SnRK1-triggered switch of bZIP63 dimerization mediates the low-energy response in plants
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 .
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 . 10 . 7554/eLife . 05828 . 003Figure 1 . 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 ½ MS agar containing 0 . 5% sucrose , transferred to ½ 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 . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 00310 . 7554/eLife . 05828 . 004Figure 1—source data 1 . ImageJ macro for determination of leaf area and green leaf area . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 00410 . 7554/eLife . 05828 . 005Figure 1—figure supplement 1 . Molecular characterization of the bzip63 line and expression of bZIP63 in the ko and ox lines . ( A ) Scheme of the genomic locus ( top ) and the protein ( bottom ) of bZIP63 . UTRs and exons are shown in grey and green , respectively . The position of the T-DNA in the first exon , as well as the position of primers used for PCR and RT-qPCR are indicated . The position of the T-DNA insertion was determined by sequencing of the PCR product in ( C ) . ( B ) PCR on genomic DNA using primers fw and rv to show the homozygous T-DNA insertion in the bzip63 line . ( C ) PCR on genomic DNA from bzip63 plants using primers fw , rv , LB , and RB to determine the localization and orientation of the T-DNA insertion . ( D ) and ( E ) Expression of bZIP63 in wt , bZIP63 ko and ox plants . RT-qPCR of bZIP63 in 5 week-old plants after 6 hr of light ( L ) or extended night ( EN ) using primers fwq and rvq . Bars represent means ± SD of 5 biological replicates and are given as fold change to Ws-2 in L . ( F ) Pictures of 5 week-old wt and mutant plants grown in a 12 hr light/12 hr dark cycle . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 00510 . 7554/eLife . 05828 . 006Figure 1—figure supplement 2 . Phenotype of bZIP63 mutants in dark-induced senescence . 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 . ( A ) Scheme of the workflow to determine the green leaf area . A detailed description and the ImageJ macro can be found in the methods section and in Figure 1—source data 1 . ( B ) Total chlorophyll ( chl ) content in µg/mg freshweight ( FW ) of plants before and after 9 days in darkness . Bars represent the mean ± SD of six biological replicates . ( C ) Representative leaf series of plants before dark treatment . ( D ) Barplot of the total green leaf area of the rosette before darkness . Values are the mean ± SD of four biological replicates . ( E ) Dark-induced senescence timecourse . ( F ) Dotplot of the green leaf area of individual leaves after 9 days in darkness . p-values from T-tests between mutants wt < 0 . 05 , < 0 . 01 , and < 0 . 001 are indicated by * , ** and *** , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 00610 . 7554/eLife . 05828 . 007Figure 1—figure supplement 3 . Expression of senescence marker genes during prolonged darkness . Chlorophyll content ( A ) and expression of senescence marker genes ( B ) in 4 . 5 week-old soil-grown wt and bzip63 plants after 0–4 days of darkness . Bars represent the mean ± SD of three biological replicates . Letters indicate significant differences as determined by ANOVA and pairwise T-testing ( p < 0 . 05 ) . ( A ) Total rosettes were harvested at the indicated time points and the chlorophyll content in µg/mg FW was determined . ( B ) From the same plants , expression of the photosynthetic marker gene CAB2 ( CHLOROPHYLL A/B-BINDING PROTEIN 2 ) and three senescence marker genes ( Dietrich et al . , 2011 ) was determined by RT-qPCR . The expression is shown relative to wt at day 0 ( before dark incubation ) . SEN1 ( SENESCENCE 1 ) and SAG103 ( SENESCENCE-ASSOCIATED GENE 103 ) are known to be strongly induced by dark-induced senescence , while YLS3 ( YELLOW-LEAF-SPECIFIC 3 ) is induced by natural , but not by dark-induced senescence ( Oh et al . , 1996; Yoshida et al . , 2001; van der Graaff et al . , 2006 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 00710 . 7554/eLife . 05828 . 008Figure 1—figure supplement 4 . Effect of sugar on bZIP63 mutants in light . Controls for the sugar rescue of the dark-induced senescence phenotype shown in Figure 1C , D . Seedlings were grown for 12 days on ½ MS agar containing 0 . 5% sucrose , transferred to ½ MS agar containing 0% or 2% glucose , and grown for another 6 days in a 12 hr light/12 hr dark cycle . ( A ) Representative seedlings . ( B ) Box-and-whiskers plot of the chlorophyll content of eight seedlings . T-tests revealed no significant differences between chlorophyll content in wt and mutants . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 008 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 ) . 10 . 7554/eLife . 05828 . 009Figure 2 . 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 . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 00910 . 7554/eLife . 05828 . 010Figure 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 . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 01010 . 7554/eLife . 05828 . 011Figure 2—figure supplement 1 . Metabolic changes in bZIP63 ko and ox plants . Table of the metabolite levels in bzip63 and ox#3 compared to their respective wt shown in Figure 2 . Values are the log-2 transformed 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 . The color gradient indicates increased ( red ) or decreased ( blue ) metabolite levels in the mutant . For relative changes between mutant and wt including the SD and p-values from T-tests see Figure 2—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 011 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 . 10 . 7554/eLife . 05828 . 012Figure 3 . 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 . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 01210 . 7554/eLife . 05828 . 013Figure 3—figure supplement 1 . Comparison of Phos-tag western blots from different figures . For better comparison of Phos-tag western blots ( αGFP ) shown in different figures of this manuscript , all Phos-tag western blots were aligned here . In case more than one image of the same lines and conditions exists , only one is shown . The bands were labeled with numbers from 1 to 8 , where 1 is the presumably non-phosphorylated state and 8 is the highest phosphorylated form . The two semicircles to the right of each blot indicate the presence of a band at this position in each of the two lanes , with the shade corresponding to the intensity of the band ( light grey for weak bands , dark for strong bands ) . The figures and figures supplements ( S ) in which the blots are shown are given below . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 01310 . 7554/eLife . 05828 . 014Figure 3—figure supplement 2 . Overview over identified phospho-peptides of bZIP63 . ( A ) Sample overview , summarizing for each of the five independent experiments: plant material , growth conditions , the number of samples digested with each proteolytic enzyme , and the identified phospho-sites ( Y ) . Experiments 1 to 3 were measured with a linear ion trap quadrupole ( LTQ ) , experiments 4 and 5 with an LTQ-Oribtrap ( OT ) . ( B ) Phospho-peptide identification frequencies for the proteolytic enzymes . The barplot shows , for each proteolytic enzyme , the number of samples in which an in vivo phosphorylation-site was covered ( black or red ) or not covered ( grey ) , and how often a phospho-peptide was identified ( red ) . ( C ) Graph showing the total protein coverage from all experiments , as well as the protein coverage that was achieved with each of the four proteolytic enzymes in % and as a sequence . Parts of the sequence that were covered and not covered are shown in black and light grey , respectively . Identified phospho-serines are red . Below the coverage , all identified phospho-peptides are listed and the instrument used for identification is specified ( LTQ or OT ) . The numbers on top indicate the position in the protein sequence . The scheme below indicates the position of the conserved bZIP domain ( green ) , including the basic domain ( dark green ) , and the N- and C-terminal extensions ( yellow ) . L , light; EN , extended night; D , dark; suc , sucrose; T , trypsin; C , chymotrypsin; L , LysC; S , subtilisin; NLS , nuclear localization signal . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 014 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 spots on the 2D gel ( Figure 3B , labelled as band 6 and band 7 , respectively ) . Under extended night conditions , an additional band appeared ( labelled as band 8 ) , indicating increased phosphorylation of bZIP63 . Notably , the ratio of the most phosphorylated form of bZIP63 ( band 8 ) to the two less phosphorylated forms ( band 6 and 7 ) was different between the ox ( ox#3 ) and a genomic complementation line of bzip63 ( GY11 , detailed description follows later ) . In the genomic complementation line , the most phosphorylated form of bZIP63 was the strongest band under extended night conditions , which might be a result of the different bZIP63 amounts in relation to the endogenous kinase ( s ) , and likely better reflects the phosphorylation state of the endogenous protein . Interestingly , in seedlings grown in liquid culture , the extended night-triggered phosphorylation of bZIP63 could be abolished by the addition of 1% of sucrose ( Figure 3B ) . In fact , phosphorylation was even lower here than in the light . These data indicate that bZIP63 has a basal level of phosphorylation in the plant and undergoes hyper-phosphorylation under starvation conditions . In contrast , addition of external sugars leads to a reduced phosphorylation status of bZIP63 . In order to identify the in vivo phosphorylated residues , bZIP63-GFP was immunoprecipitated from total leaf extracts using bead-coupled anti-GFP antibodies , subjected to proteolytic digest and analyzed by liquid chromatography coupled to tandem mass spectrometry ( LC-MS/MS ) ( Figure 3C ) . To achieve maximum sequence coverage of the protein we combined proteolytic digests from four proteolytic enzymes ( trypsin , chymotrypsin , LysC , and subtilisin ) . This approach resulted in a total sequence coverage of 93 . 6% and the identification of several phospho-peptides , indicating that bZIP63 is phosphorylated at up to seven serines ( S29 , S59 , S102 , S160 , S261 , S294 , and S300 ) in vivo ( Figure 3C; Figure 3—figure supplement 2 ) . Two of these sites—S29 and S300—were also found in a recent phospho-proteomics study ( Umezawa et al . , 2013 ) . Notably , only three of the seven sites—S29 , S294 , and S300—were found by tryptic protein digest , underpinning the advantage of alternative proteolytic digests for phospho-peptide identification in targeted proteomics . To identify potential upstream kinases of bZIP63 , we performed in-gel kinase assays with plant protein extracts from roots of hydroponically grown Arabidopsis plants or root cell culture using recombinantly expressed bZIP63 as substrate . Root material was chosen because in leaf extracts the large amount of Ribulose-1 , 5-bisphosphate carboxylase/oxygenase ( RuBisCO ) interfered with some of the kinase signals and poses a considerable problem during MS-based protein identification . Three strong bands of about 40 , 50 , and 55 kDa , respectively , were visible on the autoradiogram ( Figure 4A ) , indicating that at least three kinases can phosphorylate bZIP63 in vitro . These bands were not visible on a gel without substrate , excluding the possibility that they originate from kinase auto-phosphorylation ( Figure 4—figure supplement 1 ) . To reduce the sample complexity and to enrich low abundant bZIP63-binding proteins before kinase identification , we affinity purified plant protein extracts on immobilized bZIP63 . The eluted fractions were tested in an in-gel kinase assay for kinase activity towards bZIP63 and loaded on a normal SDS-PAGE gel without substrate for kinase identification . Bands corresponding in molecular weight ( MW ) to the signal from the in-gel kinase assay were excised from the gel , digested with trypsin and analyzed by LC-MS/MS ( Figure 4B , C ) . In total , 27 protein kinases and kinase complex subunits were identified in four independent experiments ( Figure 4—source data 1; Figure 4—source data 2 ) . From those , proteins which did not match the expected MW or for which it had been shown experimentally that they are not localized in the nucleus were excluded . Only proteins which were identified in more than one sample with at least one proteotypic peptide were considered to be high confidence candidates , resulting in six protein kinases and two kinase regulatory subunits ( Figure 4C; Figure 4—source data 1; Figure 4—source data 2 ) . We identified the two main catalytic subunits of SnRK1 , AKIN10 and AKIN11 , as well as the regulatory subunit SNF4 . Several members of the calcium dependent protein kinase ( CDPK ) family were found , but only CPK3 was identified with high confidence . In addition , two catalytic ( CKA1 and CKA2 ) and one regulatory subunit ( CKB1 ) of casein kinase II ( CKII ) were found , as well as Casein kinase 1-like protein 2 ( CKL2 ) . The SnRK1 kinases , CDPKs , and CKL2 , correspond in MW to the two upper bands , while the lower band corresponds to the CKII kinase subunits . As SnRK1 was previously 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 , 2008 ) —which could explain the observed hyper-phosphorylation of bZIP63 in extended night—we focused our further analysis on AKIN10 and AKIN11 . 10 . 7554/eLife . 