Author response: SnRK1-triggered switch of bZIP63 dimerization mediates the low-energy response in plants
2015-02-23

Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Metabolic adjustment to changing environmental conditions, particularly balancing of growth and defense responses, is crucial for all organisms to survive. The evolutionary conserved AMPK/Snf1/SnRK1 kinases are well-known metabolic master regulators in the low-energy response in animals, yeast and plants. They act at two different levels: by modulating the activity of key metabolic enzymes, and by massive transcriptional reprogramming. While the first part is well established, the latter function is only partially understood in animals and not at all in plants. Here we identified the Arabidopsis transcription factor bZIP63 as key regulator of the starvation response and direct target of the SnRK1 kinase. Phosphorylation of bZIP63 by SnRK1 changed its dimerization preference, thereby affecting target gene expression and ultimately primary metabolism. A bzip63 knock-out mutant exhibited starvation-related phenotypes, which could be functionally complemented by wild type bZIP63, but not by a version harboring point mutations in the identified SnRK1 target sites. https://doi.org/10.7554/eLife.05828.001 eLife digest Organisms need to adjust their metabolism in response to changing environmental conditions to ensure that they balance their energy intake with the demands of growth and reproduction. In plants, an enzyme called SnRK1 plays a crucial role in responses to starvation in two ways: by altering the activities of enzymes involved in metabolism and by regulating the expression of genes. To perform the second job, SnRK1 is thought to control the activity of proteins called transcription factors—which alter the expression of genes by binding to DNA—but it is not known which ones. SnRK1 has ‘kinase’ activity, that is, it can alter the activities of other proteins by adding small molecules called phosphates to them. It has been suggested that a group of transcription factors called the bZIP proteins may be regulated by SnRK1. Two bZIP proteins work together to switch on a gene, and the combination of bZIP proteins that interact can influence which genes are switched on. Here, Mair et al. studied the role of a bZIP protein called bZIP63 during starvation in the plant Arabidopsis. The experiments show that bZIP63 is involved in controlling responses to starvation. Furthermore, its activity is regulated by SnRK1, which adds phosphates to three specific locations on the protein. These phosphates alter the ability of bZIP63 to interact with other bZIP proteins, leading to changes in gene expression during starvation. Mair et al. triggered starvation in Arabidopsis plants by keeping the plants in darkness for several days. The leaves of normal plants turn yellow in response to starvation, but the leaves of mutant plants that lacked bZIP63 remained green. In contrast, plants containing higher amounts of this bZIP protein showed the opposite effect and their leaves turned yellow much more quickly than normal plants. The mutant plants that lacked bZIP63 could be rescued by the normal protein, but not by another version of the protein that SnRK1 is unable to add phosphates to. These data suggest that SnRK1 regulates bZIP63 activity to alter metabolism in response to starvation. Mair et al. propose a model in which the ability of bZIP63 to interact with other bZIPs is normally rather low. However, when the plants are starved, SnRK1 adds phosphates to bZIP63, which increases its ability to bind to other bZIP proteins and leads to changes in gene expression. The bZIP proteins are also found in animals; therefore a future challenge is to find out whether these proteins are also regulated in a similar way. https://doi.org/10.7554/eLife.05828.002 Introduction Flexibility in the regulation of gene expression is crucial for all organisms to adjust their metabolism to changing growth conditions. Particularly under stress, available energy resources need to be balanced between defense and growth. The SUCROSE NON-FERMENTING RELATED KINASE 1 (SnRK1) in plants and its orthologs, the sucrose-non-fermenting 1 (Snf1) kinase in yeast and the AMP-dependent protein kinase (AMPK) in mammals, are well-known and crucial master regulators of energy homeostasis. SnRK1 is involved in the regulation of plant metabolism, development, and stress response (Polge and Thomas, 2007; Baena-González and Sheen, 2008), Snf1 is required for the switch from fermentative to oxidative metabolism in the absence of glucose (Hedbacker and Carlson, 2008), and AMPK regulates glucose, lipid, and protein metabolism, mitochondrial biogenesis, and feeding behavior in animals (Hardie et al., 2012). They are generally activated under energy starvation conditions and trigger metabolic reprograming to slow down energy-consuming processes and turn on pathways for alternative energy production in order to survive the stress conditions (Hardie, 2007; Tome et al., 2014). This happens, in two ways: by direct phosphorylation and modulation of the activity of key enzymes in nitrogen, carbon, or fatty acid metabolism (Sugden et al., 1999; Kulma et al., 2004; Harthill et al., 2006), and by massive transcriptional reprogramming (Polge and Thomas, 2007; Baena-González and Sheen, 2008; McGee and Hargreaves, 2008). Especially in plants, the latter aspect, the regulation of transcription, is still poorly understood. In Arabidopsis protoplasts, transient overexpression of AKIN10, a catalytic subunit of the SnRK1 complex, resulted in a transcriptional profile reminiscent of various starvation conditions and led to the identification of 1021 putative SnRK1 target genes (Baena-González et al., 2007). However, the transcription factors mediating the transcriptional response of SnRK1 to energy starvation are still unknown. Based on reporter gene activation assays in protoplasts (Baena-González et al., 2007) and modelling of microarray data (Usadel et al., 2008), some members of the C/S1 group of basic leucine zipper (bZIP) transcription factors (TFs)—foremost bZIP11 and bZIP1 from the S1 group—were speculated to be involved in this process. Yet, a direct regulation of these bZIPs by SnRK1 has never been shown. bZIP proteins form a large and highly conserved group of eukaryotic TFs. They bind the DNA as dimers and are characterized by a basic region for specific DNA binding and a leucine zipper for dimerization (Deppmann et al., 2006; Reinke et al., 2013). They are involved in a multitude of cellular processes, including cell proliferation and differentiation, metabolism, stress response, and apoptosis (Mayr and Montminy, 2001; Jakoby et al., 2002; Motohashi et al., 2002; Rodrigues-Pousada et al., 2010; Tsukada et al., 2011). The diversity and flexibility of transcriptional regulation by bZIP TFs can at least partially be attributed to their potential to form variable dimer combinations, which bind to different consensus target sites (Deppmann et al., 2006; Tsukada et al., 2011). While the leucine zipper determines the possible dimer combinations (Deppmann et al., 2006; Reinke et al., 2013), the actual in vivo dimer composition is further influenced by factors such as protein availability, binding of regulatory proteins, or post-translational modifications (Kim et al., 2007; Schuetze et al., 2008; Lee et al., 2010). Since the initial discovery that the mammalian bZIP cAMP response element binding protein (CREB) is regulated by reversible phosphorylation, many bZIP TFs were reported to be phosphorylated (Holmberg et al., 2002; Schuetze et al., 2008; Tsukada et al., 2011). However, particularly in plants, the functional consequences of these phosphorylation events often remained unclear. For example, it has been known for several years that abscisic acid (ABA)-dependent phosphorylation of some ABA-responsive element binding proteins (AREBs) by SnRK2 kinases increases their transcriptional activity (Furihata et al., 2006), yet the underlying mechanism of this activation is still unknown. It is also surprising that, while many examples for phosphorylation-dependent regulation of bZIP activity, DNA-binding, subcellular localization, stability, and interaction with regulatory proteins are known (Schuetze et al., 2008; Tsukada et al., 2011), reports on the regulation of dimerization are scarce. So far, only three publications (Kim et al., 2007; Guo et al., 2010; Lee et al., 2010) showed compelling evidence for phosphorylation-dependent changes