Role of cytokinin and ethylene during senescence in broccoli (Brassica oleracea var. Italica) : a thesis submitted for the degree of Doctor of Philosophy at Massey University

Abstract

Broccoli (Brassica oleracea var. italica) deteriorates rapidly following harvest. The two plant hormones ethylene and cytokinin are known to act antagonistically on harvest-induced senescence in broccoli: ethylene acts by accelerating the process, whereas additional cytokinin delays it. The overall aim of this thesis was to gain a better understanding of how these two hormones control postharvest senescence. The effects of exogenous cytokinin (6-benzyl aminopurine, 6-BAP), 1-aminocyclopropane-1-carboxylic acid (ACC) and sucrose on senescence-associated gene expression were measured in both wild-type plants and transgenic plants harbouring an antisense tomato ACC oxidase gene (pTOM13). Exogenous cytokinin caused both a reduction (BoACO) and an increase (BoACS) in ethylene biosynthetic gene expression as well as reduced expression of genes encoding sucrose transporters and carbohydrate metabolising enzymes, indicating a significant role for cytokinin in the delay of senescence. Transgenic broccoli was produced using Agrobacterium tumefaciens-mediated transformation. Ethylene biosynthesis was targeted via an antisense BoACO2 gene fused to the harvest-induced asparagine synthetase (AS) promoter from asparagus. In addition, broccoli was transformed with constructs harbouring the Agrobacterium tumefaciens isopentenyl transferase (ipt) gene using the senescence-associated SAG12 and floral-associated MYB305 gene promoters to enhance the levels of cytokinin either during senescence or in floral tissue, respectively. The presence of the antisense AS-ACO construct was associated with an increased rate of transformation when compared to control constructs. Physiological analyses of mature plants showed that the antisense AS-ACO gene construct caused delayed senescence in both detached leaves and detached heads. Gene expression analyses of harvested floret tissue from AS-ACO lines showed decreases in transcript levels of senescence marker genes compared to wild-type and transgenic control lines, as well as a reduction in expression of sucrose transporter and carbohydrate metabolising genes, confirming the key role of ethylene in the promotion of senescence. In addition, genes involved with cytokinin biosynthesis and metabolism were isolated by PCR using primers based on Arabidopsis clones. The four broccoli ipt sequences aligned closely to four of the Arabidopsis sequences and were subsequently named BoIPT4, BoIPT5, BoIPT6 and BoIPT7. A cytokinin oxidase clone (BoCKX) was also isolated from broccoli. The four BoIPT genes were expressed in a number of different tissues, suggesting that the different genes may be involved in different biological processes in the plant. BoIPT4 was expressed early and BoCKX expressed late in florets during senescence. A model depicting the regulation of senescence in broccoli through the expression of cytokinin biosynthesis and metabolism genes, and their interaction with ethylene and carbohydrate metabolism is presented and discussed.