In order to analyze the signaling function of hydrogen peroxide (H2O2) production in senescence in more detail, we manipulated intracellular H2O2 levels in Arabidopsis thaliala (L.) Heynh by using the hydrogen-peroxide-sensitive part of the Escherichia coli transcription regulator OxyR, which was directed to the cytoplasm as well as into the peroxisomes. H2O2 levels were lowered and senescence was delayed in both transgenic lines, but OxyR was found to be more effective in the cytoplasm. To transfer this knowledge to crop plants, we analyzed oilseed rape plants Brassica napus L. cv. Mozart for H2O2 and its scavenging enzymes catalase (CAT) and ascorbate peroxidase (APX) during leaf and plant development. H2O2 levels were found to increase during bolting and flowering time, but no increase could be observed in the very late stages of senescence. With increasing H2O2 levels, CAT and APX activities declined, so it is likely that similar mechanisms are used in oilseed rape and Arabidopsis to control H2O2 levels. Under elevated CO2 conditions, oilseed rape senescence was accelerated and coincided with an earlier increase in H2O2 levels, indicating that H2O2 may be one of the signals to inducing senescence in a broader range of Brassicaceae.
As the worldwide demand for food- and fuel crops steadily rises, the improvement of productivity has been of increasing focus, not only in regards to quantity of yield, but also to the quality of the harvested products. Leaf senescence is the last stage of the the foliar lifecycle, and predominantly serves as a mechanism for annual plants to recycle nutrients out of non-reproductive tissues into developing fruits and seeds. It is a highly complex and dynamic process modulated by nutrition status and various endogenous signals, as well as by biotic and abiotic stress conditions. All these triggers have to be interconnected to a certain degree in a complicated signaling network, and integration of these signals determines onset, progression, or even reversion of senescence. When the restructuring and remobilization of nutrients are terminated, senescing leaves eventually die and are shed by the plant.
In the crop plant oilseed rape, the reutilization of nutrients, especially nitrogen compounds, is inefficient in comparison to other crops. Most of the oilseed rape leaves are shed before or during early flowering, and thus do not contribute to remobilization but instead amount to a loss of up to 15% of the plant's total N reserves (Rossato et al. 2001). Phloem loading of amino acids is not limiting for nitrogen remobilization from senescing leaves in oilseed rape; therefore, other factors have to be considered that may limit nitrogen remobilization and N efficiency. One strategy to improve the nitrogen use efficiency in oilseed rape would be to delay senescence until seed filling is completed (Tilsner et al. 2005). However, delayed senescence in wheat has been shown to correlate with low protein content (Uauy et al. 2006), whereas drought stress appears to enhance senescence and increase carbon remobilization and protection of stem tissue against oxidative stress (Bazargani et al. 2011). Even though high N availability and high CO2 prolonged flowering time of oilseed rape plants, they did not lead to the production of more ripe pods, but instead more likely prevented apical switch off, thus leading to a strong branching out (Franzaring et al. 2011). Furthermore, high CO2 caused a decline in seed oil content, indicating that nutrient remobilization appears to be very complex during senescence.
Despite the importance of senescence, our knowledge of the regulatory mechanisms of this process is still limited. Senescence is triggered by exogenous and endogenous parameters. The most important endogenous factors are the age of the leaves and the age or developmental stage of the plant. However, how these two parameters are sensed and translated into molecular signals is still unclear. The photosynthetic activity of leaves of annual plants decreases continuously after full expansion (Batt and Woolhause 1975; Hensel et al. 1993). A decline in photosynthetic activity under a certain threshold may act as a senescence-inducing signal (Matile et al. 1992; Smart 1994), but chlorophyll a/b ratio also appears to be important (Sakuraba et al. 2012). However, autumn senescence in free-growing aspen (Populus tremula) is exclusively initiated by the photoperiod (Keskitalo et al. 2005). Sugar accumulation as well as sugar starvation are believed to be signals to induce senescence; however, changes in the sugar to nitrogen content during the sink/source transition of leaves also appear to play a role in the induction of leaf senescence (Masclaux et al. 2000; van Doorn 2004; Pourtau et al. 2004; Wingler et al. 2004, 2009). Again, it remains unclear how these parameters are sensed and translated into molecular signals. Aside from photoperiod, sugars, and nitrogen, senescence is triggered by the interplay of many plant hormones acting at specific concentrations in synergistic or antagonistic ways and in concert with other signals like calcium and especially oxygen free radicals (ROS). Therefore, the complex signaling network that triggers senescence is far from being understood.
