Plant Mol Biol (2013) 83:445–458 DOI 10.1007/s11103-013-0102-8
The roles of two transcription factors, ABI4 and CBFA, in ABA and plastid signalling and stress responses Zhong-Wei Zhang • Ling-Yang Feng • Jian Cheng • He Tang • Fei Xu • Feng Zhu • Zhong-Yi Zhao • Ming Yuan • Yang-Er Chen Jian-Hui Wang • Shu Yuan • Hong-Hui Lin
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Received: 4 October 2012 / Accepted: 27 June 2013 / Published online: 6 July 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Genetic and physiological studies have revealed evidences for multiple signaling pathways by which the plastid exerts retrograde control over photosynthesis-associated-nuclear-genes. In this study we have examined the mechanisms of control of transcription by plastid signals, focusing on transcription factors. We have also further addressed the physical nature of plastid signals and the physiological role, in stress acclimation of this regulatory pathway. ABI4, a master Apetala 2 (AP2)-type transcription factor (TF), is targeted by multiple signalling pathways in plant cells, such as abscisic acid (ABA) signals, sugar signals
Zhong-Wei Zhang and Ling-Yang Feng have contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s11103-013-0102-8) contains supplementary material, which is available to authorized users. Z.-W. Zhang L.-Y. Feng S. Yuan (&) College of Resources and Environmental Sciences, Sichuan Agricultural University, Chengdu 611130, China e-mail:
[email protected] J. Cheng H. Tang F. Xu F. Zhu Z.-Y. Zhao H.-H. Lin (&) Ministry of Education Key Laboratory for Bio-Resource and Eco-Environment, College of Life Science, Sichuan University, Chengdu 610064, China e-mail:
[email protected] M. Yuan Y.-E. Chen College of Biology and Science, Sichuan Agricultural University, Ya’an 625014, China J.-H. Wang Horticulture Institute, Sichuan Academy of Agricultural Sciences, Chengdu 610066, China
and plastid signals derived from reactive oxygen species (ROS) and chlorophyll intermediates. ABI4 binds the promoter of target genes to prevent their transcription by competing with other competitive TFs. However, we found that once ABI4 bound the element (CCACGT), it may not be bound by other TFs, therefore making the signalling longlasting. Downstream of ABI4, CBFA (CCAAT binding factor A) is a subunit of the HAP2/HAP3/HAP5 (Heme activator protein) trimeric transcription complex. CBFA however is a redundant HAP3 subunit. When emergency occurs (such as herbicide treatments or environmental stresses followed by ABA and ROS accumulation), the master transcription factor ABI4 down-regulates some TFs, like CBFA, and then some other TF subunits enter the transcription complex and transcriptional efficiency of stressresponsive genes (including the transcription co-factor CBP) is improved instantaneously. abi4, cbfA and cbp mutants showed weaker drought-tolerance after a herbicide norflurazon treatment, which indicated the physiological role of these key transcription factors. Keywords Transcription factor ABI4 CBFA GUN1 Plastid signaling Tetrapyrroles Abbreviations ABA ABI4 CBFA CBP ChIP GUN1 HL Linc Mg-Proto IX
Abscisic acid Abscisic acid insensitive 4 CCAAT binding factor A Transcription co-factor CAAT binding protein Chromatin immunoprecipitation Genomes uncoupled 1 High light Lincomycin Mg-protoporphyrin IX
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NF PET PGE PhANGs ROS TF
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Norflurazon Photosynthetic electron transport chain Plastid gene expression Photosynthesis-associated-nuclear-genes Reactive oxygen species Transcription factor
