Pracownie badawcze

Badania

Najważniejsze osiągnięcia badawcze

• We functionally characterized the first plant-selective autophagy cargo receptor Joka2/NBR1.
• We identified several ABA-related transcription factors as novel targets of NBR1 and proposed that NBR1 is a novel player in the crosstalk of autophagy with ABA signaling.
• We demonstrated the involvement of proteasomal degradation in the sulfur-deficiency response and discovered that the E3 ubiquitin ligase EBF1 interacts with the main sulfur response regulator SLIM1.
• We contributed to characterization of the plant-specific LSU family by creating and mutationally verifying a structural model of LSU dimers and identified novel partners of LSUs.

Opis badań

• Plant response to sulfur deficiency stress is complex and controlled by protein degradation systems

Sulfur is an essential macronutrient for all organisms. The knowledge of plant sulfur metabolism and responses to its deficiency expanded significantly during recent decades. Our group contributed to this knowledge using tobacco and then Arabidopsis as a model. For several years, an important aim of our research was the characterization of regulatory mechanisms that are involved in plant adaptation to sulfur deficiency stress [1-3]. Interestingly, numerous data from our laboratory and other laboratories revealed that two major systems that are involved in cellular protein degradation (autophagy and proteasome) control the plant response to nutrient deficiency. This observation prompted us to focus on various aspects of ubiquitination, proteasomal degradation, and autophagy.

Ubiquitination controls many facets of plant growth and development and the response to biotic and abiotic stress. Therefore, a reasonable assumption is that some components of the ubiquitination system are involved in controlling the plant response to sulfur deficiency. Signaling processes during stress require explicit timing control, which explains why plants frequently rely on selective, ubiquitination-based protein degradation. We work on functional analyses of ubiquitin-proteasome system components that are involved in the modulation of the sulfur deficiency response and identification of target proteins. These studies include monitoring the transcriptome and proteome in response to sulfur deficiency stress, with a particular focus on ubiquitinated proteins with putative regulatory functions. We recently showed that several genes that encode E3 ligases are specifically regulated by sulfur deficiency and that a key transcription factor of the sulfur deficiency response, SLIM1, undergoes proteasomal degradation and is able to interact with the F-box protein EBF1 [4]. The plant stress response is also controlled by autophagy, an evolutionarily conserved degradation process. Surprisingly, we demonstrated the existence of the NBR1-type cargo receptor in tobacco and its link to the sulfur deficiency response [5].

• Selective autophagy cargo receptor NBR1 modulates plant growth and the response to environmental stress

Our recently published and unpublished results revealed that the selective degradation of strategic cellular targets by plant autophagy machinery is used to reprogram metabolism during nutrient deficiency. We searched for protein partners of NBR1 under normal and sulfur-deficiency conditions. We identified numerous novel candidates as NBR1 targets that play a potential strategic role in plant metabolism. For example, the interaction between NBR1 and ribosomal protein S6 (RPS6) and S6 kinase (S6K) might suggest the involvement of NBR1-mediated selective autophagy in the control of ribosome composition or activity, depending on growth conditions [6]. Moreover, the identification of ABA-responding transcription factors as novel partners of NBR1 prompted us to examine the involvement of NBR1 in the modulation of ABA signaling [7] and possibly hormonal crosstalk. To further investigate the function of NBR1, we constructed several unique plant lines with either deletion or overexpression of the NBR1 gene.

• Plant-specific family of LSU proteins

The LSU family, consisting of four members in Arabidopsis, attracted our attention several years ago [8]. LSU proteins interact with NBR1, and LSU genes are induced by sulfur deficiency in tobacco and Arabidopsis [9,10]. Additionally, LSU1 belongs to a cluster of six Arabidopsis genes that are co-regulated by an unclear mechanism that involves a small metabolite, O-acetyloserine. LSU proteins are engaged in multiple protein-protein interactions, but their function is unknown. We identified novel partners, built and analyzed an interaction network, and proposed that LSU proteins are involved in the stabilization of their partners and possibly also in the facilitation of their cellular transport [10].  This hypothesis remains to be verified.

• Future plans

The process of NBR1-mediated selective autophagy in plants and its significance in the abiotic stress response have not been sufficiently addressed to date. We want to further explore several of our preliminary findings, such as the involvement of NBR1 in coordinating hormone signaling, the feedback modulation of autophagy flux, and the adjustment of ribosome activity to growth conditions. Our aim is to obtain insights into these issues at the molecular level. Other interesting issues that require further clarification are the mechanisms of target recognition by NBR1. Proteins that are targeted by NBR1 can be divided into two categories: (i) requiring the ubiquitin-associated (UBA) domain of NBR1 for interactions and (ii) interacting in a UBA-independent way. Partners in the first category are likely “conventional” degradation cargo that is recognized via a ubiquitin tag. Partners in the second category might be “non-conventional” degradation targets, or they can serve as regulators of NBR1 activity.