ROS play an important role during leaf senescence in two different aspects: signaling and molecule degradation. In contrast to calcium signaling, which is executed by storage and release of Ca2+, ROS signaling is controlled by production and scavenging (Mittler et al. 2004). Thus, plants have developed a fine-tuned network of enzymatic and low-molecular-weight antioxidative components which act in different cellular compartments. Additionally, different plant species have developed diverse strategies to balance their redox potential and regulate their ROS status. In Arabidopsis, a network of at least 152 genes is involved in managing the ROS levels. This network is highly dynamic and redundant, and includes ROS-scavenging and ROS-producing proteins (Mittler et al. 2004).
We have already shown that Arabidopsis catalases (CAT) exhibit senescence-specific regulation. CAT2 activity decreased at a very early stage during the time of bolting. The increase in H2O2 levels is enforced by a decrease in ascorbate peroxidase 1 (APX1) activity at the same time point (Ye et al. 2000; Zimmermann et al. 2006). However, APX1 is not down-regulated at the transcriptional level during the time of bolting (Panchuk et al. 2005), but rather H2O2 itself most likely leads to the inactivation of APX1 (Miyake and Asada 1996; Zimmermann et al. 2006). Furthermore, we have shown that this inactivation is dependent on the developmental stage of the plants with the highest sensitivity during bolting time, suggesting a feedback amplification loop. This coordinated regulation of the hydrogen peroxide scavenging enzymes on the transcriptional and posttranscriptional level creates a distinct increase in H2O2 at the time point when the plants start to bolt and flower, and a coordinated senescence process of all rosette leaves should be induced (Zimmermann et al. 2006). This distinct increase in H2O2 most likely induces the expression of transcription factors and senescence-associated genes (SAGs). NAC and WRKY transcription factors constitute the two largest groups of transcription factors of the senescence transcriptome (Guo et al. 2004), and certain members of these two transcription factor families have been shown to play central roles in regulating senescence in wheat and Arabidopsis (Miao et al. 2004; Guo and Gan 2006; Uauy et al. 2006; Balazadeh et al. 2010, 2011; Breeze et al. 2011; Yang et al. 2011). Besides other triggers, the expression of these senescence-associated WRKY and NAC factors is controlled by H2O2 (Miao et al. 2007, 2008; Miao and Zentgraf 2010; Balazadeh et al. 2010, 2011).
A rapid turnover of the catalase protein necessitates continuous transcription and translation of mRNA (Feierabend et al. 1992). Using the yeast-one-hybrid system, G-box binding factor 1 (GBF1) has been identified as a negative regulator of CAT2 expression (Smykowski et al. 2010). In GBF1 knock-out (GBF1-KO) plants, CAT2 down-regulation is abolished, the H2O2 peak disappears, and plants show delayed onset of leaf senescence, clearly indicating that H2O2 is used as a signal to induce the onset of senescence. Since GBF1 also controls other important senescence-down-regulated genes like RUBISCO (Smykowski et al. 2010), we wanted to isolate the effect of H2O2 regulation from effects related to other GBF1 target genes. Therefore, we manipulated H2O2 levels by overexpression of the hydrogen-peroxide-sensitive part of the E. coli transcription regulator OxyR to confirm that the loss of the H2O2 signal is leading to the delay of senescence. Furthermore, we investigated senescence processes in Brassica napus L. cv. Mozart, focusing on the role of H2O2 and its scavenging enzymes during leaf and plant development.
