- © 2011 American Society of Plant Biologists
Abstract
Control of energy homeostasis is crucial for plant survival, particularly under biotic or abiotic stress conditions. Energy deprivation induces dramatic reprogramming of transcription, facilitating metabolic adjustment. An in-depth knowledge of the corresponding regulatory networks would provide opportunities for the development of biotechnological strategies. Low energy stress activates the Arabidopsis thaliana group S1 basic leucine zipper transcription factors bZIP1 and bZIP53 by transcriptional and posttranscriptional mechanisms. Gain-of-function approaches define these bZIPs as crucial transcriptional regulators in Pro, Asn, and branched-chain amino acid metabolism. Whereas chromatin immunoprecipitation analyses confirm the direct binding of bZIP1 and bZIP53 to promoters of key metabolic genes, such as ASPARAGINE SYNTHETASE1 and PROLINE DEHYDROGENASE, the G-box, C-box, or ACT motifs (ACTCAT) have been defined as regulatory cis-elements in the starvation response. bZIP1 and bZIP53 were shown to specifically heterodimerize with group C bZIPs. Although single loss-of-function mutants did not affect starvation-induced transcription, quadruple mutants of group S1 and C bZIPs displayed a significant impairment. We therefore propose that bZIP1 and bZIP53 transduce low energy signals by heterodimerization with members of the partially redundant C/S1 bZIP factor network to reprogram primary metabolism in the starvation response.
INTRODUCTION
Due to their phototrophic life style, plants have to steadily adjust their metabolism to day-night rhythms and environmental changes to withstand transient energy deprivation (reviewed in Baena-González and Sheen, 2008; Usadel et al., 2008). Low energy stress can easily be mimicked by the cultivation of plants in the dark. Extended dark treatment is correlated with dramatic changes in primary plant metabolism, in particular, reduced photosynthesis, degradation of proteins, amino acids, or nucleic acids, hydrolysis of polysaccharides, or oxidation of fatty acids. These physiological changes are accompanied by a massive reprogramming of transcription, which is reflected in several recent transcriptome profiling studies (Gan, 2003; Lin and Wu, 2004; Buchanan-Wollaston et al., 2005) In particular, the whole set of genes leading to the biosynthesis of Asn is reprogrammed in response to dark treatment. In comparison to Gln, the major transport form of nitrogen in the light, Asn, contains less carbon than Gln and is therefore used to store and transport nitrogen especially under stress conditions where carbon is limited (Lam et al., 1994).
Recently, the Arabidopsis thaliana SnRK1-like kinases (SNF1-related protein kinases 1) KIN10 and KIN11 have been proposed to function as central signaling integrators that mediate adaptation to low energy stress (Baena-González et al., 2007). These kinases show structural similarities to the SNF1 kinase (for SUCROSE NONFERMENTING1) in yeast and the AMP-DEPENDENT PROTEIN KINASE in mammals, which function as master regulators of the energy balance that is essential for survival under stress (Polge and Thomas, 2007). In Arabidopsis, the ASPARAGINE SYNTHETASE1 (ASN1) gene, which encodes the final step in Asn biosynthesis, was proposed to be a target of the KIN10/11 pathway that regulates the level of Asn (Baena-González et al., 2007). The dark- or stress-induced regulation of ASN1 expression is mediated specifically by a G-box cis-element (Baena-González et al., 2007; Hanson et al., 2008), which is typically recognized by basic leucine zipper (bZIP) transcription factors (TFs). bZIP proteins, which are exclusively found in eukaryotic cells, bind DNA by forming homo- or heterodimers. In the Arabidopsis genome, 75 bZIP genes have been identified and classified into 10 groups (Jakoby et al., 2002). Interestingly, only a specific subset of G-box binding bZIP factors was shown to activate ASN1 in transiently transformed protoplasts (Baena-González et al., 2007), namely, bZIP2 (GBF5), bZIP11 (ATB2), bZIP53, and bZIP1. Based on amino acid homology and specific heterodimerization properties with group C bZIPs, these proteins were classified as belonging to the S1 subgroup (Ehlert et al., 2006). These C and S1 bZIPs form a functional interlinked TF network (Weltmeier et al., 2009).
In a transcriptome analysis using plants expressing bZIP11 in a dexamethasone-inducible manner, ASN1 was shown to be regulated by bZIP11 (Hanson et al., 2008). Furthermore, bZIP53 regulates the expression of PROLINE DEHYDROGENASE (ProDH) during the hypoosmolarity response (Nakashima et al., 1998; Satoh et al., 2004; Weltmeier et al., 2006). ProDH degrades the compatible osmolyte Pro during recovery from stress.
To deal with diurnal changes in carbon supplies, plants retain some photosynthates as starch, which can be remobilized during the night (Usadel et al., 2008). However, within 2 to 4 h of an extended night, these resources are depleted, leading to severe limitation of carbohydrates. This metabolic process is demonstrated in the starchless pgm mutant from Arabidopsis, which uses up its carbohydrate resources within the first few hours of night, eventually leading to growth retardation (Usadel et al., 2008). Expression profiling of plants cultivated in an extended night regime allowed the construction of regulatory models, and these models suggest that plants respond to small changes in the carbon status in an acclimatory manner (Usadel et al., 2008). In this work, several group S1 bZIPs, including bZIP1 and bZIP53, were suggested to be involved in the plant’s response to carbohydrate starvation. Accordingly, systems biology approaches studying the integration of C- and N-derived metabolic signals proposed bZIP1 as a regulator in the nitrogen-responsive gene network, which includes the modulation of ASN1 expression (Gutiérrez et al., 2008).
Although several members of the group S1 bZIPs have been implicated in starvation responses and particularly in amino acid metabolism, experimental data are limited to gain-of-function studies in plant protoplasts. Here, we define bZIP1 and, to a minor extent, bZIP53 as transcriptionally and posttranscriptionally activated TFs in the low energy stress response of Arabidopsis. The impact of bZIP1 and bZIP53 on the starvation-induced transcription of key genes in amino acid metabolism and amino acid accumulation is demonstrated in protoplasts and transgenic plants. The results of loss-of-function approaches indicate that several partially redundant TFs of the C/S1 bZIP network cooperate to regulate plant low energy responses.
RESULTS
bZIP1 and bZIP53 Expression Is Enhanced during Dark-Induced Starvation
To identify candidate bZIP TFs, which are involved in regulating plant starvation responses, a screening of public expression databases (Hruz et al., 2008) and quantitative real-time PCR (qPCR) using RNA from plants exposed to extended darkness were performed. The expression of bZIP1 was strongly induced upon extended dark treatment and repressed by sugars (see Supplemental Figures 1A to 1C online). A minor but reproducible transcriptional induction was also observed for the closest bZIP1 homolog, bZIP53, but not for the other group S1 bZIPs. Based on these findings, bZIP1 and bZIP53 were selected as candidate transcriptional regulators to study the dark-induced starvation response of Arabidopsis.