Introduction Genetic and physiological studies have revealed evidence for multiple signalling pathways by which the plastid exerts retrograde control over photosynthesis-associatednuclear-genes (PhANGs), such as LHCB gene (encoding a light-harvesting chlorophyll a/b-binding protein) (Strand et al. 2003; Nott et al. 2006; Koussevitzky et al. 2007; Woodson and Chory 2008; Kleine et al. 2009; Jung and Chory 2010; Barajas-Lo´pez Jde et al. 2013). One of these pathways is related to some chlorophyll-biosynthetic Intermediates (called tetrapyrroles), including Mg-protoporphyrin IX (Mg-Proto IX) (Strand et al. 2003; Mochizuki et al. 2008; Moulin et al. 2008; Zhang et al. 2011a). Another signal is induced by inhibition of plastid gene expression (PGE) (Nott et al. 2006; Koussevitzky et al. 2007; Zhang et al. 2010a). Kakizaki et al. (2009) further showed that the plastid signals were activated by attenuated plastid protein import. The fourth may correlate with sugar signals (Koussevitzky et al. 2007; Zhang et al. 2010a). The fifth signal is controlled by the redox state of the photosynthetic electron transport chain (PET), and the sixth employs reactive oxygen species (ROS) (Wagner et al. 2004; Nott et al. 2006; Koussevitzky et al. 2007; Kindgren et al. 2012; Maruta et al. 2012). The plastid volatile b-carotene derivative b-cyclocitral triggers changes in the expression of nuclear 1O2-responsive genes and leads to an enhancement of photooxidative stress tolerance (Ramel et al. 2013). Thus, besides their well-known functions in light harvesting and photo-protection, carotenoids can also play a role through their non-enzymatic oxidation in the sensing and signalling of reactive oxygen species and photo-oxidative stress in photosynthetic organisms. Recently, Xiao et al. (2012) showed that plastid methylerythritol cyclodiphosphate (MEcPP), a precursor of isoprenoids produced by the plastidial methylerythritol phosphate pathway, elicits the expression of selected stress-responsive nuclear-encoded plastidial genes. Moreover, 30 -phosphoadenosine 50 -phosphate (PAP) also generates retrograde signals from the chloroplast to the nucleus (Estavillo et al. 2011). And there are some cross-talks between plastid signaling and light signalling (Ruckle et al. 2007).
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For plastid-signalling and its related stress-adaption studies, a herbicide norflurazon (NF) was used. NF inhibits phytoene desaturase by competition with the cofactors (such as NADP, Sandmann et al. 1980; Breitenbach et al. 2001), and blocks carotenoid synthesis (Oelmu¨ller and Mohr 1986). Carotenoid-deficient plants suffer severe photooxidative damage due to the production of ROS in light by excited triplet states of chlorophyll, and therefore lead to a dramatic reduction in the expression of LHCB and other PhANGs (Oelmu¨ller and Mohr 1986). To elucidate this retrograde plastid-to-nucleus signalling pathway, Arabidopsis genome uncoupled (gun) mutants gun1–gun5 were isolated that showed a de-repression of LHCB expression in the presence of NF. GUN5 encodes the H-subunit of Mg-chelatase (CHLH), and LHCB RNA in gun5 mutant could not be repressed by NF, because of its declined Mg-proto IX synthesis (Mochizuki et al. 2001). GUN4 is a Mg-chelatase cofactor (Larkin et al. 2003). gun2 (encoding heme oxygenase) and gun3 (encoding phytochromobilin synthase) mutants accumulate high levels of heme (Mochizuki et al. 2001). While Mg-Proto IX or heme accumulation in turn leads to negative feedback regulation of chlorophyll biosynthesis, including the inhibition to 5-aminolevulinic acid (ALA) synthesis (Nott et al. 2006; Kauss et al. 2012). Woodson et al. (2011) recently indentified a gain-of-function gun mutant gun6-1D, which overexpresses the conserved plastid ferrochelatase 1 (FC1, heme synthase). The treatments of plants with lincomycin (Linc), which inhibit plastid gene expression (PGE), also resulted in decreased expression levels of PhANGs. The inhibitors were effective in preventing nuclear gene expression only if applied within the first 2–3 days of Arabidopsis seedling development, indicating that PhANG expression is dependent on chloroplast biogenesis (Nott et al. 2006). Our previous studies showed that 3 % glucose with 1 mM chloramphenicol (another PGE inhibitor, like Linc) cotreatment repressed LHCB transcript significantly in mature Arabidopsis