To determine that the delayed senescence phenotype of the gbf1 mutants is mainly due to CAT2 down-regulation and to the lack of the hydrogen peroxide signal instead of due to the regulatory effects of GBF1 on other important target genes like RUBISCO, we aimed to alter the H2O2 content using a transgene which confers no alternative function in plants. To this end, we used a construct which combines the regulatory domain of the E. coli OxyR transcription factor for sensing H2O2 in bacteria with a cpYFP (Belousov et al. 2006). The E. coli OxyR transcription factor contains two domains: an H2O2- sensitive regulatory domain (amino acids 80–310, OxyR-RD), and a DNA-binding domain (amino acids 1–79). In the presence of H2O2, the reduced form of OxyR-RD is oxidized. The key residues for oxidation of OxyR-RD are Cys199 and Cys208. H2O2 converts Cys199 to a sulfenic acid derivative and forms a disulfide bond with Cys208, leading to a dramatic conformational change which allows DNA binding (Zheng et al. 1998). In vitro experiments conducted by Aslund et al. (1999) revealed that the hydrogen peroxide that they added to the reaction appears to be consumed because OxyR returns to its reduced form in the time course experiment. Thus, they proposed that OxyR is acting as a peroxidase in these in vitro reactions. This proposition is supported by the finding that significant NADPH consumption was observed (Aslund et al. 1999). Here, we used plants overexpressing OxyR-RD-cpYFP in the cytoplasm and in the peroxisomes where the CAT2 protein is predominately located. These lines were constructed by Costa and coworkers, and the YFP localization in the two different compartments was confirmed by confocal microscopy (Costa et al. 2010). Two cytoplasmic and two peroxisomal overexpressing lines with different levels of transgene expression were used for phenotypic analyses. Senescence was delayed in all lines expressing OxyR-RD-YFP. Interestingly, the delay was dependent on the compartment to which the OxyR-RD was directed, since the cytoplasmic lines (1 and 8) showed a more severe phenotype than the peroxisomal lines (2 and 9) even though OxyR-RD-YFP expression was higher in the peroxisomal lines (Figure 1). In order to determine if the senescence delay was due to altered H2O2 concentrations, we analyzed H2O2 concentrations using the fluorescent dye DCFDA for the peroxisomal line 2 (2/per) and the cytoplasmic line 8 (8/cyt), with both lines expressing OxyR-RD-YFP to approximately the same extent (Figure 1). The H2O2 peak which is observed in wildtype plants during bolting and the onset of flowering is diminished in both lines in young as well as in middle-aged leaves (Figure 2). However, the decrease was more pronounced in the 8/cyt line. This is in accordance with a more pronounced delay of chlorophyll loss and the overall senescence phenotype which is observed in 8/cyt. This clearly indicates that cytoplasmic H2O2 appears to be more effective in senescence signaling than peroxisomal H2O2.
In order to characterize the role of H2O2 as a signaling molecule during early senescence in crop plants, we investigated the H2O2 levels in oilseed rape plants for individual leaves of one plant and for the same leaf position during plant development in the spring variety cv. Mozart grown under greenhouse conditions. H2O2 levels of young oilseed rape leaves were higher than H2O2 levels in old leaves independent of the age of the plant (Figure 3A). Overall levels increased with plant age when plants started to bolt and flower as has already been shown in Arabidopsis (Miao et al. 2004; Zimmermann et al. 2006), clearly indicating that the increase in H2O2 appears to be a systemic signal which is dependent only on plant age and not on leaf age (Figure 3B). However, in very old Arabidopsis plants, H2O2 increased to very high levels (Miao et al. 2004; Zimmermann et al. 2006), a phenomenon which was not observed in oilseed rape (Figure 3C), indicating that during bolting and flowering time H2O2 appears to be used as a signaling molecule in oilseed rape. H2O2 was not observed to play a role in molecule degradation in late stages of senescence in oilseed rape plants.
The increase in H2O2 during bolting time in Arabidopsis is predominantly regulated by interplay of CAT2 and APX1 activity regulation (Zimmermann et al. 2006). Although CAT2 is down-regulated on the transcriptional level by the transcription factor GBF1 (Smykowski et al. 2010), APX1 down-regulation appears to be a secondary effect and is achieved on the post transcriptional level (Ye et al. 2000; Panchuk et al. 2005; Zimmermann et al. 2006). A matrix of CAT and APX activities of oilseed rape leaves revealed that CAT activity also declined during bolting and the onset of flowering (Figure 4A). The CAT activity pattern observed in oilseed rape leaves was very similar to that observed in Arabidopsis leaves, and by employing 3-aminotriazole (3-AT) treatment, the different protein bands in the gels stained for CAT activity could be clearly assigned to CAT2 and CAT3 isoforms (Figure 4B; Orendi et al. 2001). However, the inhibition of APX during bolting time is less pronounced than in Arabidopsis plants (Ye et al. 2000; Zimmermann et al. 2006) and is only seen in younger leaves (Nos. 10 and 12). Furthermore, no clear relation to different APX isoforms could be drawn. Therefore, only the overall activity of APX was measured in the following experiments.
Developmental H2O2 profiles were recorded in more detail at three different leaf positions (leaf Nos. 5, 8, 12) and were accompanied by analyses of chlorophyll content, ascorbate and glutathione pools, CAT and APX activities, and total carbon (C) and nitrogen (N) contents (Figure 5). In this experiment, H2O2 increased and CAT and APX activities decreased again during bolting and the onset of flowering (Figure 5D, E). This was very similar for all three leaf positions; therefore, we exemplified the data gathered from leaf No. 8. With increasing plant age, the total glutathione pool decreased, with GSSG levels decreasing more dramatically at the beginning of development (Figure 5B). The total ascorbate pool also decreased with plant age, but no obvious difference between DHAsc and Asc was observed. Remarkably, glutathione as well as ascorbate pools slightly increased again after H2O2 had reached its maximum level in 9-week-old plants (Figure 5C), which might be a counter-regulation for the increasing H2O2 levels.
