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.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 DOI:http://dx.doi.org/10.7554/eLife.05828.001 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.Arabidopsis.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 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.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.Mair et al. triggered starvation in 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.DOI:http://dx.doi.org/10.7554/eLife.05828.002 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  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 , Snf1 iset genes . Howeveret genes  and modeet genes , some mebZIP 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 . They arArabidopsis bZIPs, which was proposed to play a role in energy metabolism, seed maturation, and germination under osmotic stress , energy starvation response (DIN10 = RAFFINOSE SYNTHASE 6), and senescence (SEN1 = SENESCENCE 1)  . The C-gCENCE 1) . Three o as well . Further as well . In thatHere 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.bzip63 plants displayed a stay-green phenotype  and separated treated and untreated extracts on 2D gels by isoelectric focusing (IEF) in the first, and SDS-PAGE in the second dimension . 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  . To achiArabidopsis 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 . 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  of bZIP63, observed in the wt in extended night, was much weaker in the akin10 background  to knock down AKIN10 and AKIN11 in a genomic bzip63 complementation line (GY9). Resulting plants showed strongly reduced growth and accumulation of anthocyanins  in the extended night was now almost completely gone  was found to be more abundant in an AKIN10 ox line and less abundant in the ko line after extended night treatment, respectively .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 . DiffereArabidopsis 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  reporter , or a S/A mutation of S29, S294, and S300, respectively (GAY lines) with a C-terminal YFP tag  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 nd ProDH . In protnd ProDH . Note, tnd ProDH . In bothTo 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 . TogetheTo 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 . CompareNicotiana tabacum leaves with bZIP1 and 11 as alternative interaction partners for bZIP63. For this, we expressed all three bZIPs  from the same plasmid and co-transformed AKIN10  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.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  in Nicotd AKIN10 . bZIP11 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 , as wellSimilar 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 . This suProDH and ASN1  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 ProDH and ASN1 expression.We found that bZIP63 is highly phosphorylated in response , is the Since the discovery that AKIN10 can activate several members of the C/S1 network , it has ASN1 and ProDH  in protoplast assays , althougN. 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  in the Ws-2 ecotype is a T-DNA insertion line. Pool number CSJ1 (NASC ID: N700001) from the Arabidopsis Knockout Facility (AKF)   was screition 76 . The ko  . 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 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 . Transgene expression in GY and GAY lines was tested using RT-qPCR, western blots with an antibody against GFP, and epifluorescence microscopy , a genomic fragment containing croscopy . The S29AKIN10 ko line  in the Col-0 ecotype is a T-DNA insertion line from the GABI KAT collection . 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 , 1 part perlite , and the fertilizer Osmocote  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  with or without 0.5% sucrose and the medium was exchanged after 7 days. Seedlings on plates were grown on ½ MS  with 0.7 g/l plant agar  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)  and the See 2O (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 10–C40 , 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 . 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 . 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  buffer , including cOmplete protease inhibitor , 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 , precipitated with ammonium acetate and resuspended in 1× rehydration stock solution  CHAPS, 2% IPG buffer , 2.8 mg/ml DTT, Bromphenol blue). Protein extracts were then applied to 7 cm Immobiline DryStrips  over night and separated by IEF on an IPGphor  according to the manufacturer's instructions. For second dimension separation, the strip was incubated in SDS equilibration buffer  with 10 mg/ml DTT and then without DTT for 15 min each, followed by standard SDS PAGE and western blotting.2 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 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.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  and 50 µM MnClWestern 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 , Anti-AKIN10 , Anti-AMPKalpha-pT172 , Anti-Flag , Anti-HA High Affinity , Anti-HA , Anti-GAL4 DNA BD , 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 . 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 .2O containing 100 µM of the proteasome inhibitor MG-132  6 hr before harvesting. Protein extracts were prepared by mixing frozen and ground plant material with an equal volume of cold extraction buffer , followed by centrifugation. The supernatant was then incubated with protein A Sepharose CL-4B , which had been pre-incubated for 2.5 hr in extraction buffer with Anti-GFP antibody , or with GFP-Trap_A beads  for 1 hr at 4°C. The beads were washed 2–3 times with extraction buffer and alternatively twice with wash buffer . 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.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 . For the identification of kinases, root protein extracts were affinity purified with recombinant GST-bZIP63 , 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  in 50 mM ABC and incubated over night at 37°C. For higher sequence coverage of bZIP63 alternative proteases were used: LysC  at 37°C overnight, subtilisin  at 37°C for 0.5–2 hr, chymotrypsin  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 . Digests were loaded on a trapping column , equilibrated with 0.1% trifluoroacetic acid (TFA), and separated on an analytical column  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 , 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 . The electrospray voltage was set to 1500 V. The mass spectrometer was operated in the data-dependent mode: 1 full scan  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.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  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 (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 hosphoRS .bZIP63.2, bZIP1, bZIP2, bZIP11, bZIP44.1, bZIP55, AKIN10.1/3, and AKIN11.1/2 was obtained by PCR (see SnAK2 was in pDEST15 (AKIN10 and non-phosphorylatable (S/A) versions of bZIP63 were created by mutagenesis PCR using the primers listed in E. coli (ER2566 or BL21 strain), purified using Glutathione Sepharose 4B  according to the manufacturer's instructions, and stored at −80°C in GST elution buffer containing 10–25% glycerol.In vitro kinase assays were performed with GST-tagged recombinant proteins. The cDNA of  pDEST15 . An inac2, 100 µM EGTA, 1 mM DTT, 50 µM ATP, pH 7.5) at room temperature. For radioactive assays, 1 µCi γ-32P-labeled ATP  was added in each reaction. The reactions were then separated by SDS PAGE and exposure on a Storage Phosphor Screen  or Phos-tag gel electrophoresis and Western blotting, respectively.Kinase assays were performed by incubating the kinase and substrate for 20–30 min in kinase reaction buffer  and purified over a HiTrap column  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 , 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  was loaded on an equilibrated GSTrap FF column . 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 , wash buffer II , and denaturation buffer . Subsequently, the gel was incubated in renaturation buffer  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 , 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 .