05828 . 015Figure 4 . Several different kinases can phosphorylate bZIP63 . ( A ) In-gel kinase assay with a root protein extract from hydroponically grown wild type plants and bZIP63 as substrate . Arrows indicate the positions of potential bZIP63 kinases . ( B ) Scheme of the kinase identification process . ( C ) In-gel kinase assay with samples from affinity purification of a root protein extract with bZIP63 and bZIP63 as a substrate ( left ) and a list of catalytic and regulatory ( * ) kinase subunits identified with high confidence ( right ) . The list also contains the gene identifier ( AGI ) , molecular weight ( MW ) , and number of samples in which the protein was found . For controls and a list of all identified kinases and kinase peptides see Figure 4—figure supplement 1 and Figure 4—source data 1 , 2 . CBB , Coomassie brilliant blue . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 01510 . 7554/eLife . 05828 . 016Figure 4—source data 1 . Overview over the kinases identified by LC-MS/MS after affinity purification with bZIP63 . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 01610 . 7554/eLife . 05828 . 017Figure 4—source data 2 . Excel table containing a detailed overview over the identified kinases and analyzed samples as well as all peptides found for the kinase subunit . The tab ‘kinase overview’ contains a table showing in which samples each kinase subunit was identified , including the number of peptides , proteotypic peptides , MASCOT ( Matrix Science ) score , and expected molecular weight . The other tabs list all peptides found for the kinase subunits in each sample and state whether the peptide is proteotypic ( Y ) or not . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 01710 . 7554/eLife . 05828 . 018Figure 4—figure supplement 1 . Auto-phosphorylation from the protein extracts is negligible in in-gel kinase assays . In-gel kinase assay with samples from affinity purification of a root protein extract from hydroponically grown plants with bZIP63 . Comparison of a gel with bZIP63 as a substrate ( top , see also Figure 4C ) and a gel without substrate ( middle ) . A Coomassie brilliant blue ( CBB ) stained gel is shown at the bottom . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 018 To confirm that AKIN10 and AKIN11 phosphorylate bZIP63 , we first performed an in-gel kinase assay with protein extracts of wt and akin10 seedlings in the presence of EGTA , to reduce the signal from CDPKs of the same MW ( Figure 5A ) . In both root and leaf extracts of akin10 plants one band at the expected MW of AKIN10 nearly disappeared ( see Figure 5—figure supplements 1–3 for characterization of the akin10 line ) . As AKIN11 has approximately the same MW as AKIN10 , the remaining signal likely originates from AKIN11 . In vitro kinase assays , with equal amounts of both kinases , showed that both kinases can phosphorylate bZIP63 . However , AKIN10 phosphorylates bZIP63 much stronger than AKIN11 does ( Figure 5B ) . Addition of the SnRK1 upstream kinase SnAK2 increased the activity of AKIN10 and AKIN11 about sevenfold ( see also Crozet et al . , 2010 ) , but had no effect on the ratio between the signal intensities ( Figure 5—figure supplement 4 ) . In vivo interaction assays with the identified SnRK1 complex subunits supported the findings of the kinase assays . In both yeast two-hybrid ( Y2H ) ( Figure 5C ) and bimolecular fluorescence complementation ( BiFC ) assays ( Figure 5D ) AKIN10 and the regulatory subunit SNF4 interacted strongly with bZIP63 , to a comparable level as measured for the bZIP63 homo-dimer , which was used as positive control ( Walter et al . , 2004 ) . In contrast , AKIN11 and the two regulatory subunits AKINβ1 and AKINβ2 showed almost no interaction with bZIP63 ( Figure 5C , D; Figure 5—figure supplement 5 ) . However , it has to be considered that AKIN10 and AKIN11 are part of a trimeric complex including SNF4 ( Emanuelle et al . , 2015 ) , which is neglected in these two assays . It is therefore still possible that AKIN11 interacts indirectly with bZIP63 via the regulatory subunit of the SnRK1 complex and plays a minor role in bZIP63 phosphorylation in the plant . 10 . 7554/eLife . 05828 . 019Figure 5 . The SnRK1 kinase AKIN10 phosphorylates bZIP63 and interacts with bZIP63 in vivo . ( A ) In-gel kinase assay with protein extracts from wt and akin10 plants and bZIP63 as a substrate ( top ) , western blot against AKIN10 ( αAKIN10 , middle ) , and Coomassie brilliant blue stain ( CBB , bottom ) . Asterisks mark the position of AKIN10 . For characterization of the akin10 line see Figure 5—figure supplements 1–3 . ( B ) In vitro kinase assay with recombinant AKIN10/AKIN11 and bZIP63 as a substrate . See also Figure 5—figure supplement 4 for kinase assays including the SnRK1 upstream kinase SnAK2 . ( C ) and ( D ) Interaction of SnRK1 subunits with bZIP63 . Homo-dimerization of bZIP63 was used as a positive control . ( C ) Yeast two-hybrid ( Y2H ) assay with auto-activation in grey and interaction with bZIP63 in blue . Bars represent means ± SD of eight biological replicates . For Y2H with AKINβ1 and β2 see Figure 5—figure supplement 5 . ( D ) Laser scanning microscopy images of bimolecular fluorescence complementation ( BiFC ) in transiently transformed Nicotiana tabacum leaves ( top ) . Arrowheads indicate the position of the nucleus . Size bar = 20 µm . Expression of the fusion proteins was verified by western blots ( αHA , αFlag , bottom ) . ( E ) Phos-tag gel western blots showing the in vivo phosphorylation state of bZIP63 in 4–5 week-old soil grown plants after 6 hr light ( L ) or extended night ( EN ) in the presence and absence of AKIN10 alone or both AKIN10 and AKIN11 . Plants overexpressing bZIP63-YFP in the akin10 line were compared to plants overexpressing bZIP63-GFP in the wt background ( ox#3 ) ( left ) . Additionally , AKIN10 and AKIN11 were knocked down ( akin10/11 ) by virus-induced gene silencing ( VIGS ) in plants expressing genomic bZIP63 with a YFP-tag ( GY9 line ) . See Figure 5—figure supplement 7 for images and a western blots of the VIGS plants . 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 ) . A comparison of the phosphorylation state of bZIP63 in seedlings can be found in Figure 5—figure supplement 6 . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 01910 . 7554/eLife . 05828 . 020Figure 5—figure supplement 1 . Molecular characterization of the akin10 line . ( A ) Scheme of the genomic locus and protein of AKIN10 ( top ) and IMS2 ( bottom ) . For the genomic locus , UTRs and exons are shown in grey and green , respectively . For AKIN10 an alternative first exon for splicing form 2 ( sf2 ) is shown in light green . The confirmed ( AKIN10 ) and annotated but not detected ( ISM2 ) T-DNA insertion sites are indicated , as well as the binding sites of primers used for PCRs in ( B–D ) . For the AKIN10 protein , the positions of the kinase catalytic domain ( KD ) , the ubiquitin-associated domain ( UBA ) , the kinase-associated domain ( KA ) , as well as the binding sites for antibodies against AKIN10 and AMPK-pT172 used in ( E ) are shown . The AMPK-pT172 antibody recognizes the phosphorylated T in the T-loop of both AKIN10 and AKIN11 . The position of K48 , which was mutated in the inactive AKIN10 version used in this manuscript , is shown as well . ( B ) PCR on genomic DNA from wt and akin10 plants to confirm the T-DNA insertion in AKIN10 and homozygosity of the plant lines . ( C ) PCR on genomic DNA from wt and akin10 plants did not confirm the second T-DNA insertion in the akin10 line in the IMS2 gene . ( D ) Expression of AKIN10 and AKIN11 in rosette leaves of 5 week-old wt and akin10 plants after 6 hr of light ( L ) and extended night ( EN ) as determined by RT-qPCR . Bars represent the means ± SD of four biological replicates and are normalized to wt in L . Letters indicate significant differences as determined by ANOVA and pairwise T-testing ( p < 0 . 05 ) . ( E ) Western blots detecting AKIN10 ( αAKIN10 ) and AKIN10 and 11 phosphorylated in the T-loop ( αAMPK-pT172 ) in wt and akin10 plants . Proteins were extracted from mature soil-grown plants after 6 hr of light or extended night ( left ) or from 2 week-old seedlings treated with 6 hr of extended darkness in the presence ( + ) or absence ( − ) of 1% sucrose ( suc; right ) . CBB , Coomassie Brilliant Blue . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 02010 . 7554/eLife . 05828 . 021Figure 5—figure supplement 2 . Phenotype and gene expression of selected AKIN10 target genes in the akin10 line . ( A ) No obvious difference in growth could be observed between wt and akin10 plants grown for 7 weeks under short day conditions . The number of leaves , fresh weight ( FW ) , dry weight ( DW ) and the water content ( FW/DW ) were determined . Bars represent the means ± SD of 20 biological replicates . ( B ) Mild flowering phenotype of akin10 . Wt and akin10 plants were grown under short day conditions and the number of leaves at time of bolting was determined . Bars represent the means ± SD of 14 ( wt ) and 11 ( akin10 ) biological replicates . ( C ) Expression of selected AKIN10 target genes ( Baena-Gonzalez et al . , 2007 ) involved in amino acid metabolism ( ASN1 , ProDH , BCAT2 ( BRANCHED-CHAIN AMINO ACID TRANSAMINASE 2 ) , AA-TP family protein ( amino acid transporter family protein ) ) and sugar metabolism ( DIN10 ) after 6 hr of light ( L ) and extended night ( E ) as determined by RT-qPCR . Bars represent the means ± SD of three biological replicates . p-values from T-tests between mutants and wt < 0 . 05 , < 0 . 01 , and < 0 . 001 are indicated by * , ** and *** , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 02110 . 7554/eLife . 05828 . 022Figure 5—figure supplement 3 . Expression of bZIPs in the akin10 line . Expression of bZIP63 , bZIP1 , and bZIP11 in rosette leaves of 5 week-old wt and akin10 plants after 6 hr of light ( L ) and extended night ( EN ) as determined by RT-qPCR . Bars represent the means ± SD of four biological replicates and are normalized to wt in L . P-values from T-tests between mutants and wt < 0 . 05 , < 0 . 01 , and < 0 . 001 are indicated by * , ** and *** , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 02210 . 7554/eLife . 05828 . 023Figure 5—figure supplement 4 . SnAK2 increases the kinase activity of AKIN10 and AKIN11 but does not phosphorylate bZIP63 . ( A ) In vitro kinase assay with recombinant AKIN10 or AKIN11 and bZIP63 as a substrate in the presence of SnAK2 . ( B ) Phos-tag gel western blot of an in vitro kinase assay with inactive AKIN10 ( AKIN10 K/M ) , active AKIN10 , or AKIN11 and bZIP63 as a substrate in the presence of SnAK2 . An antibody recognizing the C-terminus of bZIP63 ( αbZIP63 ) was used ( see Figure 6—figure supplement 1 ) . ( C ) In vitro kinase assay with AKIN10 and/or SnAK2 and bZIP63 as a substrate showing the activity of AKIN10 in the presence or absence of SnAK2 . The signal intensity from the bZIP63 phosphorylation is given in % of the strongest signal . CBB , Coomassie brilliant blue . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 02310 . 7554/eLife . 05828 . 024Figure 5—figure supplement 5 . AKINβ1 and AKINβ2 do not interact with bZIP63 . Yeast two-hybrid ( Y2H ) assay showing of AKINβ1 and AKINβ2 with bZIP63 . Homo-dimerization of bZIP63 was used as a positive control . Auto-activation of BD-fusion proteins is shown in grey , interaction with bZIP63 in blue . Bars represent means ± SD of eight biological replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 02410 . 7554/eLife . 05828 . 025Figure 5—figure supplement 6 . Altered sugar-dependent in vivo phosphorylation of bZIP63 in seedlings . Phos-tag gel western ( αGFP ) blots showing the in vivo phosphorylation state of bZIP63 in plants overexpressing bZIP63-GFP/YFP in the wt or akin10 background . Proteins were extracted from seedling cultures after 6 hr extended night in the presence ( + ) or absence ( − ) of 1% sucrose . 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 ) . 8 indicates the hyper-phosphorylated form of bZIP63 . CBB , Coomassie brilliant blue . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 02510 . 7554/eLife . 05828 . 026Figure 5—figure supplement 7 . AKIN10/AKIN11 VIGS plants . AKIN10 and AKIN11 were knocked down in plants expressing a genomic fragment of bZIP63 with a c-terminal YFP tag ( GY9 line ) using VIGS . 2 week-old plants were infiltrated with the VIGS construct ( akin10/11 , right ) or not ( control , left ) and 2 weeks later plants showed a clear phenotype and a strong reduction in AKIN10 and AKIN11 protein amount . ( A ) Pictures of representative plants showing stunted growth of akin10/11 plants , as well as increased anthocyanin accumulation , leaf wilting and early senescence . ( B ) Western blot of control and akin10/11 leaf extracts with an antibody against the phosphorylated T-loop of AMPK ( αAMPK-pT172 ) , recognizing both AKIN10 and AKIN11 . Samples marked with an asterisk were used for the Phos-tag gels shown in Figure 5E . CBB , Coomassie brilliant blue . