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Gibberellic Acid-Insensitive mRNA Transport in Pyrus

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Abstract

Grafting is a common method for clonal propagation of fruit trees. Moreover, it serves as a mean to deal with abiotic stress, adjust tree growth vigor, increase yield, and improve other fruit quality traits. Investigations of rootstock and scion graft relationships have originally focused on anatomical and cellular development, nutrient transport, and hormonal movement across graft union. Discovery of long distance transport of mRNA and small RNAs in phloem tissues of rootstock and scion has provided new opportunities for investigation. In this study, we report on the endogenous transport of Gibberellic acid insensitive (GAI) across graft union of a traditional local Chinese pear cultivar, Pyrus bretschneideri cv. Yali (scion), and a wild Pyrus betulaefolia cv. Bunge (rootstock). Cleaved amplified polymorphic sequence analysis RT-PCR indicated that Pyrus-GAI can be transported within 4 and 10 days after micro-grafting, and it can also be transported to a 10–50-cm tall scion of a 2-year-old grafting tree. To further investigate the transport capacity of Pyrus-GAI transcript, a 35S:pear (P. betulaefolia)-GAI transgenic tobacco (Nicotiana tabacum L. cv. Samson.) was prepared and grafted to wild-type tobacco. RT-PCR indicated that sustained transmission of GAI mRNA through the graft union occurred from the 15th day after grafting. The results have laid a foundation for improving rootstock and regulating the properties of scion in fruit trees by transgenic technology.

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Acknowledgments

We would like to thank Song-ling Bai (Hirosaki University, Japan) and Ai-de Wang (Cornell University, USA) for the technical assistance. This work was supported by the Doctoral Program Special Fund of the Ministry of Education in China (2010000811036), National Natural Science Foundation of China (30871697) and Beijing Natural Science Foundation (6102017).

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Authors and Affiliations

  1. Laboratory of Fruit Cell and Molecular Breeding, China Agricultural University, Beijing, 100193, People’s Republic of China

    Wen-Na Zhang, Lei Gong, Chao Ma, Jian-Fang Hu & Tian-Zhong Li

  2. Laboratory of Plant Breeding and Genetics, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, 036-8561, Japan

    Hai-Yan Xu & Takeo Harada

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  1. Wen-Na Zhang
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  2. Lei Gong
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Correspondence to Tian-Zhong Li.

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Fig. S1

Nucleotide sequences of DL-GAI and YL-GAI genes. YL-GAI, P. bretschneideri GAI; DL-GAI, P. betulaefolia GAI (JPEG 946 kb)

Fig. S2

CAPS analysis of DL-GAI and YL-GAI alleles. a P. bretschneideri ‘Yali’ and Bunge cDNA sequences. b Predicted fragment sizes by HapII digestion (JPEG 30 kb)

High resolution image (TIFF 8464 kb)

Fig. S3

Evidence of no contamination of gDNA in cDNA preparations. a The genomic structure of a part of the Actin gene. Boxes and lines indicate exons and introns, respectively. The positions of primers used are shown above the structure. b PCR products when gDNA and cDNA were both used as templates. The gDNA fraction amplified the PCR product including introns, whereas the cDNA fractions amplified the 82 bp exon sequences. The scion and stock cDNA fractions were from plants sampled 5 days after grafting. M. DNA ladder 2000 (JPEG 23 kb)

High resolution image (TIFF 2360 kb)

Fig. S4

Transgenic construct of 35S:DL-GAI cDNA into pBI 121. The plant expression vector pBI 121 (GenBank ID, AF485783) contained 35S CaMV promoter, resistant tab neomycin phosphotransferaseII (NPTII), and a nopaline synthase (nos) 3’ transcriptional terminator sequence, was used for the expression studies in transgenic tobaccos. The sense primer XBAGAIF was contained in XbaIsite at the 5’ end and the antisense primer SACGAIR was contained in SacIsite at the 3’ end. Amplified DL-GAI cDNA sequences were purified and inserted into the same sites of pBI 121 without GUS. The pBI 121 without GUS gene was used as controls (JPEG 25 kb)

High resolution image (TIFF 17926 kb)

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Zhang, WN., Gong, L., Ma, C. et al. Gibberellic Acid-Insensitive mRNA Transport in Pyrus. Plant Mol Biol Rep 30, 614–623 (2012). https://doi.org/10.1007/s11105-011-0365-7

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