J. Marie Bradley

UVB RADIATION INDUCED INCREASE IN QUERCETIN : KAEMPFEROL RATIO IN WILD-TYPE AND TRANSGENIC LINES OF PETUNIA

The use of genetically modified plants offers unique opportunities to study the role of specific flavonoids in plant UVB protection. Along with a parental wild‐type Mitchell Petunia, two transgenic lines with altered flavonoids were also examined; Lc with enhanced levels of antho‐cyanins due to the action of a maize flavonoid regulatory gene Leaf color, and AFLS that carries an antisense fla‐vonol synthase construct and is known to have reduced flavonol levels in flowers. All three lines were grown in near ambient sunlight, sunlight lacking UVB (280–320 nm) radiation and sunlight with 25% added UVB. Ultra‐violet‐B radiation induced significant reductions in the rates of leaf expansion and seedling growth in all three lines. The presence of anthocyanins did not appear to afford Lc plants any special protection from UVB. Ul‐traviolet‐B treatment induced increases in total flavonol content in young plants of all three lines, and this effect decreased with increasing leaf age. Notably, increasing UVB levels led to an increase in the ratio of quercetin: kaempferol with all three cultivars. The AFLS transgenic, contrary to expectations based on its genetic construction, had normal levels of flavonols in the leaves and the highest Q:K ratio of the three cultivars. This transgenic was the least susceptible to UVB, which may indicate an enhanced protective role for quercetin. Because both quercetin and kaempferol have similar UVB screening properties, quercetin may exert this role by other means.

Enhancing anthocyanin production by altering competition for substrate between flavonol synthase and dihydroflavonol 4-reductase

Flavonoids, in particular the anthocyanins,are responsible for flower colour in manyspecies. The dihydroflavonols represent abranch point in flavonoid biosynthesis,being the intermediates for production ofboth the coloured anthocyanins, through theaction of the enzyme dihydroflavonol4-reductase (DFR), and the colourlessflavonols, produced by flavonol synthase(FLS). In this study the white-flowered,flavonol accumulating Mitchell line ofpetunia was used as a model to examine theinteraction between DFR and FLS enzymeactivities and possibilities forredirecting flavonoid biosynthesis awayfrom production of flavonols and towardsanthocyanins. Introduction of a 35SCaMV-DFR sense transgene construct causedthe production of anthocyanins, resultingin a pink-flowered phenotype. Furthermore,inhibition of FLS production throughintroduction of an FLS antisense RNAconstruct also led to anthocyaninproduction and a pink-flowered phenotype. A combination of both transgenes gave thehighest level of anthocyanin formation. Anthocyanins were produced in the DFR-senseand FLS-antisense transgenic lines in spiteof the greatly reduced levels of geneexpression in the Mitchell line for threeenzymes late in anthocyanin biosynthesis,anthocyanindin synthase, UDP-glucose:flavonoid 3-O-glucosyltransferase andUDP-rhamnose: anthocyanidin-3-glucosiderhamnosyltransferase. Thus, the level ofgene activity required for visibleanthocyanin formation is much lower thanthe high levels normally induced duringpetal development. Altering the balancebetween the DFR and FLS enzyme activities,using genetic modification, may be a usefulstrategy for introducing or increasinganthocyanin production in target ornamentalspecies.

Flavonoid biosynthesis in flower petals of five lines of lisianthus (Eustoma grandiflorum Grise.)

Abstract The biosynthesis of flavonoid pigments during petal development was investigated in five lines of lisianthus ( Eustoma grandiflorum Grise.), with purple, pink, mauve or white flowers (W42 and W84). Flavonols were found at high levels at all stages of petal development, but peaked just prior to flower opening. In the cyanic lines, anthocyanins were first detected in opening flowers, increasing in amounts thereafter. The polymerase chain reaction was used to generate cDNAs from lisianthus petal RNA for phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI) and dihydroflavonol 4-reductase (DFR), and expression of their corresponding mRNAs during petal development investigated. In the cyanic lines, PAL, CHS and CHI transcripts were present throughout petal development, with peak expression in young, acyanic buds. DFR expression was low in young buds and peaked immediately preceding anthocyanin synthesis. Thus, the activity of the biosynthetic genes correlated closely with the flavonoid content of the petals, with the synthesis of flavonols being temporally separated from that of anthocyanins. Transcripts for all four genes examined were detected in both white-flowered lines, although line W42 had only low levels for DFR. Neither white-flowered line could synthesize anthocyanins when petal tissue was fed with leucopelargonidin and leucocyanidin (fed to W42 only), products of the DFR enzyme. Thus, line W42 may lack activity for a regulatory factor that controls the expression of the genes of the later part of the flavonoid biosynthetic pathway, while W84 may contain a block in the conversion of leucoanthocyanidin to anthocyanin.

An antisense chalcone synthase cDNA leads to novel colour patterns in lisianthus (Eustoma grandiflorum) flowers

Three cultivars of lisianthus (Eustoma grandiflorum (Grise.)) were transformed with a homologous antisense CHS cDNA via Agrobacterium-mediated transformation. Over 50% of the transgenics derived from the purple flowering lines exhibited an altered flower colour pattern ranging from small streaks of white on the wild-type purple background through to completely white flowers. A significant portion of the transgenic lines showed unstable phenotypes. Northern and biochemical analysis showed that the altered flower patterns were associated with a loss of CHS gene transcript and a corresponding loss of CHS enzyme activity. In the white flowering line the level of total flavonoids was reduced to ca. 2.0% of the wild-type level. Some of the transgenic plants also exhibited alterations in flower form such as the formation of frilled petal tips and reduced flower opening. Several of the new patterned lines are being evaluated for stability and possible commercial release.

