3. Conclusion and Outlook 3.1. Future perspectives of transcriptional regulation triggered by citrate
We found good evidence that citrate can act as a signalling molecule. Specific changes in gene expression were observed upon citrate feeding that did not occur with other organic acids and feeding of external citrate showed a reprogramming of transcripts which was very similar to the response in gene expression that occurred after mitochondrial perturbation.
Especially three different groups of target genes were affected including photosynthetic light reactions, protein synthesis, and pathogen defense. It is not clear yet if citrate directly binds to TF regulating gene expression or if altered citrate concentrations are perceived by a receptor which triggers a signalling cascade. Recently, a signalling cascade for AA perception was discovered which includes activation of a rhomboid protease cleaving ANAC017 which then shuttles to the nucleus and activates AOX1 expression (Ng et al., 2013). Because citrate can also increase AOX1 transcript levels, it would be interesting to investigate if citrate also leads into the ANAC17 mediated MRR pathway. Many open questions remain to be answered in the future, e.g. how is citrate signalling transduced in the cell and what mechanisms control the transcriptional changes. As we only analyzed the change in transcript levels after external addition of citrate it would be interesting to see the effect of locally induced citrate concentration on a sub-cellular level. This could be achieved by over-expressing CS in mitochondria. Furthermore, mutants impaired in citrate transport could give deeper insights into citrate-mediated MRR. To identify novel cis-elements which are responsible for the transcriptional activation after citrate treatment promoter-luciferase constructs of citrate-inducible genes have been designed. 2000 bp of the promoter region were fused to a luciferase gene and stably transformed into Arabidopsis. To elucidate the region responsible for the citrate-dependent promoter activation, deletion constructs of the promoter region will be constructed. After narrowing down the promoter, yeast one hybrid screens and EMSA assays can be performed to identify citrate-dependent promoter motifs and their DNA-binding proteins. To identify proteins which are involved in citrate signalling, an affinity chromatography could be carried out. A citrate mimic can be coupled to an agarose column and incubated with total plant extract as well as organellar fractions to pull down citrate-binding proteins which can then be detected via MS. To uncover how changes in the appearance of key metabolites such as citrate and other carboxylic acids influence signalling within the plant cell and how they are perceived will be challenging for the future.
3.2. Research outlook for lysine acetylation on metabolic enzymes
In the second part of my work, the abundance of lysine acetylation in plant mitochondria was investigated. In total 120 proteins of several functional groups including TCA cycle enzymes as well as proteins from the OXPHOS were discovered. Until now, regulation of lysine acetylation is thought to be controlled by KATs and KDACs. While no orgarnellar KAT has been discovered in plants, here we demonstrated that lysine acetylation can occur non-enzymatically because of the basic pH in the mitochondrial matrix. Hence, it seems more likely that acetylation levels are mainly controlled by KDACs. We characterized a sirtuin-type like KDAC, SRT2, which was supposed to be the only mitochondrial sirtuin in Arabidopsis.
We could confirm the mitochondrial localization as well as the deacetylase activity of SRT2.
No obvious phenotype was visible in the knockout plants but changed lysine acetylation levels for proteins of CV as well as for the ATP/ADP carriers were observed. Strikingly, an enhanced ADP uptake in the srt2-1 knockout mutant was detected as well as changed ATP/ADP ratios. Additionally, we observed changes in metabolic levels with increased glycine and serine levels, while sugars, some amino acids and organic acids were significantly decreased in abundance. We hypothesized that SRT2 is involved in fine-tuning mitochondrial metabolism by interacting with the ATP/ADP carries. Many interesting research questions concerning mitochondrial lysine emanate from our recent findings. After the discovery of the lysine acetylome in mitochondria, the functional relevance now has to be investigated. Does lysine acetylation change activity of metabolic enzymes in plant mitochondria? In other species several mitochondrial enzymes regulated by lysine acetylation were uncovered in the last years, for example the malate dehydrogenase and the superoxide dismutase 2 in human cells (Qiu et al., 2010; Tao et al., 2010; Zhao et al., 2010); the succinate dehydrogenase complex, subunit A as well as the aldehyde dehydrogenase 2 in mouse (Cimen et al., 2010; Lu et al., 2011). One possible approach to investigate the relevance of specific lysine residues is to perform site-directed mutagenesis. The exchange of lysine to arginine is supposed to mimic the non-acetylated state and the exchange to glutamine the acetylated state. An interesting candidate for using site-directed mutagenesis is the PDC complex as it is of great importance for the TCA cycle and it controls acetyl-CoA levels in mitochondria. The next question which should be answered is, when and how does lysine acetylation vary in mitochondria. Does lysine acetylation depend on the redox state of the organelle and does it reflect the metabolic state? How does lysine acetylation change in terms of abiotic and biotic stress conditions and what is the impact on enzyme activity? To answer these questions, we have to learn more about the lysine acetylation regulating enzymes. As no orgarnellar KAT has been discovered
3. Conclusion and Outlook so far the question arises if they exist in organelles at all or if lysine acetylation only occurs non-enzymatically. We found SRT2 as the only sirtuin type KDACs in mitochondria but other KDACs might exist as well. To answer these questions it will be critical to dissect these regulatory enzymes in more detail.