Altering expression of the flavonoid 3′-hydroxylase gene modified flavonol ratios and pollen germination in transgenic Mitchell petunia plants
David Lewis A F , Marie Bradley A B , Stephen Bloor C , Ewald Swinny C D , Simon Deroles A , Chris Winefield A E and Kevin Davies AA New Zealand Institute for Crop & Food Research Limited, Private Bag 11-600, Palmerston North, New Zealand.
B Present address: Foundation for Research, Science and Technology, PO Box 12-240, Wellington, New Zealand.
C New Zealand Institute for Industrial Research and Development, Gracefield Research Centre, PO Box 31-310, Lower Hutt, New Zealand.
D Present address: Food and Biological Chemistry Laboratory, Chemistry Centre WA, 125 Hay Street, East Perth, WA 6004, Australia.
E Present address: Cell Biology Group, Agriculture and Life Sciences Division, Lincoln University, PO Box 84, Lincoln, Canterbury, New Zealand.
F Corresponding author. Email: lewisd@crop.cri.nz
Functional Plant Biology 33(12) 1141-1152 https://doi.org/10.1071/FP06181
Submitted: 24 July 2006 Accepted: 20 October 2006 Published: 1 December 2006
Abstract
Antisense technology was successfully used to reduce flavonoid 3′-hydroxylase (F3′H) gene expression and enzyme activity and to promote the accumulation of monohydroxylated flavonols in petunia flower tissue. The hydroxylation pattern of specific flavonoid groups is a target for modification because of the possible associated changes in a range of factors including colour, stress tolerance and reproductive viability. Petunia (cv. Mitchell) plants were transformed to express in the antisense orientation the sequences encoding the F3′H (asF3′H). Transformants showed a range of responses, in terms of the level of endogenous F3′H gene expression and the relative proportion of the monohydroxylated flavonol (kaempferol) glycosides that accumulated. Kaempferol glycosides increased from 7% of the total flavonols in flower limb tissue of the wild type plants, to 45% in the flower limb tissue of line 114, the transgenic line that also showed the greatest decrease in F3′H expression in flower tissue. In leaf tissue, the trend was for a decrease in total flavonol concentration, with the relative proportion of kaempferol glycosides varying from ~40 to 80% of the total flavonols. The changes in leaf tissue were not consistent with the changes observed in flower tissue of the same lines. Endogenous F3′H activity in flower limb tissue was not completely shut down, although an 80% decrease in enzyme activity was recorded for line 114. The residual F3′H activity was still sufficient that quercetin glycosides remained as the major flavonol form. Alteration of F3′H activity appears to have affected overall flavonoid biosynthesis. A decrease in total flavonol concentration was observed in leaf tissue and two other flavonoid biosynthetic genes were down-regulated. No morphological changes were observed in the transgenic plants; however, up to a 60% decrease in pollen germination was observed in line 13. Thus, the relatively small change in flavonoid biosynthesis induced by the asF3′H transgene, correlated with several other effects beyond just the specific biosynthetic step regulated by this enzyme.
Keywords: antisense, enzyme activity, kaempferol, quercetin, reproductive viability.
Acknowledgments
This work was funded by the New Zealand Foundation for Research, Science and Technology. We thank John Koolard for assistance with the statistical analyses and Steve Arathoon, Ian King, Jan Manson, Gayle Marshall and Shannon Bullock for technical assistance with various aspects of the project. We also thank Florigene Ltd (Australia) for access to their plasmids containing petunia PAL, C4H, CHS, CHI, FLS and F3′H cDNAs.
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