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Isolation and functional characterisation of banana phytoene synthase genes as potential cisgenes

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Abstract

Carotenoids occur in all photosynthetic organisms where they protect photosystems from auto-oxidation, participate in photosynthetic energy transfer and are secondary metabolites. Of the more than 600 known plant carotenoids, few can be converted into vitamin A by humans and so these pro-vitamin A carotenoids (pVAC) are important in human nutrition. Phytoene synthase (PSY) is a key enzyme in the biosynthetic pathway of pVACs and plays a central role in regulating pVAC accumulation in the edible portion of crop plants. Banana is a major commercial crop and serves as a staple crop for more than 30 million people. There is natural variation in fruit pVAC content across different banana cultivars, but this is not well understood. Therefore, we isolated PSY genes from banana cultivars with relatively high (cv. Asupina) and low (cv. Cavendish) pVAC content. We provide evidence that PSY in banana is encoded by two paralogs (PSY1 and PSY2), each with a similar gene structure to homologous genes in other monocots. Further, we demonstrate that PSY2 is more highly expressed in fruit pulp compared to leaf. Functional analysis of PSY1 and PSY2 in rice callus and E. coli demonstrates that both genes encode functional enzymes, and that Asupina PSYs have approximately twice the enzymatic activity of the corresponding Cavendish PSYs. These results suggest that differences in PSY enzyme activity contribute significantly to the differences in Asupina and Cavendish fruit pVAC content. Importantly, Asupina PSY genes could potentially be used to generate new cisgenic or intragenic banana cultivars with enhanced pVAC content.

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Abbreviations

PSY:

Phytoene synthase

pVAC:

Pro-vitamin A carotenoid

VAD:

Vitamin A deficiency

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Acknowledgments

The authors are grateful to: Doug Becker at QUT for coordinating the supply of plant material from Queensland Department of Employment, Economic Development and Innovation (DEEDI); Jeff Daniells at DEEDI (South Johnstone) for supplying Asupina green fruit tissue; Jennifer Kleidon at QUT for supplying Cavendish and Ladyfinger leaf tissue, maintenance of Asupina, Lakatan and Wain banana plants at QUT, and for training in rice tissue culture and transformation techniques; Sharon Hamill at DEEDI (Nambour) for supplying Asupina, Lakatan and Wain banana plants; Russell Reinke (Yanco Agricultural Institute) for supplying Nipponbare rice seeds; Queensland University of Technology International Doctorate Scholarship program for postgraduate scholarship support and funding. The research was funded by a grant from the Bill and Melinda Gates Foundation Grand Challenges in Global Health Initiative. The authors declare that they have no conflict of interest.

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  1. Priver Namanya

    Present address: National Agricultural Research Laboratories-Kawanda, P.O. Box 7065, Kampala, Uganda

Authors and Affiliations

  1. Centre for Tropical Crops and Biocommodities, Queensland University of Technology, 2 George Street, Brisbane, QLD, 4001, Australia

    Bulukani Mlalazi, Priver Namanya, Harjeet Khanna, R. Jason Geijskes, Mark D. Harrison, Rob Harding, James L. Dale & Marion Bateson

  2. Faculty of Biology, University of Freiburg, 79104, Freiburg, Germany

    Ralf Welsch

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  1. Bulukani Mlalazi
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Supplementary material 1 (DOC 122 kb)

425_2012_1717_MOESM2_ESM.jpg

Supplemental Fig. 2. Alignment of banana PSY partial protein sequences. Neighbour joining multiple sequence alignment of banana PSY1 and PSY2 partial protein sequences translated from the partial nucleotide sequences used in Fig. 1 (Table S3) and Cavendish PSY2b (Ma-PSY2b; GenBank Accession No. JX195666) (JPEG 1515 kb)

425_2012_1717_MOESM3_ESM.jpg

Supplemental Fig. 3. Comparative structure of PSY mRNA and genomic DNA sequences from Asupina and Cavendish cultivars. (a) Structure of putative Asupina (A) and Cavendish (Ma) PSY mRNA transcripts showing the coding region (black rectangle) between the start (AUG) and stop (UGA or UAA) codons of A-PSY1 (GenBank Accession No. JX195658), A-PSY2a (GenBank Accession No. JX195659), Ma-PSY1 (GenBank Accession No. JX195664) and Ma-PSY2a (GenBank Accession No. JX195665). Putative transcription initiation (t) and polyadenylation (*) sites are marked on the 5’ and 3’ UTRs (grey arrows), respectively. (b) Analysis of genomic sequences of Asupina and Cavendish PSY genes. Diagrammatic representation of the structure of the genomic sequences corresponding to the coding region (between the start and stop codons) of Asupina and Cavendish PSY genes A-PSY1 (GenBank Accession No. JX195661), A-PSY2a (GenBank Accession No. JX195660), Ma-PSY1 (GenBank Accession No. JX195663) and Ma-PSY2a (GenBank Accession No. JX195662) compared to that of maize (Zm) PSY1 (GenBank Accession No. AY324431.1) and PSY2 (GenBank Accession No. AY325302.1) showing the positions of the exons (black arrows) and introns (grey lines). The sizes (bp) of the exons (bold numbers) and introns (plain numbers) are shown above and below the coding region of each gene (JPEG 768 kb)

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Mlalazi, B., Welsch, R., Namanya, P. et al. Isolation and functional characterisation of banana phytoene synthase genes as potential cisgenes. Planta 236, 1585–1598 (2012). https://doi.org/10.1007/s00425-012-1717-8

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