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  and firsshown in , from roAKIN10.1/3, AKIN11.1/2, AKINβ1, AKINβ2, SNF4, and bZIP63.2 were amplified from cDNA 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 , 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 , 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  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]).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 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  and HA  epitopes.For BiFC and multi-color BiFC interaction studies in 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.Multi-color BiFC was done as described in akin10/11) or not (control) with an AKIN10-AKIN11 silencing construct according to the method described by 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 AKIN11 fragment (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  before use in Phos-tag gels . The protoplast suspension was filtered on a metal net to remove leaf debris and washed twice with 10 ml of W5 solution . 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 . 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  (bZIP63.2 or AKIN10.1/3. pBT10 containing proASN1::GUS or proProDH::GUS (2) 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  in the growth chamber in order to not affect their diurnal circle. GUS and NAN enzyme assays were performed according to Protoplast were obtained from 3 week-old soil-grown wt e method  with sma plasmid  for normosidase) . For prooDH::GUS  was usedhttp://phytozome.net). The protein sequences were aligned with ClustalΩ  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).Homologues of bZIP63 in 8 plant species were identified by blasting the protein sequence of bZIP63.2 on the Phytozome webpage (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).Gene identifiers of the 2O. 6.5 µg RNA were treated with 2u of RQ1 DNAse , 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  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 , 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 . The qPCR was performed on a Mastercycler ep realplex2 (Eppendorf) in white 96-well twin.tec real-time PCR plates  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  was used as a reference gene. For pPCR of bZIP63,  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.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  and 500 µl acidic phenol  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 , 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 H, AKIN11 ; bZIP63,, bZIP11 ; ASN1, Dnd ProDH  TBP2 , 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). review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent , a Reviewing Editor and three peer reviewers, and the decision was reached after discussions between the reviewers. Based on our discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.Thank you for choosing to send your work entitled “SnRK1-triggered switch of bZIP63 dimerization mediates the low-energy response in plants” for consideration at akin10 mutant). As the use of this mutant is crucial for major conclusions of your work, comprehensive characterization of this mutant is required before further consideration of your submission. We would welcome re-reviewing this manuscript if the major concerns listed in the reviews below were convincingly addressed.While the reviews acknowledge the overall quality of your work, questions remain about the validity of one of the major tools used in your study , PROLINE DEHYDROGENASE (PDH1/PRODH), RAFFINOSE SYNTHASE (DIN10) and SENESCENCE 1 (SEN1). To justify the statements in the Abstract and Introduction that bZIP63 is a key regulator of starvation response, the paper starts with limited characterization of a bzip63 T-DNA insertion mutant, and two bZIP63-ox overexpression lines.Plant SnRK1 kinases are structural and functional homologs of yeast Snf1 and animal AMP-activated kinases that play a central role in sensing energy depletion and maintaining cellular energy homeostasis. In this paper, a multinational consortium set out to demonstrate that bzip63 mutant (defined as stay-green phenotype), and 3-5 more leaves undergo bleaching in the bZIP63-ox plants, suggesting enhanced senescence response.The authors use a phenotypic assay by placing plants for 9 days into the dark and monitoring rosette leaf senescence/bleaching by ImageJ scanning of green versus bleached/yellow leaf areas. Whereas in wild type (wt) the first two leaves .Next, the authors compare metabolites entering or derived from glycolysis and citrate cycle in light-grown (L) and dark exposed  plants and find that the levels of “most” free amino acids is increased in the When inspecting the metabolite profiling data, it is clear that the discussion of the results is rather superficial. For example, raffinose levels are decreased, whereas trehalose levels are increased in both mutant and ox lines, similarly to glucose in the dark. In general, we do not learn much from the metabolomics data. The actual conclusion is that “misregulation of bZIP63” (in the subsection “bZIP63 controls dark induced senescence and primary metabolism”) has an effect on metabolism.On the other hand, we do not find any additional data , which would provide a further insight documenting how alteration of bZIP63 levels modulate the starvation-induced and/or senescence responses. Whether 9 days of dark treatment can be considered as starvation or general stress response remains a matter of discussion.By performing 2D and Phos-tag gel westerns , the autBy mass spectrometry analysis, seven phosphorylated residues were then identified in the immonoprecipitated bZIP63-GFP protein. Subsequent in-gel kinase tests indicate that bZIP63 is phosphorylated by multiple protein kinases showing apparent molecular masses of 50-60 and 35-38 kDa. In To isolate the kinases detected in the in-gel assay, GST-bZIP63 was immobilized on GST-Sepharose and plant protein extracts made from root cultures or root-derived cell suspension , is at least 10-20 times more active/abundant compared to its homolog AKIN11 in akin10 GABI_579E09 T-DNA insertion mutant to demonstrate that AKIN10 is indeed responsible for in vivo phosphorylation of bZIP63. Unfortunately, according to the reviewer's knowledge, this mutant has not yet been characterized by others, so, to a certain extent, this must be performed by the authors in this paper.As a key argument, the authors use the akin10 mutant extracts, and they fail to detect AKIN10 by western with the Agrisera αAKIN10 antibody in roots, but observe a lower Mw weakly cross-reacting protein band in leaf extracts. According to these results, the GABI_579E09 insertion mutant appears to produce no immunologically detectable AKIN10. Yet, the authors could successfully cultivate the mutant, which means that it must be fertile. If AKIN10 is a central regulator of starvation responses, and shows an order of magnitude higher activity compared to AKIN11 in Arabidopsis, based on the published striking AKIN10 RNAi senescence phenotype by Bhaena-Gonzalez et al., one would expect a dramatic growth phenotype. Consequently, it is absolutely necessary to show the phenotype of the akin10 mutant both in the light and dark that we can compare that to that of the bzip63 mutant.In Furthermore, the analysis of transcription of bZIP63 target genes should provide further clear evidence that post-translational regulation of bZIP63 is indeed impaired in the GABI_579E09 T-DNA insertion mutant. This is because N. tabacum protein-protein interaction assays indicating that that unlike AKIN11, the AKIN10 catalytic and SNF4 activating subunits of SnRK1 interact with bZIP63. Given the high level of amino acid homology between AKIN10 and AKIN11, it is surprising that bZIP63 fails to bind AKIN11 and provide a possible hint about the location of possible interaction sites. Did you map these?akin10 mutant plants.On the other hand, it is remarkable that the kinase-substrate interaction is so strong that this can be clearly detected by both assays. However, as these are heterologous assays based on overexpression of proteins, it is absolutely necessary to present co-immunoprecipitation data using the later described bZIP63 GENE construct (and not 35S ox lines) and αAKIN10 western from wt and According to As GST-AKIN10/11, GST-bZIP63 