To further substantiate these findings, a detailed time-course expression experiment was performed. Remarkably, a night extension of up to 4 h leads to an eightfold accumulation of bZIP1 transcripts (Figure 1A), which further increased during an extended night by up to 30-fold. bZIP53 transcripts accumulate only slightly during an extended night (three- to fourfold). Histochemical staining of plants containing promoter reporter constructs (ProbZIP1:GUS [for β-glucuronidase] and ProbZIP53:GUS) was used to demonstrate bZIP expression at the whole-plant level. Whereas the GUS staining of plants grown under a 16-h/8-h day-night cycle demonstrated a bZIP1 and bZIP53 gene activity only in young sink leaves (Weltmeier et al., 2009), the prolonged incubation in the dark led to a rapid spreading of expression patterns also into older, well-developed source leaves (Figure 1B). However, this response was not detected in transgenic lines harboring promoter:reporter constructs of other group S1 bZIPs (e.g., bZIP11 or bZIP44) (Weltmeier et al., 2009).
Analysis of bZIP1 and bZIP53 in the Low Energy Response.
(A) Expression of bZIP1 and bZIP53 increases after extended night treatment. Wild-type plants are cultivated at a day/night cycle of 16/8 h as indicated by the scheme. Day, night, and extended night phases are indicated by white, black, and gray bars, respectively. Transcript abundance as determined by qPCR is presented for bZIP1 (black bars) and bZIP53 (white bars). Rosette leaves of 10 3-week-old plants were pooled and used for RNA preparation and qPCR at the time points indicated. Fold change compared with wild-type expression is depicted at 0 h. Mean value and sd of two replicates are given.
(B) Histochemical GUS staining of transgenic plants expressing Pro-bZIP1:GUS (top panel) and ProbZIP53:GUS (bottom panel). The upstream regions of the constructs expressed in these plants (diagram above) contain the conserved system of uORFs (depicted by rectangles) that mediates sucrose-dependent posttranslational repression (Wiese et al., 2005; Weltmeier et al., 2009). GUS staining of plants grown under a 16/8-h day/night cycle (0 h) or darkness for 48 and 120 h is shown.
(C) Three-week-old plants expressing bZIP1 under control of the 35S promoter (Pro35S:bZIP1), the wild type (wt), bzip1, and the bzip1 bzip53 double mutant were analyzed for an enhanced senescence phenotype after 6 d in the dark.
(D) Relative chlorophyll content of rosette leaves of Pro35:bZIP53 and the plants depicted in (C). Plants were cultured in a normal day/night cycle (white bars) or for extended dark treatment as indicated. Significance was tested by one-way analysis of variance (ANOVA) analysis following Fisher’s LSD post-test, P < 0.05.
As demonstrated in previous studies (Wiese et al., 2004; Weltmeier et al., 2009), a posttranscriptional regulatory mechanism applies for all group S1 bZIPs, including bZIP1 and bZIP53, which leads to a sucrose-induced repression of translation mediated by a conserved system of upstream open reading frames (uORFs). With respect to bZIP1 and bZIP53, transcriptional and posttranscriptional mechanisms interact to enhance expression in response to dark treatment. By contrast, in particular bZIP11 shows an inverse regulation. Transcription is repressed by dark treatment and induced by sugar application (see Supplemental Figure 1B online). Hence, these differences in expression suggest a function for bZIP1 and bZIP53 in the dark-induced starvation response that is not shared by the other group S1 members.
Ectopic Expression of bZIP1 and bZIP53 Results in Enhanced Dark-Induced Senescence
To further study the function of bZIP1 and bZIP53 in the dark-induced starvation response, the phenotypes of plants ectopically expressing bZIP53 and bZIP1 (Pro35S:bZIP53 and Pro35S:bZIP1) and their HA-tagged versions (Pro35S:HA-bZIP53 and Pro35S:HA-bZIP1) under the control of the 35S promoter were analyzed (Weltmeier et al., 2006, 2009; see Supplemental Figure 2 online). Whereas Pro35S:bZIP1 plants grew normally under standard day-night cycles, Pro35S:bZIP53 plants showed a dwarf growth phenotype that depended on the expression level of the transgene (Alonso et al., 2009). Prolonged cultivation of the bZIP overexpressing lines in the dark resulted in an obvious phenotype (Figure 1C). In particular, the Pro35S:bZIP1 plants showed a faster dark-induced leaf yellowing, which was less pronounced for Pro35S:bZIP53 plants. Accordingly, the bZIP1 and bZIP53 overexpressing plants had significantly reduced amounts of chlorophyll after 4 d of cultivation in darkness (Figure 1D). However, bzip1 and bzip53 single and double mutants (see Supplemental Figure 2 online) did not show obvious alterations in comparison to the wild type.
The culture conditions were further analyzed using well-defined marker genes for ongoing leaf senescence (see Supplemental Figure 3 online). Whereas the chlorophyll a/b binding protein gene (CAB), a light-induced marker for photosynthetically active leaves, was transcriptionally downregulated in darkness (van der Graaff et al., 2006), the SENESCENCE ASSOCIATED GENE103 (SAG103), a marker for dark-induced senescence, was induced in wild-type plants after 24 h of extended night (at the 48-h time point). By contrast, the YELLOW LEAF SPECIFIC3 (YLS3) gene, a marker for natural senescence (van der Graaff et al., 2006), was not significantly affected in its transcription. We therefore conclude that the process observed during extended night is distinct from natural senescence and that ectopic expression of bZIP1 or bZIP53 enhances physiological responses that are correlated to dark-induced starvation.
bZIP1 and bZIP53 Regulate ProDH Transcript Level and Pro Content during Dark-Induced Starvation
ProDH, which encodes an enzyme that mediates the catabolism of Pro (Figure 2A), is a direct transcriptional target of bZIP53 in the hypoosmolarity response of Arabidopsis (Weltmeier et al., 2006). ProDH transcription was also induced after dark treatment, as demonstrated by RNA gel blot analysis (Figure 2B) and qPCR (Figure 2C). These data are in agreement with the hypothesis that, during the starvation response, amino acids are recycled to support C, N, and energy demands. Ectopic expression of bZIP1 and bZIP53 resulted in significantly higher levels of ProDH transcripts. However, whereas bZIP53 overexpression led to high ProDH transcript levels both in light and darkness, which was further enhanced by extended dark treatment, the regulation by bZIP1 differed, as ProDH transcript accumulation was preferentially enhanced in the dark (Figures 2B and 2C).
bZIP1 and bZIP53 Regulate ProDH Transcription and Pro Content during Dark Treatment.
(A) The ProDH enzyme regulates catabolism of the amino acid Pro to pyrrolin-5-carboxylate (P5C). Given is the complementary biosynthesis pathway based on Glu and making use of P5C as intermediate (Hellmann et al., 2000).
