peach. Even if transgenic lines are successfully obtained, it will be extremely difficult to evaluate fruit traits due to the long juvenile phase of the plants. To overcome this problem, usage of virus vector is one of the excellent approaches. In this study, we evaluated gene function related to fruit traits in peaches via virus-induced gene silencing (VIGS). Carotenoid cleavage dioxygenase 4 (CCD4) is proposed to be the key factor responsible for carotenoid degradation in white flesh peaches. Then, we knocked down the CCD4 gene in the white flesh peaches (“Akatsuki” and “Manami”) via VIGS system. Resultantly, yellow pigmentation and increased contents of carotenoids including lutein, s-carotene, s-cryptoxanthin, zeaxanthin, and violaxanthin were observed in the agroinfiltration portions of the white flesh peaches, which is a direct evidence that CCD4 is a determinant for yellow flesh trait of peach. Our results suggested the possible application of VIGS system for functional studies of the genes related to fruit traits.

Carnation (Dianthus caryophyllus L.) belongs to the family Caryophyllaceae in the order Caryophyllales. Plants in this order do not accumulate carotenoids in petals. To understand how carotenoid accumulation is controlled in carnation petals, we analysed the expression of genes related to carotenoid accumulation. Petals at an early stage of development accumulated small amounts of carotenoids. As petals matured, their carotenoid content decreased to extremely low levels. In contrast, carnation leaves contained substantial amounts of carotenoids that are essential for photosynthesis. Most of the carotenogenic genes were expressed in petals at levels similar to those in leaves, and the expression levels of these genes increased during petal development. Genes encoding phytoene synthase and lycopene e-cyclase were exceptions. Their expression levels in petals were very low compared with those in leaves. Expression of the gene encoding carotenoid cleavage dioxygenase 4 was detected in neither leaves nor petals. These data suggest that the low levels of carotenoids in carnation petals are caused not by enzymatic degradation but rather by low rates of carotenoid biosynthesis.

Plants belong to the section Mina showed characteristic petal pigmentation among Ipomoea plants. For example, petals of Ipomoea hederifolia var. lutea display yellow color derived from carotenoids and those of Ipomoea quamoclit display red color derived from pelargonidin-based anthocyanins. In this study, the pigment composition and the expression patterns of genes for pigment biosynthesis in the petals of I. quamoclit and I. hederifolia var. lutea were analyzed to elucidate the crucial factors that determine petal color in section Mina. The petals of white-flowered I. quamoclit lack the ability to synthesize anthocyanins and carotenoids because of the suppression of anthocyanidin synthase (ANS) gene expression and transcriptional down-regulation of multiple carotenogenic genes, respectively. In petals of I. hederifolia var. lutea, the absence of dihydroflavonol 4-reductase (DFR) gene expression is responsible for the lack of anthocyanins. All F1 progeny obtained by interspecies crossing between I. quamoclit and I. hederifolia var. lutea had scarlet petal color produced by a combination of pelargonidin-based anthocyanins and carotenoids. The flavanone 3-hydroxylase gene, DFR, and ANS were expressed in the petals of the F1 progeny. The results suggest that in these section Mina species, the carotenoid-accumulating phenotype in petals is dominant, and that pelargonidin was produced in the F1 progeny by complementary expression of DFR from white-flowered I. quamoclit and ANS from I. hederifolia var. lutea.

It has long been proposed that white-flowered chrysanthemums (Chrysanthemum morifolium Ramat.) have a single dominant gene that inhibits carotenoid formation or accumulation in ray petals. However, the precise function of the proposed gene was unknown. We previously isolated a gene encoding carotenoid cleavage dioxygenase 4, designated CmCCD4a, which is specifically expressed in the ray petals of white-flowered chrysanthemums. Because CmCCD4a was a strong candidate for the single dominant gene, we analyzed the relationship between CmCCD4a expression and carotenoid content in two sets of petal color mutants. Here, we show that CmCCD4a represents a small gene family containing at least four members. Two of them, CmCCD4a-1 and CmCCD4a-2, were highly expressed in ray petals of two taxa with low carotenoid levels. In petal color mutants derived from these taxa, increases in carotenoid levels accompanied decreases in CmCCD4a expression levels in ray petals. Two different circumstances reduced the levels of CmCCD4a expression in the mutants: either a CmCCD4a gene was lost from the genome or the expression of a CmCCD4a gene was suppressed. In the latter case, suppression may be caused by the loss of a function that normally enhances CmCCD4a transcription. A stepwise decrease in the amount of CmCCD4a expression in either L1 or L2 resulted in a corresponding stepwise increase in the carotenoid content in ray petals. From these results, we propose that CmCCD4a expression is the key factor that controls the carotenoid content in ray petals of chrysanthemum.

Japanese morning glory (Ipomoea nil) is a representative plant lacking a yellow-flowered cultivar, although a few wild Ipomoea species contain carotenoids in their petals such as Ipomoea sp. (yellow petals) and I. obscura (pale-yellow petals). In the present study, carotenoid composition and the expression patterns of carotenogenic genes during petal development were compared among I. nil, I. obscura, and Ipomoea sp. to identify the factors regulating carotenoid accumulation in Ipomoea plant petals. In the early stage, the carotenoid composition in petals of all the Ipomoea plants tested was the same as in the leaves mainly showing lutein, violaxanthin, and beta-carotene (chloroplast-type carotenoids). However, in fully opened flowers, chloroplast-type carotenoids were entirely absent in I. nil, whereas they were present in trace amounts in the free form in I. obscura. At the late stage of petal development in Ipomoea sp., the majority of carotenoids were beta-cryptoxanthin, zeaxanthin, and beta-carotene (chromoplast-type carotenoids). In addition, most of them were present in the esterified form. Carotenogenic gene expression was notably lower in I. nil than in Ipomoea sp. In particular, beta-ring hydroxylase (CHYB) was considerably suppressed in petals of both I. nil and I. obscura. The CHYB expression was found to be significantly high in the petals of Ipomoea sp. during the synthesis of chromoplast-type carotenoids. The expression levels of carotenoid cleavage genes (CCD1 and CCD4) were not correlated with the amount of carotenoids in petals. These results suggest that both I. obscura and I. nil lack the ability to synthesize chromoplast-type carotenoids because of the transcriptional down-regulation of carotenogenic genes. CHYB, an enzyme that catalyses the addition of a hydroxyl residue required for esterification, was found to be a key enzyme for the accumulation of chromoplast-type carotenoids in petals.