GDH1 and GDH2 expression depends on the intensity and duration of the light exposure
It is well known that GDH1 and GDH2 expression increases in the dark and decreases in the light (Turano et al. 1997; Lee at al. 2010), but dynamics of the expression changes during the light shifts has not been studied so far. In our experiments, the transcript levels of these genes in the dark are dozens more than in the light (Fig. 1, Fig. 3b). The decrease in GDH1 and GDH2 expression during the dark-to-light shift occurs more quickly than the increase during the light-to-dark shift (Fig. 1a, 1b). Comparison of the transcript content in plant leaves adapted to different light intensity provided the greatest differences in the range from 0 to 25 µmol * m− 2 * s− 1 of the white light, i.e. at the low light. It suggests that a regulatory mechanism providing light dependent changes in the GDH1 and GDH2 expression is highly sensitive to light.
Earlier, we have demonstrated that functionally active chloroplasts are necessary for the light-dependent repression of GDH1 and GDH2 genes in Arabidopsis (Garnik et al. 2013). It means that the light-dependent regulation of GDH1 and GDH2 genes is mediated not by cytoplasm photoreceptors, but by plastid-to-nucleus signals.
DCMU treatment results in a higher expression of GDH1 and GDH2 in the light
Among a number of currently known plastid-to-nucleus signals, redox signals respond most quickly to the light intensity changes. This group includes signals mediated by singlet oxygen and Executer1 / Executer2 proteins; signals mediated by hydrogen peroxide generated in C-ETC; unknown signals associated with changes in the plastoquinone pool redox state (Dietz et al. 2016). Our attention was drawn to the latter regulatory path, since it is known that the pathway associated with Executer1 / Executer2 chloroplast-to-nucleus proteins is activated mainly under high light and photooxidative stress (Kim and Apel 2013), and hydrogen peroxide generation is closely associated with the plastoquinone pool redox state (Mubarakshina and Ivanov 2010). To manipulate with a plastoquinone pool redox state in plant leaves in vivo, we used the classical electron transfer inhibitors, DCMU and DBMIB.
DCMU treatment in the light led to the 8-fold higher transcript content for GDH1 and 25-fold higher for GDH2 (Fig. 2b). This can be interpreted as a participation of plastoquinone-derived redox signals in the regulation of GDH gene expression. This assumption is in a good agreement with a high sensitivity of the expression of the studied genes to differences in the light intensity under the low light conditions (Fig. 1c).
The redox state of the plastoquinone pool is important for the regulation of gene expression in the plastid (Pfannschmidt et al. 1999), as well as in the nucleus (Pfannschmidt et al. 2001). It is still not completely understood how such regulation is ensured in the cases where it is not mediated by ROS, but to date it has been observed for genes encoding the C-ETC subunits, the large Rubisco subunit (Pfannschmidt et al. 1999; 2001), chlorophyll-binding proteins of light-harvesting complexes (Borisova-Mubarakshina et al. 2014), flavonoid biosynthesis enzymes (Akhtar et al. 2010), and some antioxidant enzymes (Slesak et al. 2003; Yabuta et al. 2004; Garnik et al. 2016).
It was demonstrated using microarray technology that there are many more plastoquinone redox state-dependent genes. According to Adamiec et al., expression of 50 Arabidopsis genes (among 663 genes differentially expressed depending on the light intensity) returned to the dark levels after the DCMU treatment (Adamiec et al. 2008). A number of studies have shown participation of plastoquinone redox signals in responses of Arabidopsis transcript levels to stresses associated with high light and cold (Kopriva and Rennenberg 2004; Lepetit et al. 2013; Bode et al. 2016), as well as in regulation of stomata closure (Wang et al. 2016). Thus, even processes not directly related to photosynthesis may depend on the plastoquinone pool redox state.
Changes in the expression of the GDH1 and GDH2 genes under the DBMIB treatment may be the result of its effect on mitochondrial ETC redox state
If we consider the redox state of the plastoquinone pool as the main factor influencing the expression of the GDH1 and GDH2 genes during light shifts, then we would expect that a DBMIB treatment will lead to an even greater decrease in their expression. However, the expression of the GDH1 and GDH2 genes under the DBMIB treatment was slightly higher than in the leaves of the untreated plants (Fig. 2b). The absence of a reduction can be explained by the rather high light intensity and, respectively, the low expression level of the studied genes in the control plants in the light.
