Isolation and antisense suppression of flavonoid 3', 5'-hydroxylase modifies flower pigments and colour in cyclamen

<p>Abstract</p> <p>Background</p> <p>Cyclamen is a popular and economically significant pot plant crop in several countries. Molecular breeding technologies provide opportunities to metabolically engineer the well-characterized flavonoid biosynthetic pathway for altered anthocyanin profile and hence the colour of the flower. Previously we reported on a genetic transformation system for cyclamen. Our aim in this study was to change pigment profiles and flower colours in cyclamen through the suppression of flavonoid 3', 5'-hydroxylase, an enzyme in the flavonoid pathway that plays a determining role in the colour of anthocyanin pigments.</p> <p>Results</p> <p>A full-length cDNA putatively identified as a <it>F3'5'H </it>(<it>CpF3'5'H</it>) was isolated from cyclamen flower tissue. Amino acid and phylogeny analyses indicated the <it>CpF3'5'H </it>encodes a F3'5'H enzyme. Two cultivars of minicyclamen were transformed via <it>Agrobacterium tumefaciens </it>with an antisense <it>CpF3'5'H </it>construct. Flowers of the transgenic lines showed modified colour and this correlated positively with the loss of endogenous <it>F3'5'H </it>transcript. Changes in observed colour were confirmed by colorimeter measurements, with an overall loss in intensity of colour (C) in the transgenic lines and a shift in hue from purple to red/pink in one cultivar. HPLC analysis showed that delphinidin-derived pigment levels were reduced in transgenic lines relative to control lines while the percentage of cyanidin-derived pigments increased. Total anthocyanin concentration was reduced up to 80% in some transgenic lines and a smaller increase in flavonol concentration was recorded. Differences were also seen in the ratio of flavonol types that accumulated.</p> <p>Conclusion</p> <p>To our knowledge this is the first report of genetic modification of the anthocyanin pathway in the commercially important species cyclamen. The effects of suppressing a key enzyme, F3'5'H, were wide ranging, extending from anthocyanins to other branches of the flavonoid pathway. The results illustrate the complexity involved in modifying a biosynthetic pathway with multiple branch points to different end products and provides important information for future flower colour modification experiments in cyclamen.</p

Betalain production is possible in anthocyanin-producing plant species given the presence of DOPA-dioxygenase and L-DOPA

<p>Abstract</p> <p>Background</p> <p>Carotenoids and anthocyanins are the predominant non-chlorophyll pigments in plants. However, certain families within the order Caryophyllales produce another class of pigments, the betalains, instead of anthocyanins. The occurrence of betalains and anthocyanins is mutually exclusive. Betalains are divided into two classes, the betaxanthins and betacyanins, which produce yellow to orange or violet colours, respectively. In this article we show betalain production in species that normally produce anthocyanins, through a combination of genetic modification and substrate feeding.</p> <p>Results</p> <p>The biolistic introduction of DNA constructs for transient overexpression of two different dihydroxyphenylalanine (DOPA) dioxygenases (DODs), and feeding of DOD substrate (L-DOPA), was sufficient to induce betalain production in cell cultures of <it>Solanum tuberosum </it>(potato) and petals of <it>Antirrhinum majus</it>. HPLC analysis showed both betaxanthins and betacyanins were produced. Multi-cell foci with yellow, orange and/or red colours occurred, with either a fungal DOD (from <it>Amanita muscaria</it>) or a plant DOD (from <it>Portulaca grandiflora</it>), and the yellow/orange foci showed green autofluorescence characteristic of betaxanthins. Stably transformed <it>Arabidopsis thaliana </it>(arabidopsis) lines containing <it>35S: AmDOD </it>produced yellow colouration in flowers and orange-red colouration in seedlings when fed L-DOPA. These tissues also showed green autofluorescence. HPLC analysis of the transgenic seedlings fed L-DOPA confirmed betaxanthin production.</p> <p>Conclusions</p> <p>The fact that the introduction of DOD along with a supply of its substrate (L-DOPA) was sufficient to induce betacyanin production reveals the presence of a background enzyme, possibly a tyrosinase, that can convert L-DOPA to <it>cyclo</it>-DOPA (or dopaxanthin to betacyanin) in at least some anthocyanin-producing plants. The plants also demonstrate that betalains can accumulate in anthocyanin-producing species. Thus, introduction of a DOD and an enzyme capable of converting tyrosine to L-DOPA should be sufficient to confer both betaxanthin and betacyanin production to anthocyanin-producing species. The requirement for few novel biosynthetic steps may have assisted in the evolution of the betalain biosynthetic pathway in the Caryophyllales, and facilitated multiple origins of the pathway in this order and in fungi. The stably transformed <it>35S: AmDOD </it>arabidopsis plants provide material to study, for the first time, the physiological effects of having both betalains and anthocyanins in the same plant tissues.</p

MYB and bHLH transcription factor transgenes increase anthocyanin pigmentation in petunia and lisianthus plants, and the petunia phenotypes are strongly enhanced under field conditions

Petunia line Mitchell [MP, Petunia axillaris × (P. axillaris × P. hybrida)] and Eustoma grandiflorum (lisianthus) plants were produced containing a transgene for over-expression of the R2R3-MYB transcription factor (ROSEA1) that up-regulates flavonoid biosynthesis in Antirrhinum majus. The petunia lines were also crossed with previously produced MP lines containing a Zea mays flavonoid-related bHLH transcription factor transgene (LEAF COLOR, LC), which induces strong vegetative pigmentation when these 35S:LC plants are exposed to high light levels. 35S:ROS1 lisianthus transgenics had limited changes in anthocyanin pigmentation, specifically, precocious pigmentation of flower petals and increased pigmentation of sepals. RNA transcript levels for two anthocyanin biosynthetic genes, chalcone synthase and anthocyanidin synthase, were increased in the 35S:ROS1 lisianthus petals compared to those of control lines. With MP, the 35S:ROS1 calli showed novel red pigmentation in culture, but this was generally not seen in tissue culture plantlets regenerated from the calli or young plants transferred to soil in the greenhouse. Anthocyanin pigmentation was enhanced in the stems of mature 35S:ROS1 MP plants, but the MP white-flower phenotype was not complemented. Progeny from a 35S:ROS1×35S:LC cross had novel pigmentation phenotypes that were not present in either parental line or MP. In particular, there was increased pigment accumulation in the petal throat region, and the anthers changed from yellow to purple colour. An outdoor field trial was conducted with the 35S:ROS1, 35S:LC, 35S:ROS1×35S:LC and control MP lines. Field conditions rapidly induced intense foliage pigmentation in 35S:LC plants, a phenotype not observed in control MP or equivalent 35S:LC plants maintained in a greenhouse. No difference in plant stature, seed germination, or plant survival was observed between transgenic and control plants