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Stress-inducible Arabidopsis thaliana RD29A promoter constitutively drives Citrus sinensis APETALA1 and LEAFY expression and precocious flowering in transgenic Citrus spp.

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Abstract

Transgenic ‘Duncan’ grapefruit (Citrus paradisi Macf.) and ‘Valencia’ sweet orange (Citrus sinensis [L.] Osbeck) plants ectopically expressing C. sinensis (cv. Washington navel orange) APETALA1 (CsAP1) or LEAFY (CsLFY) genes under control of the Arabidopsis thaliana stress-inducible promoter AtRD29A flowered under non-inductive (warm temperature, well-watered) greenhouse conditions, whereas their wild-type (WT) counterparts did not. The transgenic plants that flowered exhibited no altered morphological features, except the lack of thorns characteristic of juvenile WT plants. The most precocious T0 line, ‘Duncan’ grapefruit (Dun134-3) expressing the CsAP1 gene, flowered and fruited when it was 4.5 years old and the T1 siblings from this line flowered and fruited when they were just over 18 months old. In contrast, T1 seedlings from three lines of ‘Duncan’ grapefruit expressing the CsLFY gene flowered within 3 months after germination, but were unable to support fruit development. Transcript levels of corresponding transgenes in leaves were not correlated with earliness of flowering. To further study the activity of AtRD29A, leaves from three ‘Carrizo’ citrange (C. sinensis × Poncirus trifoliata) rootstock seedlings transformed with the green fluorescent protein (GFP) gene under regulation of the AtRD29A promoter were subjected to drought stress or well-watered conditions. Expression of GFP was not stress-dependent, consistent with the observation of flowering of CsAP1 and CsLFY transgenic plants under non-inductive conditions. Taken together, the results suggest that AtRD29A is constitutively expressed in a citrus background. Despite the loss of control over flowering time, transgenic citrus lines ectopically expressing C. sinensis AP1 or LFY genes under control of the A. thaliana RD29A promoter exhibit precocious flowering, fruit development and viable transgenic seed formation. These transformed lines can be useful tools to reduce the time between generations to accelerate breeding.

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Acknowledgements

The authors thank Dr. Lynn Pillitteri for the full-length gene sequences for Citrus sinensis APETALA1 and LEAFY and Dr. Martha L. Orozco, Director of the Plant Transformation Research Center, University of California, Riverside, for providing the pCAMBIA2301 plasmid and for helping with the preparation of the transformation vectors containing the CsAP1 and CsLFY genes.

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Authors and Affiliations

  1. Citrus Research and Education Center, University of Florida/IFAS, Lake Alfred, FL, 33850, USA

    Vladimir Orbović, Seyed Ali Ravanfar, Yosvanis Acanda, Benjamin A. Merritt & Amit Levy

  2. Department of Botany and Plant Sciences, University of California, Riverside, CA, 92521, USA

    Javier Narvaez & Carol J. Lovatt

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  1. Vladimir Orbović
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Contributions

CJL, VO, and YA designed the experiments, JN and YA constructed binary vectors, VO, SAR, BM, and YA preformed the experiments, all authors wrote the manuscript.

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Correspondence to Vladimir Orbović.

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Orbović, V., Ravanfar, S.A., Acanda, Y. et al. Stress-inducible Arabidopsis thaliana RD29A promoter constitutively drives Citrus sinensis APETALA1 and LEAFY expression and precocious flowering in transgenic Citrus spp.. Transgenic Res 30, 687–699 (2021). https://doi.org/10.1007/s11248-021-00260-z

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