and GST-SnAK2 have closely similar masses, it would be necessary to resolve them in a lower % of SDS-PAGE.In vitro phosphorylation of bZIP63 by AKIN10 and AKIN11 looks convincing in The referred “consensus SnRK1 sites” in ASN1/DIN6-GUS and ProDH-GUS reporters. Whereas sequential phosphorylation might indeed trigger threshold-dependent conformational change of TFs, it is premature to conclude anything about possible order of phosphorylation sites .The promoter activation assays in protoplast support the conclusion that S29 is the critical AKIN10 phosphorylation site, but in combination with the S294A and S300A amino acid exchanges, AKIN10-S29A shows lower transactivation of In the subsection “The AKIN10 target sites play an important role for bZIP63 function in planta” it is stated that: “The phosphorylation status of the wt construct after 6h of light and extended night and in the presence or absence of sugar was similar to the one observed in ox#3 plants .” ConcerIn the same subsection, I do not understand the logic of the following sentence: “Notably, hyper-phosphorylation of bZIP63 in the extended night was even stronger in the GY lines than in ox#3, probably due to lower bZIP63 expression.” Do the authors suggest that AKIN10 activity is limiting in terms of bZIP63 phosphorylation even if AKIN10 was supposed to be activated by extended night?bzip63 mutant characterization, the lack of complementation by the triple S/A bZIP63 mutant gene is documented. As the mutant gene constructs were generated by PCR of 2+xkb genomic DNA fragments, the Materials and methods should mention that all constructs were verified by sequencing.With the limitations mentioned above concerning the necessary parameters of ASN1 and DIN10. In the fold-induction curve and column diagram to the right, the standard error of Ws-2 is huge at 4h of EN, thus no significant difference between wt and bzip63 mutant can be claimed. In this figure part, either the curves shown to the left should include all four samples  or all samples at the four time points should be presented as column diagrams. Given the data presented in bzip63 mutant transformed with the triple phosphor-site mutant of bZIP63 was analysed.In Arabidopsis response regulator ARR18 involved in cytokinin signaling, whereas alone (i.e. by interacting with S-group partners in heterodimers), bZIP63 functions as negative regulator of osmotic stress responses and seed germination.Ehlert et al. reported that, when overexpressed in plant protoplasts, bZIP63 can heterodimerize with members of the S-group, and in those assays bZIP63 homodimers were barely detectable. bzip63 mutant.Several heterodimerization partners of bZIP63 are well characterized in terms of their transcription regulatory targets. Therefore, it is apparent that their regulatory functions might be modified by bZIP63, where heterodimerization capability of bZIP63 could be controlled post-translationally, e.g. by phosphorylation through SnRK1 and potentially other protein kinases. This idea should be introduced clearly, with some necessary references to the functions of suspected bZIP63 dimerization partners at the beginning of the manuscript and discussed in this term at the end. For example, the observed effect of bzip63 mutation on the levels of free amino acids could be related to the function of its heterodimerization partner bZIP1. So, probably citing a recent paper from Para et al.  may be helpful for discussion of metabolite changes observed in the bzip63 mutation could influence the function of bZIP1, i.e. if we assumed that the bZIP1 functions as heterodimer with bZIP63. It is therefore interesting that overexpression of AKIN10 in protoplasts has only a slight effect on heterodimerization of overexpressed Gal-AD-bZIP1 and Gal-BD-bZIP63. While this artificial protoplast dimerization assay in Evidently, the Although the Discussion takes care to announce that “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”, it is rather flat. Somewhere, it should mention that the current study did not answer the question of whether the observed phosphorylation sites would modify the cellular location (i.e. nuclear import) and stability of bZIP63, in addition to its heterodimerization, which should be anyhow confirmed by co-IP studies using native gene constructs.akin10 mutant (i.e. a necessary but missing control).Unfortunately, the majority of the Discussion includes high-flying general statements, such as, “Here we show that bZIP63 plays an important role in the energy starvation response and metabolic regulation…”. If this is so, the reader will ask whether transcription of bZIP63 and its heterodimerization partners is controlled by AKIN10 and is altered in the bzip63 mutant?Can the enhanced senescence caused by bZIP63-ox compensated by glucose or sucrose supply? Is there any evidence for enhanced ROS production in the bZIP63-ox lines or change or red-ox regulation in the “We measured strong differences in proline and asparagine levels in the bZIP63 ko and ox […] like altered carbon or nitrogen assimilation or protein turnover” (in the subsection “bZIP63 is an important metabolic regulator in the starvation response”). So, how would bZIP63 control these mechanisms? Through controlling genes involved in N or C signaling or uptake?You say: “We found that the SnRK1 kinase AKIN10, which was proposed to be a central regulator of transcription in starvation response , is the ASN1 and ProDH by bZIP63”. This may be, but AKIN10 alone also activates these reporter genes, probably through other factors, which according to this paper are probably not the S-group bZIPs (i.e. as they appear not to be phosphorylated by AKIN10). It would be fair to mention this somewhere.In the same subsection, you state: “Reporter activation assays in protoplasts revealed that these  sites are essential for AKIN10-dependent induction of ProDH expression in plants, as compared to the protoplast assay, is probably due to redundancy within the C/S1 group bZIPs…”. Does this sentence predict that other C-group bZIPs could be SnRK1 substrates? Would it be not necessary to discuss potential conservation of observed AKIN10 sites in other bZIPs?Still in the same subsection, the authors say: “The relatively small difference in akin10 mutant plants and that of the bZIP63 S29A/S294A/S300A protein obtained from bzip63 plus/minus sugar or the akin10 mutant.In the same part of the text, the authors state: “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.” This should clearly be demonstrated by identical Phos-tag gel patterns of bZIP63 isolated from sucrose-treated or Reviewer #2:In this manuscript, Mair et al. report the function of bZIP63 as a key regulator in plant starvation response. Furthermore, using a combination of biochemical and omics approaches, the authors demonstrate that bZIP63 is regulated by protein phosphorylation and the kinase involved is AKIN10, an AMPK/Snf1/SnRK1 member. Phosphorylation of bZIP63 by AKIN10 changed its dimerization preference, thereby affecting target gene expression and ultimately plant primary metabolism.This report identifies a missing link between energy sensing and transcriptional reprogramming of metabolism in plants under starvation conditions. The topic is of interest to a wide audience. The data are of high quality, clearly laid out, and mostly convincing. I only have one suggestion that should clarify the Phos-tag results and further enhance the quality of the paper:Phos-tag analysis of in vitro phosphorylated bZIP63 samples allows the attribution of each shifted band to the phosphorylation of a specific Ser residue . In contReviewer #3:The authors demonstrate low energy stress-dependent SnRK-catalyzed direct phosphorylation of bZIP63 transcription factor as prerequisite for differential bZIP homo/hetero dimerization. Their comprehensive clear data provide for the first time mechanistic insight in how transcriptional reprogramming of gene subsets in a given pathway is driven by a conditional differential hetero/homodimerization of key transcriptional regulators.The manuscript is well written, clear in its argumentation, and statements are well backed by an almost overly thorough analysis and a large amount of data of high quality. I would like to comment on two aspects:1) Because SnRK and bZIP are well known proteins in plant biology, and because phosphorylation of bZIPs has been