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Expression, subcellular localization, and cis-regulatory structure of duplicated phytoene synthase genes in melon (Cucumis melo L.)

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Abstract

Carotenoids perform many critical functions in plants, animals, and humans. It is therefore important to understand carotenoid biosynthesis and its regulation in plants. Phytoene synthase (PSY) catalyzes the first committed and rate-limiting step in carotenoid biosynthesis. While PSY is present as a single copy gene in Arabidopsis, duplicated PSY genes have been identified in many economically important monocot and dicot crops. CmPSY1 was previously identified from melon (Cucumis melo L.), but was not functionally characterized. We isolated a second PSY gene, CmPSY2, from melon in this work. CmPSY2 possesses a unique intron/exon structure that has not been observed in other plant PSYs. Both CmPSY1 and CmPSY2 are functional in vitro, but exhibit distinct expression patterns in different melon tissues and during fruit development, suggesting differential regulation of the duplicated melon PSY genes. In vitro chloroplast import assays verified the plastidic localization of CmPSY1 and CmPSY2 despite the lack of an obvious plastid target peptide in CmPSY2. Promoter motif analysis of the duplicated melon and tomato PSY genes and the Arabidopsis PSY revealed distinctive cis-regulatory structures of melon PSYs and identified gibberellin-responsive motifs in all PSYs except for SlPSY1, which has not been reported previously. Overall, these data provide new insights into the evolutionary history of plant PSY genes and the regulation of PSY expression by developmental and environmental signals that may involve different regulatory networks.

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Abbreviations

ABA:

Abscisic acid

ABRE-CE:

ABA-response element–coupling element

CTAB:

Cetyltrimethyl ammonium bromide

DAP:

Days after pollination

EST:

Expressed sequence tag

FPP:

Farnesyl pyrophosphate

GGPP:

Geranylgeranyl pyrophosphate

GST:

Glutathione-S-transferase

HSE:

High temperature responsive element

IPTG:

Isopropyl-β-d-thiogalactoside

LTR:

Low temperature responsiveness element

LB:

Luria Broth

NJ:

Neighbor-joining

PIF:

Phytochrome interacting factor

PSY:

Phytoene synthase

RACE:

Rapid amplification of cDNA ends

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SNP:

Single nucleotide polymorphism

TCA:

Trichloroacetic acid

TILLING:

Targeting induced local lesions IN genomes

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Acknowledgments

We thank Nadia Ono for critical reading of the manuscript. We would also like to thank Karen Arlin (Harris-Moran) for proving us with melon seeds, Dr. Florence Negre-Zakharov and Minmin Wang for providing the field-grown Navigator melon fruits, and Dr. Francis Cunningham (University of Maryland) for providing the pAC-85b and pAC-BETA plasmids. This work was supported by the California Melon Research Board and the UC Davis New Faculty Startup Fund.

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Authors and Affiliations

  1. Department of Plant Sciences, Mail Stop 3, University of California, Davis, Davis, CA, USA

    Xiaoqiong Qin, Ardian Coku, Kentaro Inoue & Li Tian

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  1. Xiaoqiong Qin
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  2. Ardian Coku
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  3. Kentaro Inoue
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  4. Li Tian
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Corresponding author

Correspondence to Li Tian.

Additional information

Genomic sequences of CmPSY1 and CmPSY2 have been deposited in the GenBank under JF745118 and JF745117, respectively.

Electronic supplementary material

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Table S1 Primer sequences used for cloning, 3’ RACE, inverse PCR and real-time qPCR analyses.

Table S2 Light and circadian regulated cis-elements identified from Arabidopsis, tomato and melon PSY promoters.

Table S3 The frequencies of cis-elements identified in the 5’-flanking region (~1,500 bp) of Arabidopsis, tomato and melon PSYs in the fully-sequenced Arabidopsis genome.

Fig. S1 Transcript levels of melon housekeeping genes in various melon tissues presented as absolute Ct values.

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Qin, X., Coku, A., Inoue, K. et al. Expression, subcellular localization, and cis-regulatory structure of duplicated phytoene synthase genes in melon (Cucumis melo L.). Planta 234, 737–748 (2011). https://doi.org/10.1007/s00425-011-1442-8

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