REGULATION OF TRANSLATION DURING ABIOTIC STRESS CONDITIONS IN PLANTS
- Medina Villar, Silvia - Technician
- Muñoz Gutiérrez, Alfonso - Visiting Scientist
- Novo Uzal, Esther - Young Investigator Researcher (YIR)
- Ochoa Herreros, Clara - Technician
- Okeke Infante, Isabel Chinyere - PhD Student
- Pulido Navarro, Aurora - Technician
- Tello Rodríguez, Sandra - PhD Student
- Toribio López, Francisco René - Postdoctoral Fellow
- Vazquez, María Magdalena - Postdoctoral Fellow
Most plants complete their life cycle in a single location and, therefore, their development and reproduction depend on the environmental conditions they are exposed to. Environmental stresses such as drought, high and low temperatures, saline soils, etc., seriously affect plant development and growth. To cope with these challenges, plants have evolved molecular strategies that allow them to survive under such adverse conditions.
One of the earliest plant responses to environmental stresses is regulation of translation. This regulation usually involves a general inhibition of protein synthesis. Under such conditions, some mRNAs able to bypass the global inhibition are selectively translated. These mRNAs are usually involved in the establishment of the stress response and are crucial regulators of plant adaptation to environmental cues. Despite these well-known outputs, the knowledge about the possible mechanisms involved in the general inhibition and the selective translation of mRNAs is very scarce in plants.
Our lab aims to contribute to understand how plants respond to stress and, from this knowledge, to provide molecular tools to improve crop security and production. To achieve this goal, our lab focuses on the study of translational regulation to identify new regulators of the stress response. In addition, one of our main interests is to uncover new mechanisms involved in translational regulation in plants.
Identification of new regulators involved in the adaptation to stress in plants
It has been widely reported that translation regulation plays an important role during the plant response to environmental cues, allowing the translation of specific mRNAs that are crucial for the response. Based on this, our lab has implemented different techniques (such as super-resolution ribosome profiling, Ribo-seq analysis, polysomal profiling, etc.) to allow the identification of those mRNAs that are translationally regulated (and so, highly susceptible to play an important function) during specific stages of development and in response to environmental stresses.
As an example of these approaches, in the recent past we carried out a high-throughput translatome analysis in Arabidopsis seedlings subjected to a moderate heat stress (38°C 1 h) (Yangüez et al., 2013). From this study, we identified different mRNAs that were selectively translated under heat stress conditions.
Heat selectively regulates translation of functionally relevant cohorts of mRNAs. (A) Density distribution of mRNA relative translation efficiencies (ΔmRNAPB/ΔmRNATOT) in response to heat-stress. mRNAs with a relative translation efficiency ≤ 1.5 fold (class I) or ≥ 1.5 fold (class III) were considered as selectively regulated at the translational level. (B) Functional analysis of the mRNAs selectively regulated at the translational level in response to heat (Yangüez et al., 2013).
In addition, and with the same objective, we did a comparative proteomics analysis of plants grown under control conditions, during a heat acclimation event and at the early stages of the recovery periods from severe heat treatments that selectively lead plants to either survival or death. This quantitative proteomics analysis allowed us to unravel new proteins specifically involved in thermotolerance (Echevarría-Zomeño et al., 2016).
Proteomic analysis to uncover new regulators involved in thermotolerance in plants. Arabidopsis seedlings were subjected to stress treatments that lead to plant survival (A) or death (B). The time-points of samples subjected to iTRAQ analysis corresponding to C (control), A (acclimated), CS (committed to plant survival) and CD (committed to plant death) are highlighted. (C) Cluster analysis of protein accumulation during the different assayed treatments. (D) Thermotolerance assay of mutants of new regulators of thermomorphogenesis that were identified for the first time in this study (Echevarría-Zomeño et al., 2016).
The combined analysis of these translatomic and proteomic approaches retrieved different new potential regulators of plant adaptation to heat stress, which are being analyzed in the lab as part of our research activity.
Characterization of HOP proteins, new mater hubs in plant response to stress
The previously described approaches allowed the identification of AtHOP3 as a potential regulator of thermotolerance in plants.