reported before, the true novelty in the manuscript is in my opinion the phosphorylation-dependent differential dimerization leading potentially to a change in transcriptional specificity plus activity toward distinct target genes (summarized in i) Differential dimerization on protein level: conducting for example transient co-localization studies based on (multi-color) fluorescent proteins/BiFC , or usinii) Show differential target gene specificity: 2) The primary identification of “in vivo” phosphorylation sites is conducted on bZIP-GFP overexpressing lines on material harvested in both light and prolonged dark. Therefore, basic constitutive bZIP phosphorylation (and the involvement of other kinases than AKIN10) cannot be excluded. Ideally, one may ask for the degree of conditional in vivo phosphorylation (in particular at S29 of endogenous bZIP) performing MRM/SRM phosphoproteomics. [Editors’ note: the author responses to the first round of peer review follow.]We have carefully read the reviewer’s comments and found that although some points that are mentioned are valid and we agree that these have to be addressed, we also saw that some parts of our manuscript were not written clearly enough and have been misunderstood. Therefore we would like to respond here directly to two issues that we think are the major issues for all three reviewers and ask furthermore for concrete feedback regarding the resubmission of a revised version of this work. To this end, we outline the experiments and further clarifications we would propose for a revision.akin 10 mutant:1) The akin10 mutant, which we used for our study, and we apologize for not including that in our manuscript. This mutant has been used – and confirmed – by three groups in our consortium independently. GABI_579E09) is indeed a valid akin10 null mutant. The T‐DNA insertion was indicated at the very 3’ end of the AKIN10 gene directly in front of the last exon  in the akin10 line. These data were completely independently obtained in the Baena‐ Gonzalez lab as well as in our lab.We checked the expression level of AKIN10 protein by Western blotting using two antibodies: firstly the AKIN10 specific antibody from Agrisera (AS 10919), and secondly an antibody against the phosphorylated T‐loop threonine (T172) of AMPKα, the mammalian AKIN10 ortholog , that specifically recognizes the phosphorylated T‐loop threonine in AKIN10 and AKIN11 . The position of the peptide recognized by each antibody is indicated in akin10 mutant, we include here also a phenotypical growth analysis of the akin10 mutant in akin10 mutant displays normal growth with respect to leaf number, fresh and dry weight as well as water content. As can be seen in akin10 mutant displays a little delay in flowering as compared to the wt, but it is flowering and produces viable seeds without problem. This is also in perfect agreement with the data on the AKIN10 RNAi line reported by AKIN10 RNAi lines described in that paper showed also normal growth. It is the akin10/11 double mutant that is apparently not viable and when an akin10/11 knockdown is generated through VIGS, the plants show premature senescence that precludes flowering. It is also shown there that AKIN10 overexpression alters inflorescence development.As reviewer 1 raised also concerns regarding a potential growth phenotype of the expression levels of AKIN10 target genes in response to six hours extended night treatments in this akin10 line (ASN1 (DIN6) and ProDH. DIN10 (raffinose synthase) was not affected under this conditions, but this time point might already be a bit too late, as we observed clear changes in the induction of all three genes already starting after two hours EN in the bzip63 mutant ; thirdly, the remaining background in target phosphorylation (i.e. bZIP63) as, for example, seen in the phos‐tag analysis  In vivo phosphorylation status of bZIP63 detected in phos‐tag gels:Reviewer 1 and 2 raised several questions about the phosphorylation status of bZIP63 as detected by Western blotting of phos‐tag gels. As reviewer 1 pointed out, so far no transcription factor has rigorously been shown to represent a direct phosphorylation target of SnRK1 in plants. Therefore, we think that the phos‐tag data, which show the in vivo phosphorylation status of bZIP63, are crucial for this manuscript and we feel that we need to explain these better in different conditions, treatments and of AKIN10.In order to better illustrate the different phosphorylation status and to allow a better comparison of the different experiments, we prepared a figure that shows a direct comparison of all phos‐tag gels from the different experiments .As control for the non‐phosphorylated bZIP63, the recombinant protein is shown in the very left panel. For all other samples we have now labelled the bands from 1‐7 according to an increase in their phosphorylation status. This is the maximum of different isoforms we could identify on different phos‐tag gels and corresponds to the seven identified in vivo phosphorylation sites from the combined MS analyses. Of course, not all isoforms are always visible on the gel as they depend on the phosphorylation level, and furthermore, the phosphorylation status is also highly dynamic, which complicates the picture. Nevertheless, the main point we want to make in the manuscript – and we think this is clearly evident from the figures – is that the level of bZIP63 phosphorylation changes in response to the different treatments and is furthermore depending on AKIN10.akin10 mutant. This might be considered as a small difference but these data should be seen in the overall context. In the genomic complementation, lines bZIP63 are almost exclusively detected in bands 5 and 6 in the light (so the same as before), and now most of the bZIP63 protein is shifted to the most phosphorylated form in band 7 in the extended night (EN). Strikingly, in the line complemented with the version of bZIP63 in which all three AKIN10 target sites are mutated to an alanine (S/A), ZIP63 is detected only in the bands 1 and 2  under all conditions. This makes clear that 1) the 3 mutated sites are the main in vivo phosphorylation sites and 2) the increased phosphorylation in EN happens at one of these sites.In the first panel from the left, it can be seen that, in the light, bZIP63 is already highly phosphorylated – which is not surprising considering seven identified in vivo phosphrylation sites and several potential kinases. However, the majority of bZIP63 is present in bands 5 and 6. In response to starvation (6 hrs extended night EN), band 7 appears as higher phosphorylated form. This new band 7 is barely detectable in the EN samples from the akin10 line. This is indeed true, however, considering again that we identified more than one kinase as potential upstream regulators of bZIP63 this is not surprising and does not mean that AKIN10 does not phosphorylate bZIP63 in vivo. As mentioned before, one likely explanation for the differences in phosphorylation is that AKIN11 might take over part of the function of AKIN10 in the akin10 mutant. This would also explain the lack of a strong phenotype in the akin10 line  could also phosphorylate one or more of the three target sites of AKIN10. We will discuss this aspect more clearly in a revised version.As reviewer 1 pointed out, the band pattern in this line (GAY14) is not the same as in the Reviewer #1:Plant SnRK1 kinases are structural and functional homologs of yeast Snf1 and animal AMP-activated kinases that play a central role in sensing energy depletion and maintaining cellular energy homeostasis. In this paper a multinational consortium set out to demonstrate that Arabidopsis SnRK1, carrying the AKIN10 catalytic subunit, phosphorylates in vivo the C-group bZIP63 transcription factor. bZIP63 has been identified to control transcription of some SnRK1-regulated target genes, involving ASPARGINE SYNTHASE 1 (ASN1/DIN6), PROLINE DEHYDROGENASE (PDH1/PRODH), RAFFINOSE SYNTHASE (DIN10) and SENESCENCE 1 (SEN1). To justify the statements in the Abstract and Introduction that bZIP63 is a key regulator of starvation response, the paper starts with limited characterization of a bzip63 T-DNA insertion mutant, and two bZIP63-ox overexpression lines.bZIP63 ko and ox lines was provided in Figure 6–figure supplement 6A-C as well as in the Methods section on plant lines in the previous version