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In Arabidopsis thaliana (A. thaliana), induction of leaf senescence is accompanied by a reduction of the transcription and activity of central hydrogen peroxide (H2O2) scavenging enzymes, which prompts the accumulation of intracellular H2 O2 (Zimmermann et al., 2006; Smykowski, 2010). The expression of multiple early senescence associated genes (SAGs) is H2 O2 inducible, and the role of H2O2 as a signalling molecule during senescence has often been demonstrated and is widely accepted. However, a central question of reactive oxygen species (ROS) mediated signal transmission is, how a specific response can arise from signalling cascades, which rely on rather simple and commonly occurring ROS. Within this work, this question has been addressed with respect to senescence inducing signalling. Besides the type of ROS, the only information directly carried by H2O2 is its concentration, rate of production, and its location. To precisely monitor these properties, we intended to use the fluorescent H2O2 sensor protein HyPer (Belousov et al., 2006). However, during the initial establishment of required measurement protocols (Wierer et al., 2011), its H2O2 scavenging and thus senescence delaying properties in A. thaliana became clear (Bieker et al., 2012). Nonetheless, because the HyPer constructs were targeted to different subcellular compartments, their respective expression lead to clearly distinguishable effects on intracellular H2O2 contents and the induced senescence phenotype (Bieker et al., 2012). In the following, I developed a new technique for the analysis of senescence induction and progression on the basis of leaf colouration (Bresson et al., 2017). This allowed me to harness HyPers’ H2O2 scavenging properties for the closer analysis of the impact the subcellular location of H2O2 signals has on the downstream triggered signalling events. I was able to demonstrate that the reduction of peroxisomal (data not shown) or cytoplasmic H2O2 contents provokes a delay of senescence induction, whereas chloroplastic scavenging has a disturbed senescence progression as consequence (unpublished, see Section 6.1). Thus, I could show, that the site of ROS generation and perception is of importance for the elicitation of specific downstream signalling cascades. Biotic and abiotic stress responses very often entail the generation of ROS, including H2O2 . Moreover, most of these stress responses also can provoke the induction of leaf senescence. This indicates an intricate connection between stress elicited and senescence inducing signalling. For example, expression of the early SAG WRKY53 is responsive to H2O2. But furthermore, we demonstrated an additional regulatory mechanism of WRKY53 expression via WRKY18, a factor also known to be involved in diverse plant stress responses (Potschin et al., 2014; Chen et al., 2010). During pre-senescent plant growth, WRKY18 acts as a homo-dimer and represses WRKY53. However, upon senescence induction, WRKY53 expression is induced and the resulting WRKY53 protein can form hetero-dimers with WRKY18. This withdraws WRKY18 proteins from the homo-dimer and reduces the transcriptional suppression of WRKY53. Moreover, the formed W53/W18 hetero-dimers now enhance WRKY53 expression (Potschin et al., 2014). Leaf senescence also is an integral element of nutrient remobilization from leaves to developing reproductive organs and seeds. For this reason, H2O2 dependent senescence induction was analysed in the agronomically relevant species Brassica napus (B. napus). As both, B. napus and A. thaliana are members of the Brassicaceae family, similarities in senescence inducing mechanisms were expected. Indeed, the induction of SAG expression and the beginning decline of chlorophyll contents were also here accompanied by the reduction of anti-oxidative capacities and an increase of intracellular H2 O2 contents. To investigate a possible influence on senescence induction exerted via the plants’ nutritional status, plants were grown under different nitrogen (N) and carbon dioxide (CO2 ) regimes. When B. napus plants were supplied with low N combined with high CO2 levels, senescence induction took place slightly earlier than in control plants (Bieker et al., 2012). Moreover, the reduction of N supply to a minimum (N starvation) provoked the induction of leaf senescence. However, despiten the measured reduction of anti-oxidative capacities during this, leaf H2O2 contents decreased severely in N starved B. napus and A. thaliana plants. This indicates a different mechanism of senescence induction during N-starvation in comparison to age-dependent leaf senescence (Bieker et al., 2019). To further elucidate the impact of the plants’ nutritional status on leaf senescence, transcriptome profiling was conducted of leaf material also analysed in Bieker et al. (2012) (380 ppm CO2, NL & NO; see Franzaring et al., 2011; Bieker et al., 2012; Safavi-Rizi et al., 2018). I processed and screened the raw data for genes displaying expression profiles (anti-) correlated to the previously measured H2O2 contents. Surprisingly, I found seed storage proteins (SSPs) to be expressed in vegetative tissue, and also to be correlating with respective H2O2 contents. Further investigation of this revealed a progressive accumulation pattern of SSPs during senescence. Starting in the oldest leaves at senescence induction, the proteins accumulated and then seemingly ascended to the next younger leaf-position, shortly before the former was shed. Moreover, the expressed SSP type varied with the nitrogen supply of the plant. Smaller 2S-SSPs were expressed under low N, bigger 12S variants under optimal N supply and none of both under N starvation conditions. Taken together, this indicates a possible mechanism supporting senescence associated nutrient (esp. N) remobilisation, which also adapts to different N supply conditions (Bieker et al., 2019). |
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