The regulation of chlorophyll levels in maturing kiwifruit

Abstract

The chlorophyll degradation pathway is central to a number of plant processes including senescence and fruit ripening. However, the regulation of the chlorophyll degradation pathway enzymes is not well understood. The aim of this thesis was to elucidate the genetic mechanisms that control changes in pigment composition leading to fruit flesh yellowing in kiwifruit. Actinidia deliciosa and A. chinensis fruit, which are green and yellow, respectively, provide an opportunity to study the regulation of chlorophyll levels.

The expression of genes that code for enzymes of the chlorophyll and cytokinin metabolic pathways was measured using qRT-PCR. Candidates for chlorophyll degradation regulatory points were then characterised for functionality by transient transformation in N. benthamiana. The endogenous cytokinin levels were measured in kiwifruit and transient activation assays were carried out with the promoters of key candidate genes.

Overall, expression of the chlorophyll degradation genes was elevated in yellow fruit and expression of biosynthetic genes was higher in green fruit. The chlorophyll degradation-associated protein, STAY-GREEN2 (SGR2), was more highly expressed in yellow fruit, and transient over-expression of SGR was sufficient to drive chlorophyll degradation. Expression of isopentenyl transferase (IPT), the rate-limiting step for cytokinin biosynthesis, showed an increase towards maturity in green fruit, but not in yellow fruit. However, both fruit had similar high levels of cytokinin nucleotides and free bases. A gene coding for O-glucosylation was also highly expressed in green fruit. Green fruit contained higher levels of cytokinin O-glucosides and ribosides towards maturity, suggesting differences in cytokinin signalling, which could lead to regulation of chlorophyll levels via activation of the SGR promoter by transcription factors.

It is likely that the chlorophyll degradation pathway and cytokinin metabolism are linked. The differential expression of cytokinin response regulators could lead to differential regulation of cytokinin levels in the fruit of the two species, and possibly differential regulation of the chlorophyll degradation pathway. Progress towards elucidation of the control of chlorophyll levels provides knowledge of this key process in kiwifruit and potentially gene-based markers for breeding new kiwifruit cultivars.