(B) RNA gel blot analysis of ProDH in the wild type (wt), Pro35S:bZIP1 (line C), and Pro35S:bZIP53 (line 10) (Weltmeier et al., 2006) in response to long-term dark treatment for 1 to 8 d. Plant material was harvested late in the afternoon (5 pm).
(C) Analysis of ProDH transcript accumulation in the wild type (black bars), Pro35S:bZIP1 (gray bars), Pro35S:bZIP53 (hatched bars), and bzip1 bzip53 (white bars) plants after short-term dark treatment as described in Figure 1A. Rosette leaves of 10 3-week-old plants were pooled and used for RNA preparation and qPCR at the time points indicated. Fold change compared with wild-type expression is depicted at 0 h. Mean value and sd of two replicates are given. Expression analysis of bZIP1 and bZIP53 is provided in Supplemental Figure 4 online. For visualizing the differences in transcript levels, the y axis is broken twice, at 1.5- and 30-fold induction.
(D) Quantification of Pro levels of the plants described in (B) and (C) after dark treatment. Given are ng Pro/mg dry weight (DW) as mean values and sd of two independent repetitions. Asterisks represent significant differences between wild-type, overexpressor, and mutant plants at the indicated time points (two-way ANOVA; *P < 0.05 and ***P < 0.001).
(E) The transcript abundance of bZIP1 and bZIP53 is regulated by sugar depletion. Three-week-old wild-type plants were cultivated in hydroponic culture as depicted in the scheme. Plants were harvested 3 h after the beginning of the light period (L). The remaining plants were incubated in darkness for 24 h (D). After 24 h, the plants were transferred to medium containing equimolar (167 mM) 3-oMG, glucose, sucrose, and PEG1000 or nonsupplemented medium as control (−). During these incubations, plants were kept in the dark. RNA was isolated from the differently supplemented cultures after 1 or 2 h, respectively. Given are RNA gel blot analyses of ProDH, bZIP1, and bZIP53 transcripts. Loading is controlled by ethidium bromide (EtBr) staining.
In addition to an overexpression analysis, we also performed loss-of-function studies using T-DNA insertion mutants of bZIP1 and bZIP53 (see Supplemental Figures 2 and 4 online). In contrast with single bzip1 and bzip53 mutants (see Supplemental Figure 5B online), a moderate but significant reduction in the dark-induced activation of ProDH transcript accumulation was observed in bzip1 bzip53 double mutants when compared with the wild type (Figure 2C; see Supplemental Figure 5B online). However, because the ProDH transcript level is still responsive to dark-induced starvation, additional, partly redundant, transcriptional regulators have to be postulated.
As the ProDH enzyme mediates Pro degradation, its activation should result in reduced Pro levels, which indeed was observed after transfer of wild-type plants to darkness (Figure 2D). Compared with the wild type and the bzip1 bzip53 double mutant, the Pro levels were significantly reduced in Pro35S:bZIP53 and Pro35S:bZIP1 plants (Figure 2D). This observation is in agreement with our postulated function of bZIP1 and bZIP53 in ProDH-mediated Pro degradation during the dark-induced starvation response.
Dark-Induced bZIP1 Expression Depends on Sugar Depletion
To elucidate whether depletion of sugars, which function as the major energy resource during night, or the absence of light function as important regulatory signals in the expression of bZIP1, bZIP53, and ProDH, we grew Arabidopsis plants in a hydroponic culture system under different carbohydrate regimes. As shown in Figure 2E, the transcripts of bZIP1, bZIP53, and ProDH coordinately accumulated after 24 h in the dark. However, transcriptional regulation of bZIP53 was always less pronounced when compared with bZIP1 (Figures 1A and 2E). The plants were then transferred to medium supplemented with equimolar concentrations of 3-ortho-methyl-glucose (3-oMG), glucose, sucrose, or polyethylene glycol (PEG) and further kept in darkness. 3-oMG serves as a control as it is taken up by the cells but does not trigger the glucose-specific sugar signaling pathways (Cortès et al., 2003). Sucrose and glucose, but not 3-oMG, repressed bZIP1, bZIP53, and ProDH transcript accumulation. These data suggest that sugar signaling and not the absence of light regulates the transcript accumulation of bZIP1 and bZIP53. As a putative target, the ProDH transcript level followed that of the two bZIP TFs, with a slower kinetic, as demonstrated by comparing the 1- and 2-h time points. It has been reported that changing of the osmolarity conditions also modulates the ProDH transcript levels (Satoh et al., 2004; Weltmeier et al., 2006). Hyperosmolarity conditions applied by PEG1000 treatment led to downregulation of ProDH but did not affect bZIP1 transcript accumulation. From these data, we conclude that, while ProDH transcript accumulation is regulated by several different stimuli, dark-induced energy starvation results in a bZIP1/bZIP53-dependent induction of Pro degradation.
bZIP1 and bZIP53 Regulate the Level of Asn and the Branched-Chain Amino Acids Leu, Ile, and Val
To determine whether bZIP1 and bZIP53 regulate amino acid metabolism in general during the dark-induced starvation response, a comprehensive amino acid analysis was performed. In wild-type plants, the total amount of amino acid increased in response to prolonged darkness. By contrast, Pro35S:bZIP53 plants showed a significantly stronger accumulation of total amino acids, whereas in the bzip1 bzip53 double mutant the increase was less pronounced (Figure 3A). During an extended night, an increase was observed especially for the levels of the branched-chain amino acids (BCAAs) Leu, Ile, and Val as well as for Asn (Figure 3B; see Supplemental Data Set 1 online). The increase in BCAA levels, in particular of Leu and Ile, was strongly repressed in the Pro35S:bZIP1 and Pro35S:bZIP53 plants, indicating that the bZIP regulators promote the degradation of Leu and Ile. The Val levels followed a similar accumulation pattern; however, it was less pronounced (Figure 3B; see Supplemental Data Set 1 online). The impact of bZIP1 and bZIP53 on Asn metabolism differed considerably from that of Leu, Ile, and Val. Dark-induced Asn levels were enhanced by the overexpression of bZIP53 but not bZIP1, whereas the bzip1 bzip53 plants displayed a slightly reduced amount of Asn (Figure 3B; see Supplemental Data Set 1 online). Together, our data suggest that bZIP1 and bZIP53 participate in the transcriptional reprogramming of amino acid metabolism during the dark-induced starvation response.
Quantitative Analysis of the Amount of Amino Acids in Dark-Treated Plants.