in bZIP dimerization in animals. Still, even in these cases it is often not entirely clear whether bZIP phosphorylation affects dimerization directly or indirectly by enhancing DNA binding. bZIP63 is a member of the C-group of Arabidopsis bZIPs, which was proposed to play a role in energy metabolism, seed maturation, and germination under osmotic stress (Jakoby et al., 2002; Correa et al., 2008; Veerabagu et al., 2014). Its transcriptional profile indicates that bZIP63 could be involved in the (energy) starvation response, as transcription and mRNA stability are repressed by sugars and ABA and mRNA levels increase in the night and even more during extended night treatments (Matiolli et al., 2011; Kunz et al., 2014). A small set of potential target genes for bZIP63 has been identified, including genes involved in amino acid metabolism (ASN1/DIN6 = ASPARAGINE SYNTHETASE 1, ProDH = PROLINE DEHYDROGENASE), energy starvation response (DIN10 = RAFFINOSE SYNTHASE 6), and senescence (SEN1 = SENESCENCE 1) (Baena-González et al., 2007; Dietrich et al., 2011; Matiolli et al., 2011; Veerabagu et al., 2014). The C-group bZIPs form a dimerization network with the S1-group in plants, in which bZIP63 can interact with all members (Ehlert et al., 2006; Kang et al., 2010). Three of its dimerization partners from the S1-group—bZIP1, bZIP11, and bZIP53—were shown to be important metabolic regulators, especially under energy starvation conditions, and to regulate the expression of ASN1 and ProDH as well (Hanson et al., 2008; Dietrich et al., 2011; Ma et al., 2011). Furthermore, bZIP1 was recently confirmed as a transcriptional master regulator of the rapid response to nutrient signals controlling mainly genes involved in amino acid metabolism and cell death/phosphorus metabolism as primary targets (Para et al., 2014). In that study the authors also speculate about a posttranslational modification of bZIP1 or its binding partners. Here we show that bZIP63 is an important metabolic regulator, especially under stress/starvation conditions, and that bZIP63 is phosphorylated at multiple sites in vivo in a sugar- and energy-dependent manner. In an unbiased approach, we identified SnRK1 as one of the kinases responsible for bZIP63 phosphorylation and found that it targets three highly conserved serine residues in the N- and C-terminus of bZIP63. Moreover, we demonstrate that the phosphorylation of these sites is crucial for bZIP63's dimerization and activity in planta and propose a molecular model for a phosphorylation-triggered switch of bZIP63 dimerization partners, which ultimately regulates metabolic reprogramming. Results bZIP63 controls dark-induced senescence and primary metabolism To better understand the role of bZIP63 in the plant we first tested whether bZIP63 has a similar phenotype as its dimerization partners bZIP1 and bZIP11. Prolonged darkness was shown to induce increased chlorophyll loss in plants overexpressing bZIP1 (Dietrich et al., 2011). Therefore, we incubated a bZIP63 knock-out (ko), two independent overexpressor (ox) lines (Figure 1—figure supplement 1), and their respective wild types (wt) in the dark and determined the percentage of the green leaf area as a measure for chlorophyll content (Figure 1; Figure 1—figure supplement 2A). This method was preferred over direct chlorophyll measurements (Figure 1—figure supplement 2B) as it is not affected by the water loss in senescing leaves. While no differences were observed before dark treatment, significant differences were visible after 9 days in darkness (Figure1—figure supplement 2C–E). Similar to the bZIP1 ox, the bZIP63 ox lines (ox#2 and ox#3) had a significantly higher percentage of yellow leaf area than the wt. In contrast, bzip63 plants displayed a stay-green phenotype (Figure 1A,B; Figure1—figure supplement 2F). RT-qPCRs of different senescence marker genes confirmed that the phenotype is due to dark-induced and not to natural senescence (Figure 1—figure supplement 3). Transcriptional responses in dark-induced senescence show clear similarities to starvation-induced senescence in cell suspension culture, which likely results from carbon depletion in both systems (Buchanan-Wollaston et al., 2005). We therefore tested whether addition of sugar could rescue