Functional analysis of plant idioblasts (Myrosin cells) and their role in defense, development and growth.

Abstract

Glucosinolates are natural plant products known as flavor compounds, cancerpreventing agents, and biopesticides. There is a strong interest in controlling the level of glucosinolates to improve flavor and nutritional qualities of food crops and to study the physiological role of glucosinolates in plants. The role of the myrosinaseglucosinolate system in plant defense-related reactions of Cruciferae is accepted. As metabolism of glucosinolates in healthy intact plants has been reported, this indicates that products produced by hydrolysis of glucosinolates may also be important in the growth and development of the plant.

The main focus in this thesis has been to study the effect of preventing glucosinolate hydrolysis by removal of the hydrolyzing enzyme, myrosinase. By the use of the Myr1.Bn1 promoter, we have made myrosinase free seeds of Brassica napus by controlled cell death (genetic ablation) of myrosin cells. The glucosinolate substrates were mainly unaffected by this ablation supporting their localization in non-myrosin cells and a cellular separation of enzyme and substrate. GC and GC-MS analysis confirmed that seeds with ablated myrosin cells had lost the ability to produce isothiocyanates. The effects of genetically ablating myrosin cells were tested for two other components in this complex enzyme system. The epithiospecifier protein (ESP) had lost the expression of one of its isoforms (39 kDa ESP) by the ablation indicating a cellular co-localization with myrosinase in myrosin cells for this specific isoform.

The expression of myrosinase binding proteins (MBPs) was not severely affected supporting localization to non-myrosin cells in the B. napus seed. Plants grown from seeds with ablated myrosin cells (MINELESS) showed a bushy phenotype and morphological abnormalities in flower and silique. Seed maturation was delayed and seed production reduced. Parallel experiments performed in our lab with Arabidopsis thaliana produced similar results. The tissues affected were consistent with the expression pattern directed by the myrosinase gene promoter used.

Ablating myrosinase proteins affected the behavior of the specialist cabbage aphid Brevicoryne brassicae and the generalist peach potato aphid Myzus persicae. When seedlings of MINELESS and wild type were challenged by these aphid species, a difference in preference was observed. B. brassicae preferred wild type seedlings with high levels of myrosinase proteins, while M. persicae preferred MINELESS seedlings with a reduced level of myrosinase compared to wild type. Statistical analysis revealed that the MINELESS seedlings were more susceptible to attacks by aphids, regardless of aphid species. Furthermore, the number of aphids established on the seedling influenced the myrosinase activity and the height of the plant. Thus, by ablating myrosinase proteins, new insights into the functions of the myrosinase-glucosinolate system have been elucidated. This complex system seems important not only in the defense against pests, but also show potential to influence the growth and development of the plant.