Furthermore, total C and N contents and the C/N ratio were determined as additional senescence parameters. C and N contents continually dropped during progression of development and senescence. During senescence, N content decreased more dramatically leading to an increase in the C/N ratio (Figure 5F), which has been reported to be a senescence signal (Wingler et al. 2009). However, these changes can be observed only in later stages of senescence in 13-week-old plants when 40% of the chlorophyll has already been lost (Figure 5G). Interestingly, the C/N ratio dropped again in 14-week-old plants.
Gene expression analyses of SAGs and SDGs using material derived from leaf No. 8 revealed that with the maximum of H2O2 expression of early SDGs like CAB, RUBISCO and CAT2 (Figure 6B) started to decline while early SAGs like SAG13 and CAT3 were up-regulated (Figure 6A). In contrast, expression of the late senescence marker SAG12 and of glutamate synthase (GLS) was still low when chlorophyll content had not yet started to decrease (Figures 5G, 6A). The expression profile of the transcription factor WRKY53, which plays a central role in early senescence regulation in Arabidopsis, followed the H2O2 profile (Figure 6C). Taken together, hydrogen peroxide levels in oilseed rape appear to be regulated in a very similar way as observed in Arabidopsis, and might also be used as a signal in senescence regulation.
Since oilseed rape is an important crop plant, developmental senescence of oilseed rape was also analyzed in field-near conditions and under three different N (N min, N opt, N plus) and two different CO2 (380 ppm and 550 ppm) regimens in fully-programmed growth chambers (Franzaring et al. 2011, 2012). Plants were grown under conditions simulating the seasonal increments of the day length and temperature of South-Western Germany. Here, we focused on H2O2 levels and chlorophyll content as determined by SPAD readings. In contrast to our greenhouse conditions, plants developed fewer leaves before flowering; therefore, leaf No. 5 was used for H2O2 measurements in this case (Figure 7). In leaf No. 5, H2O2 started to increase with bolting and flowering and reached its maximum level when full flowering was observed. The increase was more prominent in N plus and N min conditions compared to an optimal N supply. As a phenotypic indicator for senescence, chlorophyll content started to decrease when H2O2 reached its maximum under all three N conditions. In a high CO2 atmosphere, chlorophyll started to decline one week earlier under low and optimal N conditions, and H2O2 also reached its peak one week earlier, indicating that under field-near conditions H2O2 is most likely used as a signal for the onset of senescence (Figure 7).
Many different agriculturally-important traits are affected by plant senescence, so understanding and modulating senescence processes might help solve mounting problems such as drought stress that are due to global climatic changes. ROS appear to play an important role during senescence, since many senescence mutants are also linked to antioxidative capacity. The delayed leaf senescence mutants ore1, ore3, and ore9, exhibit increased tolerance to various types of oxidative stress (Woo et al. 2004). The Arabidopsis crp5/old1 mutants exhibit early senescence through deregulation of the cellular redox balance, since transcriptome data suggest that presymptomatic cpr5/old1 plants are in a state of high cellular oxidative stress (Jing et al. 2008). It is speculated that CPR5 behaves as a shuttling inner nuclear membrane-bound transcription co-factor that possibly interacts with multiple partners to induce pleiotropic effects (Perazza et al. 2011). Furthermore, old5 mutants displaying a higher respiration rate also show an increased expression of oxidative stress markers (Schippers et al. 2008). Moreover, the vtc1 mutant, being deficient in ascorbic acid, exhibits early senescence (Barth et al. 2004). Kurepa et al. (1998) also showed a strong correlation between longevity and resistance to oxidative stress.
Obviously, plants have developed a very fine-tuned network of enzymatic and low molecular weight antioxidative components in different cell compartments, and different plant taxa have developed different strategies to balance their redox potential and regulate their ROS status. The network managing the ROS balance is highly dynamic and redundant, and includes ROS-scavenging and ROS-producing proteins.
More or less all cellular compartments produce ROS, but they also have their own scavenging systems. However, the types of ROS and the amounts produced can vary between those compartments in which H2O2 is able to pass through membranes and can be released from different compartments. Mitochondria, chloroplasts and peroxisomes carry out important functions associated with the senescence process; however, it is believed that chloroplasts play a principal role in the regulation of the leaf senescence process (Zapata et al. 2005; Martínez et al. 2008) and are the main target of age-associated oxidative stress in plants (Munné-Bosch and Alegre 2002). However, peroxisomes and ROS generated in these organelles were shown to play a central role in natural and dark-induced senescence in pea plants (del Río et al. 1998), and it has been discussed that peroxisomes could act as subcellular sensors of plant stress and senescence by releasing NO, superoxide, and H2O2 as signaling molecules into the cytosol, thereby triggering the expression of specific genes (Corpas et al. 2001, 2004; del Rio et al. 2006). The down-regulation of CAT2 expression by the transcription factor GBF1 induces the onset of leaf senescence (Smykowski et al. 2010), and assigns a regulatory role to peroxisomal H2O2.