The levels of total amino acid content (A) and of Leu, Ile, Val, and Asn (B). Amino acid levels of the wild type (wt) (black bars), Pro35S:bZIP1 (gray bars), Pro35S:bZIP53 (hatched bars), and bzip1 bzip53 (white bars) after 0, 1, 4, and 6 d of dark treatment are calculated as ng amino acid/mg dry weight (DW). Given are mean values and sd of two independent experiments. Asterisks represent significant differences between wild-type, overexpressor, and mutant plants at the indicated time points (two-way ANOVA; *P < 0.05, **P < 0.01, and ***P < 0.001).
bZIP1 and bZIP53 Regulate the Expression of Genes Involved in the Dark-Induced Synthesis of Asn and BCAAs
As bZIP1 and bZIP53 are responsible for modulation of amino acid levels, we tested the expression of genes that are related to the corresponding metabolic pathways. Asn is the major source for N/C transport in darkness (Lam et al., 1994, 1998, 2003). Asn biosynthesis is derived from pyruvate and requires the coordinated, transcriptional upregulation of several genes (Lin and Wu, 2004; Figure 4A). Expression of these genes during dark-induced starvation was substantiated by public expression data (see Supplemental Figure 6A online) and confirmed by RNA gel blot analysis (Figure 4B). The tested genes encoding enzymes of the Asn biosynthetic pathway, such as GLUTAMATE DEHYDROGENASE2 (GDH2), ASPARTATE AMINOTRANSFERASE3 (ASP3), GLUTAMATE SYNTHASE (GLNS), and ASN1, were induced during 8 d of dark treatment (Figure 4B). The overexpression of bZIP1 resulted in an enhanced or more rapid transcript accumulation of these biosynthetic genes, whereas overexpression of bZIP53 caused constitutively high transcript levels. Accumulation of PepCK transcripts, which encode PEP CARBOXYKINASE, the first enzymatic step in Asn biosynthesis, was neither induced by darkness nor by bZIP1 overexpression. A slight accumulation of PepCK transcript was only observed when bZIP53 was overexpressed (Figure 4B).
bZIP1 and bZIP53 Regulate Gene Expression of Asn Metabolism during Extended Night Treatment.
(A) The Asn biosynthesis pathway according to Lin and Wu (2004). αKG, α-keto-glutarate; Pyr, pyruvate; PEP, phosphoenolpyruvate; PPDK, pyruvate orthophosphate dikinase.
(B) RNA gel blot analysis of the indicated genes corresponding to the enzymatic steps depicted in the Asn biosynthesis pathway in (A) after long-term dark treatment for 0 to 8 d. Compared are wild-type (wt), Pro35S:bZIP1, and Pro35S:bZIP53 plants. During dark induction, plants were harvested at the indicated days at 5 pm. As a loading control, ethidium bromide stainings are provided for each hybridization experiment.
(C) and (D) Induction of ASN1 (C) or ANS (D) after short-term dark treatment. Rosette leaves of 10 3-week-old plants were pooled and used for RNA preparation and qPCR at the time points indicated. Fold change compared with wild-type expression is depicted at 0 h. Mean value and sd of two replicates are given. The wild type (black bars), Pro35S:bZIP1 (gray bars), Pro35S:bZIP53 (hatched bars), and bzip1 bzip53 (white bars) analyzed by qPCR as described in Figure 1A. For visualizing the differences in transcript levels of ASN1, the y axis is broken twice, at two- and 140-fold induction. The y axis of the ANS plot is broken once at 10-fold induction.
A detailed time course of an extended night treatment was performed for the central Asn biosynthesis pathway gene ASN1. Comparable to ProDH, rapid induction of ASN1 transcript accumulation was detected within 4 h of the extended night treatment (Figure 4C). This response was strongly enhanced in Pro35S:bZIP1 plants but not in Pro35S:bZIP53 plants. These expression data seem to contradict the metabolic analysis because the Asn levels were higher in Pro35S:bZIP53 than in Pro35S:bZIP1 plants. However, in contrast with bZIP53, bZIP1 also activated an ASPARAGINASE (ANS) gene (Figure 4D), which participates in the degradation of Asn (Bruneau et al., 2006). Our observations suggest that bZIP1 and bZIP53 have partly overlapping but also distinct functions in the regulation of Asn metabolism.
Ectopic expression of bZIP1 and bZIP53 leads to reduced levels of BCAA, indicating their involvement in the corresponding catabolic pathway. The mitochondrial BCAA TRANSAMINASE1 (BCAT1) gene was proposed to encode the central catabolic enzyme (Diebold et al., 2002; Schuster and Binder, 2005; see Supplemental Figures 6A and 6B online). However, the analysis of the six Arabidopsis BCAT genes revealed that BCAT2 and to a minor extent BCAT1 were induced during dark treatment. As depicted in Supplemental Figure 6 online, bZIP1 strongly enhances BCAT2 transcript accumulation in the dark. The BCAT2 enzyme is localized in the chloroplasts, where it contributes to Leu and Glu biosynthesis (Schuster and Binder, 2005). Therefore, the plastidic deamination reaction of BCAT2 in the dark might supply the cell with Glu, which in turn is essential for Asn biosynthesis (see Supplemental Figure 6B online). In conclusion, the dark-induced accumulation of BCAT2 transcript is rather linked to the dark-induced Asn biosynthesis than to dark-induced BCAA degradation.
Promoters of Amino Acid Metabolic Genes Are Regulated by bZIP1 and bZIP53 in Response to Energy Starvation in Protoplasts
To assess the direct impact of bZIP1 and bZIP53 on gene regulation, the activity of ASN1 and ProDH promoter:reporter constructs (ProASN1:GUS and ProProDH:GUS) was studied in transiently transfected protoplasts. Starvation was induced by either the transfer of light-cultivated protoplasts to darkness or treatment of light-cultivated protoplasts with the photosystem II inhibitor DCMU. Both starvation treatments induced the transcriptional activity of the ProASN1:GUS and ProProDH:GUS reporter genes, demonstrating that the protoplast system can be used to analyze starvation-induced transcription (see Supplemental Figure 7 online). bZIP53 upregulated the activity of the ProDH promoter both after light and dark cultivation. In contrast with the results obtained in transgenic plants, expression of bZIP1 in protoplasts did not induce both reporter constructs, indicating that additional factors are needed to fulfill its function in plants, which are not present in protoplasts. The differences observed in bZIP1 and bZIP53 function in protoplasts were not due to different protein levels as confirmed by immunoblot analysis (see Supplemental Figure 7C online).
bZIP1 and bZIP53 Directly Regulate ASN1 and ProDH Promoter Activity via G-Boxes or ACT cis-Elements in the Starvation Response
Using the protoplast transfection system, we analyzed whether the starvation response is mediated by ACGT motifs, which represent typical binding sites for bZIP TFs (Jakoby et al., 2002). As summarized in Supplemental Table 1 online, all promoters of the Asn biosynthesis genes and of ProDH and BCAT2 harbor at least one ACGT motif. In the ASN1 promoter, two G-boxes (CACGTG) were found, and G-box 1 was identified as the crucial cis-element in mediating SnRK1 responses (Baena-González et al., 2007). Sequential mutation in the ProASN1:GUS reporter gene demonstrated that the dark-induced transcription and the bZIP1/bZIP53-mediated enhancement of transcription depended exclusively on G-box 1 (Figure 5A). No alteration in the ProASN1:GUS reporter gene activity was observed with a loss-of-function mutation in G-box 2, indicating that the position of the hexameric CACGTG sequence within the promoter is important to mediate the starvation-related gene expression in protoplasts.
bZIP1 Binds Directly to the ASN1 and ProDH Promoters and Mediates Starvation Responses via G-Box (CACGTG) or ACTCAT cis-Elements.
