Abstract
The apple TFL1 genes, MdTFL1-1 and MdTFL1-2, were tried to be silenced using the opposing dual-promoter gene construct 620p9U10-HSP::MdTFL1-HSP containing a 323 bp fragment of MdTFL1-1 flanked by two inverted sequences of the Gmhsp17.5-E heat-inducible promoter. This construct was used for transformation experiments with the apple cultivars ‘Gala’ and ‘Pinova’. Three (T967, T973, and T985) and two (T976 and T987) transgenic lines were obtained for ‘Pinova’ and ‘Gala’, respectively. Single heat shock treatments at 42 °C for 1 and 2 h, respectively, were applied to plants of transgenic lines and non-transformed controls grown in vitro or in the glasshouse. Heat shock treatments induced a short-term activation of the transgene construct. Silencing of MdTFL1-1 and MdTFL1-2 was detected in transgenic plants, but also in non-transformed controls, highlighting a similar heat response. The transcription of both MdTFL1 genes was repressed for 24 h post heat shock induction. In parallel, we treated shoots of glasshouse-grown plants at 42 °C for 2 h daily for the duration of 28 days. This treatment resulted in silencing of MdTFL1 in leaves of transgenic and non-transformed plants. However, repeated heat shock treatments failed to induce flower development in plants. Notwithstanding, we recorded a number of deformed leaves on transgenic shoots in comparison to non-transformed ones. This has lead to the conclusion that the transgene construct is functional in down-regulation of TFL1 homologs. The heat shock treatments demonstrated a major role of both MdTFL1 genes in plant’s heat response.
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Abbreviations
- HSP:
-
Gmhsp17.5-E heat shock promoter from Glycine max
- MdTFL1 :
-
Malus × domestica TERMINAL FLOWER 1
- SAM:
-
Shoot apical meristem
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Acknowledgments
This work was funded by the EU Seventh Framework Programme, Project FRUIT BREEDOMICS No. 265582 titled “Integrated approach for increasing breeding efficiency in fruit tree crop”. The views expressed in this work are the sole responsibility of the authors and do not necessarily reflect the views of the European Commission. Furthermore, we are grateful to A. Lauber, U. Hille, I. Hiller, I. Polster, T. Strunz, and M. Tronicke for technical assistance.
Authors contribution
All authors contributed equal to the conception and design of the experiments. K. W. performed the experiments, the analysis and interpretation of data and partly wrote the manuscript. H. F., A. P. and M.-V. H. contributed to the manuscript and critical revised the article.
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Table S1
Primers sequences and amplicon characteristics used in PCR and qRT-PCR analyses. (DOCX 15 kb)
Figure S1
Molecular detection of nptII copies within the plant genome of transgenic HSP::MdTFL1-1 apple lines by Southern blotting. Hybridization of BamHI (a) or HindIII (b) digested genomic DNA of the transgenic and control apple genotypes using a digoxygenin-labeled probe amplified with nptII F/R primers. Lines which were generated by transformation of ‘Gala’ were illustrated bold, ‘Pinova’ and its lines of were not. WM DNA Molecular Weight Marker VII (Roche). (TIFF 2385 kb)
Figure S2
Induction of the HSP promoter in leaf tissue of transgenic glasshouse apple genotypes by a single heat shock treatment. qRT-PCR analysis of HSP::MdTFL1-1 transcription before and post heat shock treatment at 42 °C for 1 h. The qRT-PCR analysis results were normalized using EF1α as reference gene. Graphs represent the average of the relative transcript level from three biological replicates per genotype. (TIFF 421 kb)
Figure S3
The effect of heat shock treatment on MdTFL1 transcription in leaves of glasshouse apple genotypes during the first day of heat shock treatment. qRT-PCR analysis of MdTFL1-1 transcription before and post a single heat shock treatment at 42 °C for 1 h. The qRT-PCR analysis results were normalized using EF1α as reference gene. Graphs represent the average of the relative transcript level from three biological replicates per genotype. (TIFF 509 kb)
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Weigl, K., Flachowsky, H., Peil, A. et al. Heat mediated silencing of MdTFL1 genes in apple (Malus × domestica). Plant Cell Tiss Organ Cult 123, 511–521 (2015). https://doi.org/10.1007/s11240-015-0855-7
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DOI: https://doi.org/10.1007/s11240-015-0855-7