By using Arabidopsis plants overexpressing the fusion protein E. coli OxyR-RD-YFP in peroxisomes and the cytoplasm, we intended to alter H2O2 levels in these two compartments independent of other cellular functions to evaluate the specific impact of these H2O2 molecules on leaf senescence. Our results show that altered cytoplasmic H2O2 levels appear to have a higher impact on senescence than altered H2O2 levels in peroxisomes (Figure 1), because the delay of senescence was more pronounced in the cytoplasmic lines even though expression of the transgene was higher in peroxisomes. H2O2 levels measured with a canonical technique were lower in transgenic lines and correlated with the decrease in chlorophyll and the severity of the phenotype (Figure 2). Since senescence is predominantly regulated on transcriptional level, the cytoplasmic compartment might have a direct influence on redox regulation of transcription factors like WRKY53. Moreover, cytoplasmic H2O2 can also directly activate a specific Arabidopsis MAP triple kinase, ANP1, which initiates a phosphorylation cascade involving two stress MAPKs, AtMPK3 and AtMPK6 (Kovtun et al. 2000). Expression of the MAP triple kinase1 (MEKK1) of Arabidopsis can also be induced by H2O2 and shows its expression maximum during onset of leaf senescence (Miao et al. 2007). Whether H2O2 induced expression of SAGs is transduced by MAPK signaling or directly by redox-sensitive transcription factors has yet to be elucidated. However, it is reasonable to speculate that eukaryotic cells also evolved nuclear antioxidant systems distinct from the cytosolic ones, since nuclear redox-states influence the activity of a large number of nuclear proteins including transcription factors, and oxidative injury or DNA replication errors caused by ROS are serious problems for aerobic organisms. At least one family member of the peroxiredoxins has been reported to be localized in the nucleoplasm of rice and Arabidopsis (Dietz 2011).
In order to see whether H2O2 also serves as a senescence signal in the crop plant oilseed rape, we established a matrix of H2O2 levels and CAT and APX activities in oilseed rape plants. As for Arabidopsis, the hydrogen peroxide levels increased during bolting and flowering; however, in very late stages of senescence when H2O2 levels again increased substantially in Arabidopsis rosettes (coinciding with increased lipid peroxidation and fatty acid degradation), no such increase could be observed in oilseed rape, indicating that mobilization of the carbon skeletons of lipids during membrane deterioration might be less efficient. With increasing H2O2 levels, CAT and APX activity decreased, with CAT2 down-regulation being relatively severe and APX down-regulation being less pronounced and only observable in young oilseed rape leaves. Although CAT2 down-regulation in Arabidopsis is achieved through transcriptional down-regulation by GBF1 (Smykowski et al. 2010), APX1 down-regulation appears to be posttranscriptional, most likely involves H2O2, and is dependent on the developmental stage; however, the precise mechanism is still unknown (Ye et al. 2000; Zimmermann et al. 2006). Down-regulation of CAT and APX activity and the increase of H2O2 are not restricted to Brassicaceae, and have also been observed in sunflower senescence (Agüera et al. 2010).
Total glutathione and ascorbate pools decreased during development, and interestingly, the reduction rate of both pools increased with the progression of senescence in oilseed rape leaves. In 6 and 7-week-old plants, the oxidized forms reached their maximum values, maintained comparable levels from weeks 8 to 12, and then decreased again to nearly undetectable levels of GSSG in 12 and 13-week-old plants, which coincides with low H2O2 levels in these final stages. This is in agreement with the characteristic high reduction state of the glutathione pool. In the absence of stress, tissues such as leaves typically maintain measurable GSH:GSSG ratios of approximately 20:1 with considerable variation in specific subcellular compartments (Meyer et al. 2007; Queval et al. 2011). In cat2 mutants, the conversion of GSH to GSSG within h after the onset of H2O2 production was followed by a subsequent increase of the total glutathione pool over 3–4 d (Queval et al. 2009). The GSH:GSSG ratio of leaves dropped from approximately 20:1 to almost 1:1 after induction of endogenous H2O2 production at moderate rates (Mhamdi et al. 2010). Such effects were not observed during the developmental increase of H2O2 in oilseed rape. Only a slight increase in the glutathione pool was observed in 10-week-old plants after H2O2 had reached its maximum level (Figure 5A).