(A) Arabidopsis protoplasts were transformed with a ProASN1:GUS reporter construct or the indicated promoter mutations (see diagrams beneath the x axis). After cotransformation with the effector plasmids (Pro35S:bZIP1 or Pro35S:bZIP53), reporter induction was compared in constant darkness (black bars) or in constant light (white bars) conditions. Given is the fold change with respect to the empty vector control experiment without any bZIP construct added (−) under constant light. Four transfection experiments were used to calculate mean values and sd. Significance was tested by one-way ANOVA analysis following Tukey’s post-test (P < 0.05).
(B) Direct binding of bZIP1 to the ASN1 promoter as demonstrated by ChIP. ASN1 promoter structure and primer binding sites are indicated on the left. Chromatin extracts from wild-type (wt) plants and Pro35S:HA-bZIP1 were subjected to qPCR analysis with ASN1 promoter-specific primers after immunoprecipitation with an anti-HA antibody (α-HA). Ct values for Pro35S:HA-bZIP1 samples were subtracted from the Ct values of the equivalent wild type. For normalization, an actin (ACT7) gene was used. Calculated are induction levels with respect to the wild-type samples. Given are mean values and sd of three independent experiments.
(C) Analysis of the ProProDH:GUS constructs as described in (A). Additional promoter analyses are provided in Supplemental Figure 8 online.
(D) ChIP experiment of wild-type and Pro35S:HA-bZIP1 plants as described in (B). Chromatin was isolated from 3-week-old plants grown under normal light/dark cycle (white bars) or plants cultivated in an extended night for 4 d (black bars). Given are mean values and sd of three repetitions.
(E) Immunoblot analysis of chromatin derived from wild-type and Pro35S:HA-bZIP1 plants detected with an αHA antibody indicates a comparable HA-bZIP1 protein abundance in light- and dark-treated plants. As a loading control, Ponceau staining of the protein preparation is given (bottom panel).
To define whether the identified genes involved in amino acid metabolism are direct targets of the bZIP factors, chromatin immunoprecipitation (ChIP) experiments were performed with transgenic Arabidopsis lines expressing the HA-tagged version of bZIP1. Using primers that amplify the G-box 1/2 promoter region, we could show direct binding of HA-bZIP1 proteins to the ASN1 promoter (Figure 5B).
Previous results revealed pronounced differences in the regulation of ProDH and ASN1. The ProDH promoter harbors no G-box, but a C-box (GACGTC) and two ACT elements (ACTCAT), which are proposed to be bZIP binding sites involved in ProDH regulation (Satoh et al., 2004; see Supplemental Table 1 online). Whereas single mutations in the ACTCAT elements (Figure 5C) or C-box (see Supplemental Figure 8 online) resulted in minor but significant effects on dark-induced ProDH activation, multiple mutations in two cis-elements completely abolished inducibility of the ProDH promoter. From these data, we propose a crucial combinatorial in vivo function of these elements in the dark-induced ProDH activation.
Recently published ChIP experiments demonstrated the in vivo binding of bZIP53 to the ProDH promoter (Weltmeier et al., 2006). In addition, ChIP analyses using primers surrounding the ACT elements and Pro35S:HA-bZIP1 plants also revealed a direct binding of bZIP1 to the ProDH promoter (Figure 5D). Immunoblot analysis of chromatin derived from light- and dark-grown plants showed equal amounts of HA-tagged bZIP1 protein in the ChIP assays (Figure 5E). Therefore, the binding activity of bZIP1 to the ProDH promoter was independent of the light/dark regime.
Multiple bZIP Mutants and Plants Expressing EAR Repressor Fusions of bZIP Factors Are Partially Impaired in Dark-Induced Transcription of Amino Acid Metabolic Genes
The bzip1 bzip53 double mutant showed only limited impairment in dark-induced ProDH and ASN1 transcript accumulation (Figures 2C and 4C; see Supplemental Figure 5 online). Therefore, we applied an alternative loss-of-function approach. Fusions between bZIP53 and bZIP1 and the EAR repressor domain (Hiratsu et al., 2003) were generated and tested for their impact on the ProDH reporter in protoplasts. As shown in Figure 6A, the light- and dark-induced activation of the ProProDH:GUS reporter was completely abolished by EAR-bZIP1 and strongly reduced by EAR-bZIP53. Expression of the fusion proteins was confirmed by immunoblot analysis as demonstrated in Supplemental Figure 9A online. These data further substantiate our hypothesis that bZIP1 and bZIP53 play a crucial role in the regulation of dark-induced ProDH transcription. However, due to their heterodimerization properties, other bZIPs, presumably members of the C/S1 network, are likely candidates for mediating the dark-induced starvation response (Ehlert et al., 2006). We therefore included quadruple T-DNA mutants of bZIP1 and bZIP53 with different group C bZIPs (bzip1 bzip53 bzip9 bzip63 and bzip1 bzip53 bzip10 bzip25) in our study. As demonstrated in Supplemental Figures 9B and 9C online, depending on the particular bZIP gene, complete null alleles or knockdown alleles were obtained in the respective mutant lines. The accumulation of ASN1, ProDH, and BCAT2 (Figure 6B) transcripts was considerably impaired during extended dark treatment in the quadruple mutants, although no complete loss of transcript accumulation was observed. Surprisingly, after long-term dark treatment, gene expression was partially restored, indicating that the plant harbors regulatory mechanisms to substitute for the loss of particular bZIP proteins.
Impact of bZIP Factors on Target Gene Expression Using bZIP-Specific Loss-of-Function Approaches.
(A) EAR repressor fusions of bZIP1 and bZIP53 reveal a regulatory function in dark-induced ProDH transcription. Arabidopsis protoplasts were transiently transformed with a ProProDH:GUS reporter and cotransfected with Pro35S-driven reporters (HA-bZIP1, HA-bZIP53, HA-EAR-bZIP1, and HA-EAR-bZIP53). Induction by cultivation in constant dark (black bars) conditions is compared with expression in constant light (white bars). Depicted is the fold change compared with the promoter in the light. Four transfection experiments were used to calculate mean values and sd. Different letters indicate significant differences, tested by one-way ANOVA analysis following Tukey’s post-test (P < 0.05). Expression of the effector constructs was confirmed by immunoblot analysis (see Supplemental Figure 9A online).