of the manuscript. To make the information more accessible, we moved the characterization of the plant lines to a new figure in the beginning of the manuscript  caused by energy depletion (see qPCR data) and that this phenotype can be rescued by addition of sugar.The authors use a phenotypic assay by placing plants for 9 days into the dark and monitoring rosette leaf senescence/bleaching by ImageJ scanning of green versus bleached/yellow leaf areas. Whereas in wild type (wt) the first two leaves (numbered 3 and 4 in) show bleaching, they remain greenish in the bzip63 mutant (defined as stay-green phenotype), and 3-5 more leaves undergo bleaching in the bZIP63-ox plants, suggesting enhanced senescence response.true leaves. Cotyledons were not included. Therefore the leaves numbered with 3 and 4 are not leaf 1 and 2 but really leaves 3 and 4.We would like to correct the statement of the reviewer. As indicated in the text, all leaves used for senescence analysis were When grown in the light, the bzip63 and bZIP63-ox plants do not differ from wt.Next, the authors compare metabolites entering or derived from glycolysis and citrate cycle in light-grown (L) and dark exposed  plants and find that the levels of “most” free amino acids is increased in the bzip63 mutant and decreased in bZIP63-ox plants, whereas sugar levels are stated to show an opposite trend of changes. Based on these data the authors conclude that bZIP63 is a key regulator of starvation responses (see Abstract and Introduction).When inspecting the metabolite profiling data, it is clear that the discussion of the results is rather superficial. For example, raffinose levels are decreased, whereas trehalose levels are increased in both mutant and ox lines, similarly to glucose in the dark. In general, we do not learn much from the metabolomics data. The actual conclusion is that “misregulation of bZIP63” (in the subsection “bZIP63 controls dark induced senescence and primary metabolism”) has an effect on metabolism.We agree with this and have intensively studied the literature and found a number of relevant links of our metabolite data to starvation and senescence. We have therefore completely re-written this part and are fully confident that the data now do clearly support the DIS phenotype and the role of the AKIN10-bZIP63 module in regulating primary metabolism in the low-energy response.On the other hand, we do not find any additional data , which would provide a further insight documenting how alteration of bZIP63 levels modulate the starvation-induced and/or senescence responses.We now include quantitative chlorophyll measurements of plants before and after 9 days in darkness . ResultsCAB2), as a marker for active photosynthesis was quickly downregulated, while SENESCENCE 1 (SEN1) and SENESCENCE ASSOCIATED GENE 103 (SAG103), were strongly upregulated. Both genes are known to be regulated by dark-induced senescence. In contrast, YELLOW-LEAF-SPECIFIC 3 (YLS3), which is induced by natural senescence, was not increased. We could not observe any strong differences in expression of these genes between wt and bzip63 plants, indicating that bZIP63 is not involved in the signaling pathways upstream of these senescence markers.Further, to confirm that the phenotype we observe is related to dark-induced and not age-induced senescence we did RT-qPCR of marker genes for dark-induced and natural senescence . Early tbzip63 plants could be related to the metabolic phenotype we observed. Amino acids can serve as alternative energy under starvation conditions . We therefore tested whether the dark-induced senescence phenotype is energy related and could be complemented by the addition of sugars. We found that glucose can indeed almost completely rescue the dark-induced senescence phenotype of bZIP63 mutant in seedlings  isoforms of bZIP63-GFP immunoprecipitated from overexpressing plants. On the Phos-tag gel, they show a rather weak band termed “supershift” of bZIP63, which appears in dark-treated plants, and detect somewhat weaker bZIP63-GFP bands with altered pattern plants grown with 1% sucrose. They use the latter result, without explicit explanation or demonstration, to suggest that the observed phosphorylation changes reflect inhibition of SnRK1 by 1% sucrose.not claim at any point in the text that this is the case. We merely suggest that sugar feeding counters the starvation resulting from cultivation in extended dark conditions.Although it is tempting to suggest a connection between the reduced phosphorylation of bZIP63 in the presence of sucrose and the activity of SnRK1, we do However, it is possible that some of the difference in bZIP63 phosphorylation between seedlings incubated in the presence and absence of sucrose is due to altered SnRK1 activity. T6P levels increase and decrease in parallel to sucrose levels and are rapidly depleted in the presence of sucrose  and it has been shown that in seedlings and young leaves T6P has an inhibitory effect on SnRK1 activity .By mass spectrometry analysis, seven phosphorylated residues were then identified in the immonoprecipitated bZIP63-GFP protein. Subsequent in-gel kinase tests indicate that bZIP63 is phosphorylated by multiple protein kinases showing apparent molecular masses of 50-60 and 35-38 kDa. In, it should be clearly indicated that the plant extract used for in-gel kinase assays derived from hydroponic root cultures , along with the conditions used for root culture in the case of the experiment inWe assume the reviewer means 3A in the original manuscript .To isolate the kinases detected in the in-gel assay, GST-bZIP63 was immobilized on GST-Sepharose and plant protein extracts made from root cultures or root-derived cell suspension  were subjected to affinity binding followed by NaCl elution. The eluted proteins resolved between 30 and 60 kDa were isolated from in-gel kinase PAGE (if I understood well), and identified by mass spectrometry. From 28 candidates only 7 kinases  were identified by 2-9 peptides. It should be noted in the Materials and methods how the excess of His6-bZIP63 was filtrated out during the mass spectrometry analysis.The reviewer misunderstood the use of in-gel PAGE for protein identification. Proteins were isolated from a non-substrate gel. This is shown in the scheme in In addition, it would be worth knowing whether the GST-alone matrix had retained any of these kinases.Unfortunately, we never tested whether any of the kinases phosphorylating bZIP63 binds to a GST-coupled column. Affinity purification was employed in the first place to reduce the complexity of the sample and at the same time enrich for bZIP63 binding proteins before MS analysis. For kinase identification we cut out only those regions giving a signal in the in-gel kinase assay and considered only kinases with the correct molecular weight as a further restrictive step.It is possible that one of the kinases identified by MS binds to GST and not bZIP63. However, for the “hot candidates” among the kinases we conducted additional assays  to confirm their interaction with bZIP63 and we tested phosphorylation of bZIP63 by these kinases. Results for SnRK1 subunits can be found (among others) in Furthermore, αAKIN10 western showing an enrichment of AKIN10 on the GST-bZIP63 matrix versus GST would be more convincing.We did not do a western blot as we did not have an antibody against AKIN10 at the time we undertook the affinity purifications (2008). However, we are routinely doing proteomics analysis in our department and SnRK1 peptides are usually not detected in total protein extracts with shotgun proteomics. As we repeatedly found a large number of peptides for AKIN10 and AKIN11 in our affinity purification samples, this is a very good indication that these kinases have been enriched.From here on, the authors concentrate on the analysis of bZIP63 phosphorylation by the SnRK1 catalytic subunits AKIN10, which according to Baena-Gonzalez et al. and Jossier et al. , is at least 10-20 times more active/abundant compared to its homolog AKIN11 in Arabidopsis.illustrates that GST-AKIN11 purified from E. coli also phosphorylates bZIP63 less efficiently compared to GST-AKIN10 in vitro. It is strange that no autophosphorylation of AKIN10 was ever detected by the authors either in normal or in-gel kinase assays. Any explanation?We assume the reviewer means 4B in the original manuscript . They