Endogenous cytokinin in developing kiwifruit is implicated in maintaining fruit flesh chlorophyll levels

Background and Aims

Green kiwifruit (Actinidia deliciosa) retain high concentrations of chlorophyll in the fruit flesh, whereas in gold-fleshed kiwifruit (A. chinensis) chlorophyll is degraded to colourless catabolites during fruit development, leaving yellow carotenoids visible. The plant hormone group the cytokinins has been implicated in the delay of senescence, and so the aim of this work was to investigate the link between cytokinin levels in ripening fruit and chlorophyll de-greening.

Methods

The expression of genes related to cytokinin metabolism and signal transduction and the concentration of cytokinin metabolites were measured. The regulation of gene expression was assayed using transient activation of the promoter of STAY-GREEN2 (SGR2) by cytokinin response regulators.

Key Results

While the total amount of cytokinin increased in fruit of both species during maturation and ripening, a high level of expression of two cytokinin biosynthetic gene family members, adenylate isopentenyltransferases, was only detected in green kiwifruit fruit during ripening. Additionally, high levels of O-glucosylated cytokinins were detected only in green kiwifruit, as was the expression of the gene for zeatin O-glucosyltransferase, the enzyme responsible for glucosylating cytokinin into a storage form. Season to season variation in gene expression was seen, and some de-greening of the green kiwifruit fruit occurred in the second season, suggesting environmental effects on the chlorophyll degradation pathway. Two cytokinin-related response regulators, RRA17 and RRB120, showed activity against the promoter of kiwifruit SGR2.

Conclusions

The results show that in kiwifruit, levels of cytokinin increase markedly during fruit ripening, and that cytokinin metabolism is differentially regulated in the fruit of the green and gold species. However, the causal factor(s) associated with the maintenance or loss of chlorophyll in kiwifruit during ripening remains obscure.     

Investigation of cytokinin-deficient phenotypes in Arabidopsis by ectopic expression of orchid DSCKX1

The plant hormone cytokinin plays a major role in regulating plant growth and development. Here we generated cytokinin-reduction Arabidopsis plants by overexpressing a heterologous cytokinin oxidase gene DSCKX1 from Dendrobium orchid. These transgenic plants exhibited reduced biomass, rapid root growth, decreased ability to form roots in vitro, and reduced response to cytokinin in growing calli and roots. Furthermore, the expression of KNAT1, STM, and CycD3 genes was significantly reduced in the transgenic plants, suggesting that cytokinin may function to control the cell cycles and shoot/root development via regulation of these genes.     

Analysis of IIId, IIIe and IVa group basic-helix-loop-helix proteins expressed in Arabidopsis root epidermis

Differentiation of Arabidopsis epidermal cells into root hairs and trichomes is a functional model system for understanding plant cell development. Previous studies showed that one of the Arabidopsis basic-helix-loop-helix (AtbHLH) proteins, GLABRA3 (GL3), is involved in root-hair and trichome differentiation. We analyzed 11 additional AtbHLH genes with homology to GL3. Estimation of the phylogeny based on amino acid sequences of the bHLH region suggests that 11 AtbHLH genes used in this study evolved by duplications of a single common GL3 ancestor. Promoter-GUS analysis showed that AtbHLH006, AtbHLH013, AtbHLH017 and AtbHLH020 were expressed in roots. Among them, AtbHLH006 and AtbHLH020 were preferentially expressed in root epidermal non-hair cells. Consistent with the expression patterns from promoter-GUS analysis, GFP fluorescence was observed in the nuclei of root epidermal non-hair cells of AtbHLH006p::AtbHLH006:GFP and AtbHLH020p::AtbHLH020:GFP transgenic plants. However, AtbHLH006 and AtbHLH0020 proteins did not interact with epidermis-specific MYB proteins and TTG1. Taken together, AtbHLH006 and AtbHLH020 may function in root epidermal cells, but other GL3-like bHLH proteins may have evolved to regulate different processes.