seedlings, while effective solo glucose treatment needed a concentration of 7 % (Zhang et al. 2010a). Koussevitzky et al. (2007) also indicated a relationship between PGE signals and sugar signals. GUN1 is a plastid protein and integrates multiple plastid signals (Koussevitzky et al. 2007). GUN1 may bind to plastid DNA (Koussevitzky et al. 2007), and plastid signals affect RNA editing in plastids, although the editing is not always related to GUN1 protein (Kakizaki et al. 2012). In response to the GUN1-mediated signals ABI4 (an Apetala 2-type transcription factor) binds the promoter of LHCB1 and this is proposed to prevent GBF (a G-box binding factor required for tissue-specific, light-induced expression of LHCB) from binding (Koussevitzky et al. 2007). A chloroplast envelope-bound plant homeodomain transcription
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factor PTM may connect GUN1-pathway in the plastid and ABI4-pathway in the nucleus (Sun et al. 2011). ABI4 also is a key component of the ABA signaling pathway (Niu et al. 2002). Germination sensitivity of gun4 and gun5 mutant seeds to ABA was increased compared with the wild-type seeds (Voigt et al. 2010). gun1 seedlings were more sensitive than wild-type seedlings to the inhibition of seedling growth and development by ABA (Cottage et al. 2010). All the data imply that tetrapyrrole, plastid signalling and ABA signalling may be interconnected. However, the potential role of GUN5 as an ABA receptor (Shen et al. 2006) is controversial because that some laboratories reported that GUN5 does not bind ABA (Mu¨ller and Hansson 2009; Tsuzuki et al. 2011). And the role of GUN5 in ABA-regulated processes is distinct from its role in chlorophyll biosynthesis and plastid-to-nucleus signalling (Du et al. 2012). Interestingly, it was very difficult to revert the plastid signals once changes in nuclear gene expression have been triggered (Nott et al. 2006). However, why the signalling is long-lasting is still unknown. Considering that ABAinduced LHCB repression can not be reversed either (see the Results), and ABI4 but not GUN1 is required for ABAinduced LHCB repression (Koussevitzky et al. 2007), we presume that the ABI4 is the master factor controlling over the long-lasting signalling. Previously, we found that among the multiple signals, Mg-proto IX, heme and a high level of sugar (over 7 %) doubled cellular total RNA within 48 h. This rapid RNA multiplication is important for effective cellular resistance to oxidative stress, such as high-light and herbicide costress conditions, where the plastid-signalling defective mutant gun1 shows severe photobleaching (Zhang et al. 2011b). Considering that mRNA, tRNA and rRNA were all increased after the treatments, one could speculate that some transcription co-factors promoted by the treatments. Our previous results showed that except for the transcription co-factor CBP (CAAT binding protein, At1g15780), all other transcription factor transcripts could not be enhanced by plastid signals, including four transcription co-factors similar to CBP (Zhang et al. 2011b, 2011c), and RNA multiplication can not be induced in the cbp mutant, indicating the importance of CBP in this RNA doubling. Both GUN1 and ABI4 are required for this rapid RNA multiplication and high-light stress adaption (Zhang et al. 2011b). However, ABI4 appears to be a negative regulator of PhANGs expression (Koussevitzky et al. 2007). How it increases CBP expression and what positive transcription factors participate in the signalling are almost unknown. Here we show that a CCAAT binding factor CBFA, which could be repressed by the binding of ABI4 to its promoter, may be the key regulator. Furthermore, gun1, cbfA and cbp mutants showed impaired basal drought–tolerance, while
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gun1, abi4, cbfA and cbp mutants showed hampered NFmediated drought-tolerance, which indicated the physiological role of these key transcription factors in stress adaption.