also showed akin10 and akin11 single mutants, which did not have a striking phenotype. Single mutants of AKIN10 (RNAi) were not smaller than wt and flowered normally, which is probably due to the action of AKIN11. Therefore, the lack of a strong phenotype in our akin10 T-DNA insertion line is not in contrast to the data published in Baena-Gonzalez, but rather supports the findings described there. A molecular and phenotypic characterization of the GABI-KAT akin10 line can be found in The reviewer confuses the plant lines described in Baena-Gonzalez et al. . The mutant showing the strong phenotype in the Baena-Gonzalez paper is an Furthermore, the analysis of transcription of bZIP63 target genes should provide further clear evidence that post-translational regulation of bZIP63 is indeed impaired in the GABI_579E09 T-DNA insertion mutant.ASN1, DIN10, and ProDH in the akin10 line after 6h of light and extended night .Not many bZIP63 target genes are known. We tested the expression of ed night . ASN1 anThis is becausedoes not really show a very convincing change in the Phos-tag pattern of phosphorylated bZIP63 despite a lower level of “supershift” indicated by asterisks .akin10 background in the light and in extended night. Only the upper band is reduced . As SNF4 interacts strongly with bZIP63, it might act as a link to bring bZIP63 and AKIN11 together.No, we did not map the interaction sites. We observed a very weak interaction with AKIN11 in tobacco and no detectable interaction in yeast. This probably reflects the affinity of the kinase subunit alone to the bZIP and fits to the observed reduction in phosphorylation of bZIP63 by AKIN11. However, in vivo AKIN11 probably still plays a role in bZIP63 phosphorylation. SnRK1 forms a trimeric complex, which always includes SNF4  and αAKIN10 western from wt and akin10 mutant plants.We tried co-IPs from the genomic complementation lines. However, we could not successfully pull down AKIN10. One possible explanation for this is that, as the reviewer stated above, interactions between a kinase and its substrate are mostly not very strong and often not very long lived. We can therefore expect that only a fraction of bZIP63 is bound to AKIN10 at any given time and that there is not enough bound AKIN10 to be detected in the western blot. This analysis is furthermore complicated because bZIP63 is expressed at very low levels and very hard to detect , therefore it is again not surprising that we could not detect it in a co-IP where most likely only a fraction will bind very transiently to the kinase.According to, the putative upstream activating kinase SnAK2 enhances the activity of AKIN10 (and may be that of AKIN11). SnAK2 is supposed to phosphorylate the T-loop of both AKIN10 and AKIN11. Yet, we do not see enhanced phosphorylation of GST-AKIN10/11 in this figure. Any explanation?The phosphorylation of AKIN10 and AKIN11 in the presence of SnAK2 is enhanced, as can be seen in As GST-AKIN10/11, GST-bZIP63 and GST-SnAK2 have closely similar masses, it would be necessary to resolve them in a lower % of SDS-PAGE.The bands are close together, but still distinguishable. Only SnAK2 is very close in size to bZIP63. We provide all controls to show that the substrate, and not the kinases, is highly phosphorylated.To better separate the bands, it would be necessary to run the gels longer, which we would like to avoid as otherwise radioactive nucleotides run into the running buffer.In vitro phosphorylation of bZIP63 by AKIN10 and AKIN11 looks convincing in, so this approach should have provided a simple means to determine both AKIN10 and AKIN11 phosphorylation sites comparatively in bZIP63 by mass spectrometry. Yet, in, a complicated intuitive combination of alanine replacements of in vivo observed seven serine phosphorylation sites is used for demonstration that GST-AKIN10 probably phosphorylates only S29, S294 and S300 residues of bZIP63.Kinase assays with non-phosphorylatable mutants of potential kinase target sites are a standard technique to pin down the likely kinase target sites. We do not think that this strategy is complicated and unusual.Furthermore, as was mentioned in our manuscript, it is extremely difficult to cover the total protein with MS as some areas do not produce suitable tryptic peptides. Therefore an MS based approach was not our first choice.The referred “consensus SnRK1 sites” inrequire a corresponding reference either in the text or figure legend.The reference was added in the figure legend.The promoter activation assays in protoplast support the conclusion that S29 is the critical AKIN10 phosphorylation site, but in combination with the S294A and S300A amino acid exchanges, AKIN10-S29A shows lower transactivation of ASN1/DIN6-GUS and ProDH-GUS reporters. Whereas sequential phosphorylation might indeed trigger threshold-dependent conformational change of TFs, it is premature to conclude anything about possible order of phosphorylation sites .We removed speculations about the order of phosphorylation.In the subsection “The AKIN10 target sites play an important role for bZIP63 function in planta“ it is stated that: “The phosphorylation status of the wt construct after 6h of light and extended night and in the presence or absence of sugar was similar to the one observed in ox#3 plants .” Concerning the reproducibility and similarity of Phos-tag gels, would it be possible to run on the same gel the reactions shown inand/orwith, as well as Figure 4–figure supplement 3 and, to have an idea about the identity/mobility of phosphorylated bands in the respective reactions? Probably, it would also be beneficial to compare the Phos-tag assays on the same gel with the reactions shown in. This would be necessary because the Phos-tag gel depicted inlooks dramatically different for the GY11 line in L and EN compared to the 35S:bZIP63-GFP line in.Unfortunately, it is impossible to run all samples on one gel due to technical limitations, i.e. loading capacities. As the identity of the bands on the Phos-tag gels seem to be too confusing, and comparison between figures is difficult, we have first aligned all Phos-tag gels and put them in a supplementary figure for convenient comparison .Further, we marked all bands and labeled them persistently with numbers from 1-8 in each of the figures according to an increase in their phosphorylation status. This is the maximum of different isoforms we could identify on different Phos-tag gels and corresponds to the nonphosphorylated form plus the seven identified in vivo phosphorylation sites from the combined MS analyses. Of course, not all isoforms are always visible on the gel as they depend on the phosphorylation level and, furthermore, the phosphorylation status is also highly dynamic, and not every phosphorylation causes a similar strong shift, which complicates the picture. Nevertheless, the main point we want to make in the manuscript – and we think this is clearly evident from the figures – is that the level of bZIP63 phosphorylation changes in response to the different treatments and is furthermore depending on AKIN10.In the same subsection, I do not understand the logic of the following sentence: “Notably, hyper-phosphorylation of bZIP63 in the extended night was even stronger in the GY lines than in ox#3, probably due to lower bZIP63 expression.” Do the authors suggest that AKIN10 activity is limiting in terms of bZIP63 phosphorylation even if AKIN10 was supposed to be activated by extended night?The expression of bZIP63 in the genomic line is of course lower than in the ox lines. Therefore the ratio between bZIP63 and AKIN10 is also lower in these plants, which means that there is more AKIN10 per bZIP63 in the wt situation, which corresponds to the Phos-tag gel, where more bZIP63 is shifted to the hyper-phosphorylated form.With the limitations mentioned above concerning the necessary parameters of bzip63 mutant characterization, the lack of complementation by the triple S/A bZIP63 mutant gene is documented. As the mutant gene constructs were generated by PCR of 2+xkb genomic DNA fragments, the Materials and methods should mention that all constructs were verified by sequencing.The constructs were of course verified by sequencing. We have added this information in the Materials and methods section.In, it is rather surprising that the GY11 wt complementing line shows significantly higher induction