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 026 To verify that SnRK1 plays an important role in the in the in vivo phosphorylation of bZIP63 we compared the phosphorylation state of bZIP63 in plants overexpressing bZIP63-GFP or -YFP in the wt and akin10 background , respectively . As SnRK1 has been suggested to act as a major regulator in the energy deprivation response , we again compared leaf protein extracts after 6 hr light and extended night and found that the hyper-phosphorylated form ( band 8 ) of bZIP63 , observed in the wt in extended night , was much weaker in the akin10 background ( Figure 5E ) . The same effect was observed in seedling cultures after 6 hr of extended night ( Figure 5—figure supplement 6 ) . To see whether the weak phosphorylation remaining in the akin10 mutant would be further reduced in an akin10/11 double mutant , we employed virus-induced gene silencing ( VIGS ) to knock down AKIN10 and AKIN11 in a genomic bzip63 complementation line ( GY9 ) . Resulting plants showed strongly reduced growth and accumulation of anthocyanins ( Figure 5—figure supplement 7 ) , as previously described in Baena-González et al . ( 2007 ) . Like in the akin10 line , there was no difference in bZIP63 phosphorylation between akin10/11 and control plants in light , but the hyper-phosphorylated form of bZIP63 ( band 8 ) in the extended night was now almost completely gone ( Figure 5E ) . This shows clearly , that AKIN10 and—to a lower extent—AKIN11 are the major kinases responsible for bZIP63 hyper-phosphorylation under starvation conditions . Additional evidence for in vivo phosphorylation of bZIP63 by SnRK1 emerges from a recent phosphoproteomics study in which one of the in vivo phosphorylation sites in bZIP63 ( S300 ) was found to be more abundant in an AKIN10 ox line and less abundant in the ko line after extended night treatment , respectively ( Nukarinen et al . , unpublished ) . Next , to elucidate which of the seven in vivo phosphorylation sites can be phosphorylated by AKIN10 , we performed in vitro kinase assays using the wt version of bZIP63 and different serine to alanine ( S/A ) mutants as substrates ( Figure 6A; Figure 6—figure supplements 1 , 2 ) . Differences in phosphorylation were observed for proteins with mutations in S29 , S294 , and S300 . In detail , the signal from S29A was strongly decreased in full length bZIP63 and completely gone in the N-terminal peptides that appeared as lower MW degradation products in these assays . The S300A mutation led to an even stronger decrease of the signal , comparable to the S294/300A double mutant . Even though the S294A single mutant showed a similar signal as the wt , all three serines had to be mutated to completely abolish phosphorylation , suggesting that all of them are in vitro targets for AKIN10 , with S294 being the weakest . Importantly , all three sites match the SnRK1 consensus sequence ( Huang and Huber , 2001 ) ( Figure 6B ) . 10 . 7554/eLife . 05828 . 027Figure 6 . AKIN10 targets three highly conserved and functionally important serine residues in bZIP63 . ( A ) In vitro kinase assay of wt and S/A mutants of GST-tagged bZIP63 with AKIN10 . Positions of full length ( FL ) and N-terminal fragments of bZIP63 are marked by black arrows . The scheme on the right shows the position of the in vivo phosphorylation sites and the in vitro target sites of AKIN10 ( red asterisk ) on bZIP63 . See Figure 6—figure supplements 1 , 2 for controls and Phos-tag gel of kinase assays , respectively . ( B ) Conservation of phosphorylation sites in bZIP63 . Sequences of bZIP63 homologues from eight species were aligned with ClustalΩ . The scheme on top indicates the positions of the in vivo phosphorylation and AKIN10 target sites on bZIP63 . The histogram below shows the sequence identity ( red/black ) and similarity ( grey ) to A . thaliana bZIP63 . Red bars represent the in vivo phosphorylation sites . Below , the alignment of the sequence surrounding the AKIN10 target sites and the SnRK1 consensus motif ( Huang and Huber , 2001 ) are shown . The grey/black shading indicates the degree of conservation , phosphorylation sites are in red . For alignment of non-AKIN10 target sites and full sequence alignment see Figure 6—figure supplement 3 , for sequences in fasta format see Figure 6—source data 1 . ( C ) Promoter activation assays in protoplasts with an ASN1/ProDH promoter-driven GUS reporter . Activation by bZIP63 wt and S/A mutants without ( grey ) or with ( blue ) co-transformation of AKIN10 is shown . Bars are means ± SD of 4 biological replicates , given in % of the activity of wt bZIP63 with AKIN10 . Horizontal dashed lines indicate the signal in the control without bZIP63 . Letters indicate significant differences as determined by ANOVA and pairwise T-testing ( p < 0 . 05 ) . See Figure 6—figure supplement 4 for a western blot control . CBB , Coomassie brilliant blue . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 02710 . 7554/eLife . 05828 . 028Figure 6—source data 1 . Sequences of the bZIP63 homologues . Text file containing the sequences of the bZIP63 homologes used for ClustalΩ alignment in Figure 5B and Figure 5—figure supplement 3 in fasta format . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 02810 . 7554/eLife . 05828 . 029Figure 6—figure supplement 1 . AKIN10 phosphorylates bZIP63 but not GST . ( A ) In vitro kinase assay with recombinant AKIN10 and GST-tagged bZIP63 or GST as a substrate . Positions of full length ( FL ) and N-terminal fragments of bZIP63 are marked by black arrows . The scheme on the right shows the position of the in vivo phosphorylation sites and the in vitro target sites of AKIN10 ( red asterisk ) in GST-bZIP63 , as well as the approximate length of the N-terminal fragments . ( B ) Western blot ( αbZIP63-N and -C ) of GST-tagged bZIP63 , used as substrate for the kinase assays . The scheme on the right indicates the binding sites of the two antibodies on bZIP63 and the approximate length of the detected fragments . CBB , Coomassie brilliant blue . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 02910 . 7554/eLife . 05828 . 030Figure 6—figure supplement 2 . AKIN10 phosphorylates S29 , S294 , and S300 on bZIP63 . Phos-tag gel western blot ( αbZIP63-C ) of an in vitro kinase assay with active ( wt ) and inactive ( K/M ) AKIN10 and wt and S/A mutants of GST-tagged bZIP63 as substrate . The scheme on the bottom shows which of the three AKIN10 target sites can be phosphorylated ( P ) or are mutated ( X ) . The scheme on the right indicates the likely phosphorylated sites for each band . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 03010 . 7554/eLife . 05828 . 031Figure 6—figure supplement 3 . The AKIN10 target sites and S160 in the bZIP domain are highly conserved . ( A ) and ( B ) Sequence conservation of bZIP63 . Sequences of bZIP63 homologues from eight species were aligned with ClustalΩ . ( A ) Conservation of non-AKIN10 target sites . The scheme on top indicates the positions of the in vivo phosphorylation and AKIN10 target sites on bZIP63 . The histogram below shows the sequence identity ( red/black ) and similarity ( grey ) to A . thaliana bZIP63 in each position . Red bars represent the in vivo phosphorylation sites . Below , an alignment of the sequences surrounding the phosphorylation sites not targeted by AKIN10 is depicted . The grey/black shading indicates the degree of conservation , phosphorylation sites are in red . ( B ) Full sequence alignment . Species names are abbreviated . The grey/black shading indicates the degree of conservation . Red arrows indicate the positions of the in vivo phosphorylation sites . Numbers on top indicate the position in the alignment , numbers on the right indicate the position in each sequence . A consensus sequence is given below the alignment . The sequence identity matrix is at the bottom right . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 03110 . 7554/eLife . 05828 . 032Figure 6—figure supplement 4 . Expression of bZIP63 and AKIN10 in the promoter activation assays . Exemplary western blot ( αHA ) of protoplasts co-transformed with AKIN10 and wt or S/A mutants of bZIP63 . CBB , Coomassie brilliant blue . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 032 A comparison of Arabidopsis bZIP63 with orthologs from eight other plant species , ranging from mosses to higher plants , showed that the three putative AKIN10 target sites are highly conserved throughout evolution ( Figure 6B; Figure 6—figure supplement 3 and Figure 6—source data 1 ) . As the origin of SnRK1 dates back even further , to the common ancestor of plants and animals ( Bayer et al . , 2014 ) , it is likely that phosphorylation of bZIP63 by AKIN10 poses an ancient and important regulatory mechanism . We therefore set out to test the functional relevance of AKIN10-mediated phosphorylation of bZIP63 . To this end , we tested the transcriptional activity of bZIP63 in protoplast-based promoter activation assays using the ASN1 or ProDH promoter fused to the beta galactosidase ( GUS ) reporter ( Figure 6C; Figure 6—figure supplement 4 ) . In both cases , co-transformation of wt bZIP63 and AKIN10 strongly induced reporter gene expression , while transformation of bZIP63 alone was not sufficient for significant induction . Transformation of AKIN10 alone also led to a weak induction of the reporters , which could be explained by the action of endogenous bZIPs . Mutation of S29 or all three AKIN10 target sites on bZIP63 to alanine reduced the reporter activation almost to background level . In contrast , mutation of the two C-terminal serines , S294 and S300 , had only a weak negative effect on ASN1 and no significant effect on ProDH activation . Taken together , our data suggest that AKIN10 phosphorylates bZIP63 at up to three conserved sites , namely S29 , S294 , and S300 and phosphorylation by AKIN10 , especially at S29 , is crucial for bZIP63 TF activity . To determine the impact of bZIP63 phosphorylation in planta , we transformed the bzip63 mutant with genomic constructs of bZIP63 containing either the wt sequence ( GY lines ) , or a S/A mutation of S29 , S294 , and S300 , respectively ( GAY lines ) with a C-terminal YFP tag ( Figure 7A; Figure 7—figure supplement 1A–C ) . The wt construct showed strong phosphorylation in the light ( bands 6 and 7 ) and an even stronger phosphorylation in extended night ( band 8 ) , as well as reduced phosphorylation in the presence of sucrose ( bands 1 , 2 , and 4 ) ( Figure 7B; Figure 7—figure supplement 1D ) . In contrast , the S/A construct showed only weak phosphorylation ( only bands 1 and 3 visible ) and most importantly no difference between all tested conditions . This indicates that S29 , S294 and S300 are the major in vivo phosphorylation sites on bZIP63 , which are also responsible for the observed condition-dependent shift in bZIP63 phosphorylation . 10 . 7554/eLife . 05828 . 033Figure 7 . The bzip63 phenotype can be complemented by wt bZIP63 , but not by bZIP63 harboring S/A mutations of the AKIN10 target sites . ( A ) Genomic complementation constructs . Exons are green , introns black . See Figure 7—figure supplement 1 for characterization of the complementation lines . ( B ) Phos-tag gel western blots ( αGFP ) showing the in vivo phosphorylation state of bZIP63 in the complementation lines after 6 hr light ( L ) or extended night ( EN ) in 5 week-old soil-grown plants . 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 ) . ( C ) and ( D ) Dark-induced senescence phenotype of 4 . 5 week-old soil-grown plants after 9 days in darkness . ( C ) Leaves 3–7 of one representative plant per line . ( D ) Box-and-whiskers plot of the total green leaf area of eight biological replicates . Letters indicate significant differences as determined by ANOVA and pairwise T-testing ( p < 0 . 05 ) . See Figure 7—figure supplement 2 for untreated plants and green leaf area of individual leaves . ( E ) and ( F ) Metabolite profile . ( E ) Hierarchical clustering of log-2 fold changes of metabolite concentrations compared to wt . Values are means of five biological replicates . ( F ) Principal component analysis ( PCA ) . PC1 is plotted against PC2 . The proportion of variance in % is indicated . The red line surrounds bzip63 and GAY samples , the green line wt , GY , and ox#3 samples . For relative metabolite concentrations and PCA loadings see Figure 7—source data 1 . ( G ) Relative expression of potential bZIP63 target genes in 5 week-old plants during early extended night as determined by RT-qPCR . Values are means ± SD of four biological replicates and are given as fold change compared to Ws-2 at 0 hr ( left ) or 4 hr ( right ) . p-values from T-tests between mutants and wt < 0 . 05 , < 0 . 01 , and < 0 . 001 are indicated by * , ** and *** , respectively . Letters indicate significant differences as determined by ANOVA and pairwise T-testing ( p < 0 . 05 ) . CBB , Coomassie brilliant blue . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 03310 . 7554/eLife . 05828 . 034Figure 7—source data 1 . Excel table containing the relative metabolite levels of the complementation lines and the PCA loadings . The tab ‘metabolites normalized to wt’ contains the relative metabolite levels , which were used to calculate the fold changes shown in Figure 7E , as well as p-values from statistical tests . Values are the mean ± SD of five biological replicates given as fold change to the corresponding wt . The tab ‘PCA—variance and loadings’ contains the SD , proportion of variance , and loadings of all principal components from the PCA analysis shown in Figure 7F . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 03410 . 7554/eLife . 05828 . 