(B) qPCR analysis of ProDH, ASN1, and BCAT2 after extended night treatment as described in Figure 1. Rosette leaves of 10 3-week-old plants were pooled and used for RNA preparation and qPCR at the indicated time points. Fold change compared with the wild type (wt) is indicated at 0 h. Mean value and sd of two replicates are given. Asterisks represent significant differences between wild-type and mutant plants at the indicated time points (two-way ANOVA; *P < 0.05, **P < 0.01, and ***P < 0.001). Impaired expression of the corresponding bZIP genes is demonstrated in Supplemental Figures 9B and 9C online.
DISCUSSION
In this work, we identified two bZIP TFs, namely, bZIP1 and bZIP53, that translate low energy signals into an altered transcriptional pattern of amino acid metabolic genes in Arabidopsis. As outlined in the model in Figure 7, starvation activates in particular bZIP1 transcriptionally and posttranscriptionally, the latter by a conserved system of uORFs (Wiese et al., 2004; Weltmeier et al., 2009). Presumably by heterodimerization with other members of the C/S1 bZIP TF network, bZIP1 and bZIP53 initiate the change in transcriptional activity by binding to ACGT or ACTCAT-like cis-elements within the promoters of metabolic target genes. In conclusion, bZIP1 and bZIP53 are proposed to mediate transcriptional metabolic reprogramming in response to starvation.
Model Summarizing the Function of bZIP Factors in the Energy Deprivation Response.
Starvation activates bZIP1 and bZIP53 transcriptionally and posttranscriptionally, the latter by a conserved system of uORFs. Presumably by heterodimerization with other members of the C/S1 bZIP network, bZIP1 and bZIP53 initiate a change in transcriptional activity by binding to ACGT or ACTCAT-like cis-elements within the promoters of metabolic target genes, causing a reprogramming of primary metabolism in response to low energy stress.
The bZIP TFs bZIP1 and bZIP53 Are Regulated by Energy Deprivation, Both at the Transcriptional and Posttranscriptional Level
In this work, bZIP1, and to a minor extent also bZIP53, was found to be transcriptionally upregulated by conditions that lead to energy deprivation. Feeding experiments with sucrose and glucose, but not 3-oMG, repress bZIP1 and bZIP53 transcription (Figure 2E). 3-oMG is taken up by the cells but is not metabolized and appears not to signal via the hexokinase-dependent sugar signaling pathway (Cortès et al., 2003). These data indicate that sugar signaling regulates the transcription of bZIP1 and bZIP53, supporting recent findings by Kang et al. (2010). Since long-time dark treatments, which have frequently been applied for dark-induced senescence studies (Gan, 2003; Lin and Wu, 2004; Buchanan-Wollaston et al., 2005), do not reflect natural environmental conditions, we performed short-term experiments that described the detailed expression changes after extended night treatments and defined bZIP1 and bZIP53 as putative transcriptional regulators in the starvation response (Figure 1A).
For assaying starvation responses, plant and protoplast systems were applied using culture conditions in the dark or incubation with the photosystem II inhibitor DCMU (e.g., Figure 1A; see Supplemental Figures 7A and 7B online). Recently, other stresses, such as anaerobic conditions, have been found equally useful (Baena-González et al., 2007). Altogether these treatments let to comparable responses with respect to transcription of starvation-induced genes. Environmental stresses, which also lead to a low energy status of the cell, can be assumed to interfere with primary metabolism and therefore might also input into the starvation signaling network (Baena-González and Sheen, 2008).
Recent bioinformatic network analyses suggest that bZIP1 is regulated by the circadian clock (Gutiérrez et al., 2008). Interestingly, CIRCADIAN CLOCK ASSOCIATED1, which is an integral component of the Arabidopsis clock (Yakir et al., 2007), was shown to directly bind to the bZIP1 promoter (Gutiérrez et al., 2008). Although a detailed molecular analysis is still elusive, it is tempting to speculate that a regulatory energy management network exists in plant cells, which integrates C and N availability and clock-initiated day/night rhythms to regulate bZIP1-dependent gene expression.
In addition to transcriptional regulation, further posttranscriptional mechanisms might account for bZIP1 and bZIP53 regulation. For all group S1 bZIPs, a posttranscriptional repression by sucrose (sucrose-induced repression of translation) was demonstrated (Weltmeier et al., 2009). With respect to bZIP1 and bZIP53, these mechanisms result in an additive effect, leading to high intracellular levels of the bZIP TFs during energy deprivation (Figure 7). Thus, bZIP1 and bZIP53 are candidates to execute a crucial function in metabolic reprogramming during the starvation response.
bZIP1 Is Regulated Posttranslationally by a Starvation-Derived Signal
Although bZIP1 is strongly upregulated by transcriptional and posttranscriptional mechanisms, its function in gene regulation depends on an additional signal, which is initiated by dark-induced energy deprivation. Importantly, this observation resulting from studies in transgenic plants was not detectable in the protoplast system: Arabidopsis bZIP1 overexpression leads only to a minor change in target gene activation. Apparently, an unknown component is missing in the protoplast system. Therefore, these findings emphasize the importance of studying the whole plant system.
bZIP53 is predominantly regulated on the level of protein amount. When overexpressed, it activates transcription independently of the energy status of the cell, whereas bZIP1 activity is further fine-tuned at the posttranslational level by a starvation-dependent signal. Recently, KIN10 and KIN11, two kinases of the SnRK1 family, have been demonstrated to orchestrate starvation responses, in particular on the transcriptional level (Baena-González et al., 2007; Baena-González and Sheen, 2008). It remains to be analyzed whether the starvation signal regulating bZIP1 function is directly or indirectly mediated by these kinases.
bZIP1 and bZIP53 Show Overlapping but Distinct Functions in the Low Energy Response
In this work, ectopic overexpression of bZIP1 results in an early senescence phenotype in the dark, which is characterized by rapid loss of chlorophyll (Figures 1C and 1D). As defined by marker gene expression, this phenotype reflects a dark-induced starvation response, but not the natural senescence (see Supplemental Figure 3 online; van der Graaff et al., 2006). This early senescence phenotype is less pronounced in Pro35S:bZIP53 plants. Furthermore, distinct differences in the function of bZIP1 and bZIP53 become obvious when these plants were studied under a normal day/night cycle. As confirmed by immunoblot analysis, high-level expression of bZIP1 did not lead to obvious phenotypic changes, whereas medium-level expression of bZIP53 resulted in significantly reduced plant growth (Alonso et al., 2009). These findings indicate pronounced differences in bZIP1 and bZIP53 function.
