Biochemistry and inheritance of pollen/petal colour mutants in Californian poppy - investigating the role of carotenoid pigments in pollen

Abstract

This thesis reports the biochemical and genetic characterisation of several pollen/petal colour mutants of Californian poppy (Eschscholzia californica Cham.). This characterisation provided a base for experiments investigating hypotheses relating to the function of pigments in pollen: are pigments essential for pollen physiology and/or protection against potentially harmful UV damage? Although most pollen has a yellow/orange colour due to the presence of flavonoid and/or carotenoid pigments, no direct evidence has been published linking pigments found in pollen with a specific function.

A fertile white pollen mutant of Californian poppy was observed growing at Lake Benmore in the South Island of New Zealand. Open pollinated seed was collected from it, and subsequent plants were used to establish a glasshouse population for analysis. The mutation to white petals and pollen was due to a single recessive allele (b), with the white mutant allele recessive to the wildtype orange allele. Carotenoids were absent from the mutant pollen and only present in traces in petals. Flavonoid levels (as measured by total phenolics and absorbance) and composition (analysed by TLC and HPLC) in the mutant pollen and petals were not significantly different to the orange wild-type.

An extensive search of wild populations and screening of commercial cultivars found thirteen more white pollen variants and seven pale yellow to yellow pollen colour variants. Genetic complementation analysis established that additional white mutants were mutated in the same gene as the Benmore white mutant, whereas the yellow variants were mutated in different genes to the white mutant. Carotenoid and flavonoid pigment analyses of these mutants (HPLC) showed no variation in flavonol quantity or composition, and considerable variation in carotenoids among the different yellow mutants. Yellow variants IncY and BIF3, and pale yellow variants Hur I, Sel l and Ben Y were all inherited as single recessive alleles.

Having established that the white pollen mutant was deficient in carotenoids, and that white pollen mutants were rare in wild populations, the physiology of this pollen was compared to the orange wild-type, to test the hypothesis that carotenoids have a physiological role in pollen. Fitness tests of pollen viability, germination and tube growth in vitro showed a significant difference only in tube growth, where white pollen grew significantly faster than orange. However, this difference in tube growth was not found when pollen tube growth was again compared between mutant and wild-type pollen in the experiment to test whether carotenoids had a protective role against UV light in pollen. White and orange pollens' in vitro or in vivo fitness did not differ significantly after exposure to UV light.

Inbreeding was found to significantly decrease both fresh and dry foliage weights of Californian poppy. To assess whether inbreeding depression was affecting wild populations, a semi-dominant flower pattern character (j) was scored in eleven South Island populations. In ten out of eleven geographically distant populations, heterozygous forms of the j locus were present in significant excesses relative to expected frequencies based on the Hardy-Weinberg equilibrium. This indicates a high selection against homozygotes in natural populations of Californian poppy, which probably accounts for the rare frequency of the white pollen/petal mutants in nature.