035Figure 7—figure supplement 1 . Characterization of the bzip63 complementation lines . ( A ) and ( B ) Expression of bZIP63 in different plant lines . ( A ) RT-qPCR of bZIP63 in 5 week-old plants after 6 hr of light ( L ) or extended night ( EN ) . Bars represent means ± SD of five biological replicates and are given as fold change to Ws-2 in L . The 6 hr L samples were also used for the metabolic profiling shown in Figure 7E , F and Figure 7—source data 1 . ( B ) Western blot ( αGFP ) detecting YFP-tagged bZIP63 in transgenic plant lines after 6 hr of L and EN . ( C ) Epifluorescence microscopy images of soil-grown seedlings of the GY9 and GAY14 lines showing expression of bZIP63-YFP . From top to bottom: leaf epidermis , leaf veins , roots , lateral root tips . Scale bar is 20 µm . ( D ) Phos-tag gel western blots ( αGFP ) showing the in vivo phosphorylation state of bZIP63 in the complementation lines . Proteins were extracted from seedling cultures after 6 hr extended night in the presence ( + ) or absence ( − ) of 1% sucrose . 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 ) . CBB , Coomassie brilliant blue . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 03510 . 7554/eLife . 05828 . 036Figure 7—figure supplement 2 . Complementation of the dark-induced senescence phenotype of bzip63 . ( A ) Representative leaf series of 4 . 5 week-old plants before and after 9 days in darkness . ( B ) Barplot of the total green leaf area of the rosette before darkness . Values are the mean ± SD of four biological replicates . ( C ) Dotplot of the green leaf area of individual leaves after 9 days in darkness . Values are the mean ± SD of eight biological replicates . The insertion at the right side shows the values from leaves 3–7 as a barplot ( framed by dotted line ) . Letters indicate significant differences as determined by ANOVA and pairwise T-testing ( p < 0 . 05 ) . The p-values of the F-test for each leaf are given . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 036 Next , we tested whether complementation of the observed bzip63 phenotypes depends on the bZIP63 phosphorylation status , using two independent GY and GAY lines . The dark-induced senescence phenotype of bzip63 was complemented in the GY lines , but not in the GAY lines ( Figure 7C , D; Figure 7—figure supplement 2 ) . After 9 days in darkness , the ko and GAY lines showed visibly less chlorosis and had a higher percentage of green leaf area as compared to the wt and GY lines . Metabolite profiling of leaves harvested after 6 hr of light also revealed marked differences between the GY and GAY lines ( Figure 7E , F; Figure 7—source data 1 ) . The metabolite profile of the GAY lines was similar to that of bzip63 plants . In contrast , in the GY lines the metabolic changes between mutant and wt were mostly weaker than in bzip63 or even resembled those observed for ox#3 ( Figure 7E ) . In a principal component analysis the GAY lines grouped together with bzip63 , while the GY lines were closer to the two wt lines and the ox ( Figure 7F ) . To test the effect of the S/A mutation on the expression of bZIP63 target genes , we performed RT-qPCR of ASN1 , DIN10 , and ProDH ( Figure 7G ) —three suggested AKIN10 target genes ( Baena-González et al . , 2007 ) . The expression of all three genes increased steadily during a 4 hr extended night treatment , but the increase was delayed in the bzip63 mutant as compared to the wt . At the 4 hr time point we could observe a clear difference between wt and ko . We therefore chose this time point to quantify ASN1 , DIN10 , and ProDH transcripts in one GY and GAY line . As expected , the GY line had the same or even more transcript than the wt for all three genes , while the GAY line had significantly lower expression levels , similar to bzip63 . In summary , expression of wt bZIP63 but not of the S/A mutant—which cannot be phosphorylated by AKIN10—in the bzip63 background led to complementation of the bzip63 phenotypes . Together , these experiments demonstrate that phosphorylation of bZIP63 at the AKIN10 target sites is essential for the function of bZIP63 in the plant . Our findings show that bZIP63 phosphorylation , at residues distant from the central bZIP domain , strongly regulates its activity . As we did not observe any changes in localization in the bZIP63 mutants and also no change in DNA-binding activity ( of the bZIP63 homodimer ) towards a C-box motif ( GACGTC ) as canonical bZIP target site ( data not shown ) , we suspected that the observed effect on transcription would be due to changes in dimerization preferences of bZIP63 . Therefore , we tested the effect of AKIN10-mediated phosphorylation on bZIP63 homo- and hetero-dimerization with bZIP1 and bZIP11 . Both bZIP1 and bZIP11 are metabolic regulators and mediate transcription of ASN1 and ProDH ( Hanson et al . , 2008; Dietrich et al . , 2011 ) . In protoplast two-hybrid ( P2H ) assays , the addition of exogenous AKIN10 resulted in a clear enhancement of dimerization in all cases—homo-dimerization as well as hetero-dimerization with bZIP1 and bZIP11 ( Figure 8A; Figure 8—figure supplement 1A ) . Note , that the very strong effect observed with bZIP11 in these assays is misleading because bZIP11 is a much stronger activator of transcription as compared to bZIP1 and 63 ( see also ‘Discussion’ ) . From that we concluded that the phosphorylation of bZIP63 by AKIN10 is required for dimerization and set out to test the effect of S/A mutations of the AKIN10 target sites on bZIP63 homo- and hetero-dimerization with bZIP11 where we had observed the strongest effect before ( Figure 8B; Figure 8—figure supplement 1B ) . In both cases , the signal was reduced to about 30–40% of the signal obtained from dimerization with wt bZIP63 when S29 or all three serines were mutated to alanine . Mutation of one or two of the C-terminal sites decreased bZIP63 homo-dimerization weakly but had no visible effect on bZIP63-11 dimerization , indicating that these sites play , at most , a minor role in regulation of dimer formation . 10 . 7554/eLife . 05828 . 037Figure 8 . AKIN10-mediated phosphorylation of bZIP63 promotes its dimerization . ( A ) and ( B ) Protoplast two-hybrid ( P2H ) assays . ( A ) Interaction of bZIP63 fused to the Gal4-AD ( activation domain ) with bZIP63 , bZIP1 , and bZIP11 fused to the Gal4-BD ( binding domain ) without and with co-transformation of AKIN10 . Bars represent the mean normalized GUS activity ± SD of 3–4 biological replicates . p-values from T-tests < 0 . 05 and < 0 . 01 are indicated by * and ** , respectively . ( B ) Interaction of AD-bZIP63 ( left ) or AD-bZIP11 ( right ) with wt and S/A mutants of BD-bZIP63 without and with co-transformation of AKIN10 . Values are given in % of the signal with wt bZIP63 and AKIN10 . Letters indicate significant differences as determined by ANOVA and pairwise T-testing ( p < 0 . 05 ) . For western blots for ( A ) and ( B ) see Figure 8—figure supplement 1 . ( C ) In vitro kinase assay of bZIP63 , 1 , 2 , 11 , 44 , and 53 with AKIN10 and the SnRK1 upstream kinase SnAK2 . For a kinase assay with the bZIPs and SnAK2 alone see Figure 8—figure supplement 2 . ( D ) . Phos-tag gel western blots ( αGFP ) showing the in vivo phosphorylation state of S29 in bZIP63 after 6 hr light ( L ) or extended night ( EN ) . 5 week-old soil-grown plants of two genomic bzip63 complementation lines harboring different S/A mutations were used . In line GAY14 ( left ) none of the AKIN10 target sites ( S29/294/300 ) can be phosphorylated , while in the S294/300A ( right ) line S29 can still be phosphorylated . 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 ) . The likely phosphorylation state of the bands in the western blot is shown on the right . X stands for one of the non-mutated serines ( 59 , 102 , 160 , or 261 ) . CBB , Coomassie brilliant blue . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 03710 . 7554/eLife . 05828 . 038Figure 8—figure supplement 1 . Western blots and controls for the protoplast two-hybrid ( P2H ) assays . ( A ) Western blot showing the expression of AD ( activation domain ) -bZIPs and AKIN10 ( αHA ) and BD ( DNA binding domain ) -bZIP63 ( αBD ) in the P2H assay in Figure 8A . ( B ) Exemplary western blot showing the expression of BD-bZIP63 in the P2H assays in Figure 8B . CBB , Coomassie brilliant blue . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 03810 . 7554/eLife . 05828 . 039Figure 8—figure supplement 2 . SnAK2 does not phosphorylate bZIP63 or the S1 class bZIPs . In vitro kinase assay of bZIP63 , 1 , 2 , 11 , 44 , and 53 with SnAK2 . CBB , Coomassie brilliant blue . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 039 To exclude the possibility that the observed effects on dimerization are due to phosphorylation of the hetero-dimerization partner rather than bZIP63 itself , we tested whether AKIN10 is able to phosphorylate any of the S1 group bZIPs . To increase the phosphorylation efficiency of AKIN10 we included SnAK2 in the reactions . In contrast to bZIP63 , none of the S1 group bZIPs were phosphorylated by AKIN10 ( Figure 8C; Figure 8—figure supplement 2 ) . Together , these data indicate that AKIN10-mediated phosphorylation of bZIP63—especially at S29—strongly enhances its ability to form homo- as well as hetero-dimers . To further substantiate the relevance of S29 phosphorylation in vivo , we compared the phosphorylation patterns of the S29/S294/S300A mutant with a S294/S300A mutant in the light and after extended night treatments in the genomic complementation lines ( Figure 8D ) . Compared to the triple mutant , that did not show any change in the phosphorylation pattern in response to starvation , bZIP63 phosphorylation was clearly increased in the S294/S300A double mutant in the extended night . As S29 is the only AKIN10 target site left in this version , it must indeed be S29 that is phosphorylated by AKIN10 under starvation conditions . As we saw that AKIN10-dependent phosphorylation promotes dimerization of bZIP63 with all tested partners we wanted to analyze whether phosphorylation has an effect on the dimerization partner preference . Because protoplast two-hybrid assays only allow to analyze the effect of AKIN10 on a single dimer we set up a multicolor BiFC approach ( Waadt et al . , 2008 ) in Nicotiana tabacum leaves with bZIP1 and 11 as alternative interaction partners for bZIP63 . For this , we expressed all three bZIPs ( 1 , 11 , and 63 ) from the same plasmid and co-transformed AKIN10 ( Figure 9A ) . bZIP11 was fused to CFPN , bZIP1 to VENUSN , and bZIP63 to CFPC . Accordingly , the bZIP63-11 hetero-dimer generates a blue ( cyan ) signal and the bZIP63-1 hetero-dimer a yellow signal , whereas the bZIP63 homo-dimer cannot be detected . To compare the dimerization preference of phosphorylated and non-phosphorylated bZIP63 we used wt and a S29/294/300A mutant of bZIP63 . Quantification of the ratio of the VENUS/CFP signal in more than 100 nuclei clearly showed a shift towards the VENUS emission when wt bZIP63 was compared to the S/A mutant . This demonstrates that phosphorylation by AKIN10 triggers the preferential formation of bZIP63-1 over bZIP63-11 dimers in a competitive in vivo assay . 10 . 7554/eLife . 05828 . 040Figure 9 . Phosphorylation of bZIP63 shifts its dimerization preferences . ( A ) Multi-color bimolecular fluorescence complementation ( BiFC ) in transiently transformed N . tabacum leaves to test the effect of bZIP63 phosphorylation on its dimerization preference . A cassette containing bZIP63 ( wt or S29/294/300A ) , bZIP11 , and bZIP1—tagged with the C-terminal moiety of CFP and the N-terminal moieties of CFP and VENUS , respectively—was co-transformed with mCherry-tagged AKIN10 . CFP ( bZIP63-11 ) and VENUS ( bZIP63-1 ) fluorescence was detected on a confocal laser scanning microscope and quantified for nuclei showing co-expression of AKIN10 . Top: scheme of the multi-color BiFC construct and principle . Bottom center: representative microscopy pictures . Size bar = 50 µm . Bottom right: box-and-whiskers plot of the VENUS/CFP ratio of 115–118 nuclei , normalized to the median of the S29/294/300A bZIP63 construct . T-test p-value was < 0 . 001 , as indicated by *** . ( B ) Model of the regulation of bZIP63 dimerization and activity by AKIN10 . Energy deprivation triggers activation of AKIN10 , which phosphorylates S29 on bZIP63 . This leads to increased formation of specific bZIP63 dimers and altered expression of dimer-specific genes . DOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 040