Since glutathione status is often used as a marker for oxidative stress, it can be speculated that the long-term increased H2O2 levels during development are not recognized as “oxidative stress” but as a developmental signal. However, the glutathione redox potential is not only governed by the GSH:GSSG ratio, but also depends on the absolute concentration of the total glutathione pool (Mullineaux and Rausch 2005; Meyer 2008). A decrease in a total glutathione pool with a constant GSH:GSSG ratio leads to an increase in redox potential, meaning that no clear conclusion can be drawn in regards to the actual redox potential (Queval et al. 2011). Decreasing glutathione pools in senescent leaves might also indicate sulfur remobilization, since glutathione is one of the major forms of organic sulfur translocated to the phloem, and high concentrations of glutathione have also been observed in the phloem sap of Brassica napus plants (Mendoza-Cózatl et al. 2008). Furthermore, sulfur availability and senescence are interconnected in oilseed rape; a transient S limitation in oilseed rape plants is compensated for by means of a fine management of leaf N-S remobilization, and causes delayed leaf senescence (Abdallah et al. 2011). The transport protein OPT6 might play a role in long-distance transport. The corresponding Arabidopsis gene is highly expressed in the vasculature, and has been discussed to transport GS-conjugates and GS-cadmium complexes as well as GSH and GSSG (Cagnac et al. 2004), although GSSG transport is still under debate (Pike et al. 2009). Moreover, glutathione is involved in various signaling processes, but the study of glutathione-dependent signaling is still at early stages (Noctor et al. 2012). Even though ascorbate and glutathione pools are often considered as interchangeable antioxidants and both pools decreased with oilseed rape plant age, glutathione or ascorbate deficiency mutants of Arabidopsis revealed that these two compounds have specific functions. Furthermore, ascorbate and glutathione are differentially influenced by environmental factors, and cat2 mutants have been shown to produce oxidized glutathione after the onset of H2O2 production while ascorbate remains highly reduced (Queval et al. 2007; Mhamdi et al. 2010; Foyer and Nocter 2011). Although considerable amounts of oxidized glutathione can be measured in young oilseed rape plants which are reduced again with development, the ascorbate reduction state did not change significantly with age until week 13. Remarkably, the ascorbate pool increased slightly, though not significantly, after H2O2 levels had reached their maximum point in 9-week-old plants (Figure 5C). A change in SDG and SAG expression coincided with the H2O2 maximum, and preceded chlorophyll breakdown. Early SAGs like SAG13 increased, whereas early SDGs like CAB or RUBISCO decreased. Of particular note, the expression of the transcription factor WRKY53 paralleled the H2O2 profile. For Arabidopsis, expression of WRKY53 also parallels H2O2 profiles and can be induced within h by H2O2 treatment in presenescent plants. In addition, expression of the upstream and downstream regulating factors of WRKY53 is also controlled by H2O2, indicating that the transcriptional reprogramming is at least in part regulated by H2O2 (Miao et al. 2007, 2008; Miao and Zentgraf 2010). There is some evidence that WRKY53 itself is involved in the H2O2 response of the WRKY53 promoter, in which case the Zn-finger in the DNA binding region of the WRKY proteins could serve as the structural domain for direct redox-regulation (Arrigo 1999). Under close to field conditions, H2O2 levels also increased during flowering. Under low and excess nitrogen supplies, H2O2 production appeared to be slightly increased, but these slight differences had no impact on leaf senescence. In a high CO2 atmosphere, low and optimal nitrogen supply led to an earlier decline of chlorophyll and N contents (Franzaring et al. 2011, 2012), and H2O2 also peaked earlier, indicating that H2O2 maxima correlated with the onset of senescence and senescence is accelerated under high CO2 conditions. Accelerated senescence could also be observed in flag leaves of barley in a high CO2 atmosphere (Fangmeier et al. 2000). However, under high N conditions, the H2O2 increase was exactly the same as under ambient air, but the decline was steeper for H2O2 as well as for chlorophyll. Even though high N availability and high CO2 prolonged flowering time, they did not lead to the production of more ripe pods, but more likely prevented apical switch off leading to a strong branching out. In addition, a slight reddening of the youngest stem leaves and pod stalks was observed after flowering under high N availability and high CO2, probably indicating nutrient deficiencies or imbalances. Furthermore, high CO2 caused a decline in seed oil contents contents. Nevertheless, remobilization appears to remain effective since macronutrients in the seeds increased slightly, and a lower N content was measured in senescent leaves (Franzaring et al. 2011, 2012). Leaf senescence in sunflower plants was also accelerated by nitrogen deficiency, and also correlated with an early decrease in the antioxidant enzymes activities of CAT and APX (Agüera et al. 2010). In conclusion, H2O2 appears to be used as a signal to promote senescence in different plant species, and to be part of a complex regulatory network. Remarkably, transcriptome responses to both CO2 and H2O2 are highly dependent on the photoperiod (Queval et al. 2012).