Overexpression of bZIP1 and bZIP53 in transgenic plants revealed that both TFs are capable of regulating the amino acid metabolic genes proposed to be upregulated during the dark-induced starvation response, such as genes involved in Asn biosynthesis and Pro and BCAA metabolism (e.g., Figures 2C, 4B and 4C; see Supplemental Figure 6C online). ASN1, the key gene of the Asn biosynthesis pathway, is characterized by a complex transcriptional regulation (Lam et al., 1998, 2003). Transcription of the Asn pathway genes, such as ASP3, GDH2, and ASN1, appears to be coregulated and is induced after dark treatment but repressed when sugar is available (Lin and Wu, 2004). In the light, ectopic expression of bZIP53 leads to constitutive activation of ProDH, ASP3, GDH2, and ASN1, whereas bZIP1 provokes only minor effects.
Changes in the transcriptional levels of amino acid metabolic genes are well reflected on the amino acid level. For instance, the amount of Pro is significantly reduced in Pro35S:bZIP1 and Pro35S:bZIP53 plants (Figure 2D). Contrarily, the amount of Asn is induced exclusively in Pro35S:bZIP53 plants (Figure 3B). As demonstrated on the transcriptional level, bZIP1 but not bZIP53 enhances expression of an ANS (Figure 4D), which leads to the degradation of Asn (Bruneau et al., 2006). Again, bZIP53 and bZIP1 show distinct differences in target gene selection. This might explain the observed differences in the metabolite profiles.
Ectopic expression of bZIP1 and bZIP53 leads to reduced levels of BCAAs (Figure 3B), indicating the involvement of both TFs in a BCAA degradation pathway. BCAT2 transcription follows the pattern of the other analyzed, dark-induced amino acid metabolic genes, and bZIP1 and bZIP53 enhance BCAT2 transcription (see Supplemental Figure 6C online). However, it is not clear whether the BCAT2 enzyme is involved in an anabolic or catabolic context. In the dark, chloroplastic proteins, such as ribulose-1,5-bisphosphate carboxylase/oxygenase, are degraded to provide amino acid to the starved nitrogen metabolism. It was speculated that the deamination reaction of BCAT2 in the dark supplies the cell with Glu, which is essential for Asn biosynthesis (Schuster and Binder, 2005). Hence, dark-induced BCAT2 expression as well as the BCAA degradation might be closely linked to the dark-induced Asn biosynthesis and consequently appear to be regulated in a coordinated fashion by the same set of bZIP TFs.
Differences in function have already been described for bZIP53 and bZIP1 (Weltmeier et al., 2009). For instance, heterodimers that include bZIP53 regulate the expression of seed maturation genes involved in desiccation tolerance, storage compound synthesis, and source-sink regulation, such as ASN1 (Alonso et al., 2009). Although bZIP1 heterodimers share the capacity to activate seed maturation genes in protoplasts, bZIP1 appears not to be involved in the regulation of these genes during seed maturation (Weltmeier et al., 2009). Thus, bZIP53 and bZIP1 have partially overlapping but distinct functions that are probably defined by expression patterns and/or posttranslational mechanisms.
Transcriptional Control of Amino Acid Metabolic Genes by bZIP1 and bZIP53 Is Mediated by Binding to G-Box, C-Box, or ACTCAT cis-Elements
As demonstrated in transiently transfected protoplasts, one of the two G-boxes (G-box 1) in the promoter of ASN1 is essential for the dark activation of this gene (Figure 5A). Our promoter deletion experiments further substantiate the hypothesis that bZIP1 and bZIP53 signal via the G-box 1 in the ASN1 promoter. G-box-like cis-elements, which are characterized by their ACGT core, are typical binding sites for bZIP TFs as demonstrated for bZIP53 by in vitro and in vivo binding assays (Alonso et al., 2009). We used HA-tagged bZIP1 in ChIP experiments to confirm its direct binding to ProDH and ASN1 promoter regions (Figures 5B and 5D). Although, due to overexpression, the ChIP data have to be interpreted with care, they are supported by results of the protoplast assays. Our combined data suggest that the analyzed ProDH and ASN1 promoters are direct in vivo targets of bZIP1. Dark-induced recruitment of bZIP1 to its target promoters is one possible regulatory mechanism that would explain stimulus-induced target gene activation. However, dark-induced enhancement of bZIP1 DNA binding to the ProDH promoter was not detected in the ChIP experiments, at least at the time points used in this study. Surprisingly enough, the ProDH promoter harbors no G-box but a closely related, ACGT core-containing C-box and two ACTCAT motifs. The ACTCAT motifs were shown to be bound by group S1 bZIPs and are involved in the hypoosmolarity response (Satoh et al., 2002, 2004; Weltmeier et al., 2006). Multiple mutations in ACTCAT and C-box elements confirm a crucial and additive impact of all these cis-elements on basic and inducible ProDH promoter activity. These data suggest that differences in ASN1 and ProDH expression patterns are caused by the combination and location of the identified cis-elements in their promoter structures. Furthermore, bZIP heterodimerization might alter target site recognition; therefore, it is tempting to speculate that different promoters will recruit particular sets of bZIP heterodimers.
Redundant bZIP Factors Can Partially Substitute for Loss of bZIP1 and bZIP53
Expression of bZIP1 and bZIP53 fusion proteins containing a C-terminal EAR repressor domain (Hiratsu et al., 2003) completely abolishes or significantly reduces dark-induced ProDH expression in protoplasts (Figure 6A). Since the repressor-modified TF blocks specific promoter binding sites when overexpressed, this method was applied to compete with redundantly active TFs to interfere with their function. As single bzip1 and bzip53 T-DNA mutants and even a bzip1 bzip53 double mutant do not lead to dramatic impairment of amino acid target gene expression (see Supplemental Figure 5 online), the protoplast data support the view that a functional redundant bZIP TF network is operating in the starvation response. bZIP2 expression patterns are slightly similar to the one observed for bZIP53. However, since bZIP2 T-DNA insertion lines are not available and bZIP2 overexpression results in severely dwarfed, sterile plants, bZIP2 could not be included in the experimental setup. Strong interference with normal plant growth was also observed by overexpression of bZIP11 (Hanson et al., 2008). Inducible expression combined with transcriptome analysis was used to identify ProDH and ASN1 as bZIP11-regulated genes. However, although bZIP11 has the capacity to regulate these metabolic genes, it is probably not involved in the starvation response as its expression is upregulated by sugar and downregulated by darkness. As depicted in Supplemental Figure 1 online, several bZIP genes, such as bZIP41 or bZIP54, show appropriate expression patterns to fulfill a function in the energy deprivation response. Also, the group C factors bZIP63, bZIP9, and bZIP25, which form heterodimers with group S1, might be candidates. Specific bZIP heterodimerization has been shown to be important for bZIP53 activity and function (Ehlert et al., 2006; Weltmeier et al., 2006). Consequently, quadruple C/S1 T-DNA insertion mutants show strongly impaired dark-induced target gene expression (Figure 6B; see Supplemental Figure 9D online). Surprisingly, this effect is only transient and the plant can partially compensate for the loss of bZIP gene activity during long-term dark adaptation. Overlapping functional redundancy appears to be a frequently observed feature in particular in signaling networks. The regulation of target genes by C/S1 bZIPs is complex; however, it is more stable with respect to mutations and more flexible in terms of its potential to fine tune regulation. Although the plant benefits from this flexibility, the regulatory circuits controlling this TF network remain elusive.