Here we show that bZIP63 plays an important role in the energy starvation response and metabolic regulation . This is in accordance with the sugar/energy-dependent expression of bZIP63 ( Matiolli et al . , 2011; Kunz et al . , 2014 ) , as well as with the fact that several of its proposed target genes are involved in the low-energy response and metabolism ( Matiolli et al . , 2011; Veerabagu et al . , 2014 ) . Furthermore , three members of the S1-group of plant bZIPs—hetero-dimerization partners of bZIP63 ( Ehlert et al . , 2006; Kang et al . , 2010 ) —have also been linked to energy starvation response and metabolism . Inducible bZIP11 ox lines exhibit a severe dwarf phenotype ( Hanson et al . , 2008 ) and a metabolic profile resembling that of carbon starved plants ( Ma et al . , 2011 ) , while overexpression of bZIP1 and bZIP53 results in enhanced dark-induced senescence and reduced levels of proline and branched-chain amino acids ( Dietrich et al . , 2011 ) . Similar to the bZIP1 ox , bZIP63 ox plants showed increased chlorosis after 9 days of darkness , while the bZIP63 ko displayed a clear stay-green phenotype under these conditions . In contrast , neither the single , nor the double ko of bZIP1 and 53 showed reduced dark-induced senescence ( Dietrich et al . , 2011 ) . This suggests that other bZIPs can take over the function of bZIP1 in starvation-induced leaf yellowing , while bZIP63 plays a more unique role . Looking at primary metabolism , we found that misregulation of bZIP63 expression has a strong effect , especially on amino acids , which was further enhanced under starvation conditions . In line with the finding that bZIP63 is a positive regulator of ProDH and ASN1 ( Matiolli et al . , 2011; Veerabagu et al . , 2014 , and this work: Figures 6C , 7G ) and the changes in proline levels in bZIP63 mutants reported by Veerabagu et al . ( 2014 ) , we measured the strongest differences in proline and the entire glutamate family as well as in aspartate and asparagine levels in the bZIP63 ko and ox . Notably , there is a strong correlation between the amino acid profiles of the bZIP63 and the bZIP1 ox lines ( Dietrich et al . , 2011 ) . A similar accumulation of amino acids during dark-induced senescence has frequently been observed and was attributed to enhanced protein degradation ( reviewed in Araújo et al . , 2011 ) . Particularly under low carbon conditions or during senescence , alternative energy sources need to be used in plant cells , and the important role of proline and branched-chain amino acids in this process has been highlighted in a number of studies ( reviewed in: Szabados and Savouré , 2010; Szal and Podgórska , 2012 ) . Thus also the altered amino acid levels could contribute to the observed dark-induced senescence phenotype of the bZIP63 mutants . We found that bZIP63 is highly phosphorylated in Arabidopsis . By applying different proteolytic digests we were able to identify seven in vivo phosphorylated serine residues , distributed all over the protein . While exogenous sucrose decreased the global phosphorylation level of bZIP63 , extended night treatment further increased its phosphorylation , thus supporting the idea that bZIP63 plays a role in energy signaling . Moreover , we found that the SnRK1 kinase AKIN10 , which was proposed to be a central regulator of transcription in starvation response ( Baena-González and Sheen , 2008 ) , is the major kinase responsible for the starvation-induced hyper-phosphorylation of bZIP63 . Therefore , bZIP63 presents the first TF acting as direct target of SnRK1 in the transcriptional energy deprivation response . In vitro , AKIN10 phosphorylated three highly conserved and functionally important residues in the N- and C-terminus of bZIP63—S29 , S294 , and S300 , respectively . Reporter activation assays in protoplasts revealed that these sites—especially S29—are essential for AKIN10-dependent induction of ASN1 and ProDH by bZIP63 . Remarkably , the phosphorylation patterns of different bZIP63 mutants on Phos-tag gels pointed towards S29 as the main target site for AKIN10 under extended night conditions . The importance of bZIP63 phosphorylation at the SnRK1 target sites for its in planta function was further underlined by complementation of the bzip63 mutant with genomic bZIP63 constructs . Wt , but not a S29/S294/S300A mutant of bZIP63 , could complement the metabolic and senescence phenotypes . Likewise , also the strong delay in extended night-triggered induction of ASN1 , DIN10 , and ProDH in bzip63 plants was complemented with wt bZIP63 , but not the ( triple ) S/A mutant . The relatively small difference in ProDH expression in plants , as compared to the protoplast assay , is probably due to redundancy within the C/S1 group bZIPs , as it was shown by Dietrich et al . ( 2011 ) , that only ko of multiple members of the C/S1 group leads to a strong reduction in ProDH and ASN1 expression . Since the discovery that AKIN10 can activate several members of the C/S1 network ( Baena-González et al . , 2007 ) , it has been speculated ( Baena-González and Sheen , 2008; Usadel et al . , 2008 ) , but never shown experimentally , that they are downstream targets of SnRK1 . Our data provide compelling evidence that bZIP63 , but none of the S1 group bZIPs , is a bona fide in vivo target of SnRK1 in low-energy signaling . Differential dimerization is a well-known mechanism for changing the target recognition site , and thereby the target genes of bZIP TFs ( Tsukada et al . , 2011 ) . For the S1-group bZIP1 is has been shown that dimerization with C-group bZIP10 or bZIP63 affects its in vitro binding to ACGT-based motifs differently ( Kang et al . , 2010 ) . The notion that different C/S1 dimers have different target genes is further supported by a recent transcriptomics study in protoplasts . Overexpression of four C/S1 group bZIPs ( bZIP1 , bZIP10 , bZIP11 , and bZIP63 ) , individually or in combination of two , revealed overlapping but distinct gene expression patterns ( Ma , 2012 ) . This means , that although they regulate a core set of common genes , such as ASN1 and ProDH ( Baena-González et al . , 2007; Dietrich et al . , 2011; Ma et al . , 2011 ) , different dimers also have distinct functions , and switching of dimerization partners can have a considerable impact on gene expression . Our data indicate that dimerization and activity of bZIP63 strongly depend on its phosphorylation status . In general , the dimerization of bZIP63 with bZIP1 , bZIP11 , and bZIP63 , was boosted by AKIN10-mediated phosphorylation . Like in the promoter activation assays , the main influence on dimerization came from S29 phosphorylation , while phosphorylation of S294 and S300 showed a mild effect at most . Incidentally , this enhanced dimerization with bZIP63 would explain why AKIN10 was found to activate the S1 group bZIPs ( bZIP1 , 2 , 11 , and 53 ) in protoplast assays ( Baena-González et al . , 2007 ) , although they do not present direct targets of AKIN10 . At first sight , the phosphorylation triggered boost of dimerization seemed to be strongest for hetero-dimerization with bZIP11 . However , it has to be considered that bZIP11 is a very strong activator of transcription as it harbors an activation domain ( Ehlert et al . , 2006 ) and was recently also shown to recruit the histone acetylation machinery ( Weiste and Droege-Laser , 2014 ) . The same is not the case for bZIP1 and bZIP63 . As the readout of the protoplast two-hybrid interaction assay is transcription-based it is therefore not possible to quantitatively compare the effect of phosphorylation on the formation of different dimers and to draw conclusions about the in vivo dimerization preference . Therefore we used a multi-color BiFC assay with bZIP1 and bZIP11 as alternative partners for bZIP63 to test the effect of bZIP63 phosphorylation by AKIN10 on its dimerization preference . The three bZIPs were co-expressed with AKIN10 in N . tabacum leaves and the formation of bZIP63-1 and bZIP63-11 dimers was quantified . Comparison of wt and S/A mutated bZIP63 revealed that , phosphorylation of bZIP63 shifts the dimerization preference from bZIP11 towards bZIP1 . This trend fits well to the observation that both TFs are strongly upregulated in the night and extended night , while bZIP11 is downregulated under these conditions ( see Figure 5—figure supplement 3 ) . Furthermore , increased expression of bZIP63 and bZIP1 under conditions when bZIP63 is phosphorylated might further enhance the observed shift in the dimerization towards bZIP63-1 dimers . Based on the data presented in this study , we propose a simplified model for the regulation of bZIP63 dimerization by AKIN10 ( Figure 9B ) . When bZIP63 is not phosphorylated , its capacity to dimerize with other bZIPs is rather low . Under starvation conditions , bZIP63 is phosphorylated at S29 by AKIN10 , favoring the formation of bZIP63 homo- and specific hetero-dimers , particularly of bZIP63-1 dimers . This ultimately results in the induction of a different set of target genes and thereby mediates the transcriptional reprogramming of metabolism . However , it is clear that in the plant additional factors like bZIP expression , stability and interaction with other components add more complexity to the situation . Surprisingly , to date only a small number of papers have reported an influence of phosphorylation on bZIP dimerization ( Kim et al . , 2007; Guo et al . , 2010; Lee et al . , 2010 ) . Moreover , to our knowledge this is the first time that phosphorylation outside the bZIP domain was shown to affect dimerization with different partners in a distinct way . While there are numerous reports on phosphorylation-mediated changes in bZIP activity in animals , plants , and yeast , in many cases the underlying mechanism is still unknown . We therefore believe that this novel mechanism of phosphorylation-triggered switch of bZIP dimerization partners could play a substantial role in the regulation of bZIP TF activity in all higher organisms , and should be further addressed in future studies .
The lines ox#2 and ox#3 are bZIP63 ox lines in the Col-0 background , expressing bZIP63 . 2 with a C-terminal GFP tag under the control of the 35S promoter . Generation of these plant lines was previously described in Veerabgu et al . ( 2014 ) . Overexpression was confirmed by RT-qPCR of bZIP63 mRNA ( Figure 1—figure supplement 1E ) . The bZIP63 ko line ( bzip63 ) in the Ws-2 ecotype is a T-DNA insertion line . Pool number CSJ1 ( NASC ID: N700001 ) from the Arabidopsis Knockout Facility ( AKF ) ( Sussman et al . , 2000 ) was screened for a T-DNA insertion in bZIP63 and homozygous plants were selected using Kanamycin . Sequencing of the flanking regions revealed that the T-DNA is inserted in the first exon at position 76 ( Figure 1—figure supplement 1A ) . The ko was confirmed by PCR and RT-qPCR of bZIP63 ( Figure 1—figure supplement 1B–D ) . The same line was used by Veerabgu et al . ( 2014 ) . For the complementation lines ( GY9 + GY11 = wt , GAY4 + GAY14 = S29/294/300A , S294/300A ) , a genomic fragment containing bZIP63 and 2 kb of upstream sequence was obtained by PCR on Col-0 genomic DNA and ligated into pCRBlunt ( Invitrogen , Austria ) . For the S/A constructs , first S294 and S300 were mutated to alanine by mutagenesis PCR and the plasmid was subsequently used to mutate S29 to alanine ( see Table 1 for primers ) . Correct sequences were confirmed by restriction digests and sequencing . The genomic fragments were then cloned KpnI/NotI into modified pBIN19 , containing a BASTA resistance for plant selection and a C-terminal YFP tag , before transformation into the Agrobacterium tumefaciens strain GV3101 ( pMP90 ) . Homozygous bzip63 plants were transformed with the floral dip method and selected for positive transformation events by spraying seedlings with 200 mg/l BASTA solution ( Bayer , Germany ) . Transgene expression in GY and GAY lines was tested using RT-qPCR , western blots with an antibody against GFP , and epifluorescence microscopy ( Figure 7—figure supplement 1A–C ) . The S294/300A shown in Figure 8D was at the time of submission still heterozygous and in the T2 generation . Therefore , expression of the transgene was checked by epifluorescence microscopy ( not shown ) , but no quantification by RT-qPCR or western blotting was done . 10 . 7554/eLife . 05828 . 041Table 1 . List of primersDOI: http://dx . doi . org/10 . 7554/eLife . 05828 . 041GeneForward primerReverse primerPrimers for cloninggenomic bZIP63GGTACCAAAACTATAAATTTCTTGTAGGACAGTGTTGCGGCCGCCCTGATCCCCAACGCTTCGAATACGbZIP63 . 2GGGCCCATGGAAAAAGTTTTCTCCGACGAAGAAATCTCCTTGCGGCCGCCCTGATCCCCAACGCTTCGAATACGAKIN10 . 1+3GGGCCCATGGATGGATCAGGCACAGGCAGTAGCGGCCGCAGAGGACTCGGAGCTGAGCAAAKIN11 . 1+2GGGCCCATGGATCATTCATCAAATAGGCGGCCGCAGATCACACGAAGCTCTGTAASNF4GGGCCCATGTTTGGTTCTACATTGGAGCGGCCGCAAAGACCGAGCAGGAATTGGAAAKINβ1 . 1GGGCCCATGGGAAATGCGAACGGCAAAGCGGCCGCACCGTGTGAGCGGTTTGTAGAKINβ2 . 1+2GGGCCCATGTCTGCTGCTTCTGATGGTGCGGCCGCACCTCTGCAGGGATTTGTAGbZIP1CGATGGGCCCCATGGCAAACGCAGAGAAGCGATGCGGCCGCTGTCTTAAAGGACGbZIP2AAAACCATGGCGTCATCTAGCAGCACTGCGGCCGCTATACATATTGATATCATTAGbZIP11TAGGGCCCATGGAATCGTCGTCGTCGGGAATGGCGGCCGCAATACATTAAAGCATCAGAAGbZIP44 . 1CAGGGCCCATGAATAATAAAACTGCTGCGGCCGCAACAGTTGAAAACATCbZIP53AAAACCATGGGGTCGTTGCAAATGCAAACTGCGGCCGCTGCAATCAAACATATCAGCAGAKIN10 VIGSGGAATTCACTTTTTCAGCTCAGAAAATTTTGGGGGTACCCCCTCGAGCCACTCCTGATATTATCTGCTGAKIN11 VIGSCCTCGAGGTTTCTGTATATTTCTTCGCTCGGGGTACCCAGTACTCTACACCAGATATTATCBiFC MCS1TCTAGAGGGCCCATGGGCCACAGACACAGCCCGGGGCGGCCGCACATTCTGCGTCBiFC MCS2AAAAGGATCCGGGCCCATGGGCCACAGACACAGCAAGTCCAAATCCTCCGCCCGGGGCGGCCGCACATTCTGCGTCmulti-color BiFC bZIP1TCGGTACCAAGCTTAGCTTGCATGCCTGCAGGCAGGATCCAGCTGGCGAAAGGGGGATGTGCTGmulti-color BiFC bZIP11CAGGATCCTTAGCTTGCATGCCTGCAGGTCCCTAGGCCTAAGCTTCGCTATTACGCCAGCTGGCGAAAGGMutagenesisbZIP63S29ACGTCGTTGAATCGCTCGGCCGCCGAATGGGCATTCAATCACGTCATTCCATTAACCGACCAGTGbZIP63S59AGTGTGGTGTTTCCGTCTCCGCTCCTCCTAATGTTCCTGCACACGCCGTCGTAGATTCTCCGTCGTCbZIP63S102AGATACTTCTGGTAGAGCTGACAATGGTGGAGCGTATCCTGAGGTTTGATGAAAGTTCbZIP63S160ACTGATTCTCTATTAGCTAGCATCCTTTTAACGATCAGCTAGACGGTCCAGAAGAAGbZIP63S261ACAGAGACATCAAATGCTCCAGACACTACAAGCTTGTAGTGTCTGGAGCATTTGATGTCTCTGbZIP63S294AGAACAGAACAGCTGCCATGCGTAGAGTTGAGTGTTCATCTTGCACCCTATCAAGGCbZIP63S294/300AGAACAGAACAGCTGCCATGCGTAGAGTTGAGGCCTTGGAACATCTGCAGTGTTCATCTTGCACCCTATCAAGGCAKIN10K48MGGTTGCTATCATGATCCTCAATCGTCGGCAACCTTATGTCCTGTCAATGCqPCRbZIP63GAAGAAATCTCCGGTAACCATCACGATTCTCCGTCGTCTGCAGCbZIP1AACGCGGGTCTTAGATCGGAGAAGTCAGCGTTAAACTCGTCGTAGCAAbZIP11TCGTCAGGATCGGAGGAGAGTGATCGTCTAGGAGCTTTTGTTTCTTCCAB2TCAATCTTTTGAATTCGAGTGAGATCCACCACAAACACAAACCTACSEN1CAGAGTCGGATCAGGAATGGATTATGATTTCATCGTGTTTCCSAG103AGCTCGAGTGCTGGGATGCGGATTCACAGATCCTTCCTYLS3GACATCACTAAGTGCCCTGCTACTGTTTCGTTCAGACCTTTAGCAKIN10ACTGGATTTGCAGAGAGTACAAGGTCCTCAGAGGACTCGGAGCTGAGCAAKIN11GCTCGTAACTTTTTCCAGCAGATTCAGGTCTCTATGGACAACCAASN1GTCGCAAGATCAAGGCTCTGAAGTCTTTGTCAAGGAAAGGDIN10GCTTGTATTGCCTGATGGAATCTTTAGCAAGCTGACACCProDHCGCCAGTCCACGACACAATTCACGAATCAGCGTTATGTGTTGCGBCAT2AAGGTTATCAGGTAGTAGAGAAGGTTCCTGATATGTGATAGTGCCAA-TP family proteinGTTCTTGGGATCAACTTCTACAGAACCATTTACATTCCGTAGGACTBP2TGCAAGAAAGTATGCTCGGACATGAGCCTACAATGTTCTGMHF15GTTTCCTGAGCTTCTCCACTGGTCGCTTCATCTTGAGCharacterization of plant lines ( PCR ) bZIP63TTGCGGCCGCCCTGATCCCCAACGCTTCGAATACGAACCATGGATAATCACACAGCTAAAGACATTGGbzip63 RBTGGCGAATGAGACCTCAATTGCGAGCTTTbzip63 LBCATTTTATAATAACGCTGCGGACATCTACAKIN10CCAGCATAATAGAGAACGAAGCGTCCGGTTTAGTATTCAGAGGakin10 LBATATTGACCATCATACTCATTGC The AKIN10 ko line ( akin10 , GABI_579E09 ) in the Col-0 ecotype is a T-DNA insertion line from the GABI KAT collection ( Kleinboelting et al . , 2012 ) . For this line , two T-DNA insertions were suggested , one in AKIN10 and another one in IMS2 ( 2-ISOPROPYLMALATE SYNTHASE 2 , At5g23020 ) . The insertion in AKIN10 in front of the last exon was confirmed by PCR , but the second insertion in IMS2 could not be detected and was thus assumed not to be present ( Figure 5—figure supplement 1A–C ) . The ko of AKIN10 was further confirmed by RT-qPCR and western blotting , respectively . No significant amount of transcript or protein could be detected ( Figure 5—figure supplement 1D , E ) . The akin10 line was further tested for obvious growth and developmental phenotypes by comparing leaf number , fresh and dry weight , water content , and flowering time in soil-grown wt and mutant . Only a slight delay in flowering was observed ( Figure 5—figure supplement 2A , B ) . Expression of selected AKIN10 target genes after 6 hr of light or extended night was tested by RT-qPCR . Some genes showed a slight , but not dramatic reduction in expression ( Figure 5—figure supplement 2C ) . For the line expressing bZIP63-GFP in akin10 , bZIP63 . 