this phenotype by performing the dark-induced senescence experiment with seedlings grown on agar plates with and without addition of sugar. Indeed, we found that the phenotype could be rescued by the addition of glucose (Figure 1C,D Figure 1—figure supplement 4), supporting the suggested role of bZIP63 in energy/carbon starvation response. Figure 1 with 4 supplements see all Download asset Open asset bZIP63 mutants have a phenotype in dark-induced senescence. (A) and (B) Dark-induced senescence phenotype of 4.5 week-old soil grown plants. Comparison of a bzip63 line in the Wassilewskya (Ws-2), and two bZIP63 ox lines in the Columbia (Col-0) background, after 9 days in darkness. (A) Representative leaf series. (B) Box-and-whiskers plot of the total green leaf area of eight biological replicates as determined with ImageJ. See Figure 1—figure supplement 1 for molecular characterization of the bZIP63 mutant lines, Figure 1—figure supplement 2 for a scheme of the determination of the green leaf area, quantitative chlorophyll measurements, controls and green area of individual leaves, Figure1—source data 1 for the used ImageJ macro, and Figure 1—figure supplement 3 for expression of senescence marker genes in wt and bzip63. (C) and (D) Sugar rescue of the dark-induced senescence phenotype. Seedlings were grown for 12 days on 1⁄2 MS agar containing 0.5% sucrose, transferred to 1⁄2 MS agar containing 0% or 2% glucose, and grown for another 6 days before incubation in the dark for 7 days. (A) Representative seedlings. (B) Box-and-whiskers plot of the chlorophyll content of 8 seedlings per line and condition. See Figure 1—figure supplement 4 for pictures and chlorophyll content of seedlings before dark incubation. p-values from T-tests between mutants and wt < 0.05, < 0.01, and < 0.001 are indicated by *, **, and ***, respectively. https://doi.org/10.7554/eLife.05828.003 Figure 1—source data 1 ImageJ macro for determination of leaf area and green leaf area. https://doi.org/10.7554/eLife.05828.004 Download elife-05828-fig1-data1-v3.txt The notion that several hetero-dimerization partners of bZIP63, including bZIP1 and bZIP11, are important metabolic regulators under starvation conditions (Dietrich et al., 2011; Ma et al., 2011) prompted us to perform an unbiased metabolomics analysis of bzip63 and ox#3 plants and their respective wt lines. Leaves of 5 week-old plants were harvested after 6 hr of light and extended night and analyzed for changes in the primary carbon and nitrogen metabolism using gas chromatography coupled to mass spectrometry (Figure 2; Figure 2—figure supplement 1 and Figure 2—source data 1). Intriguingly, almost all amino acid levels were increased in the bZIP63 ko and decreased in the ox plants. This effect was even more pronounced after the extended night treatment. The biggest differences were observed for proline and the entire glutamate family. This accumulation of amino acids, particularly of proline, is striking in view of the observed senescence phenotype as it has been suggested that proline serves as an alternative energy source, especially under low carbon conditions (Szabados and Savouré, 2010; Szal and Podgórska, 2012). Figure 2 with 1 supplement see all Download asset Open asset bZIP63 mutants have an altered primary metabolism. Metabolic phenotype of 5 week-old soil grown plants after 6 hr light (L) and extended night (E). Log-2 fold changes of metabolite levels in ko and ox compared to their respective wt, displayed on a simplified map of the central primary metabolism. Values are means of five biological replicates. p-values from T-tests between mutants and wt < 0.05, < 0.01, and < 0.001 are indicated by *, ** and ***, respectively. For more details including mean values and SD see Figure 2—figure supplement 1 and Figure 2—source data 1. https://doi.org/10.7554/eLife.05828.009 Figure 2—source data 1 Excel table of relative metabolite levels in bZIP63 mutants and p-values from T-tests. Table of relative metabolite levels in Ws-2, bzip63, Col-0 and ox#3 after 6 hr in light or extended night, which were used to calculate the log2-fold changes shown in Figure 2 and Figure 2—figure supplement 1. Mean values and SD of five biological replicates are given as fold changes to the corresponding