Knowledge of the transcriptional regulators is crucial for understanding the plant energy control system and is a first step to establishing biotech approaches to increase yield and stress tolerance of crop plants.
METHODS
Plant Cultivation and Treatment
For plant and protoplast transformation, amino acid measurement, qPCR, RNA gel blot, and ChIP experiments, Arabidopsis thaliana ecotype Columbia (Col-0) was grown on soil under long-day conditions of 16-h-light/8-h-dark cycles. Dark treatment was performed for 4 h to 8 d using 3-week-old soil-grown plants. For RNA gel blot analysis, the first harvesting time point (0 d) was at 5 pm, during the light period. Material for qPCR was harvested as indicated, starting at the beginning of the light period (8 am). For hydroponic culture, the procedure described by Gibeaut et al. (1997) has been modified. Three-week-old plants were grown on a mesh support under short-day conditions. The media were supplemented with sugars according to the description in Figure 2E. Floral dip transformations were performed using the Agrobacterium tumefaciens strain GVG3101 (Weigel and Glazebrook, 2002). Transgenic plants and T-DNA insertion lines are summarized in Supplemental Table 2 online. Homozygous mutants were identified by PCR as described (http://signal.salk.edu/tdnaprimers.2.html) using the primers described in Supplemental Table 3 online.
Molecular Biology and Physiology Methods
RNA gel blot analysis and histochemical GUS staining were described in Weltmeier et al. (2006, 2009). Hybridization probes were PCR generated using the primers summarized in Supplemental Table 3. RNA gel blot analysis was repeated three times with comparable results. Immunoblot analysis was performed according to Weltmeier et al. (2006) using αHA antisera (Santa Cruz Biotechnology). Aa analysis was performed as described in Pilot et al. (2004).
qPCR
RNA was isolated from pooled material of the rosettes of 10 3-week-old plants. cDNA synthesis and qPCR analysis were performed as described by Fode and Gatz (2009). Cycling conditions were as follows: 10 min at 95°C, 40 cycles of 20 s at 95°C, 10 s at 55°C, and 30 s at 72°C, followed by a default dissociation stage program to detect nonspecific amplification. Amplification products were visualized by SYBR green. The ubiquitin (UBI5) gene was used for sample normalization. Other gene-specific oligonucleotides are described in Supplemental Table 3 online. Statistical analysis was performed with the GraphPad Prism or OriginPro 8.1 software using the tests indicated in the figure legend.
Transient Expression Assays and Constructs
Protoplast isolation, transformation, construction of effector plasmids, and immunoblot analysis were performed according to Ehlert et al. (2006). ProProDH:GUS and ProASN1:GUS reporter constructs were obtained by inserting the PCR-amplified promoters into the pBT10-TATA-GUS vector (Sprenger-Haussels and Weisshaar, 2000) using PstI/NcoI or XbaI/NcoI restriction sites, respectively. The PCR primers are given in Supplemental Table 3 online. Mutation of promoter cis-elements was performed using the Quick Change site-directed mutagenesis kit (Stratagene). For reporter gene assays, 5 μg reporter and 14 μg of effector were used, unless stated otherwise. One microgram of Pro35S:NAN plasmid was added for normalization (Kirby and Kavanagh, 2002). Protoplasts were incubated overnight for 16 h in constant light or constant darkness. For treatment with DCMU, 20 μM DCMU was added to the solution for incubation overnight. Four transfection experiments were used to calculate mean values and standard deviations of relative GUS/NAN activities as described by Ehlert et al. (2006).
ChIP
ChIP was performed as described by Weltmeier et al. (2006) and Alonso et al. (2009) using an HA-specific antibody (Abcam). Primers are given in Supplemental Table 3 online. The difference between the resulting C(t) values of wild-type and Pro35S:HA-bZIP1 overexpressor was calculated and normalized with the input controls of these samples, which were analyzed with the same primers. For further normalization, PCR was performed with unspecific actin (ACT7) promoter primers (see Supplemental Table 3 online).
Accession Numbers
Arabidopsis Genome Initiative identifiers for the genes mentioned in this article are as follows: bZIP53 (At3g62420), bZIP1 (At5g49450), bZIP63 (At5g28770), bZIP10 (At4g02640), bZIP25 (At3g54620), bZIP9 (At5g24800), ProDH (At3g30775), ASN1 (At3g47340), GDH2 (At5g07440), ASP3 (At5g11520), GLNS (At5g37600), PepCK (At5g65690), ANS (At3g16150), CAB (At1g29920), SAG103 (At1g10140), YLS3 (At2g44290), BCAT2 (At1g10070), LEA76 (At3g15670), UBI5 (At3g62250), and ACT7 (At5g09810).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Expression of bZIP Genes during Energy Starvation.
Supplemental Figure 2. Molecular Characterization of Transgenic Plants Altering the Amount of bZIP1.
Supplemental Figure 3. Expression of Senescence Marker Genes Has Been Analyzed in Plants That Have Been Cultivated under Extended Night Treatment.
Supplemental Figure 4. Expression of bZIP1 and bZIP53 after Extended Night Treatment.
Supplemental Figure 5. Expression Analysis of Genes Involved in Amino Acid Metabolism Analyzed in Single and Double T-DNA Insertion Mutants of bZIP1 and bZIP53.
Supplemental Figure 6. Regulation of Transcription in Branched-Chain Amino Acid (BCAA) Metabolism during Dark Treatment.
Supplemental Figure 7. Analysis of Energy Deprivation Induced Transcription in Protoplasts.
Supplemental Figure 8. Mutation in the C-Box Effects ProDH Promoter Activity.
Supplemental Figure 9. Immunoblot Analysis of the EAR Repressor Approach and Characterization of Multiple T-DNA Insertion Mutants Used in This Study.
Supplemental Table 1. Summary of Putative bZIP Binding Motifs (ACGT-Like Elements) Found.
Supplemental Table 2. Summary of the T-DNA Insertion Lines Used in This Study.
Supplemental Table 3. Summary of the Oligonucleotide Primers Used in This Study.
Supplemental Data Set 1. Summary of Amino Acid Analysis of 3-Week-Old Arabidopsis Rosettes.
Acknowledgments
We thank Jennifer Krüger, Anna Herman, and Bettina Stadelhofer for their valuable technical assistance and Johannes Hanson for critical reading of the manuscript.
Footnotes
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Wolfgang Dröge-Laser (wolfgang.droege-laser{at}uni-wuerzburg.de).
↵1 These authors contributed equally to this work.
↵[W] Online version contains Web-only data.
- Received March 17, 2010.
- Revised December 21, 2010.
- Accepted January 12, 2011.
- Published January 28, 2011.
References