2 was amplified from Col-0 cDNA ( see Table 1 for primers ) and cloned ApaI/NotI into modified pBIN19 containing the UBI10 promoter , a BASTA resistance for plant selection , and a C-terminal YFP tag . Homozygous akin10 plants were transformed and selected like the GY and GAY lines . Arabidopsis seeds were surface sterilized with chlorine gas before sowing on soil or growth medium and then vernalized at 4°C for 2 days . Plants were grown in a growth chamber in a 12 hr light/12 hr dark regime with day temperatures between 20 and 22°C and night temperatures between 18 and 20°C and a light intensity of 60–150 μmol/m2s unless specified otherwise . Plants grown under short or long day conditions were cultivated with 8 hr or 16 hr of light per day , respectively . The soil mixture consisted of 4 parts Huminsubstrat N3 ( Neuhaus , Germany ) , 1 part perlite ( Gramoflor , Germany ) , and the fertilizer Osmocote ( Substral/Scotts , Germany ) according to manufacturer's instructions . For hydroponic cultures , plants were grown with their roots in light-tight box filled with liquid ½ Hoagland medium ( 2 mM Ca ( NO3 ) 2 , 0 . 25 mM K3PO4 , 3 mM KNO3 , 1 mM MgSO2 , 45 μM NaFeIII EDTA , 5 μM H3BO3 , 1 μM MnCl2 , 0 . 15 μM ZnSO4 , 0 . 1 μM CuSO4 , 7 nM MoO3 , 4 . 5 nM Co ( NO3 ) ) . Seedling cultures in liquid medium were grown in ½ Gamborg medium ( Duchefa , Harlem , The Netherlands ) with or without 0 . 5% sucrose and the medium was exchanged after 7 days . Seedlings on plates were grown on ½ MS ( Duchefa , Harlem , The Netherlands ) with 0 . 7 g/l plant agar ( Duchefa , Harlem , The Netherlands ) and a pH of 5 . 8 . The root cell suspension culture was grown in MS medium containing 30 g/l sucrose and 2 . 5 µM 2 , 4D at 22°C in the dark under constant shaking . The medium was exchanged every 7 days by transferring ⅓ to ½ of the culture to a fresh flask and addition of fresh medium . 4 . 5 week-old soil-grown plants were incubated in the dark—in a box with tubes allowing for gas exchange—for 9 days . Before and after incubation the true leaves of 4–8 representative plants were harvested , stuck on white paper with double sided tape , scanned with a flatbed scanner without color correction at a resolution of 600 dpi , and saved as TIFF files . Images were then imported in ImageJ ( FIJI ) ( Schindelin et al . , 2012 ) and the total and green leaf area of each leaf were quantified using the built-in threshold and color threshold function , respectively . The green leaf area in % was then calculated by dividing the green area of a leaf or the whole plant by the respective total area . See Figure 1—source data 1 for the ImageJ macro for semi-automatic image processing . A scheme of this method can be found in Figure 1—figure supplement 2A . Alternatively , chlorophyll content was determined according to Porra et al . ( 1989 ) . Total rosettes or seedlings were weighed and frozen in liquid nitrogen . Rosettes were crushed crudely with a spatula to increase extraction efficiency . Chlorophyll was extracted by adding 1–5 ml of a mixture of 80% Acetone and 20% 12 . 5 mM Hepes KOH pH 8 . 2 and incubation in the dark for at least 12 hr . Absorbance at 646 . 6 nm , 663 . 6 nm , and 750 nm was measured in a quartz cuvette and the chlorophyll content in µg chlorophyll/mg fresh weight was determined as follows: ( 12 . 5* ( A663 . 6 − A750 ) − 2 . 55* ( A646 . 6 − A750 ) + 20 . 31* ( A646 . 6 − A750 ) − 4 . 91 ( A663 . 5 − A750 ) ) /FW . For sugar rescue assays seeds were germinated on ½ MS agar plates containing 0 . 5% sucrose and grown in a 12 hr light/12 hr dark cycle for 12 days before transfer to ½ MS agar plates containing 0% or 2% glucose . After 6 additional days seedlings were either harvested or put in a dark box for 7 days . The chlorophyll content in µg/mg FW of individual seedlings was determined as described above . Metabolites were extracted from leaves of 5 week-old plants , derivatized and measured as described in Naegele et al . ( 2014 ) with minor variations . Approximately 80 mg frozen and ground plant material were extracted with 1 ml –20°C cold MeOH:chloroform:H2O ( 2 . 5:1:0 . 5 ) by mixing and incubation on ice . The supernatant after centrifugation was mixed with 400–500 µl H2O and centrifuged . For the experiment shown in Figure 2 ( 2 ) the polar phase was split into 2 aliquots , spiked with 1 µg C13 labeled Sorbitol ( Campro Scientific , Berlin , Germany ) and dried . For the experiment shown in Figure 7 ( 7 ) the polar phase was not split and 2 µg of Sorbitol were added . For derivatization , metabolites were first dissolved in 10 µl ( 2 ) or 30 µl ( 7 ) pyridine containing 40 mg/ml methoxyamine hydrochloride by 90 min incubation at 30°C . Then , 40 µl ( 2 ) or 120 µl ( 7 ) of N-methyl-N-trimethylsilyltrifluoroacetamid ( MSTFA; Macherey–Nagel , Düren , Germany ) , spiked with 60 µl/ml of an Alkane Standard Mixture C10–C40 ( Fluka , Vienna , Austria ) , were added and the samples were incubated for 30 min at 37°C . GC–MS measurements were carried out on an Agilent 6890 gas chromatograph coupled to a LECO Pegasus 4D GCxGC-TOF mass spectrometer ( LECO , USA ) . Injection volume was 1 µl . In the GC step , the initial oven temperature was 70°C , which was held for 1 min , followed by a 9°C/min temperature increase until the final temperature of 350°C was reached , which was held again for 8 min . Metabolites were measured in splitless mode ( 2 and 7 ) and alternatively also in split mode with a split ratio of 5 ( 7 ) . In the MS step the data acquisition rate was set to 20 spectra/s , the detector voltage to 1550 V and the mass range to 40–600 m/z . Raw data were processed with the LECO Chroma-TOF software ( LECO , USA ) . Peak areas were normalized to the area of the internal standard and to the fresh weight before statistical analysis . Outliers , as determined by Grubb's test , were removed from the dataset . For Hierarchical clustering , log-2 transformed fold change values were imported into MeV ( MultiExperimentViewer , version 4 . 9 . 0 , Saeed et al . , 2003 ) and clustering was done using the standard settings with gene tree optimization , Pearson correlation , and average linkage clustering . For the PCA plot , normalized data were imported into R ( RStudio , version 0 . 98 . 507 ) , missing values were replaced with the k nearest neighbor ( knn ) method using the ‘impute . knn ( ) ’ function and data were Z-transformed . PCA analysis was done using the ‘prcomp ( ) ’ function and scores for PC1 and PC2 were plotted against each other . For 2D gels , proteins were extracted from 5–7 week-old soil-grown ox#3 plants which were grown in short day . 4 ml of 1× lambda phosphatase ( λPP ) buffer ( NEB , Frankfurt am Main , Germany ) , including cOmplete protease inhibitor ( Roche , Vienna , Austria ) , were added to 2 ml frozen and ground plant material , followed by vortexing and centrifugation . 0 , 30 or 50 µg of λPP were added to 1 . 5 ml of the supernatant , followed by 15 min incubation at 30°C . Proteins were extracted with phenol ( Carl Roth , Karlsruhe , Germany ) , precipitated with ammonium acetate and resuspended in 1× rehydration stock solution ( 7 M urea , 2 M thiourea , 2% ( wt/vol ) CHAPS , 2% IPG buffer ( GE Healthcare , Vienna , Austria ) , 2 . 8 mg/ml DTT , Bromphenol blue ) . Protein extracts were then applied to 7 cm Immobiline DryStrips ( GE Healthcare , Vienna , Austria ) over night and separated by IEF on an IPGphor ( GE Healthcare , Vienna , Austria ) according to the manufacturer's instructions . For second dimension separation , the strip was incubated in SDS equilibration buffer ( 6 M urea , 75 mM TrisCl , 29 . 3% glycerol , 2% SDS , Bromphenol blue , pH 8 . 8 ) with 10 mg/ml DTT and then without DTT for 15 min each , followed by standard SDS PAGE and western blotting . Phos-tag gel electrophoresis was done according to the manufacturer's instructions . Proteins were separated on SDS PAGE gels containing 8% SDS , 25 µM Phos-tag ( WAKO , Neuss , Germany ) and 50 µM MnCl2 with an amperage of 15 mA/gel for 1 . 25 hr . Before semi dry blotting , the gels were incubated in transfer buffer containing 1 mM EDTA , followed by washing with transfer buffer without EDTA . Recombinantly expressed bZIP63-YFP was used as a size marker for the nonphosphorylated fusion protein . For the expression , bZIP63 was amplified from cDNA ( see Table 1 for primers ) and cloned NcoI/NotI into pTWIN ( NEB , Frankfurt am Main , Germany ) containing YFP , to create a c-terminal YFP fusion . The protein was expressed in Escherichia coli ( ER2566 strain ) and purified according to the manufacturer's instructions . Total plant proteins were extracted with phenol . For light/extended night comparison , rosettes of 5 week old plants were collected after 6 hr of light or extended night . For ± sucrose comparison , seedlings were first germinated and grown for 1 week in liquid ½ Gamborg medium containing 0 . 5% sucrose , followed by 1 week in medium without sucrose . For treatment , at the onset of light 1% suc was added to half of the cultures and all cultures were kept in the dark for 6 additional hours . For Phos-tag gels from kinase assays , the reactions were mixed with 2× Laemmli sample buffer and loaded on the gel . Western blotting was done by semi dry transfer onto a PVDF membrane , antibody incubation , and detection with an ECL kit following standard procedures . The following primary antibodies were used: Anti-GFP ( Roche , Vienna , Austria; or ChromoTek , Munich , Germany ) , Anti-AKIN10 ( Agrisera , Sweden ) , Anti-AMPKalpha-pT172 ( Cell Signaling , Leiden , The Netherlands ) , Anti-Flag ( Sigma–Aldrich , Vienna , Austria ) , Anti-HA High Affinity ( Roche , Vienna , Austria ) , Anti-HA ( Santacruz , Heidelberg , Germany ) , Anti-GAL4 DNA BD ( Sigma–Aldrich , Vienna , Austria ) , polyclonal peptide antibodies against bZIP63: peptide antibodies against an N-terminal ( EKVFSDEEISGNHHWSVNGM ) and a C-terminal ( SLEHLQKRIRSVGDQ ) peptide were raised in rabbit and affinity purified ( Davis biotechnology , Regensburg , Germany ) . The bZIP63 antibodies were tested on protein extracts from wt , bZIP63 ko and ox plants . While the antibodies recognized recombinant protein and bZIP63 in the ox extracts well , they failed to detect endogenous levels of bZIP63 , possibly due to the low abundance of the protein ( not shown ) . The following HRP-coupled secondary antibodies were used: Anti-mouse IgG , Anti-rabbit IgG , Anti-rat IgG ( GE Healthcare , Vienna , Austria ) . To identify in vivo phosphorylation sites on bZIP63 , bZIP63-GFP was immunoprecipitated from ox#3 seedlings grown on ½ MS agar plates or leaves of mature soil-grown ox#3 plants , harvested at different time points in the light cycle and in extended night ( see Figure 3—figure supplement 2A for growth and harvesting conditions ) . In one experiment , leaves were infiltrated with H2O containing 100 µM of the proteasome inhibitor MG-132 ( Calbiochem/Merck Millipore , Vienna , Austria ) 6 hr before harvesting . Protein extracts were prepared by mixing frozen and ground plant material with an equal volume of cold extraction buffer ( 25 mM TrisCl , 10 mM MgCl2 , 15 mM EDTA , 150 mM NaCl , 1 mM DTT , 1 mM NaF , 0 . 5 mM Na3VO4 , 15 mM β-glycerophosphate , 0 . 1% Tween20 , Complete protease inhibitor , pH 7 . 5 ) , followed by centrifugation . The supernatant was then incubated with protein A Sepharose CL-4B ( GE Healthcare , Vienna , Austria ) , which had been pre-incubated for 2 . 5 hr in extraction buffer with Anti-GFP antibody ( Roche , Vienna , Austria ) , or with GFP-Trap_A beads ( ChromoTek , Munich , Germany ) for 1 hr at 4°C . The beads were washed 2–3 times with extraction buffer and alternatively twice with wash buffer ( 50 mM TrisCl , 250 mM NaCl , 0 . 1% NP–40 , 0 . 05% sodium deoxycholate , pH 7 . 