Materials and methods Plant materials and growth conditions Arabidopsis mutants were in the Col-0 background. Plants homozygous for the T-DNA insertion were identified based on the PCR analysis. Seeds of gun1-9 mutant were a gift from Prof. Joanne Chory (The Salk Institute, USA). The seed stock numbers for abi4, cbfA, gbf1, At1g198050, At4g01720 and At4g36990 mutants are CS8104, SALK_ 150879C, SALK_027691C, CS8147, SALK_000313C and CS817229 respectively. lec1 mutant is usually embryo lethal (West et al. 1994), and therefore was not used. For over-expressing transgenic plants, cDNA fragments of ABI4 (At2g40220), GBF1 (At4g36730), CBFA (At4g14540) and LEC1 (At1g21970) including the ORF sequences were amplified by PCR and cloned into pBIBBASTA-35S-GWR vectors carrying 35S promoter by the Gateway method (He et al. 2007). Wild-type plants were transformed by the floral-dip method (He et al. 2007) and transgenic lines were selected on Glufosinate-ammonium (Sigma; St Louis, MO, USA). Eight transgenic lines for each gene were confirmed by RT-PCR. All seedlings (including over-expressing transgenic plants) were grown under 12 h/12 h light/dark cycles of medium light (100 lmol m-2 s-1) at 21 °C for 3 weeks. To induce RNA multiplication and oxidative stress acclimation, plants were pretreated with 50 lM NF for 48 h (spraying the leaves with 1/2 Hoagland solution with 50 lM NF, every 24 h and 2 times total, under the growth condition) and then subjected to drought treatment (withholding water) for 14 days (under the growth condition). For the wild-type plants, 14-day drought treatment resulted in a half decline of relative water content [RWC, the ratio of (fresh mass - dry mass)/(water-saturated mass - dry mass)]. Chemical and light treatments The herbicide norflurazon (NF) is the most common trigger for plastid signaling (Nott et al. 2006). However, NF is a strong photobleaching agent and NF treatments to newlygerminated seedlings would result in great damages to renascent plastids, and therefore many unpredictable sideeffects (Kleine et al. 2009). Therefore, we used mature Arabidopsis seedlings for this investigation. And the effective NF concentration 50 lM for mature plants was
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defined by our previous studies (Zhang et al. 2011a; Zhang et al. 2011b). All Arabidopsis seedlings grown for 3 weeks were removed from the soil, washed with tap water and dried briefly with paper towels to remove surface water. Chemical treatments and porphyrin feedings were performed by submerging the roots of seedlings into 1/2 Hoagland solution with different chemicals for 24 h under a low light (about 20 lmol m-2 s-1). For higher porphyrin incorporation efficiency, vacuum infiltration to the whole seedling (two times, 2 min each) was performed before the root feeding. Mg-protoporphyrin IX (Mg-ProtoIX) 50 lM; Hemin 50 or 200 lM; Sucrose 3–7 %; lincomycin 500 lM; abscisic acid (ABA) 50 lM. All porphyrins were purchased from Frontier Scientific (Logan, UT, USA). Then some of chemical treated seedlings were transferred to high-light (HL) treatment (1/2 Hoagland solution and illumination about 250 lmol m-2 s-1) for additional 48 or 96 h. This light fluence rate was not so high that the chlorophyll level and LHCB expression could be downregulated dramatically (Karpinski et al. 1997; Ruckle et al. 2007).
RNA quantitation DNAs and RNA were isolated simultaneously with the TRIzol DNA/RNA kit (Invitrogen, Carlsbad, CA, USA) to guarantee the uniform extraction efficiency (Zhang et al. 2011b). The purification of RNA samples was detected by measuring the absorbance ratios of A260/A280, which in all the samples were about 1.9. All RNA samples were treated with DNase I before RT-PCR. It may be difficult to normalize the total RNA in the measurements. Regardless of whether the cell cycles could be affected by the treatments, DNA is the template of RNA. Therefore, DNA (template) content is the most reliable reference parameter, and the RNA levels were presented as the ratio of total RNA to total DNA (Zhang et al. 2011b, 2011c).