On the other hand, it is known that in mitochondria DBMIB can bind to the bc1 complex and inhibit the ubiquinol-cytochrome reductase activity, which leads to a reduced redox state of the mitochondrial ubiquinone pool (Surkov and Konstantinov 1980; Degli Esposti et al. 1984). We have shown previously that over-reduction of the ubiquinone pool by antimycin A treatment leads to induction of GDH2 gene in Arabidopsis heterotrophic cell culture (Tarasenko et al. 2009). It is possible that DBMIB at the used concentration exerted an inhibitory effect on the mitochondrial ETC, which masked the effect of the plastoquinone pool redox state on the expression of the studied genes.
The lower glucose level in the plants treated with DCMU or DBMIB does not correlate with the changes in the GDH1 and GDH2 genes expression
The change in the plastoquinone pool redox state is not the only factor that can affect genes expression in the presence of DCMU or DBMIB. Each of these inhibitors blocks the light stage of photosynthesis and can lead to a decrease in glucose level in the cell. According to the Miyashita and Good hypothesis it is the level of soluble carbohydrates that is the main regulator of the glutamate dehydrogenase genes expression (Miyashita and Good 2008). Therefore, we estimated the glucose content in the leaves of the same plants in which the transcripts of GDH genes were determined.
After the treatment with DCMU or DBMIB, the glucose content in the leaves of plants become significantly lower than in the control. There was no significant difference between the glucose content in the leaves of plants treated with DCMU and the plants treated with DBMIB (Fig. 2d). Therefore, if in this experiment the transcripts levels depended mainly on the glucose content in the leaves, then the same increase in the GDH1 and GDH2 expression should be expected both after DCMU or DBMIB treatments. However, we observed the significantly higher transcripts levels after the DCMU treatment (Fig. 2b). It suggests that glucose is not the only and not the main factor that regulates the expression of the studied genes.
ROS generation cannot be a factor leading to a decrease in the GDH1 and GDH2 genes expression
ROS mediated signaling pathways take part in regulation of a huge number of plant genes (Dietz et al. 2016). The plastoquinone pool is one of the main ROS generation sites in the light, so treatment with photosynthesis inhibitors significantly changes ROS level in plant cells, and it could be the reason for the observed GDH1 and GDH2 expression changes. The first ROS resulting from the leakage of electrons from a reduced or over-reduced plastoquinone pool is the singlet oxygen, which turns then into the superoxide radical O2− and the hydrogen peroxide H2O2 (Mubarakshina and Ivanov 2010).
We evaluated changes in the O2− content in Arabidopsis leaves after the DCMU or DBMIB treatment. In the presence of DBMIB, it was as high as in the light control. In the presence of DCMU in the light, it was much lower, comparable to the content in the dark (Fig. 2d). So, the lower level of superoxide radical corresponds to the higher level of GDH1 and GDH2 expression.
However, both our own results and the literature data clearly show that ROS can only increase the expression of glutamate dehydrogenase genes but not lower it (Scopelitis et al. 2006). In our experiments, when plants were exposed to 900 µmol * m− 2 * s− 1 of the white light, which is excessive for Arabidopsis and leads to photooxidative stress, the levels of GDH1 and GDH2 transcripts did not decrease, but increased (Fig. 3a). The treatment of plants with exogenous hydrogen peroxide also did not lead to a decrease in these genes expression neither at the initially high (in the dark), nor at the initially low (in the light) transcripts levels (Figs. 3c, 3d). This is in a good agreement with reports of increased expression of plant glutamate dehydrogenase during abiotic stresses. An increase in the GDH activity or in the expression of its genes during hypoxia-reoxygenation (Tsai et al. 2016), drought (Sun et al. 2013), temperature stresses (Goel and Singh 2015), and salt stress (Terce-Laforgue et al. 2015) was shown. Thus, ROS have an inducing effect on the GDH genes expression and cannot be the reason for a decrease in their expression during the dark-to-light shifts.
