Discovering new genes affecting the colour of plants

Supervisors: Dr Mandy Walker (08 8303 8629) Dr Tim Holton, QDPI & Professor Mark Tester ACPFG, University of Adelaide
Location: Adelaide
Financial support: To be confirmed

Anthocyanins are secondary metabolites present in most higher plants and contributing to the red and purple colours of leaves, flowers and fruit, and to processed products such as red wine, jam and fruit juice. Anthocyanins are synthesised by a well-characterised enzymatic pathway which also gives rise to other flavonoid compounds such as tannins, providing the characteristic astringent taste to fruit and wine.

Anthocyanin colour in fruit and flowers (Grotewold, 2006) depends on:

1. vacuolar pH where anthocyanins are stored
2. anthocyanin modification (addition of sugars, hydroxyl, acyl and methyl groups)
3. cofactors such as metal ions
4. cell shape which reflect or magnify the light.

Vacuolar pH is important because anthocyanins are generally only coloured at acidic pH and uncoloured or yellow/brown at neutral pH. To date only three genes have been identified that alter vacuolar pH thereby affecting flower colour (Fukada-Tanaka et al., 2000). While two of these genes are transcription factors, the third belongs to a class of Na⁺/H⁺ antiporters.

Aims:

1. Identify novel genes that alter the hue of transgenic Arabidopsis flower petals.
2. Test known antiporters for their ability to alter flower colour in transgenic Arabidopsis.
3. Use transgenic grape roots to further characterise these genes.

Experimental approach: Transgenic Arabidopsis with purple flowers will be mutagenised and screened for altered flower colour. Candidate genes will be isolated by a positional cloning approach and characterised by altered expression studies using tools such as RNAi constructs and overexpression vectors.  A reverse genetic approach will be utilised to examine the role of Na⁺/H⁺ antiporters in maintaining the pH in Arabidopsis flower petals. Arabidopsis with insertions in the genes of interest will be crossed to the purple flowered Arabidopsis to determine the role they play in flower colour. Both approaches will utilise trans-activated promoter trap lines of Arabidopsis (Kiegle et al., 2000) in which the expression of a gene can be controlled specifically in petal tissue. Confocal microscopy can be used to verify the expression pattern of the GFP reporter gene in petal cells. This technique will allow the analysis of genes that might otherwise be essential for normal plant growth.

Another method available to test the activity of these genes to alter colour is to use hairy root transformation of transgenic grapevine roots, which synthesise anthocyanins as shown in the picture above. This has the advantage of producing relatively large amounts of material of a single tissue type in a short time for biochemical analysis such as HPLC.

References:

• Fukada-Tanaka S, Inagaki Y, Yamaguchi T, Saito N and Iida S (2000) Colour-enhancing protein in blue petals - Spectacular morning glory blooms rely on a behind-the-scenes proton exchanger. Nature 407: 581-581.
• Grotewold E (2006) The genetics and biochemistry of floral pigments. Annual Review of Plant Biology 57: 761-780.
• Kiegle E, Moore CA, Haseloff J, Tester MA and Knight MR (2000) Cell-type-specific calcium responses to drought, salt and cold in the Arabidopsis root. Plant Journal 23: 267-278.