Ph.D. Topics

1. Investigating pre-mRNA alternative splicing during the embryonic thermoresponse in Arabidopsis thaliana

Supervisor: Helene Robert Boisivon, Ph.D.

Annotation:

Transcript variants generated by alternative splicing (AS) of a precursor mRNA transcript expand the cellular protein catalog for the plant response to various stresses without the requirement of de novo transcription. AS is a molecular strategy taken by plants under temperature stress to limit the adverse effects of high temperatures and to adjust their growth and physiology during the stress period. Spliced variants may be targeted for degradation by non-sense mediated decay (NMD) or encode alternative proteins to feedback on the stress response. Heat Shock Proteins (HSP) and Heat Shock Factors (HSF) are among the targets of temperature-induced AS. Our lab investigates how high temperatures affect embryo morphogenesis and seed development. We identified that AS regulators are upregulated in seeds of plants grown at high temperatures. The project will investigate the implications of AS in the morphogenic alterations of Arabidopsis embryos when developing at high temperatures. The proposed experimental approaches include profiling of AS events in high-temperature seeds by nanopore RNA sequencing. Specific candidates whose transcripts are targeted by AS at high temperatures, and known for their involvement in embryo morphogenesis, will be functionally characterized: expression analysis (microscopy), phenotyping (genetics), ectopic expression (cloning, generation of transgenics), etc.

The Ph.D. topic is part of a collaborative project with the research team led by Dr. Said Hafidh in the lab of Prof. Honys at the Institute of Experimental Botany in Prague.

Requirements on candidates:

The candidate will have notions of bioinformatics. The candidate has experience in molecular biology (cloning, RT-qPCR), expression analysis (microscopy), and masters the notions of plant genetics and epigenetics.

Recommended literature:

  1. John, S., Olas, J. J. & Roeber, B. M. Regulation of alternative splicing in response to temperature variation in plants. J Exp Bot erab232 (2021) doi:10.1093/jxb/erab232.
  2. Kulichová, K. et al. PRP8A and PRP8B spliceosome subunits act coordinately to control pollen tube attraction in Arabidopsis thaliana. Development (Cambridge, England) 147, dev186742 (2020).
  3. Mácová, K. et al. Effects of long-term high-temperature stress on reproductive growth and seed development in development in Brassica napus. Biorxiv 2021.03.11.434971 (2021) doi:10.1101/2021.03.11.434971.
  4. Robert, H. S. et al. Local auxin sources orient the apical-basal axis in Arabidopsis embryos. Current Biology 23, 2506–2512 (2013).
  5. Verma, S., Attuluri, V. P. S. & Robert, H. S. An Essential Function for Auxin in Embryo Development. Csh Perspect Biol 13, a039966 (2021).

Keywords:      Arabidopsis thaliana, embryogenesis, high temperatures, alternative splicing


2. Role of the AUXIN RESPONSE FACTOR5 and miRNAs in embryogenic transition in Arabidopsis

Supervisor: Helene Robert Boisivon, Ph.D.

Annotation:

Somatic embryogenesis is the plant's unique developmental capacity to switch on an embryonic developmental program in somatic cells. It is a process widely used for the micropropagation of various plant species. The somatic embryogenesis process is also a valuable working model to decipher the molecular mechanisms that determine the capacity of somatic cells to reprogram into embryonic cells. The plant hormone auxin is one key regulator and inducer of the process. And the AUXIN RESPONSE FACTOR5 plays a fundamental role in regulating many aspects of the somatic embryogenesis response. ARF5 may regulate the expression of auxin biosynthetic YUCCA enzymes and post-transcriptional modulators such as miRNA390s. This project will contribute to gain a better understanding of their regulatory interaction during the somatic embryogenesis process. The project will include the functional analysis of the MIR390 genes during the embryogenic transition and the analysis of the functional relationship between ARF5 and selected YUCCAs. The proposed experimental approaches include the preparation of molecular reporters and various other constructs (cloning and generation of transgenics), the preparation of CRISPR-based mutants and their phenotypic analysis (genetics, tissue culture), and the analysis of expression (microscopy, RT-qPCR), among others.

The Ph.D. topic is part of a bilateral collaborative project with the Polish research team led by Dr. Barbara Wójcikowska in the lab of Prof. Gaj at the University of Silesia in Katowice.

Requirements on candidates:

The project implementation will necessitate intense tissue culture work. However, this is not the main selection criteria for the suitable candidate. The candidate will have some expertise in molecular biology (cloning, RT-qPCR), expression analysis (microscopy), and master the notions of plant genetics and epigenetics.

Recommended literature:

  1. Robert, H. S. et al. Local auxin sources orient the apical-basal axis in Arabidopsis embryos. Current Biology 23, 2506–2512 (2013).
  2. Verma, S., Attuluri, V. P. S. & Robert, H. S. An Essential Function for Auxin in Embryo Development. Csh Perspect Biol 13, a039966 (2021).
  3. Wójcikowska, B. et al. LEAFY COTYLEDON2 (LEC2) promotes embryogenic induction in somatic tissues of Arabidopsis, via YUCCA-mediated auxin biosynthesis. Planta 238, 425–440 (2013).
  4. Wójcikowska, B., Wójcik, A. M. & Gaj, M. D. Epigenetic Regulation of Auxin-Induced Somatic Embryogenesis in Plants. Int J Mol Sci 21, 2307 (2020).

Keywords:  Auxin, Arabidopsis thaliana, somatic embryogenesis, miRNA