wt. p-values from T-tests between wt and mutant are listed. https://doi.org/10.7554/eLife.05828.010 Download elife-05828-fig2-data1-v3.xlsx bZIP63 is phosphorylated at multiple sites in an energy-dependent manner Kirchler et al. (2010) showed that bZIP63 can be phosphorylated in vitro by crude Arabidopsis extracts. To test whether bZIP63 is also phosphorylated in vivo, we treated total leaf protein extracts of ox#3 plants, expressing GFP-tagged bZIP63, with lambda protein phosphatase (λPP) and separated treated and untreated extracts on 2D gels by isoelectric focusing (IEF) in the first, and SDS-PAGE in the second dimension (Figure 3A). λPP treatment induced a clear shift of bZIP63 towards the basic region of the IEF strip, thus indicating dephosphorylation of the protein. Figure 3 with 2 supplements see all Download asset Open asset bZIP63 is phosphorylated at multiple sites in an energy-dependent manner in vivo. (A) 2D gel western blots (αGFP) of lambda phosphatase (λPP)-treated protein extracts from adult soil-grown plants expressing bZIP63-GFP (ox#3). (B) Phos-tag gel western blots showing the in vivo phosphorylation state of bZIP63 in seedlings after 6 hr extended night in the presence (+) or absence (−) of 1% sucrose and in 5 week-old soil-grown plants after 6 hr light (L) or extended night (EN). Plants either expressed 35S::bZIP63-GFP (ox#3) or a genomic fragment of bZIP63 (GY11) with a YFP-tag. Recombinant bZIP63-YFP was used as a nonphosphorylated control. Numbered arrowheads on the right mark the position of each observed bZIP63 band for easy reference with other figures (see Figure 3—figure supplement 1 for a comparative image of all Phos-tag western blots). For more information on the genomic line see Figure 7 and Figure 7—figure supplement 1. (C) Identification of in vivo phosphorylation sites by immunoprecipitation (IP) and tandem mass spectrometry (MS/MS). An exemplary western blot of the IP is shown. The scheme at the bottom shows the positions of the identified in vivo phosphorylation sites and the total sequence coverage reached with each proteolytic enzyme (grey bars). Asterisks mark the identification of a phospho-site. For more information on samples and (phospho-) peptides see Figure 3—figure supplement 2. IEF, isoelectric focusing; CBB, coomassie brilliant blue; BD, basic domain; ZIP, leucine zipper; LTQ, linear ion trap quadrupole; chym., chymotrypsin. https://doi.org/10.7554/eLife.05828.012 As bZIP63 expression is strongly regulated by the day/night cycle and by sugars (Matiolli et al., 2011; Kunz et al., 2014), we investigated its phosphorylation status under these conditions, applying the Phos-tag technique to enhance phosphorylation-induced mobility shifts in 1D SDS-PAGE (Kinoshita et al., 2006). Comparison of protein extracts from seedling cultures after 6 hr extended night in the presence or absence of sucrose or leaves harvested after 6 hr of light or extended night revealed strong differences in the phosphorylation patterns of bZIP63 (Figure 3B; Figure 3—figure supplement 1). Compared to the recombinantly expressed (not phosphorylated) bZIP63-YFP, the majority of plant-expressed bZIP63-GFP appeared in several slower migrating forms, thus indicating multiple and differentially phosphorylated forms. In total, we were able to distinguish eight different forms on the Phos-tag gels, depending on the conditions (Figure 3B; Figure 3—figure supplement 1). However, it is important to note, that not every phosphorylation event results in an equal shift. In the light, two strong bands were visible, possibly reflecting the two on the 2D gel (Figure as band 6 and band extended night conditions, an band appeared as band indicating increased phosphorylation of bZIP63. the of the phosphorylated form of bZIP63 to the two phosphorylated forms 6 and was different between the ox (ox#3) and a genomic line of bzip63 In the genomic the phosphorylated form of bZIP63 was the band under extended night conditions, which be a of the different bZIP63 amounts in to the and likely better the phosphorylation state of the protein. in seedlings grown in culture, the extended phosphorylation of bZIP63 could be by the addition of 1% of sucrose (Figure In phosphorylation was even than in the These data that bZIP63 has a