Physiological significance of the light-dependent regulation of the GDH1 and GDH2 genes expression
Turano et al. (1997) showed that the higher or lower GDH1 and GDH2 transcripts levels correlated with the higher or lower GDH enzyme activity, respectively. Thus, fluctuations in the transcript levels have a physiological significance.
We have here demonstrated that some chloroplast signals arising from changes in the C-ETC redox state can participate in the regulation of the Arabidopsis thaliana GDH1 and GDH2 genes expression. We believe that this regulation depends on the redox state of the plastoquinone pool, but is not mediated by ROS.
In the majority of the proven cases of gene expression regulation mediated by the plastoquinone pool redox state, the observed expression changes are aimed at adapting of the photosynthetic apparatus to changing light conditions, or at providing antioxidant protection. GDH, however, is a mitochondrial enzyme that is not directly related either to photosynthesis nor to other processes occurring in chloroplasts. However, the substrate of this enzyme, L-glutamate, also serves as a substrate for the biosynthesis of chlorophylls and heme groups of cytochromes belonging to the C-ETC (Tanaka et al. 2011).
L-glutamate is also necessary for synthesis of glutathione, which is necessary to provide antioxidant protection (Noctor et al. 2012). In plants, biosynthesis of both tetrapyrroles and glutathione is associated with chloroplasts and activated in the light (Tanaka et al. 2011; Noctor et al. 2012; Heyneke et al. 2013). Among the three metabolic pathways that use L-glutamate as the primary substrate (2-oxoglutarate formation; chlorophyll biosynthesis; glutathione biosynthesis), the second and the third are of a great importance in the light. On the other hand, the possibility of using L-glutamate in the TCA cycle after converting it to 2-oxoglutarate is important in cases of a long-term absence of photosynthesis (e.g. overnight).
We think that the physiological purpose of the light-dependent regulation of the GDH1 and GDH2 genes expression is to avoid a competition for the substrate between GDH (deamination of L-glutamate to 2-oxoglutarate), glutamyl-t-RNA synthetase (the first reaction of the tetrapyrroles biosynthesis path) and γ-glutamylcysteine synthetase (the first reaction of the glutathione biosynthesis path). In the light, expression of GDH genes is rapidly repressed, and L-glutamate can be used for biosynthesis of chlorophyll, heme and glutathione, which are more necessary at that time. After hours of absence of light, it becomes more necessary to use L-glutamate as a source of energy, that is ensured by increasing the expression of the GDH genes. The redox state of the C-ETC and the plastoquinone pool as a highly sensitive C-ETC component are ideal candidates to be the main regulators of light-dependent expression of the GDH genes.
Our hypothesis does not contradict the Miyashita and Good hypotheses about the participation of sugars in the light-dependent GDH regulation. However, Miyashita and Good suggested that the soluble sugars content should be the main regulatory factor for GDH genes expression changes, but we believe that the redox state of the plastoquinone pool is the factor playing the key role. Sugar accumulation as a GDH genes expression regulatory factor acts in the same direction as the plastoquinone pool reduction, but apparently makes a smaller contribution to their light-dependent repression.
Interestingly, glucose accumulation in the light itself can lead to a partial plastoquinone pool reduction due to a slowdown in the NADPH oxidation in the Calvin cycle (Ma et al. 2008). In this case, the decrease in the GDH genes expression in the presence of exogenous sucrose or glucose in the light can be explained not only by their direct regulatory action, but also by the glucose-dependent change in the plastoquinone redox state.
We have previously demonstrated that the expression of the GDH2 gene is induced under the over-reduced state of the mitochondrial ubiquinone pool in a heterotrophic culture of Arabidopsis cells, and this induction was independent of ROS production (Tarasenko et al. 2009). There are many parallels in the structure and functioning of the respiratory and photosynthetic ETCs, and it is even more interesting that the reduction of the ubiquinone pool leads to the induction of GDH genes, and the reduction of the plastoquinone pool leads to their repression. It cannot be excluded that these are manifestations of the same regulatory mechanism mediated by redox signals emanating from the two largest quinone pools of a plant cell.