of ASN1 and DIN10.ASN1.The line is somewhat over-expressing see , which cIn the fold-induction curve and column diagram to the right, the standard error of Ws-2 is huge at 4h of EN, thus no significant difference between wt and bzip63 mutant can be claimed.We did the appropriate statistical tests and found significant differences. Looking at standard deviations alone is not sufficient to deduce significant differences. Also, the error bars are the standard deviations and not standard errors.In this figure part, either the curves shown to the left should include all four samples  or all samples at the four time points should be presented as column diagrams.The two halves of the figure serve different purposes. We first did a time course (left) with wt and ko lines to determine the best time point for comparison of the gene expression. This was done with only two lines to reduce sample numbers. The 4h time point was chosen to test also the complementation lines.Given the data presented in, I wonder why did the authors use the low expressing GY11 line for comparison with GAY14.The lower expressing line was chosen, because bZIP63 expression is more similar in these lines .Fromit is also apparent that the GAY4 line shows no expression of triple S/A mutant bZIP63-YFP, so only one bzip63 mutant transformed with the triple phosphor-site mutant of bZIP63 was analysed.The RT-qPCR in Ehlert et al. reported that, when overexpressed in plant protoplasts, bZIP63 can heterodimerize with members of the S-group, and in those assays bZIP63 homodimers were barely detectable.reported later that bZIP63 also interacts with the Arabidopsis response regulator ARR18 involved in cytokinin signaling, whereas alone (i.e. by interacting with S-group partners in heterodimers), bZIP63 functions as negative regulator of osmotic stress responses and seed germination.Arabidopsis Response Regulator ARR18 with bZIP63 Mediates the Regulation of PROLINE DEHYDROGENASE Expression).This was reported by Veerabagu et al.  may be helpful for discussion of metabolite changes observed in the bzip63 mutant.We agree, and have introduced that reference in the Introduction and indeed re-written the entire discussion of the metabolite data and highlighted the similarities and complementary aspects with bZIP1. We are confident that in the revised version the entire story is indeed much more stringent and fits nicely together also with respect to the other published data on bZIP1.Evidently, the bzip63 mutation could influence the function of bZIP1, i.e. if we assumed that the bZIP1 functions as heterodimer with bZIP63. It is therefore interesting that overexpression of AKIN10 in protoplasts has only a slight effect on heterodimerization of overexpressed Gal-AD-bZIP1 and Gal-BD-bZIP63. While this artificial protoplast dimerization assay inindicates over 60-fold enhancement of Gal-AD-bZIP11+Gal-BD-bZIP63 dimerization, at the level of GAL-UAS4:GUS reporter, about 15-fold increase is observed, which is reduced to about 2-3-fold by triple mutation of the identified S29/S294/S300 bZIP63 phoshorylation sites. In case ofcould not fully understand why the authors used two different (i.e. GAL-UAS4:GUS and GAL-UAS4:LUC) reporters in the dimerization and reporter activation assays. This correlative evidence between the dimerization and reporter activation assay should definitely be strengthened by performing similar dimerization assays with bZIP63 carrying the S29A/S294/S300 phospho-site replacements. This appears to be obligatory.We agree that mixing the LUC and GUS system is not optimal and have therefore repeated the P2H assays of bZIP63 with bZIP1, 11, and 63 as interaction partners in the GUS system .While the general trend (addition of AKIN10 leads to stronger dimerization) remained the same, the magnitude of the change – especially for bZIP11 – is lower and fits better to the assays shown in Although the Discussion takes care to announce that “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”, it is rather flat. Somewhere, it should mention that the current study did not answer the question of whether the observed phosphorylation sites would modify the cellular location (i.e. nuclear import) and stability of bZIP63, in addition to its heterodimerization, which should be anyhow confirmed by co-IP studies using native gene constructs.We did not observe an effect of S/A mutation on the localization of bZIP63 . It is therefore not likely that phosphorylation alters the subcellular localization of bZIP63. We have stated this now clearly in the manuscript.Arabidopsis bZIP transcription factors). Again, we did not see any difference between wt and mutant bZP63 in these assays, which we also mention in the text.Concerning DNA binding, we tested whether S/A or S/D mutation of the N- and C-terminal serines would have an effect on binding to the C-box consensus motif as it was shown for S160, 164, and 168 in the DNA binding domain .bZIP63, bZIP1, and bZIP11 are differentially expressed in wt and akin10 plants by RT-qPCR . So, how would bZIP63 control these mechanisms? Through controlling genes involved in N or C signaling or uptake?“As already mentioned before, the Discussion has been rewritten to emphasize that these higher amino acids levels do indeed fit very nicely to starvation and have also been observed in dark-induced senescence. One explanation might be the increased turnover (degradation) of proteins and their role as alternative energy source, particularly under low-carbon conditions. This is now discussed in the manuscript.You say: “We found that the SnRK1 kinase AKIN10, which was proposed to be a central regulator of transcription in starvation response , 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 subsection “bZIP63 function is regulated by SnRK1-dependent phosphorylation”). Yes, but the hyper-phosphorylation of bZIP63 under extended night  caused the appearance of only a weak band marked by asterisks on the images of Phos-tag gels, which represents the only evidence supporting this statement.This is a misunderstanding. Phos-tag gels were done after 6h of extended night not after 9 days of darkness. The extended night treatment represents a starvation condition for the plant.akin10 as well as snrk1 VIGS plants after 6h of extended night can be seen as good evidence that bZIP63 is a direct target of SnRK1.Further, although the hyper-phosphorylation of bZIP63 is sometimes not so strong in the ox plants, it is very strong in the genomic complementation lines, which likely better reflect the phosphorylation state of bZIP63 in the wt. We think that the reduced phosphorylation of this band in In total, we provide evidence for this statement by:(i) identifying AKIN10 as kinase for bZIP63 in an unbiased approach after affinity purification;akin10 mutant;(ii) a missing band in in-gel kinase assays with extracts from the (iii) (various) direct interaction and phosphorylation assays;akin10 mutant;(iv) a clearly reduced appearance of the hyper-phosphorylated form in the (v) even clearer results of this effect after VIGS of AKIN10 and AKIN11;bzip63 mutants, which cannot be complemented by a bZIP63 version lacking the AKIN10 target sites;(vi) starvation (low-energy) related phenotypes of the (vii) a missing shift in response to EN treatments in the S29/S294/S300 mutant.In the same subsection, you state: “Reporter activation assays in protoplasts revealed that these  sites are essential for AKIN10-dependent induction of ASN1 and ProDH by bZIP63”. This may be, but AKIN10 alone also activates these reporter genes, probably through other factors, which according to this paper are probably not the S-group bZIPs (i.e. as they appear not to be phosphorylated by AKIN10). It would be fair to mention this somewhere.The activation of the reporter genes by AKIN10 and a possible explanation had been mentioned in the text .Still in the same subsection, the authors say: “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…”. Does this sentence predict that other C-group bZIPs could be SnRK1 substrates? Would it be not necessary to discuss potential conservation of observed AKIN10 sites in other bZIPs?ProDH and therefore bZIP63 might not be necessarily required for ProDH expression.No, this sentence does not imply that other C-group bZIPs are SnRK1 targets . We rather wanted to underline that several bZIPs have been found to regulate In the