of phosphorylation in the plant and under starvation conditions. In contrast, addition of sugars leads to a phosphorylation status of bZIP63. In order to the in vivo phosphorylated bZIP63-GFP was from total leaf extracts using to proteolytic digest and analyzed by chromatography coupled to tandem mass spectrometry (Figure To sequence coverage of the protein we proteolytic from proteolytic enzymes and This resulted in a total sequence coverage of and the identification of several indicating that bZIP63 is phosphorylated at to and in vivo (Figure Figure 3—figure supplement Two of these and also found in a study et al., 2013). only three of the and found by protein the of alternative proteolytic for identification in SnRK1, and are potential kinases of bZIP63 To potential kinases of bZIP63, we kinase assays with plant protein extracts from of grown Arabidopsis plants or cell using recombinantly expressed bZIP63 as was in leaf extracts the large of with some of the kinase signals and a during protein Three strong bands of about and were visible on the (Figure indicating that at least three kinases can bZIP63 in These bands were not visible on a gel without the that they from kinase (Figure supplement 1). To the and to low proteins before kinase we plant protein extracts on bZIP63. The were tested in an kinase for kinase activity towards bZIP63 and on a normal SDS-PAGE gel without for kinase corresponding in molecular to the from the kinase were from the with and analyzed by (Figure In total, protein kinases and kinase were identified in independent experiments (Figure data 1; Figure data proteins which not the or for which it had been shown that they are not in the were proteins which were identified in more than one with at least one were to be in protein kinases and two kinase regulatory (Figure Figure data 1; Figure data We identified the two catalytic of SnRK1, and as well as the regulatory subunit members of the protein kinase were but only was identified with In two catalytic and and one regulatory subunit of kinase were as well as kinase protein 2 The SnRK1 and in to the two while the band to the kinase As SnRK1 was reported to enhance the activity of several C/S1 group bZIPs (Baena-González et al., 2007) and was suggested to be activated under energy starvation conditions (Baena-González and Sheen, could the observed of bZIP63 in extended further analysis on and Figure 4 with 1 supplement see all Download asset Open asset different kinases can bZIP63. (A) kinase with a protein from grown wild type plants and bZIP63 as the positions of potential bZIP63 (B) of the kinase identification process. (C) kinase with samples from of a protein with bZIP63 and bZIP63 as a and a of catalytic and regulatory kinase identified with The also the gene molecular and of samples in which the protein was For controls and a of all identified kinases and kinase peptides see Figure supplement 1 and Figure data 1, 2. CBB, brilliant Figure data 1 over the kinases identified by after with bZIP63. Download Figure data 2 Excel table containing a over the identified kinases and analyzed samples as well as all peptides found for the kinase The a table showing in which samples each kinase subunit was identified, including the of and molecular The other all peptides found for the kinase in each and state whether the is or Download The SnRK1 kinase with and bZIP63 in vivo To that and bZIP63, we first an kinase with protein extracts of wt and seedlings in the presence of to the from of the (Figure In both and leaf extracts of plants one band at the of (see Figure supplements for characterization of the As has the as AKIN10, the likely from In vitro kinase with equal amounts of both showed that both kinases can bZIP63. However, bZIP63 much than (Figure of the SnRK1 kinase increased the activity of and about (see also et al., but had no effect on the between the (Figure supplement In vivo interaction assays with the identified SnRK1 the of the kinase In both yeast (Figure and assays (Figure and the regulatory subunit strongly with bZIP63, to a as for the bZIP63 which was used as control et al., In contrast, and the two regulatory and showed almost no interaction with bZIP63 (Figure Figure supplement However, it has to be that and are part of a