• Bibliography

1. Wawrzynska et al. 2015. Front Plant Sci, 6, 1053, doi:10.3389/fpls.2015.01053.
2. Wawrzynska et al. 2014. Front Plant Sci, 5, 575, doi:10.3389/fpls.2014.00575.
3. Wawrzynska et al. 2020. International Journal of Molecular Sciences 21, doi:10.3390/ijms21082771.
4. Wawrzynska et al. 2020. Plant & cell physiology, 61, 1548-1564, doi:10.1093/pcp/pcaa076.
5. Zientara-Rytter et al. 2011. Autophagy, 7, 1145-1158, doi:10.4161/auto.7.10.16617.
6. Tarnowski et al. 2020. Overexpression of the selective autophagy cargo receptor NBR1 modifies plant response to sulfur deficit. Cells, 9, 669, doi:10.3390/cells9030669.
7. Tarnowski et al. 2020. Sci Rep, 10, 7778, doi:10.1038/s41598-020-64765-z.
8. Sirko A et al. 2015. Frontiers in Plant Science, 5, doi:10.3389/fpls.2014.00774.
9. Lewandowska et al. 2010. Mol Plant, 3, 347-360, doi:10.1093/mp/ssq007.
10. Niemiro et al. 2020. Frontiers in Plant Science, 11, 1246, doi:10.3389/fpls.2020.01246.

Metodologia

We use various molecular biology and biochemistry techniques and immunoprecipitation techniques and transcriptome analysis. We successfully used CRISPR/Cas9 technology to create numerous novel deletion mutants in Arabidopsis thaliana. In collaboration with other colleagues from IBB, we often apply more complex biophysical methods and bioinformatic approaches for data acquisition and analysis. We use different bacterial, yeast, and plant (transgenic plants, plant viruses) systems to produce recombinant proteins.

Wybrane publikacje

• Identification and functional analysis of Joka2, a tobacco member of the family of selective autophagy cargo receptors. Zientara-Rytter K, Lukomska J, Moniuszko G, Gwozdecki R, Surowiecki P, Lewandowska M, Liszewska F, Wawrzynska A, Sirko A. Autophagy. 2011. doi: 10.4161/auto.7.10.16617
• Overexpression of the selective autophagy cargo receptor NBR1 modifies plant response to sulfur deficit. Tarnowski L, Rodriguez MC, Brzywczy J, Cysewski D, Wawrzynska A, Sirko A. Cells. 2020. doi: 10.3390/cells9030669.
• A selective autophagy cargo receptor NBR1 modulates abscisic acid signalling in Arabidopsis thaliana. Tarnowski L, Rodriguez MC, Brzywczy J, Piecho-Kabacik M, Krckova Z, Martinec J, Wawrzynska A, Sirko A. Sci Rep. 2020. doi: 10.1038/s41598-020-64765-z.
• Proteasomal degradation of proteins is important for the proper transcriptional response to sulfur deficiency conditions in plants. Wawrzynska A, Sirko A. Plant Cell Physiol. 2020. doi: 10.1093/pcp/pcaa076.
• Similar but not identical-binding properties of LSU (Response to Low Sulfur) proteins from Arabidopsis thaliana. Niemiro A, Cysewski D, Brzywczy J, Wawrzynska A, Sienko M, Poznanski J, Sirko A. Plant Sci. 2020, 11, doi: 10.3389/fpls.2020.01246.

Współpraca

• Rüdiger Hell, Centre for Organismal Studies (COS), University of Heidelberg, Germany, https://www.cos.uni-heidelberg.de/index.php/r.hell?l=_e
• Céline Masclaux-Daubresse, Institut Jean-Pierre Bourgin, INRAE Centre de Versailles-Grignon, France, https://www-ijpb.versailles.inra.fr/en/nap/equipes/recyclazote/index.html
• Henri Batoko, Louvain Institute of Biomolecular Science and Technology, UCLouvain, Belgium, https://uclouvain.be/en/research-institutes/libst/henri-batoko.html
• Stan Kopriva, Institute for Plant Sciences, University of Cologne, Germany, https://ag-kopriva.botanik.uni-koeln.de/
• Rainer Hoefgen, Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany, https://www.mpimp-golm.mpg.de/10698/Rainer_Hoefgen
• Jan Martinec, Institute of Experimental Botany of the Czech Academy of Sciences, Prague, Czech Republic, http://www.ueb.cas.cz/en/users/martinec
• Said El Alaoui, Covalab, Biotechnology company in Bron, France, https://www.covalab.com/