Materials and Methods
Plant material and cultivation
Arabidopsis thaliana (L.) Heynh ecotype Columbia was grown in a climate chamber at 22 °C, illuminated for 16 h at moderate light intensity (60 μmol s−1 m−2). Under these conditions, the plants developed flowers within 5–6 weeks and the mature seeds were harvested after 12 weeks. During growth and development of the leaves, the respective positions within the rosette were color-coded with different colored threads, so that even in very late stages of development, individual leaves could be analyzed according to their age. Brassica napus L. cv. Mozart was grown in greenhouses at 19–22 °C during a 16 h photoperiod of approximately 150 μmol s−1 m−2 light intensity. Near-field conditions for Brassica napus L. cv. Mozart were simulated in large growth chambers at the University of Hohenheim, using the seasonal increments of day length and the temperature of South-Western Germany as described in Franzaring et al. (2011). Plants were harvested on a weekly basis, and samples were always taken at the same time in the morning to avoid circadian effects.
Leaf discs (approximately 1 cm in diameter) were taken from approximately the same position of the respective leaf and incubated for exactly 45 min in DCFDA working-solution (Dichloro-fluorescein-Diacetate, 200 μg in 40 mL MS-Medium pH 5.7–5.8). Discs were then rinsed with water and frozen in liquid nitrogen. After homogenization on ice, 500 μL 40 mM Tris pH 7.0 was added, and the samples were centrifuged at 4 °C for 30 min. Fluorescence (480 nm excitation, 525 nm emission) of the supernatant was measured in a Berthold TriStar LB941 plate reader. All samples were taken and processed at one time point and incubated in one DCFDA working-solution to avoid staining solution- and diurnal-variability.
SPAD measurements and phenotypic analysis
For assessment of the leaf senescence state, color-coded leaves were grouped into three categories: (leaf Nos. 1–3), middle aged (leaf Nos. 4–7) and young (leaf Nos. 8–12) leaves. Chlorophyll content was estimated with a Atleaf + SPAD-Meter or a Konica-Minolta SPAD 502. Each leaf was measured in triplicate at different positions, and values were averaged. For evaluation of leaf senescence phenotypes, all leaves were categorized into four groups according to their leaf color: 1) “green”, 2) leaves starting to display yellowing as “yellowgreen”, 3) completely yellow leafs as “yellow”, and 4) dry and/or brown leafs as “brown/dry”.
C and N content analysis
Whole leaves were homogenized in liquid nitrogen and 200 mg of the homogenized powder was lyophilized. Total carbon and nitrogen contents of the lyophilized material were determined with a CN-element analyzer (Elementar Vario EL III) via heat combustion at 1 150 °C and thermal conductivity detection.
Catalase and APX zymograms
Leaf discs (approximately 1 cm in diameter) were taken from approximately the same position of the respective leaf and frozen in liquid nitrogen. After homogenization on ice, a protein extraction buffer was added (100 mM Tris, 20% Glycerol (v/v), 30 mM DTT, pH 8) and the samples were centrifuged for 15 min at 14 000 rpm and 4 °C. Total protein concentration of the supernatant was determined via Bradford assay (Bio-Rad Protein Assay), and 15 μg total protein was separated on native PAA gels (7.5% PAA, 1.5 M Tris, pH 8.8; running buffer: 25 mM Tris, 250 mM Glycin, pH 8.3). Subsequently, the gels were rinsed twice with water, incubated for exactly 2 min in a 0.01% H2O2 solution, again rinsed twice with water and stained in a solution containing 1% FeCl3 and 1% K3(Fe(CN)6); (w/v) until activity bands became visible (approximately 4 min). The staining reaction was stopped by decanting staining solution and rinsing gels with water. For isoform identification, slices of gels containing the same samples and sample amounts were incubated for 2–10 min in 10 mM 3-amino triazol (3-AT) solution prior to activity staining.
