Anne M. Johnson-Flanagan

Degreening in canola (Brassica napus cv. Westar) embryos under optimum conditions.

Summary An HPLC method for rapid identification and quantification of picogram quantities of chlorophylls, chlorophyllides, pheophytins and pheophorbides in canola seeds is described. With this method, pigment changes during seed maturation in canola (Brassica napus cv. Westar) were analyzed. During seed maturation, the embryos undergo controlled degradative processes resulting in loss of chlorophyll. Both chlorophyll a and b degradation is biphasic. The breakdown products, chlorophyllide a and pheophytin a reach a peak during the period of rapid chlorophyll a degradation. Thereafter, loss of these pigments and chlorophyll a are linearly correlated. The relationship between chlorophyll b and its degradation products is not clear. However, this may reflect the low levels of these pigments in the seed rather than a lack of causal relationship. Despite the fact that green canola oil contains a high proportion of pheophytin, neither pheophytin nor pheophorbide accounted for a large proportion of the pigments in the seed. Electrophoretic profiles and EM micrographs indicate the presence of normal chloroplasts with pigments bound in chlorophyll-protein complexes. Furthermore, there is a gradual breakdown of the chlorophyllprotein complexes during degreening. The mature nongreen seed does not contain any components of the photosystems.

Ethylene Production during Development of Mustard (Brassica juncea) and Canola (Brassica napus) Seed.

An open, continuous flow system was used to investigate ethylene production during degreening of maturing seed of mustard (Brassica juncea cv Cutlass and cv Lethbridge 22A) and canola (Brassica napus cv Westar and cv Alto). Isolated mustard seed evolved higher amounts of ethylene than those of canola, and this was particularly evident both early in embryogeny and later during the desiccation phase of seed maturation. The silique walls produced negligible amounts of ethylene in both species. The concentrations of ethylene surrounding seed as they matured within siliques were significantly higher in mustard than in canola, and this interspecies difference was greatest during the seed desiccation phase. In mustard, a 4-fold increase in silique internal ethylene levels was apparent during desiccation. In comparison, only a moderate increase in silique-derived ethylene occurred in canola.

The Impact of Sublethal Freezing during Maturation on Pigment Content in Seeds of Brassica napus

Summary Degreening in canola seeds ( Brassica napus cv. Westar) is disrupted by sublethal freezing. Both the minimum temperature reached and the moisture content at the time of freezing determine the impact on the seed. In general, the pigment content in the mature seed increases as the final minimum temperature decreases. The relationship between pigment content and the seed moisture content at the time of freezing is not as clear, as there is a peak in pigments associated with freezing in the 50% moisture range. Closer examination of the pigments shows a high proportion of chlorophylls and pheophytins, with relatively low amounts of dephytylated pigments. In comparison, freezing at the higher seed moisture contents produces mature seeds with higher concentrations of dephytylated pigments. These results, in addition to the changes noted in the chlorophyll-protein complexes, suggest that freezing at lower moisture contents induces pigment synthesis. Thus, there is evidence that sublethal freezing not only inhibits degreening, it actually exacerbates the problem by inducing renewed synthesis.

Characterization of freezing tolerance and vernalization in Vern-, a spring-type Brassica napus line derived from a winter cross

Abstract. Exposure of winter brassicas to periods of low temperature decreases the transition required from vegetative to reproductive growth. This low-temperature exposure also results in the acquisition of freezing tolerance. To investigate the relationship between vernalization and freezing tolerance, homozygous microspore-derived (DH) lines were developed from reciprocal crosses between two winter cultivars of Brassica napus L., cv. Cascade and cv. Rebel. A resulting line, termed Vern-, expressed a high degree of freezing tolerance and was selected for study. Analysis of growth and development revealed that Vern- is a true spring type, having completely lost the vernalization requirement. Using markers previously mapped in B. rapa, it was concluded that Vern- inherited freezing-tolerance alleles from Cascade. With regard to vernalization and flowering time, Rebel, although traditionally classified as a winter type, carries spring alleles at the major and some of the minor loci. Vern- inherited these alleles from Rebel, in addition to one spring allele from Cascade. Thus, spring alleles from both parents were critical in establishing the spring phenotype observed in Vern-.

Co-ordination of photosynthetic gene expression during low-temperature acclimation and development in Brassica napus cv. Jet Neuf leaves

Abstract The effect of low-temperature acclimation and leaf age on the expression of two photosynthetic genes was examined in mature and young leaves of Brassica napus cv. Jet Neuf. Nuclear-encoded (ssu) and chloroplast-encoded (lsu) genes were used to examine the changes at the DNA, mRNA and protein level. After acclimation, expression of lsu and ssu genes was low in mature leaves and high in young leaves. Corresponding Western blots showed a similar pattern for polypeptide accumulation with lower amounts of SSU and LSU polypeptides in mature leaves and higher amounts in young leaves when compared to their non-acclimated counterparts. However, in acclimated mature leaves relatively more LSU and SSU polypeptides accumulated than did the corresponding transcript. Despite the disparities between protein and message levels during acclimation, protein turnover kinetics showed that LSU and SSU had similar stability in acclimated and non-acclimated leaves of both ages. From Western blots, the ratio of LSU:SSU showed that both polypeptides accumulated in proportion during cold acclimation. Methylation studies at various restriction sites of lsu revealed alteration at PstI and HindIII sites with acclimation.

A method to study seed degreening using haploid embryos of Brassica napus cv. Topas

Summary This study explores the potential of using haploid embryos of Brassica napus cv. Topas as a model system for study of seed degreening and further, as a means of selecting for rapid degreening and freezing tolerant degreening. Microspore-derived embryos of T opas showed similar pigment composition and content as the developing (75% moisture) zygotic seed of Westar. Canola seed degreening could be mimicked in these haploid embryos in the light by the application of 50 IlM abscisic acid or by dark exposure, providing the embryos were under 5 weeks old and had not been greened for over 1 week. These treatments had little effect on the regenerative ability of the embryos. Exposure of seed, in planta , to -5 °C could not be mimicked directly in the haploid embryos as nucleated freezing was lethal, while supercooling had no effect on degreening. Thus we attempted to desiccate embryos during degreening. This proved to be lethal to dark-degreened embryos, but those embryos that were degreened in the light with 50 µM ABA showed only a slight decrease in viability. The pigment changes after controlled desiccation were similar, but not identical, to freezing-induced changes in the seed. We conclude that microspore-derived haploid embryos constitute a valid system to study seed degreening.