same part of the text, the authors state: “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.” This should clearly be demonstrated by identical Phos-tag gel patterns of bZIP63 isolated from sucrose-treated or akin10 mutant plants and that of the bZIP63 S29A/S294A/S300A protein obtained from bzip63 plus/minus sugar or the akin10 mutant.We do not agree with this statement. We believe that bZIP63 is phosphorylated by several different kinases, which makes the phosphorylation pattern more difficult to interpret.akin10 and akin10/11 mutants showed that bZIP63 is phosphorylated by more than just AKIN10, as phosphorylation was not abolished completely in these mutants  and cannot be simplified as the reviewer suggests.Reviewer #2:Phos-tag analysis of in vitro phosphorylated bZIP63 samples allows the attribution of each shifted band to the phosphorylation of a specific Ser residue . In contrast, the same analyses of in vivo samples showed variable banding patterns that are not easy to reconcile .As the comparison of different Phos-tag gels was difficult for several reviewers, we have aligned all Phos-tag gels in one figure  and intrThis could be a result of phosphorylation of bZIP63 by other kinase(s) on S59, S102, S160, and S261, four sites that are not phosphorylatable by AKIN10. I would suggest the authors to determine whether the mutation of these four Ser residues to Ala will affect the function of bZIP63. If it does, the data would reveal that bZIP63 is regulated by multiple kinase pathways to carry out its function in the energy transition response. If it does not, the data would demonstrate that AKIN10-phosphorylation on S29, S294 and S300 is the major phosphorylation event that controls the activity of bZIP63.We do believe, as the reviewer suggested, that other kinases also phosphorylate bZIP63. However, as we focused on SnRK1 regulation of bZIP63 in this manuscript, we have not analyzed the functionality of these other sites. This would be a time-consuming process and is well beyond the focus of this manuscript. We have therefore not included additional data on the non-AKIN10 target sites.Arabidopsis bZIP transcription factors), but for the other sites, no data on functionality are available.For S160 it has been shown that it can potentially reduce DNA binding of bZIP63 when phosphorylated . It would be wrong to conclude that only AKIN10 would phosphorylate the remaining three sites in this mutant, which would again complicate the interpretation of the results. This is based on the fact, that AKIN10 ko leads to the vanishing of only one band, while mutation of the three sites leads to a strong reduction in phosphorylation. In fact, in the S29/294/300A mutant, only two bands remain (the non-phosphorylated form and a presumably single phosphorylated form). Therefore, the phosphorylation pattern of S29+294+300 would again be a combined effect from more than one kinase.akin10/akin11 background, we added Phos-tag gels from VIGS ko of these two kinases  Because SnRK and bZIP are well known proteins in plant biology, and because phosphorylation of bZIPs has been reported before, the true novelty in the manuscript is in my opinion the phosphorylation-dependent differential dimerization leading potentially to a change in transcriptional specificity plus activity toward distinct target genes (summarized in– model). Data inrely on a protoplast two-hybrid assay, allowing a transcriptional readout .We have added a western blot to show the amounts of bZIP1, bZIP11, bZIP63, and AKIN10 in the P2H assay shown in To more prominently support the key message of the manuscript will have to address:i) Differential dimerization on protein level: conducting for example transient co-localization studies based on (multi-color) fluorescent proteins/BiFC , or using biochemical means to compare phosphorylation-dependent bZPI63/bZIP63 with bZIP63/bZIP11 formation, and in case even include bZIP S29D (or other) phospho-mimic constructs.We did pick up this suggestion by the reviewer, particularly as we noticed that the protoplast two-hybrid data do not allow a conclusion on preferred dimerization of different dimers because that assay is based on a transcriptional readout and leads to over-estimation of the effect on bZIP11 as mentioned before and also discussed in the revised manuscript. Therefore – as suggested by the reviewer – we have conducted a multi-color BiFC experiment to analyze the dimerization preferences of bZIP63 in a comparative in vivo assay .As exemplary dimerization partners for bZIP63, we have chosen bZIP1 and bZIP11. It is important to note here that of course many other bZIPs will also affect dimerization under the ‘real’ conditions in the wt. All three dimerization partners  were expressed under control of the 35S promoter from the same plasmid to ensure that all transformed cells express all three partners. A scheme of the BiFC experiment is shown in Dimerization of bZIP63 with bZIP1 reconstructs the VENUS construct and leads to ‘yellow’ fluorescence. Dimerization of bZIP63 with bZIP11 reconstructs the CFP construct and leads to ‘cyan’ fluorescence. Homo-dimerization of bZIP63 or dimerization of any of the three partners with an endogenous (untagged) bZIP does not lead to a signal.To see if there is a difference between bZIP63 dimerization with bZIP1 and 11, we co-transformed AKIN10 and compared the resulting signals obtained with the wt and the S29/294/300A version of bZIP63. This experiment allowed us to study the dimerization preference of phosphorylated and non-phosphorylated bZIP63. Accordingly, the bZIP63-11 hetero-dimer will generate a blue (cyan) signal and the bZIP63-1 hetero-dimer a yellow signal, whereas the bZIP63 homo-dimer cannot be detected. A quantification of the ratio of the VENUS/CFP signal obtained from more than 100 nuclei did clearly show that the co-transformation of AKIN10 did shift the signal towards the VENUS emission, thus showing that AKIN10 does preferentially trigger the formation of bZIP63-1 hetero-dimers in a competitive in vivo assay.At first glance, this seems to stand in contrast to the data obtained in the protoplast two-hybrid assays, but as already pointed out, these assays cannot be compared. As we have been mistaken by not realizing this problem before, we had also to revise our model accordingly . Howeverii) Show differential target gene specificity:contains as marker for transcriptional activity a gene which is target to all three bZIPs . Even more convincing will be phosphorylation/heterodimerization-dependent activity toward a distinct bZIP11- or bZIP63-controlled gene, respectively.Yes, we agree, this would indeed be a nice experiment and we would like to do that. Unfortunately, we do not know yet any target gene, which would be specific for only one of the different bZIP dimers. Microarray data are available for bZIP1 and bZIP11, but not for bZIP63. Moreover, also from these data, such genes cannot be deduced as the other different dimerization partners are still present. Maybe ChIP experiments would be a solution here, but that would clearly be beyond the scope of this study.2) The primary identification of “in vivo” phosphorylation sites is conducted on bZIP-GFP overexpressing lines on material harvested in both light and prolonged dark. Therefore, basic constitutive bZIP phosphorylation (and the involvement of other kinases than AKIN10) cannot be excluded. Ideally, one may ask for the degree of conditional in vivo phosphorylation (in particular at S29 of endogenous bZIP) performing MRM/SRM phosphoproteomics.To address this question we generated a genomic complementation line expressing the S294/300A version of bZIP63 in the ko background. In this line, S29 can be phosphorylated, but none of the other potential AKIN10 target sites. Due to the short time we had for the revision, this plant line is still heterozygous and cannot be used for phenotypic analysis at this point. However, we could use the line for Phos-tag gels and we here we saw indeed a striking effect: Compared to the triple mutant, that did not show any change in the phosphorylation pattern in response to starvation, the S294/S300 double mutant did still show a strong shift 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. We think therefore we did add really significant novel information, and more is simply not possible in the few months we had for the revision.