HOPs (HSP70-HSP90 organizing proteins) are a highly conserved family of cytosolic cochaperones, whose role in adaptation to stress was not previously evaluated in plants. In a recent study (Fernández-Bautista et al., 2017), we showed that HOP3 is induced during the unfolded protein response (UPR), interacts with the endoplasmic reticulum (ER)-resident protein BiP and plays an essential role during ER stress in plants (Fernández-Bautista et al 2017).
HOP3 is localized at the ER and plays an important role during the ER stress in plants. (A) HOP3 colocalizes at the ER with RFP-ER, an especific ER marker protein. (B) hop3 mutants show a hypersensibility to ER stress inducer agents, demonstrating the main role of HOP3 in the alleviation of ER stress in plants.
In addition, in a different report (Fernández-Bautista et al 2018), we described, for the first time in plants, that the three members of the AtHOP family act redundantly to promote long term acquired thermotolerance (LAT) in Arabidopsis. Our results demonstrated that HOP proteins are involved in two important processes associated to LAT, the full establishment of the transcriptional response during the acclimation period and the quality control maintenance during severe heat conditions. These data revealed that HOP family modulates the plant capacity to acclimate to high temperatures for long periods.
HOP proteins play a major role in long term-acquired thermotolerance (LAT) in plants. Growth of the hop1 hop2 hop3 triple mutant, the hsfa2 mutant (a mutant with a well-known hypersensitive to heat stress, included as a positive control) and Col-0 (the wild-type genotype) under control (A) and under LAT conditions (B) (Fernández-Bautista et al 2018).
Recent results obtained in the lab suggest that the role of HOPs is not only circumvented to these two processes, but that HOPs are master hubs in the plant response to different stresses and during specific developmental programs. In this sense, one of our current interests is the identification of the possible targets of HOP and the analysis of how this interaction modifies their activity during the studied processes.
Finally, our lab is also exploring the use of some of the potential regulators identified in previous studies to enhance stress tolerance in crops.
Characterization of the molecular mechanisms that regulate translation initiation under environmental and developmental cues
Translation regulation takes place mainly at the initiation step. In metazoans, one of the main mechanisms for translation regulation is the control of eIF4E activity by interaction with other proteins (known as 4E-BPs and 4E-binding partners). Despite the broad function of these eIF4E interacting proteins in the regulation of general and specific inhibition of translation in other eukaryotes, no orthologues have been found in plants (Sesma et al., 2017). In our lab, we are interested in characterising the mechanisms involved in the regulation of translation initiation in plants, paying a special attention to those mechanisms related to eIF4E regulation. Our data suggest that, in contrast to the general view, the regulation of translation initiation is not so conserved in plants as in other eukaryotes, highlighting the extraordinary importance of identifying and characterising these mechanisms in the plant kingdom.
Mechanisms for inhibition of general and specific mRNA translation through the interaction of different proteins with eIF4E in mammals. Under control conditions eIF4E interacts with the 7mGpppN cap structure of the mRNA and eIF4G, allowing mRNA initiation of translation. However, under different stress conditions and developmental programs, different proteins (4E-BPs and 4E-binding partners) interact with eIF4E leading to a general and selective inhibition of mRNA translation, respectively. Although, the main players in the initiation of translation are conserved among eukaryotes, no ortologues of the cited eIF4E interacting proteins have been identified in plants, rendering the knowledge about how plant eIF4E regulates mRNA translation completely unexplored.
1. S2013/ABI-2734. Tradaptación. “Translational regulation and its biotechnological use to enhance plant adaptation to biotic and abiotic stresses-TRADAPTACION”. Funding organization: CAM. Coordinator and PI: Mar Castellano.
2. RTA2013-00027-00-00. “Study of the biotechnological potential of 3P protein to enhance plant tolerance to high temperature and pathogens”. Funding organization: INIA-MINECO. 2014-2017. PI: Mar Castellano.
3. StG260468. “Plant cIRES Biotech”. “Functional characterization of plant cIRES and their use as biotechnological tools”. Funding organization: ERC. 2010-2017. PI: Mar Castellano.
4. BIO2010-15751. “Study of the regulation of the translation initiation factor eIF4E in response to abiotic stress in plants”. Funding organization: MICINN. 2011-2014. PI: Mar Castellano.
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