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Identification of miRNA Targets by AtFT Overexpression in Tobacco

  • Ahamed Khan
  • Ankita Shrestha
  • Mukundan Shaju
  • Kishore C. PanigrahiEmail author
  • Nrisingha DeyEmail author
Original Paper
  • 59 Downloads

Abstract

The onset of flowering is regulated by complex gene networks that integrate multiple genetic cues to floral transition in plants. The highly conserved florigen FLOWERING LOCUS T (FT) functions at the core of this process. Here, we demonstrate that overexpression of Arabidopsis FT in tobacco (oxAtFT) leads to precocious flowering in a photoperiod-independent manner. Strikingly altered phenotypes such as stunted growth, increased number of axillary branches, change in leaf morphology, and elongated style were observed in oxAtFT transgenic tobacco plants. In addition, the generation time of oxAtFT tobacco was drastically reduced by 8–10 weeks. We observed defects in lateral root formation in both oxAtFT tobacco and oxFT Arabidopsis lines. Interestingly, oxAtFT tobacco displayed significant heat and drought tolerance as compared to the wild type. Through small RNA profiling, we observed the upregulation of a conserved microRNA (nta-miR393a-5p) of miR393 family that targets the TRANSPORT INHIBITOR RESPONSE 1-like (TIR1) gene. Overall, the heterologous expression of AtFT in tobacco may have potential in imparting abiotic stress tolerance in other plant species through plant biotechnology–based approaches.

Keywords

FLOWERING LOCUS T Lateral root Stress tolerance Micro-RNA TIR1 

Notes

Acknowledgments

We are grateful to Dr. Indu B. Maiti, Scientist, University of Lexington, Kentucky, USA, for kindly providing us with the AtFT codon-optimized clone. We thank Dr. Ajay Parida, Director, Institute of Life Sciences, Bhubaneswar, for his interest and suggestions in this study. We would also like to thank Mr. Arup Ghosh for helping in the DGE analysis and Mr. Abhimanyu Das for his technical assistance.

Author Statement

Ahamed Khan: conceptualization, performing experiments, analyzing data, and writing the manuscript. Ankita Shrestha: data curation, writing—original draft preparation. K C Panigrahi: data analysis and correction of the manuscript. Nrisingha Dey: data curation/analysis, manuscript finalization, and overall supervision.

Abbreviations

Nt, Nicotiana tabacum; FT, FLOWERING LOCUS T; TIR1, TRANSPORT INHIBITOR RESPONSE 1-like; SOC1, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1; AP1, APETLA 1; LA, Lateral root

Funding Information

This study was supported by the funds from Government of India under grant number BT/HRD/35/01/05/2015.

Supplementary material

11105_2019_1180_Fig8_ESM.png (112 kb)
Supplementary Fig. 1.

Expression patterns of tobacco floral identity genes in WT and 35S::AtFT homozygous T3 lines tobacco lines. The transcript level of NtSOC1(A, B) and NtAP1(C, D) was found to be nearly 2 - 4 fold upregulated in 35S::AtFT lines (#2 and #8) under both LD and SD conditions. Data are mean ± standard deviation of three trials. P-value top on the bars indicate significant differences from WT to 35S::AtFT by the Student’s t test. (PNG 111 kb)

11105_2019_1180_MOESM1_ESM.tif (176 kb)
High resolution image (TIF 176 kb)
11105_2019_1180_Fig9_ESM.png (397 kb)
Supplementary Fig. 2:

Grafting experiments to demonstrate the bidirectional and long-distance movement of FT as florigen in tobacco. Cleft grafting models representing the movement of FT from apex (scion) to base (stock) and base (stock) (A) to apex (base) (B) respectively. Here, the black dot represents the FT as a florigen. (C) The 35S::AtFT/WT, grafting of AtFT transgenic scion onto WT stock and (D) WT/35S::AtFT, grafting of WT scion over 35S::AtFT stock. (E) Flowering time for both 35S::AtFT/WT and WT/35S::AtFT grafts showing relatively reduced number of “days to flowering” recorded from the initial day of grafting. All grafting experiments were conducted along with WT/WT and non-grafted WT as controls. The red arrow indicates the graft junction. The different P-value at the top of the bars (mean ± standard deviation) indicate statistically significant differences between the WT stock to WT/35S::AtFT and WT scion over 35S::AtFT stock with the Student’s t test. (PNG 396 kb)

11105_2019_1180_MOESM2_ESM.tif (1.6 mb)
High resolution image (TIF 1618 kb)
11105_2019_1180_Fig10_ESM.png (239 kb)
Supplementary Fig. 3:

Length distribution of small RNA reads. (A) Distribution of small RNAs with trimmed unique reads based on their length in bp (minimum length 16 bp and maximum 40 bp). Venn diagrams representing a total of 37 miRNA families (B) and 78 known miRNAs (C) that were common among both 35S::AtFT and WT tobacco. (D) miRNA family distribution showing the number of miRNAs that fall in each known miRNA family across the oxAtFT and WT tobacco. (PNG 238 kb)

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High resolution image (TIF 1144 kb)
11105_2019_1180_Fig11_ESM.png (311 kb)
Supplementary Fig. 4.

Gene ontology (GO) distribution of miRNA targets. Gene functional annotation was performed and used to characterize the known (A) and novel (B) miRNA targets based on their involvement in biological processes, cellular components and molecular functions. (PNG 310 kb)

11105_2019_1180_MOESM4_ESM.tif (1.3 mb)
High resolution image (TIF 1288 kb)
11105_2019_1180_MOESM5_ESM.xlsx (190 kb)
ESM 1 Supplementary excel file: The raw data of small RNA sequencing are given in single excel file with total number of 13 sheets that includes: Sheet 1: Read statistics; Sheet 2: known common family; Sheet 3: known common miRNA; Sheet 4; GO for known miRNA; Sheet 5: GO for novel miRNA; Sheet 6: known miRNA only in WT; Sheet 7: known miRNA only in 35S::AtFT; Sheet 8: known miRNA in both sample; Sheet 9: Novel miRNA only in WT; Sheet 10: Novel miRNA only in 35S::AtFT; Sheet 11: Novel miRNA in both sample; Sheet 12: Total Novel miRNA in both samples; Sheet 13: Total Novel miRNA in both samples. (XLSX 190 kb)
11105_2019_1180_MOESM6_ESM.docx (18 kb)
ESM 2 (DOCX 18 kb)
11105_2019_1180_MOESM7_ESM.mov (2.6 mb)
ESM 3 Electronic Supplementary file: Video clip demonstrating the heat shock tolerance assay (at 50 °C) with AtFT transgenic and WT tobacco. (mov 2628 kb)

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Authors and Affiliations

  1. 1.Division of Plant and Microbial BiotechnologyInstitute of Life SciencesBhubaneswarIndia
  2. 2.National Institute of Science Education and ResearchJatniIndia

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