5 ) . Finally , the beads were resuspended in 1× Laemmli sample buffer , boiled for 5 min at 95°C , and centrifuged . The supernatant was separated by SDS PAGE and bands were excised for LC-MS/MS analysis . For the identification of phosphorylation sites , bZIP63-GFP was immunoprecipitated from leaves of ox#3 plants ( see ‘Immunoprecipitation of bZIP63-GFP’ ) . For the identification of kinases , root protein extracts were affinity purified with recombinant GST-bZIP63 ( see Figure 4B for a scheme and the methods section ‘Kinase assays’ for detailed description of the affinity purification ) . Proteins were first separated by SDS PAGE . Bands of interest were then excised from Coomassie-stained gels and gel sections were chopped , washed with 50 mM ammonium bicarbonate ( ABC , pH 8 . 5 ) , and dried with acetonitrile ( ACN ) . Disulfide bonds were reduced by incubating in 200 µl of 10 mM DTT for 30 min at 56°C . DTT was washed off and cysteines were alkylated by incubation with 100 µl of 54 mM iodoacetamide for 20 min at RT in the dark . Gel pieces were dried with ACN , then swollen in 10 ng/µl trypsin ( recombinant , proteomics grade , Roche , Vienna , Austria ) in 50 mM ABC and incubated over night at 37°C . For higher sequence coverage of bZIP63 alternative proteases were used: LysC ( MS grade , WAKO , Neuss , Germany ) at 37°C overnight , subtilisin ( Sigma–Aldrich , Vienna , Austria ) at 37°C for 0 . 5–2 hr , chymotrypsin ( sequencing grade , Roche , Vienna , Austria ) at 25°C for 4 hr . Digestion was stopped by adding formic acid to a final concentration of approximately 1% and peptides were extracted by sonication . Peptides were separated on an UltiMate 3000 HPLC system or on a U3000 nano HPLC ( both Dionex , Thermo Fisher Scientific ) . Digests were loaded on a trapping column ( PepMap C18 , 5 µm particle size , 300 μm i . d . × 5 mm , Thermo Fisher Scientific ) , equilibrated with 0 . 1% trifluoroacetic acid ( TFA ) , and separated on an analytical column ( PepMap C18 , 3 μm , 75 μm i . d . × 150 mm , Thermo Fisher Scientific ) by applying a 60 min linear gradient from 2 . 5% up to 40% ACN with 0 . 1% formic acid , followed by a washing step with 80% ACN and 10% trifluoroethanol ( TFE ) on the U3000 HPLC . The UltiMate 3000 HPLC was directly coupled to a linear ion trap ( LTQ , Thermo Fisher Scientific ) , which was operated in a data-dependent MS3 method for the phosphorylation analysis . One full scan ( m/z: 450–1600 ) was followed by maximal 4 MS/MS scans . If in the MS/MS scan a fragment corresponding to a neutral loss from the precursor of 98 , 49 , or 32 Th was observed among the top 8 peaks , a MS3 scan was triggered . Fragmentation energy was set at 35% , Q-value at 0 . 25 , and the activation time at 30 ms . High resolution measurements were acquired on an LTQ-Orbitrap Velos mass spectrometer ( Thermo Fisher Scientific ) , equipped with a nanoelectrospray ionization source ( Proxeon , Thermo Fisher Scientific ) . The electrospray voltage was set to 1500 V . The mass spectrometer was operated in the data-dependent mode: 1 full scan ( m/z: 350–1800 , resolution 60 , 000 ) with lock mass enabled was followed by maximal 12 MS/MS scans . The lock mass was set at the signal of polydimethylcyclosiloxane at m/z 445 . 120025 . Monoisotopic precursor selection was on , precursors with charge state 1 were excluded from fragmentation . The collision energy was set at 35% , Q-value at 0 . 25 , and the activation time at 10 ms . Fragmented ions were set onto an exclusion list for 60 s . When ETD ( electron transfer dissociation ) was applied , the top 6 peaks from the full scan where fragmented with CID ( collision-induced dissociation ) and subsequently with ETD . For ETD , the energy parameters were as for the CID except the activation time was set to 80 or 120 ms . Data interpretation: raw spectra for the kinase identification were interpreted by Mascot 2 . 2 . 04 ( Matrix Science ) using Mascot Daemon 2 . 2 . 2 . Spectra were searched against the Arabidopsis thaliana entries in the nr-database with the following parameters: the peptide tolerance was set to 2 Da , MS/MS tolerance was set to 0 . 8 Da , carbamidomethylcysteine was set as a static modification , oxidation of Met as variable modification . Trypsin was selected as the protease allowing two missed cleavages . Mascot score cut-off was set to 30 , except for the low abundance sample 1 , where the cut-off was set to 20 . For the phosphorylation analysis of purified bZIP63 , either Mascot or Sequest ( Proteome Discoverer 1 . 2; Thermo Scientific ) were used . The search was extended to the phosphorylation of Ser , Thr , and Tyr . High resolution data were searched with 3 ppm precursor mass tolerance . Proteolytic specificity was defined according to the digest . Results were manually validated including comparison of the fragmentation pattern and the relative retention of the nonphosphorylated counterpart . Site localization was checked by manual inspection at the spectrum level in the first place and was confirmed by the site-localization algorithm PhosphoRS ( Taus et al . , 2011 ) . In vitro kinase assays were performed with GST-tagged recombinant proteins . The cDNA of bZIP63 . 2 , bZIP1 , bZIP2 , bZIP11 , bZIP44 . 1 , bZIP55 , AKIN10 . 1/3 , and AKIN11 . 1/2 was obtained by PCR ( see Table 1 for primers ) and cloned ApaI/NotI or NcoI/NotI into pGEX-4T . SnAK2 was in pDEST15 ( Crozet et al . , 2010 ) . An inactive version ( K/M ) of AKIN10 and non-phosphorylatable ( S/A ) versions of bZIP63 were created by mutagenesis PCR using the primers listed in Table 1 . The proteins were expressed in E . coli ( ER2566 or BL21 strain ) , purified using Glutathione Sepharose 4B ( GE Healthcare , Vienna , Austria ) according to the manufacturer's instructions , and stored at −80°C in GST elution buffer containing 10–25% glycerol . Kinase assays were performed by incubating the kinase and substrate for 20–30 min in kinase reaction buffer ( 20 mM Hepes , 20 mM MgCl2 , 100 µM EGTA , 1 mM DTT , 50 µM ATP , pH 7 . 5 ) at room temperature . For radioactive assays , 1 µCi γ-32P-labeled ATP ( NEN/PerkinElmer , Waltham , MA , USA ) was added in each reaction . The reactions were then separated by SDS PAGE and exposure on a Storage Phosphor Screen ( GE Healthcare , Vienna , Austria ) or Phos-tag gel electrophoresis and Western blotting , respectively . For in-gel kinase assays , bZIP63 with an N-terminal 6xHis tag was used as a substrate . To construct the expression vector , bZIP63 . 2 was amplified from cDNA ( see Table 1 ) and first cloned ApaI/NotI into pRSETa-QM ( Invitrogen , Germany ) . From there the expression cassette was excised by SalI digest , followed by a fill in with DNA Polymerase I ( Klenow fragment , NEB , Frankfurt am Main , Germany ) and XbaI digest . The cassette was ligated with pTXB3 ( NEB , Frankfurt am Main , Germany ) , which was first digested with BamHI , followed by a fill in and XbaI digest . The protein was expressed in E . coli ( ER2566 strain ) and purified over a HiTrap column ( GE Healthcare , Vienna , Austria ) according to the manufacturer's instructions . Plant protein extracts for the in-gel kinase assays were made from different plant material . For the identification of bZIP63 kinases , proteins were extracted either from roots of 8 week-old plants that were grown in hydroponic culture in short day and collected in the light phase ( shown in Figure 4A , C ) , from root cell suspension culture , or from seedlings grown on ½ MS agar plates , which were harvested in the dark phase . 4 independent experiments were conducted . For specifications on the plant material used in each of the experiments and the kinases identified please refer to Figure 4—source data 2 . For the comparison of bZIP63 phosphorylation with wt and akin10 plant extracts , roots and leaves of 2 week-old seedlings grown in liquid culture in a 12 hr light/12 hr dark cycle and collected after 4 hr of extended night were used . Extraction was done by mixing the frozen and ground plant material with an equal volume of cold protein extraction buffer ( 25 mM TrisCl , 15 mM EGTA , 10 mM MgCl2 , 75 mM NaCl , 1 mM NaF , 0 . 5 mM NaVO3 , 15 mM beta-glycerophosphate , 0 . 1% Tween20 , 1 mM DTT , Complete protease inhibitor , pH 7 . 5 ) , followed by centrifugation . Protein amounts were determined by Bradford assay . For affinity purification of bZIP63-binding proteins , GST-tagged bZIP63 was expressed in E . coli and the cell lysate of up to 1l culture in GST binding buffer ( 50 mM TrisCl , 20 mM MgSO4 , 2 mM DTT , 5 mM EDTA , 0 . 5% Tween20 , pH 8 ) was loaded on an equilibrated GSTrap FF column ( GE Healthcare , Vienna , Austria ) . The column was then washed with 5 ml cold GST binding buffer and protein extraction buffer , respectively , and 2–5 ml of total root protein in cold protein extraction buffer were loaded . Subsequently , proteins were eluted from the column by repeated washing with 5 ml cold protein extraction buffer with increasing salt concentrations , and concentrated with Amicon Ultra Centrifugal Filter Units ( Millipore ) . 8–20 µg total protein extracts and up to 40 µg affinity purified proteins were loaded on standard SDS PAGE gels containing 1 mg/ml substrate ( 6× His-bZIP63 ) in the separating gel and run at 4°C with 20 mA per gel . The gel was then incubated 3 times for 20 min , respectively , at room temperature in each of the following buffers: wash buffer I ( 50 mM TrisCl , 20% isopropanol , pH 8 ) , wash buffer II ( 50 mM TrisCl , 1 mM DTT , pH 8 ) , and denaturation buffer ( 50 mM TrisCl , 1 mM DTT , 6 M guanidinium HCl , pH 8 ) . Subsequently , the gel was incubated in renaturation buffer ( 50 mM TrisCl , 0 . 05% Tween 20 , pH 8 ) at 4°C for 12–18 hr . In this period , the buffer was exchanged 10 times after at least 30 min . After renaturation , the gel was incubated 2 times for 30 min , respectively , in kinase buffer ( 20 mM HEPES , 20 mM MgCl2 , 50 µM CaCl2 or 500 µM EGTA , 1 mM DTT , 0 . 05% Tween 20 , pH 7 . 5 ) , followed by 30 min incubation in kinase reaction solution ( kinase buffer containing 50 µM ATP and 100 µCi γ-32P-labeled ATP ) . Finally , the gel was washed 2 times for 15 min , respectively , in 5% TCA and several times in 5% TCA containing 1% sodium pyrophosphate , until the wash solution was only weakly radioactive . The dried gels were exposed on a Storage Phosphor Screen and the signal was recorded on a Typhoon 8600 ( GE Healthcare , Vienna , Austria ) . AKIN10 . 1/3 , AKIN11 . 1/2 , AKINβ1 , AKINβ2 , SNF4 , and bZIP63 . 2 were amplified from cDNA ( see Table 1 for primers ) and cloned ApaI/NotI or NcoI/NotI into pBTM117 and bZIP63 . 2 was cloned into pACTIIJ to generate N-terminal fusions with the LexA-BD and the GAL4-AD , respectively . The yeast strain L40 ( MATα hisΔ200 trp1-900 leu2-3 . 112 ade2 LYS2:: ( lexA op ) 4HIS3 URA3:: ( lexA op ) 8lacZ Gal4 gal80 ) was transformed with empty pACTIIJ or bZIP63 . 2-containing pACTIIJ in combination with different pBTM117 vectors . Freshly grown L40 was mixed gently with 1 ml transformation mix ( 800 µl 50% PEG 3600 , 100 µl 2 M LiAc , 100 µl 1 M DTT , 10 µl bacterial RNA [10 µg/µl] ) , to get a cloudy suspension . 2 . 5 µg of each plasmid were added to 125 µl transformation mix , followed by 20 min incubation at 30°C and 44°C , respectively , addition of 1 ml H2O , and 1 min centrifugation at 3500×g . The cells were resuspended in a small volume and plated on SD medium without Leu and Trp to select for successful transformation . Single colonies were inoculated in SD medium without Leu and Trp and proteins were extracted from 2 ml of an over-night culture . The yeast cells were resuspended in 200 µl enzyme lysis buffer ( 25 mM TrisCl , 20 mM NaCl , 8 mM MgCl2 , 5 mM DTT , 0 . 1% NP-40 , pH 7 . 5 ) , 200 µl glass beads ( 0 . 4–0 . 6 mm diameter ) were added , the cells were frozen in N2 , thawed , and broken by vigorous shaking on a Vibrax at 4°C for 20 min . The supernatant after 10 min centrifugation was transferred to a fresh tube and kept on ice . The protein concentration of the extract was determined by Bradford assay . The GUS activity was determined by mixing 50 µl of the extract with 650 µl Z-buffer ( 60 mM Na2HPO4 , 40 mM NaH2PO4 , 10 mM KCl , 10 mM MgSO4 , 0 . 25% beta mercaptoethanol ) and 100 µl ONPG ( 4 mg/ml ) , and incubating for up to 10 min at room temperature . The reaction was stopped by adding 400 µl 1 M Na2CO3 and the extinction at 420 nm was measured on a photometer . The GUS activity in U/mg protein was calculated as follows: ( A420 × 24 × 1000 ) / ( 45 × incubation time [min] × protein concentration [mg/ml] ) . For BiFC and multi-color BiFC interaction studies in N . tabacum , bZIP63 . 2 , AKIN10 . 1/3 , AKIN11 . 1/2 and SNF4 were cloned into modified pBIN19 vectors , containing either the N- or C-terminal moieties of the split CFP system as N-terminal fusions as described in Waadt et al . ( 2008 ) ( see Figure 5D for scheme ) . To generate BiFC plasmids for ApaI/NotI and NcoI/NotI cloning , CPK3 , harboring XbaI , NcoI , and ApaI ( for plasmids with MCS [multi cloning site] 1 ) or ApaI and NcoI ( for plasmids with MCS 2 ) in the terminus and NotI and SmaI in the C-terminus was generated by PCR from cDNA ( see Table 1 for primers ) . It was then cloned XbaI/SmaI or ApaI/SmaI into the different BiFC casettes in pUC19 from Waadt et al . ( 2008 ) . The cassettes were then cloned into pBIN19 with HindIII/EcoRI and CPK3 was exchanged for other genes using ApaI and NotI . A . tumefaciens ( AGL1 strain ) was transformed with the resulting plasmids by electroporation and further used for transient transformation of tobacco leaf epidermis . For transformation , 5 ml agrobacterium overnight cultures were filled to 50 ml with fresh LB medium containing 50 µg/ml Kanamycin and 10 µg/ml Gentamicin and grown for 4 hr at 30°C . The cells were pelleted by centrifugation at 3500×g , resuspended in LB containing 150 µM acetosyringone , and grown for another 2 hr . The cultures were then pelleted again and resuspended in 5% sucrose solution to reach a final OD600 of 2 . For co-infiltration , equal volumes of agrobacteria suspensions , containing the respective constructs , were mixed and infiltrated into the leaves of 5 week-old plants . 