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Induction of anthocyanin in the inner epidermis of red onion leaves by environmental stimuli and transient expression of transcription factors

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Abstract

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Novel imaging approaches have allowed measurements of the anthocyanin induction in onion epidermal cells that can be induced through water stress or transient expression of exogenous transcription factors.

Abstract

Environmental and genetic mechanisms that allow the normally colourless inner epidermal cells of red onion (Allium cepa) bulbs to accumulate anthocyanin were quantified by both absorbance ratios and fluorescence. We observed that water-stressing excised leaf segments induced anthocyanin formation, and fluorescence indicated that this anthocyanin was spectrally similar to the anthocyanin in the outer epidermal cells. This environmental induction may require a signal emanating from the leaf mesophyll, as induction did not occur in detached epidermal peels. Exogenous transcription factors that successfully drive anthocyanin biosynthesis in other species were also tested through transient gene expression using particle bombardment. Although the petunia R2R3-MYB factor AN2 induced anthocyanin in both excised leaves and epidermal peels, several transcription factors including maize C1 and Lc inhibited normal anthocyanin development in excised leaves. This inhibition may be due to moderate levels of conservation between the exogenous transcription factors and endogenous Allium transcription factors. The over-expressed exogenous transcription factors cannot drive anthocyanin biosynthesis themselves, but bind to the endogenous transcription factors and prevent them from driving anthocyanin biosynthesis.

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Acknowledgements

This research was funded by the University of Canterbury College of Science (DAC), Plant & Food Research (CCE), and a grant from Invitrogen NZ Ltd (EJW). EJW thanks the New Zealand Federation of Graduate Women (NZFGW) for financial support. From Plant & Food Research, Palmerston North, we also thank Kevin Davies and Andy Allan for their comments on drafts of the manuscript, and Nick Albert for comments and assistance with phylogenic trees.

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Author notes

  1. Colin C. Eady

    Present address: New Zealand Agriseeds, 2547 Old West Coast Road, RD1, Christchurch, 7671, New Zealand

Authors and Affiliations

  1. School of Biological Sciences, The University of Canterbury, Private Bag 4800, Christchurch, 8140, New Zealand

    Elizabeth J. Wiltshire & David A. Collings

  2. The New Zealand Institute for Plant & Food Research Limited, PO Box 4704, Christchurch, 8140, New Zealand

    Colin C. Eady

  3. School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW, 2308, Australia

    David A. Collings

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  1. Elizabeth J. Wiltshire
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Corresponding author

Correspondence to David A. Collings.

Ethics declarations

These experiments were conducted in accordance with New Zealand regulations concerning genetic modification of organisms, and NZ EPA decision GMD08056.

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Communicated by Inhwan Hwang.

Electronic supplementary material

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299_2017_2132_MOESM1_ESM.tif

Fig. S1 Induced anthocyanin shows spectral variations. Spectral properties were tested in the outer epidermis (a, d), naturally red inner epidermis (b), and inner epidermis induced for 4 days (c, e). While fluorescence emission spectra were similar for the three different cell types using excitations at 405, 488 and 561 nm (see Fig. 3), when cells were photo-activated with 5 min of 405 nm excitation, changes in fluorescence were induced at 488 nm. a–c Normalised emission spectra at 488 nm before and after photo-activation. e, f Representative paired images of cells before (left) and after 5 min 405 nm photo-activation (right) of the boxed areas demonstrated the yellow shift in fluorescence from outer (e) but not inner epidermal cells (f). Images were recorded with blue excitation and a long pass filter, and with a Leica DFC310FX colour camera. Bar in F 100 µm (TIF 3050 KB)

299_2017_2132_MOESM2_ESM.tif

Fig. S2 R2R3-MYB sequences from subgroups 5 and subgroup 6 that are known to induce anthocyanin formation were aligned in ClustalW, and a phylogenic tree created. Two subgroup 4 sequences were used as an outgroup. Several families for which multiple R2R3-MYBs grouped together are highlighted, including the Solanaceae, Brassicaceae and Rosaceae. Monocots are indicated in green, sequences tested in this study highlighted in red, and the native AcMYB1 from Allium is highlighted in magenta. This figure demonstrates the complexities of R2R3-MYB-driven anthocyanin formation with respect to taxonomy. Onion (Allium) which is in the order Asparagales is in a different order to Lilium and Iris (Liliales) whose R2R3-MYB sequences AcMYB1 most closely resembles whereas the orchids (Phalaenopsis, Vanda and Oncidium) that are also in the Asaparagales use a subtype 5 R2R3 MYBs to drive anthocyanin induction. Information on the different gene sequences used and their Genbank accession details are included in Supplementary Table 1 (TIF 615 KB)

Supplementary material 3 (DOC 82 KB)

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Wiltshire, E.J., Eady, C.C. & Collings, D.A. Induction of anthocyanin in the inner epidermis of red onion leaves by environmental stimuli and transient expression of transcription factors. Plant Cell Rep 36, 987–1000 (2017). https://doi.org/10.1007/s00299-017-2132-1

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