PUB - Publikationen an der Universität Bielefeld

The molecular mechanisms by which plants respond to their changing environment without the ability to move away from potentially harmful stimuli are subject of intensive research. Light in this context takes a special role, since it represents the ultimate source of energy for autotrophic growth. In addition, light-driven photosynthesis is inevitably linked to oxidative stress in a stressful environment, so suitable coping mechanisms must be at place to balance both processes.

This work delivers evidence that 10 min of low light to high light transfer (10 min LL $\\rightarrow$ HL) and the associated high light stress (oxidative stress) significantly influences the cytosolic translation process as decisive part of the cellular stress response. Evident unloading of polysomes indicates that oxidative stress caused by the 10 min LL $\\rightarrow$ HL prevails over the energizing effects the sudden light increase has on shade-adapted plants in the time frame of the given treatment. Stress-induced unloading of polysomes and subsequent selective loading of specific transcripts represents an efficient and rapid mechanism to adjust protein synthesis and participates decisively in gene expression regulation. The selection of the specific transcripts which get preferentially translated upon high light stress is controlled on multiple regulatory levels and plays an integral role for the required tailored response.

Several retrograde signals and signal transduction elements affect the cytosolic translational machinery. They include i.a. energy signaling pathways employing MITOGEN-ACTIVATED KINASE (MPK) and signaling via altered ratios of reduced (GSH) to oxidized forms (GSSG) of the low molecular weight antioxidant glutathione. Analyses of polysome-association of STRESS-ASSOCIATED PROTEIN (SAP) 2 and 3, both actively translated in the 10 min LL $\\rightarrow$ HL-treatment of Arabidopsis thaliana wildtype, show similar deviant behavior in the signaling-deficient mutants ARABIDOPSIS KIN 10 (akin10), mpk6 and phytoalexin-deficient 2-1 (pad2-1, knock-down). The knockout (KO) of SUCROSE-NON FERMENTING KINASE 1 (SnRK1) subunit AKIN10 had significant impact on polysomal loading, indicating that the abundance of CBB Cycle products together with the redox state of the cell are critical to drive the capacity of the translational initiation process. As all three mutations lead to a decrease of polysome-associated transcripts, chloroplast signals presumably set the first, yet possibly unspecific early layer in the journey to a tailored translatome upon high light stress. More specific regulatory processes evidently affect translation downstream of stress signaling, targeting differential mRNA characteristics, specific mRNA recognition via interaction with RNA-binding proteins (RBPs) and redox state-based conformational changes. Although the composition of polysomal messenger ribonucleoproteins (pmRNPs) does not significantly change in the 10 min LL $\\rightarrow$ HL, a sequence motif identified in actively translated transcripts in HL is shown to bind proteins in vitro. Most proteins sequestered by the corresponding native motif sequences of SAP2 and SAP3 were lacking classic RNA-recognition domains, indicating that these proteins have a potential moonlighting function as RBP during translational regulation for stress acclimation. In this role, the interaction with the sequence motif in most cases implied a repressive effect on translation in LL, as most proteins were captured in LL rather than HL conditions.

Besides specific interaction with known and previously unknown RBPs, the translation initiation of mRNAs in the 10 min LL $\\rightarrow$ HL is determined by interaction with EUKARYOTIC INITIATION FACTOR 4E (eIF4E) or its angiosperm-exclusive isoform eIFiso4E. Analysis of translatome compositions in eif4e and eifiso4e-KO lines revealed a high degree of deregulation in the 10 min LL $\\rightarrow$ HL, especially for HL-induced transcripts. Here, a high proportion of deregulated transcripts was specifically affected by the loss of eIF4E or eIFiso4E. However, translatome in wildtype (WT) plants indicates that a certain ratio of both factors is needed for a physiologically normal translational response. Analysis of 5′- and 3′-untranslated regions (UTRs) of differentially regulated transcripts identified predominant sequence motifs. Significant differences in folding energies might determine their interaction with eIF4E/eIFiso4E. Whether these interactions are direct or facilitated by additional RBPs needs further investigation. A clear preference of either factor for short or long UTR-length was not observed. A tight relation between the plant-specific eIFiso4E and photosynthesis performance can be assumed, as eIFiso4E was able to increase initiation events in HL relative to LL-conditions, which was not visible in eifiso4e. Photosynthesis-related transcripts however, were deregulated in both KO-lines. Cells perceive oxidative stress as consequence of HL by thiol-based redox switches in proteins. This role imposes evolutionary pressure on cysteine (Cys) residues to be conserved in protein primary sequences. By employing the ConCysFind algorithm, conserved Cys were identified in the majority of plant translation factors, indicating the whole process to be potentially regulated directly by redox cues. Especially high Cys-conservation is evident for EURKARYOTIC ELONGATION FACTORS (eEFs) and EUKARYOTIC RELAESE FACTORS (eRFs). ConCysFind analyses were validated in vitro by redox-environment dependent oligomerization of eRF1-1 and RECEPTOR FOR ACTIVATED KINASE 1 (RACK1A), which were dependent on Cys conserved throughout the plant kingdom.