ROS visualization Superoxide and H2O2 levels were visually detected with nitro blue tetrazolium (NBT) and 3,3-diaminobenzidine (DAB), respectively, as described previously (Yang et al. 2004). Seedlings were excised at the base with a razor blade and supplied through the cut ends with NBT (0.5 mg ml-1) or DAB (2 mg ml-1) solutions for 8 h. Leaves were then decolorized in boiling ethanol (95 %) for 20 min. At least three leaves were used for each treatment.
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Real-time quantitative PCR LHCB (LHCB1.1, At1g29920), ABI4, GBF1, CBFA, LEC1 and CBP (At1g15780) expression levels were detected by quantitative real-time PCR analysis. The cDNA was amplified by using SYBR Premix Ex Taq (TaKaRa). The Ct (threshold cycle), defined as the PCR cycle at which a statistically significant increase of reporter fluorescence was first detected, was used as a measure for the starting copy numbers of the target gene (Czechowski et al. 2005). Three technical replicates were performed for each experiment. ACTIN1 gene (At2g37620) was used as internal controls. The expression levels of the wild-type seedlings without any treatment are normalized to 100 %. All primers are shown in Supplemental Table 1. Histochemical GUS staining The fragments of 800 bp upstream of the translational start sites of AtCBFA and AtCBP were fused independently to the GUS reporter gene in pBI121 vector. For cis-element analysis, the CCACGT box in the promoter of CBFA gene was mutated to TTACAA; the CCAAT box in the promoter of CBP gene (complementary strand) was mutated to AAGGT. The GUS constructs were then used to transiently transform to mature (3 weeks) Arabidopsis seedlings. About 1 L of Agrobacterium suspension was added to a 4 L vacuum jar (12 cm diameter). Three plastic pots containing plants were placed on the jar grooves. The pots were transferred upside-down into the jar so that all the plant material was in contact with the Agrobacterium solution. The vacuum jar was covered and the vacuum (400 mmHg) was applied. The vacuum was kept for 2 min and then release immediately. The vacuum infiltration was repeated once. After 48 h incubation in darkness, the seedlings were subjected to various treatments. Histochemical localization of GUS was carried out in a solution containing 5 mg mL-1 5-bromo-4-chloro-3-indolyl-b-D-glucuronide as the substrate in a buffer containing 100 mm phosphate buffer (pH 7.0), 0.5 mm potassium ferrocyanide, 0.5 mm potassium ferricyanide, 10 mm EDTA, and 0.3 % (v/v) Triton X-100 and incubated at 37 °C overnight and wrapped in aluminum foil (Jefferson et al. 1987). After staining, tissue was incubated in 70 % (v/v) ethanol to remove chlorophyll and reduce background. GUS activity was quantitated as described by Zhang et al. (2010b). Antibody generation and western blotting The whole coding sequences of ABI4, GBF1, CBFA and LEC1 were amplified by PCR using specific complete CDS primers (Supplemental Table 1). The whole coding
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sequences were cloned into pGEX-4T-2 (Pharmacia). The reconstructed pGEX-4T-2 plasmid containing the coding sequences was then transformed into Escherichia coli strain BL21 (DE3) to express the proteins by induction for 8 h at 37 °C. The resulting glutathione S-transferase (GST) fusion proteins were purified using Glutathione Sepharose 4B (Pharmacia). E. coli cells from a 50 ml culture (with 1 mM isopropyl b-D-thiogalactopyranoside) were harvested by centrifugation, and resuspended in 2.5 ml of 19 PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3). Then the cell lysate was sonicated on ice with three 5-s pulses at high intensity. The lysate was centrifuged at 3,0009g for 15 min to pellet the cellular debris. A 50 % Glutathione Sepharose 4B slurry (thoroughly washed with 19 PBS) for the purification was prepared. The cell lysate was added to the prepared Glutathione Sepharose 4B and incubate for 30 min at room temperature. The