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Isolation of a LEAFY homolog from Populus tomentosa: expression of PtLFY in P. tomentosa floral buds and PtLFY-IR-mediated gene silencing in tobacco (Nicotiana tabacum)

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Abstract

To understand the genetic and molecular mechanisms underlying floral development in Populus tomentosa, we isolated PtLFY, a LEAFY homolog, from a P. tomentosa floral bud cDNA library. DNA gel blot analysis showed that PtLFY is present as a single copy in the genomes of both male and female individuals of P. tomentosa. The genomic copy is composed of three exons and two introns. Relative expression levels of PtLFY in tissues of P. tomentosa were estimated by RT-PCR; our results revealed that PtLFY mRNA is highly abundant in roots and both male and female floral buds. A low level of gene expression was detected in stems and vegetative buds, and no PtLFY-specific transcripts were detected in leaves. PtLFY expression patterns were analyzed during the development of both male and female floral buds in P. tomentosa via real-time quantitative RT-PCR. Continuous, stable and high-level expression of PtLFY-specific mRNA was detected in both male and female floral buds from September 13th to February 25th, but the level of PtLFY transcripts detected in male floral buds was considerably higher than in female floral buds. Our results also showed an inverted repeat PtLFY fragment (PtLFY-IR) effectively blocked flowering of transgenic tobacco plants, and that this effect appeared to be due to post-transcriptional silencing of the endogenous tobacco LFY homologs NFL1 and NFL2.

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Abbreviations

E. coli :

Escherichia coli

SDS:

Sodium dodecyl sulfate

SSC:

Saline-sodium citrate

WT:

Wild type

qRT-PCR:

Quantitative reverse transcription polymerase chain reaction

IR:

Inverted repeat

PTGS:

Post-transcriptional gene silencing

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Acknowledgments

We gratefully acknowledge Prof. Phillip F. Elliott, East Hartford University, CT, USA, and Dr. Jean W. H. Yong, Nanyang Technological University, Singapore, for invaluable advice and assistance. We thank Huiquan Zheng, a Ph.D. student currently in our laboratory, for assistance with figure preparation. This work was supported by grants from the Natural Science Foundation of China (No. 30571511), National High-tech R&D Program of China (No. 2009AA10Z107) and Key Project of Education of Chinese Ministry of Education (No. 108017).

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Authors and Affiliations

  1. National Engineering Laboratory for Tree Breeding, Beijing Forestry University, Beijing, 100083, People’s Republic of China

    Xin-Min An, Dong-Mei Wang, Ze-Liang Wang, Bo Li, Wen-Hao Bo, Guan-Lin Cao & Zhi-Yi Zhang

  2. Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants, Ministry of Education, Beijing Forestry University, Beijing, 100083, People’s Republic of China

    Xin-Min An, Dong-Mei Wang, Ze-Liang Wang, Bo Li, Wen-Hao Bo, Guan-Lin Cao & Zhi-Yi Zhang

  3. The Tree and Ornamental Plant Breeding and Biotechnology Laboratory of State Forestry Administration, Beijing Forestry University, Beijing, 100083, People’s Republic of China

    Xin-Min An, Dong-Mei Wang, Ze-Liang Wang, Bo Li, Wen-Hao Bo, Guan-Lin Cao & Zhi-Yi Zhang

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Fig. S1

The predicted secondary and tertiary structures of the PtLFY protein. (A) The secondary structure of PtLFY. The blue, yellow, green and light blue colors indicate the helix, sheet, turn and coil structure, respectively. (B) Putative proportion of helix, sheet, turn and coil in PtLFY. (C) The tertiary structure of PtLFY. The highly conserved PtLFY C-terminal is composed of α1, α2, α3, α4, α5, α6 and α7 helices (TIFF 4662 kb)

Fig. S2

The alignments of C-terminal amino acid sequences (A) and nucleotide sequences (B) of PtLFY (GenBank accession no. AY211519) with NFL1 (U16172) and NFL2 (U15799). PtLFY shares 94.7% amino acid identity with NFL1 and NFL2 at C-terminal designed as the target of PtLFY-IR structure (A). Nucleic acid of PtLFY shows 82.1% similarity to that of NFL1 and NFL2 at C-terminal designed as the target of PtLFY-IR structure (B) (TIFF 1128 kb)

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An, XM., Wang, DM., Wang, ZL. et al. Isolation of a LEAFY homolog from Populus tomentosa: expression of PtLFY in P. tomentosa floral buds and PtLFY-IR-mediated gene silencing in tobacco (Nicotiana tabacum). Plant Cell Rep 30, 89–100 (2011). https://doi.org/10.1007/s00299-010-0947-0

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