For APX activity, two leaf discs (approximately 1 cm in diameter) were taken from approximately the same position of the respective leaf and frozen in liquid nitrogen. After homogenization on ice, a protein extraction buffer was added (50 mM potassium phosphate pH 7.8, 2% Triton-X 100 (v/v), 5 mM ascorbic acid, 35 mM β-mercapto ethanol, 2% polyvinylpyrrolidon (w/v)) and the samples were centrifuged for 15 min at 14 000 rpm and 4 °C. Protein concentration of the supernatant was determined via Bradford assay (Biorad Protein Assay). The native PAA gels (10% PAA, 1.5 M Tris pH 8.8, 13% Glycerol (v/v); running buffer: 25 mM Tris, 250 mM Glycin, 2 mM ascorbic acid, pH 8.3) were run for 30 min at 120 V before samples were loaded, and then 30 μg of protein was loaded and the electrophoresis was conducted for approximately 3 h (120 V, 4 °C). After electrophoresis, the gels were incubated three times in 50 mM potassium phosphate buffer pH 7 containing 2 mM ascorbic acid for 10 min, and once in 50 mM potassium phosphate buffer pH 7 containing 4 mM ascorbic acid and 0.5 μM H2O2 for 10 min. Gels were rinsed twice with water after each incubation step. Gels were equilibrated for 1–2 min in 50 mM potassium phosphate buffer pH 7.8 before the staining reaction was started by adding 50 mM potassium phosphate buffer pH 7.8 containing 14 mM TEMED and 2.45 mM NBT. The staining reaction was stopped when the first bands became visible (after approximately 5–10 min) by decanting the staining solution and rinsing the gels with water.
APX activity measurement
Total protein extracts were obtained from homogenized leaf discs and stored in a 50 mM potassium phosphate buffer (pH 7.8) with 0.25 mM EDTA, 2% PVP (w/v), 10% Glycerol (v/v) and 5 mM ascorbic acid. 100 μL of these extracts were used for activity measurements in a 25 mM phosphate buffer (pH 7) with 0.1 mM EDTA, 1 mM H2O2 and 0.25 mM ascorbic acid in a total volume of 1 mL. The reaction was started by the addition of H2O2, and a decrease in ascorbic acid was recorded at 290 nm. For the final calculation, an ascorbic acid extinction coefficient of 2.8 mM * cm−1 was used.
GSSG/GSH and DHAsc/Asc analytics
Four leaf discs were homogenized in 300 μL 5 N HCl in 15% methanol, sonicated for 3 min, and incubated for 15 min on ice. Subsequently, the samples were centrifuged at 4 °C for 15 min at 14 000 rpm. The supernatant was removed and directly used for LC/MS analysis (Acquity UPLC, SynaptG2 HDMS; Waters, Manchester) of ascorbic acid and glutathione pools. For the analysis, 5 μL of the supernatant was injected onto a 2.1 mm × 100 mm, 1.8 μm Acquity UPLC HSS T3 column operating at a flow rate of 0.3 mL/min. For chromatographic separation, an 8 min gradient from 99% water to 99% methanol (both solvents with 0.1% formic acid) was used. The mass spectrometer was equipped with an ESI source and operated in resolution scanning mode with a scan time of 0.2 s. For quantification, bracketing calibration was performed.
Protein extraction, western blot and immunodetection
150–300 mg leaf material was homogenized on ice with 300 μL protein extraction buffer (100 mM potassium phosphate, 1 mM EDTA, 1% Triton-X 100 (v/v) and 10% Glycerol (v/v)). After 15 min centrifugation at 14 000 rpm and 4 °C, protein concentration of the supernatant was determined. For western blotting, 30 μg of protein was loaded on a SDS-PAGE gel and transferred onto a nitrocellulose membrane by semi-dry blotting. The membrane was washed twice with Tris-buffered saline (TBS), and then blocked with 3% milk powder in TBS. The detection was performed with polyclonal Anti-GFP antibodies in 1.5% milk powder. After washing twice with TBS-Tween 20 (TBS-T), peroxidase-conjugated anti-mouse antibodies were used for the chemiluminescence detection.
Primer design for qRT-PCR was done via QuantPrime (Arvidsson et al. 2008). RNA extraction and cDNA synthesis were conducted with the InviTrap Spin Universal RNA Mini Kit (Invitek) and qScript cDNA SuperMix (Quanta Biosciences), respectively. qPCR was performed with Perfect CTa SybrGreen Fast Mix (Quanta Biosciences) in an iCycler iQ System (Biorad). Relative quantification to ACTIN2 was calculated with the ΔΔCT– Method (Pfaffl et al. 2001). ACTIN2 was chosen as a reference gene for Senescence, since the variation of ACTIN2 expression over different leaf and plant stages in Arabidopsis was very low in contrast to that of other housekeeping genes (Panchuk et al. 2005).
(Co-Editor: Hai-Chun Jing)
We thank Alex Costa from the University of Padua, Italy, for providing the plants overexpressing OxyR-RD-cpYFP in the cytoplasm and peroxisomes. We thank Gabriele Eggers-Schumacher and Bettina Stadelhofer (ZMBP, University of Tuebingen, Germany) for their excellent technical assistance. This work was financially supported by the DFG (FOR948).