Expression of an Antirrhinum majus UDP-glucose : flavonoid-3-O-glucosyltransferase transgene alters flavonoid glycosylation and acylation in lisianthus (Eustoma grandiflorum Grise. )

Abstract A binary vector containing an Antirrhinum majus UDP-glucose:flavonoid-3- O -glucosyltransferase (UFGT) cDNA under the control of the cauliflower mosaic virus 35S promoter was used to transform lisianthus ( Eustoma grandiflorum Grise.). Of four independent transgenic lines recovered, one produced high levels of the UFGT transcript and synthesized 3- O -glucosylated anthocyanins novel to lisianthus, as well as enhanced levels of 3- O -glucosylated flavonols. The novel 3- O -glucosylated anthocyanins accounted for approximately 30% of the total anthocyanins in the petals. The level of 3- O -glucosylated flavonols increased by approximately 4.8 x , representing a change from the production of 3- O -galactosylated flavonols to 3- O -glucosylated flavonols of approximately 32%. In addition, the increase in glucosylation of the flavonols resulted in a corresponding decrease in acylation. Modifications at other positions of the flavonoid nuclei were unaffected. The results indicate that in lisianthus there is a high specificity shown by some flavonoid modifying enzymes, such as that for flavonol acylation, while other enzymes, such as those involved in modifications at the C-5 of the anthocyanin, are more flexible in their choice of substrate. The results also provide information on the role of flavonoid glycosylation and acylation in copigmentation in lisianthus.

Variation in the ability of the maize Lc regulatory gene to upregulate flavonoid biosynthesis in heterologous systems

Abstract We have previously shown that overexpression of the maize basic helix-loop-helix (bHLH) regulatory gene, Leaf colour (Lc) in petunia enhanced pigmentation through the upregulation of the flavonoid biosynthetic pathway (Bradley et al. Plant J. (13) (1998) 381–392) [1] . Here we report on the effect of Lc expression in two popular ornamental species, lisianthus (Eustoma grandiflorum) and a regal pelargonium cultivar (Pelargonium X domesticum Dubonnet). A number of transformants expressing the Lc cDNA under the control of the cauliflower mosaic virus (CaMV) 35S promoter were generated in three different lisianthus cultivars, including purple, pink and cream flowered lines, and the pelargonium cultivar Dubonnet. Unlike transgenic Lc petunia, which had enhanced pigmentation in foliage and flowers, no visible phenotypic alteration in vegetative or floral pigmentation was observed in either the pelargonium or lisianthus lines expressing Lc. A detailed analysis of flavonoid content and expression of a number of flavonoid biosynthetic genes in the leaves of these lines indicated that, unlike in petunia, Lc alone was unable to transcriptionally upregulate the flavonoid biosynthetic genes of lisianthus or pelargonium. This suggests there may be a divergence of the regulatory mechanisms in different dicots and that a combination of introduced bHLH and MYB factors may be required to increase pigmentation in some plant species. This report extends the study of bHLH regulatory gene action in several Solanaceae and a single Brassiceae species to include members of the Gentianaceae and Geraniaceae.

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 [TF; ROSEA1 (ROS1)] 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 basic helix-loop-helix TF 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 in the petal throat region, and the anthers changed from yellow to purple pigmentation. 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.

Identification of flavonol and anthocyanin metabolites in leaves of petunia Mitchell and its LC transgenic

Abstract The leaves of Petunia Mitchell, a model species employed for transgenic manipulation, contain a wider array of acylated flavonol glycosides than occur in the petals. Three new acylated flavonol glycosides are described, kaempferol-3- O -(2- O -feruloyl- β - d -glucosyl(1→2)6- O -malonylglucoside), quercetin-3- O -(2- O -caffeoyl- β - d -glucosyl(1→2)6- O -malonylglucoside), and quercetin-3- O -(2- O -feruloyl- β - d -glucosyl(1→2)glucoside). A transgenic Mitchell line expressing the maize Leaf colour (Lc) cDNA had enhanced levels of anthocyanins, particularly in their leaves. These anthocyanins were determined to be the same acylated petunidin glycosides as those which produce a slight red colouration in the tube of the flowers of Petunia Mitchell.

Flower pattern stability in genetically modified lisianthus (Eustoma grandiflorum) under commercial growing conditions.

Abstract Lisianthus (Eustoma grandiflorum Grise.) with novel flower colour patterns have been generated by genetic modification techniques that alter flavonoid biosynthesis. As described previously, the level and pattern of pigmentation in purple‐flowered lisianthus cultivars was altered by the introduction of a lisianthus chalcone synthase (CHS) cDNA in an antisense orientation. As the next step towards commercial development of these new cultivars, the consistency of altered flower patterns in the progeny and the performance of these lines under conditions resembling those used by commercial lisianthus growers in New Zealand have now been assessed. The introduced antisense CHS transgene had no apparent effect on germination rates or plant survival. Altered flower patterns observed in the progeny correlated with the inheritance of the transgenes, as measured by kanamycin resistance and reduction in CHS expression. At least two of the lines showed sufficient consistency in flower pattern to warrant continued development. Additionally, no transfer of the introduced DNA between transgenic and neighbouring non‐transformed lisianthus was observed during this trial.