48 hr after infiltration equally sized leaf sections were analyzed for their CFP fluorescence signal with an LSM510 confocal laser scanning microscope ( Zeiss ) and the corresponding ZEN software ( Zeiss ) . The same settings were used for each construct to allow comparison of the signal intensities . To verify that the fusion proteins were expressed and to determine the relative amount of each interaction partner , proteins were extracted from the leaf sections used for microscopy and subjected to Western blot analysis with antibodies against the Flag ( N-terminal CFP moiety ) and HA ( C-terminal CFP moiety ) epitopes . Multi-color BiFC was done as described in Waadt et al . ( 2008 ) with some modifications . bZIP11 fused to the N-terminal moiety of CFP ( CFP-N ) , bZIP1 fused to the N-terminal moiety of VENUS ( VENUS-N ) , and bZIP63 ( wt or S29/294/300A ) fused to the C-terminal moiety of CFP ( CFP-C ) were expressed from one plasmid to have co-expression at equal amounts in all transformed cells ( for a scheme of the construct see Figure 9A ) . To generate the construct , bZIP1 and bZIP11 were first amplified from cDNA by PCR ( see Table 1 for primers ) and cloned ApaI/NotI into pBIN19 containing a c-terminal fusion of CFP-C and CFP-N , respectively ( plasmids described above ) . From there , 35S::bZIP1 was cloned HindIII/NotI into pUC19 containing CPK3 with a c-terminal VENUS-N fusion ( described above ) to exchange the shorter 35S primer in the VENUS construct . Then , the two cassettes including the 35S promoter , bZIP1 or 11 and the VENUS-N or CFP-N tag were amplified by PCR introducing KpnI/HindII in the front and BamHI in the back of the bZIP1 cassette and BamHI in the front and HindIII in the back of the bZIP11 cassette ( see Table 1 for primers ) . The PCR products were ligated into pCRBlunt ( Invitrogen , Germany ) and the bZIP1 cassette was then cloned KpnI/BamHI in front of the bZIP11 cassette . Finally , the combined cassette with bZIP1 and 11 was cloned HindII into pBIN19 containing bZIP63 wt or S29/294/300A ( mutated by PCR ) with an n-terminal CFP-C fusion . AKIN10 was amplified from cDNA by PCR ( see Table 1 for primers ) and cloned ApaI/NotI into pBIN19 containing a c-terminal mCherry tag . A . tumefaciens ( AGL1 strain ) was transformed with the resulting plasmids by electroporation and further used for transient transformation of tobacco ( N . tabacum ) leaf epidermis as described above . 48 hr after infiltration equally sized leaf sections were analyzed for their VENUS , CFP , and mCherry fluorescence signal with a TCS SP5 DM-6000 confocal laser scanning microscope ( Leica ) and the corresponding Leica software . CFP , VENUS , and mCherry were excited with 458 nm ( Ar laser ) , 514 nm ( Ar laser ) , and 574 nm ( white light laser ) , respectively . The fluorescence was detected at 461–495 nm , 520–550 nm , and 600–615 nm , respectively . To avoid bleed-through of the CFP signal into the VENUS channel CFP and VENUS were excited and detected separately . Pictures were taken with a 20× air objective and the same settings for both combinations ( wt bZIP63 and S29/294/300A ) . The pinhole was set to 1 . 5 . To determine the relative VENUS/CFP signal ratio , images were processed in the LasX software from Leica . Nuclei showing a CFP/VENUS as well as mCherry signal were selected and the signal intensity was determined with the histogram tool . For each nucleus a ratio of the background corrected pixel sum from VENUS and CFP was built . All values for one leaf were divided by the median of the ratio from the bZIP63 S29/294/300A construct to allow comparison between experiments . In total , six leaves from two infiltrations were analyzed with similar results . 2 week-old GY9 plants were infiltrated ( akin10/11 ) or not ( control ) with an AKIN10-AKIN11 silencing construct according to the method described by Burch-Smith et al . ( 2006 ) . For the AKIN10-AKIN11 construct two gene-specific fragments corresponding to AKIN10 and AKIN11 were cloned in tandem into the TRV-based vector pYL156a . First , a 480 bp AKIN10 fragment was amplified from cDNA using the AKIN10 VIGS primers ( see Table 1 for primers ) and cloned into the pYL156/pTRV2 vector using EcoRI/KpnI . A 503-bp AKIN11 fragment ( Baena-González et al . , 2007 ) was thereafter amplified from cDNA using the AKIN11 VIGS primers ( see primer list ) and cloned into the pYL156/pTRV2 vector harboring the KIN10 fragment using XhoI/KpnI . 2 weeks after infiltration the AKIN10-AKIN11 knock-down plants displayed visible growth defects and anthocyanin accumulation . Silencing of AKIN10 and AKIN11 in these plants was confirmed by western blotting with an antibody against phosphorylated AMPK alpha ( Anti-AMPKalpha-pT172 ) before use in Phos-tag gels ( see Figure 5—figure supplement 7 ) . The VIGS experiments were repeated 4 times and 3 biological replicates were analyzed in each experiment with consistent results . Protoplast were obtained from 3 week-old soil-grown wt Arabidopsis plants and transformed according to the guide method ( Yoo et al . , 2007 ) with small modifications . Leaves were harvested 1 hr after onset of the light phase , cut into tiny stripes , and digested for 30 min under vacuum and 3 hr at atmospheric pressure , respectively , with enzyme solution ( 1 . 25% [wt/vol] Cellulase R-10 , 0 . 3% Macerozyme R-10 , 400 mM mannitol , 20 mM KCl , 10 mM CaCl2 , 20 mM MES , pH 5 . 7 ) . The protoplast suspension was filtered on a metal net to remove leaf debris and washed twice with 10 ml of W5 solution ( 2 mM MES , 154 mM NaCl , 125 mM CaCl2 , 5 mM KCl , pH 5 . 7 ) . Protoplasts were resuspended in 10 ml of W5 , incubated on ice for at least 1 hr and subsequently resuspended to a final concentration of 1 × 105 cells/ml in MMg buffer ( 4 mM MES , 400 mM mannitol , 15 mM MgCl2 , pH 5 . 7 ) . Protoplasts were then co-transformed with 10 µg each of up to three effector plasmids , 7 µg of a reporter plasmid , and 3 µg of a 35S::NAN transfection control reporter plasmid ( Kirby and Kavanagh , 2002 ) for normalization . For P2H assays , the effector plasmids were pHBTL containing bZIP63 . 2 , bZIP1 , or bZIP11 with an N-terminal GAL4-AD or GAL4-BD fusion ( described in detail in Ehlert et al . , 2006 ) and alternatively pHBTL containing AKIN10 . 1/3 . The reporter plasmid contained GAL-UAS4::GUS ( beta galactosidase ) ( Ehlert et al . , 2006 ) . For promoter activation assays , effector plasmids were pHBTL containing HA-tagged bZIP63 . 2 or AKIN10 . 1/3 . pBT10 containing proASN1::GUS or proProDH::GUS ( Dietrich et al . , 2011 ) was used as a reporter plasmid . For transformation , 200 μl of the protoplast suspension were gently mixed with the DNA and 220 μl of PEG ( 40% PEG 4000 , 200 mM mannitol , 100 mM CaCl2 ) were added , followed by gentle mixing , and 10 min incubation at room temperature . The protoplasts were then washed by addition of 800 µl W5 and 1 min centrifugation at 300×g . The supernatant was removed and the protoplasts were incubated for 16 hr in 200 µl of WI solution ( 4 mM MES , 500 mM mannitol , 20 mM KCl , pH 5 . 7 ) in the growth chamber in order to not affect their diurnal circle . GUS and NAN enzyme assays were performed according to Kirby and Kavanagh ( 2002 ) and the relative activity of GUS and calculated as GUS/NAN . The expression of the effector constructs was confirmed by Western blot analysis . Homologues of bZIP63 in 8 plant species were identified by blasting the protein sequence of bZIP63 . 2 on the Phytozome webpage ( http://phytozome . net ) . The protein sequences were aligned with ClustalΩ ( http://www . ebi . ac . uk/Tools/msa/clustalo/ ) using the default settings and the Clustal output file was imported into GeneDoc ( http://www . psc . edu/biomed/genedoc ) for visualization and minor adjustments of the alignment . The identity matrix for the alignment was calculated using the PID3 method in the SIAS webmask ( http://imed . med . ucm . es/Tools/sias . html ) . Gene identifiers of the bZIP63 homologues ( numbers are the Phytozome10 references ) : Medicago truncatula ( Medtr7g115120 ) , Glycine max ( Glyma . 10G162100 ) , Populus trichocarpa ( Potri . 013G040700 ) , Vitis vinifera ( GSVIVG0102179000 ) , Zea mays ( GRMZM2G007063 ) , Oryza sativa ( LOC_Os03g58250 ) , Selaginella moellendorffii ( 270282 ) , Physcomitrella patens ( Phpat . 009G02690 ) . RNA was extracted with phenol from total rosettes of 4 . 5 week-old soil-grown plants at the indicated time points . 500 µl RNA extraction buffer ( 1% SDS , 10 mM EDTA , 200 mM NaAc , pH 5 . 2 ) and 500 µl acidic phenol ( pH 4 , Carl Roth , Karlsruhe , Germany ) were added to 100 mg frozen and ground plant material , followed by 1 min vortexing and 10 min centrifugation . The aqueous phase was extracted twice with an equal volume of PCI ( Phenol:Chloroform:Isoamylalcohol ( 25:24:21 ) , Carl Roth , Karlsruhe , Germany ) by vortexing for 30 s and 2 min centrifugation , and twice with chloroform . Then , the RNA was precipitated by adding ⅓ volume 10 M LiCl and incubating at 4°C for at least 2 hr , followed by 15 min centrifugation at 4°C . The pellet was washed once with 2 . 5 M LiCl and then with 70% EtOH , dried and resuspended in 50 µl RNAse free H2O . 6 . 5 µg RNA were treated with 2u of RQ1 DNAse ( Promega , Mannheim , Germany ) , according to the manufacturer's instructions and precipitated again for at least 2 hr with ⅓ volume of 10 M LiCl to remove the DNAse from the solution . The solution was then centrifuged for 15 min , washed once with 70% EtOH , dried and resuspended in RNAse free H2O . 1 . 5 µg RNA were reverse transcribed using M-MLV RT [H-] Point Mutant ( Promega , Mannheim , Germany ) according to the manufacturer's instructions and diluted 1:4 with RNAse free H2O . 2 µl of cDNA ( 15 ng/µl ) or H2O ( for the no template control ) were added to 18 µl of a PCR reaction mix containing 0 . 25u DreamTaq polymerase ( Thermo Scientific , Vienna , Austria ) , 1× Dream Taq buffer , additional 3 mM MgCl2 , 100 µM dNTPs , 400 nM of each primer , and 0 . 4× SYBR Green I nucleic acid gel stain ( Sigma–Aldrich , Vienna , Austria ) . The qPCR was performed on a Mastercycler ep realplex2 ( Eppendorf ) in white 96-well twin . tec real-time PCR plates ( Eppendorf , Vienna , Austria ) with the following program: 1 cycle with 3 min at 95°C , followed by 40 cycles with 20 s at 95°C , 20 s at 59/60°C , and 12 s at 72°C . 2–3 technical replicates were used . A 20 min melting curve was added in the end . For data evaluation , raw data were imported into LinRegPCR ( version 2014 . 02 , Ramakers et al . , 2003 ) , where the PCR efficiency was checked and the N0 ( staring concentration of cDNA ) was calculated with a common baseline setting for each primer pair . Samples excluded by LinReqPCR were not used . In case only two technical replicates were used , samples for which the dCq between the two technical replicates was bigger than 0 . 5 were also excluded from the calculations . The mean N0 of the technical replicates was calculated for each sample and primer pair . The resulting mean N0 of the test genes was then normalized by dividing by the mean N0 of the reference genes . For qPCR of AKIN10 , AKIN11 ( Figure 5—figure supplement 1D ) ; bZIP63 , bZIP1 , bZIP11 ( Figure 5—figure supplement 3 ) ; ASN1 , DIN10 , and ProDH ( Figure 7G ) TBP2 ( Tata binding protein 2 ) was used as a reference gene . For pPCR of bZIP63 , ( Figure 1—figure supplement 1D , E , Figure 7—figure supplement 1A ) ; CAB2 , SEN1 , SAG103 , and YLS3 ( Figure 1—figure supplement 3B ) ; ASN1 , ProDH , BCAT2 , AA-TP family protein , and DIN10 ( Figure 5—figure supplement 2C ) the geometric mean of the values for TBP2 and MHF15 ( Thioredoxin superfamily protein ) were used . The normalized N0 values were then used to calculate the mean and SD . Outliers were determined with the Grubb's test and removed . Statistical tests were performed in excel or R . Equality of variances was tested with Levene's test . This was followed by unpaired t-tests or multivariate statistics . In case of equal variance ANOVA followed by Bonferroni or TukeyHSD corrected pairwise comparison was chosen . In case of unequal variance Welch corrected ANOVA was applied , followed by Games Howell test of all samples or pairwise comparison of samples with equal variance . bZIP63 ( At5g28770 ) , bZIP1 ( At5g49450 ) , bZIP11 ( At4g34590 ) , bZIP2 ( At2g18160 ) , bZIP44 ( At1g75390 ) , bZIP53 ( At3g62420 ) , AKIN10/SnRK1 . 1 ( At3g01090 ) , AKIN11/SnRK1 . 2 ( At3g29160 ) , AKINβ1 ( At5g21170 ) , AKINβ2 ( At4g16360 ) , SNF4/AKINβγ ( At1g09020 ) , SnAK2/GRIK1 ( At3g45240 ) , ASN1/DIN6 ( At3g47340 ) , DIN10 ( At5g20250 ) , ProDH/ERD5 ( At3g30775 ) , BCAT2 ( AT1G10070 ) , AA-TP ( amino acid transporter ) family protein ( AT2G40420 ) , TBP2 ( At1g55520 ) , MHF15 ( At5g06430 ) , CAB2 ( At1g29920 ) , SEN1/DIN1 ( At4g35770 ) , SAG103 ( At1g10140 ) , YLS3 ( At2g44290 ) .
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 .
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 .