Peroxisomal-dependent signalling in plant response to abiotic stress

Resumen

There are numerous environmental conditions that are continuously changing and that affect all organisms, including plants. As an evolutionary adaptation, plants have developed specific mechanisms that allow them to cope with these adverse conditions. Environmental changes cause stress in plants and their response usually begins with the perception of the stress, followed by changes in metabolism and accompanied by gene expression adjustments and protein modifications. All these processes are expected to induce an efficient response to the stress. Key players in orchestrating this response are reactive oxygen and nitrogen species (ROS/RNS), which build signalling networks with external and internal signals. ROS/RNS are generated during cell metabolism. Under normal conditions antioxidant system controls ROS/RNS production, however at high or uncontrolled concentrations, a rapid accumulation takes place, leading to cellular damage. One of the main sites for ROS/RNS generation in the cell are peroxisomes. These organelles are highly dynamic and metabolically active and are found in almost all eukaryotic cells. Peroxisomes are closely linked to mitochondria and chloroplasts, sharing metabolic pathways, as well as the import and transport of proteins. Organelles/compartments-dependent signalling communication to the nucleus, termed retrograde signalling, from mitochondria and chloroplast in stress response are more studied and then better understood than in peroxisomes. Different types of stresses and the subsequent response of the plant are studied in our group, being the common link the peroxisome and the production of the signal molecules ROS/RNS. Thus, the present Thesis aims to elucidate peroxisomal-dependent signalling in plant response to abiotic stress as general objective. In Chapter 1, we have analyzed plant response to the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D). This auxin is used as an herbicide at high concentrations, and induces ROS production in plant cells and an epinastic phenotype (downward bending of leaves). We found that acyl CoA oxidase 1 (ACX1), the first enzyme in the fatty acid β-oxidation occurring in the peroxisome, is one of the main sources of ROS production after 2,4-D treatment. Transcriptomic analyses of WT plants exposed to this stress revealed two different responses. An early response, in which a ROS-related peroxisomal footprint was detected and later responses, in which other organelles, such as mitochondria and chloroplast, are involved. We also determined that peroxisomal ROS derived from ACX1 regulated a large number of genes previously associated with epinasty and also the expression of the auxin receptor AUXIN SIGNALLING F-BOX 3 (AFB3) at early times. AFB3 together with the SCF (ASK-cullin-F-box) E3 ubiquitin ligase complexes, was shown to mediate auxins degradation by the 26S proteasome downstream of ACX1, and we have found that AFB3 in an ACX1-dependent way, is involved in the epinastic phenotype induced by 2,4-D. Later ACX1-dependent genes in plant response to 2,4-D are related with proteasome, which we have shown to be also involved in epinasty development. Adjustments in gene expression are essential to trigger a suitable response to environmental cues, being the peroxisomes a key source of signalling molecules. In Chapter 2, and given the scarcity of information related to retrograde signalling of the peroxisome under stress conditions, we carried out a meta-analysis to try to bring some light on this topic. After collecting data from different public and in-house transcriptomes with mutants and/or stresses leading to disturbances of peroxisomal-dependent ROS production, we identified a data set of common and peroxisome-specific genes regulated by ROS from this organelle, under different conditions. Thus, we found 101 and 86 genes commonly regulated at short-time and long-time stress treatments, respectively. Enrichment analysis with early peroxisomal-dependent genes showed their involvement in response to stress/stimulus, and a high co-expression, suggesting an early coordinated peroxisomal-dependent plant response to stress. In particular, these genes clustered in two main nodes related to heat shock factors (HSFs) and jasmonic acid (JA) biosynthesis and signalling. Genes commonly regulated at long-time were enriched in terms also related to stress and clustered in a gene network related to JA biosynthesis, suggesting that peroxisomal retrograde signalling is a coordinated response to avoid damages in the cell and to protect proteins under stress conditions. Plasticity of peroxisomes enables them to adapt their morphology, number and movement to changes in their surroundings. Although peroxisomal proliferation has been described for a long time, many aspects of this process remain undiscovered. Peroxins 11a-e (PEX11a-e) are proteins involved in the first stage of peroxisomal proliferation. Furthermore, PEX11a have been shown to be essential for peroxule production, very dynamic extensions produced by peroxisomes and regulated by ROS and nitric oxide in response to cadmium (Cd). However, functionality of PEX11a and therefore, of peroxules, is not well known. In Chapters 3 and 4 we have tried to expand the knowledge about functionality of peroxules under control and stress conditions. For this purpose, we have generated mutant lines: 1) by CRISPR/Cas9 altering PEX11a gene (pex11a-CR) and 2) by cross-polination, generating double mutants with a T-DNA insertion in PEX11a locus and with a CFP (px-ck) located in peroxisomes (pex11a-SKI x px-ck). As a result of changes in protein sequences, a fragment in the C-terminal is absent in both pex11a mutants, being the functional protein shorter in pex11a-CR lines. Despite of peroxisomal phenotype observed in each mutant was different, both pex11a mutants were unable to produce peroxules in response to Cd, confirming PEX11a involvement in peroxule formation and fast response to stress. An in silico analysis of PEX11a expression, regulation and putative post-translational modifications (PTMs) of the protein was carried out. We suggest that the presence or not of target aminoacids for different PTMs in pex11a-CR and pex11a-SKI x px-ck sequence, could explain the differences observed in the peroxisomal phenotype between them. We also carried out a phenotypical characterization revealing that early germination was altered in pex11a-CR, which could be due to changes in fatty acid metabolism. Lateral roots, as well as foliar area, were reduced in pex11a-CR mutants. Furthermore, we checked Cd response in pex11a mutants fluorescently labelled and studied by confocal laser scanning microscopy (CLSM), and using histochemical technique (DAB) followed by light microscopy analysis. A proliferation of these organelles in response to Cd in px-ck seedlings was observed and curiously, pex11a-CR lines did not display a statistically significant peroxisomes increment by CLSM, but different results were observed by histochemistry. To take a deep insight into the role of PEX11a in plant response to Cd stress, we performed transcriptomic analysis included in Chapter 4. px-ck and pex11a-CR seedlings were treated with Cd (100 μM) for 1 and 24 h. Functional annotation analysis of genes differentially expressed (DEGs) revealed numerous alterations related to morphology and metabolism of chloroplasts in pex11a-CR under control conditions. In addition, DEGs in pex11a-CR in non-treated plants were assigned to pathways such as photosynthesis, porphyrin and chlorophyll metabolism, glutathione metabolism and starch and sucrose metabolism. Experimental data of pigment content and organelle ultrastructure confirmed transcriptional results, showing alterations of thylakoid/stroma rate and reduction of chlorophylls and carotenoids content in the mutants, respect to the WT background. Under control conditions we also determined a reduction in starch accumulation. In response to Cd 1 h we found a higher number of DEGs in pex11a-CR compared to px-ck (6,192 vs. 3,485). We filtered the early PEX11a-dependent genes regulated after Cd 1 h, and their enrichment showed categories related to iron homeostasis and transport. Enrichment of the later transcriptional response to Cd (24 h) displayed a link with nucleus, ribosomes, translation and peptide metabolic and biosynthetic processes. These results together support the key function for peroxisomes in plant development and plant response to stress being able to regulate different processes such as protein protection networks under stress, hormonal-dependent signalling and biosynthesis, such as for auxins and jasmonic acid, nutrition and development.