medium was sedimented by centrifuging at 5009g for 5 min. Then the column was washed twice 10 bed volumes of 19 PBS. The bound protein was eluted by adding 1 ml of elution buffer (50 mM Tris–HCl, 10 mM reduced glutathione, pH 8.0) per 1 ml bed volume of the original slurry. The eluted protein was incubated at room temperature for 10 min. The medium was sedimented by centrifugation at 5009g for 5 min. The polyclonal antibodies against ABI4, GBF1, CBFA or LEC1 generated by inoculation of rabbits, were purified using the Amino Link Plus Immobilization Kit (Pierce) (Chen et al. 2009). The immune specificity of each antibody was verified by Western blotting to the wild-type, mutant, and over-expressing transgenic plants (see the blots in Supplemental Fig. 4). For protein extraction, 7-day old seedlings were homogenized on ice with 5 mL of buffer containing 50 mM Tris–HCl (pH 6.8), 4 % SDS, 6 % b-mercaptoethanol, 4 M urea, 10 % (v/v) glycerol and 1 mM phenylmethanesulfonyl fluoride (PMSF). The homogenates were centrifuged at 10,000g for 10 min. SDS-PAGE and western blotting analysis of the extracts was processed according to the method as described previously (Liu et al. 2009). For Western blots, 50 lg seedling total proteins were loaded for each sample. ChIP-qPCR assay The chromatin samples for chromatin immunoprecipitation (ChIP) experiments were obtained following the methods by Saleh et al. (2008). The plants seedlings were first cross-linked by formaldehyde, and the purified cross-linked nuclei were then sonicated to shear the chromatin into suitably sized fragments. The antibody that specifically recognizes the recombinant ABI4-GST, GBF1-
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GST, CBFA-GST or LEC1-GST was used to immunoprecipitate DNA/protein complexes from the chromatin preparation. The DNA in the precipitated complexes was recovered and analyzed by qPCR methods. The chosen primer combinations (Supplemental Table 1) could amplify fragments of 50–400 bp within the promoters of LHCB, CBFA and CBP genes. To ensure the reliability of ChIP data, the input sample and no antibody (NoAB) control sample were analyzed with each primer set. Rabbit control IgG was purchased from Abcam Comp. (Cambridge, UK). The results were quantified with a calibration line made with DNA isolated from cross-linked and sonicated chromatin (Haring et al. 2007). Statistics analysis All experiments were repeated three times, and average results are presented and the standard deviations (n = 3) were shown. Student’s t test was used for comparison between different treatments. A difference was considered to be statistically significant when p \ 0.05.
Results Exogenous hemin represses LHCB gene independent of ROS Firstly, we re-validated the action of norflurazon (NF, a herbicide and plastid signalling trigger), Mg-Proto IX, 7 % Sucrose, 3 % Sucrose ? lincomycin (Linc) and ABA on LHCB repression and RNA multiplication (Fig. 1a and Supplemental Fig. 1). Inhibition of plastid gene expression (such as Linc) increases cellular sensitivity to sugar. Thus the sucrose threshold for LHCB repression decreased from 7 % in seedlings without Linc treatment to 3 % in seedlings treated with Linc (Zhang et al. 2010a). 50 lM hemin treatment under a low light (20 lmol m-2 s-1) doubled the cellular total RNA level but did not repress LHCB expression. Nevertheless when the hemin level was increased further to 200 lM, LHCB transcripts were significantly down-regulated (Fig. 1a and Supplemental Fig. 1). 50 lM hemin treatment under a high light (HL, 250 lmol m-2 s-1) produced a similar result as 200 lM hemin in the low light (Fig. 1a). NF, ABA and 50 lM Hemin in HL generated superoxide and hydrogen peroxide apparently, but the other treatments could not (Fig. 1b). It is known that ROS act downstream of NF (Kleine et al. 2009; Zhang et al. 2011a) and ABA signalling (Zhang et al. 2006). 200 lM hemin under the low light could not induce ROS obviously (Fig. 1b), suggesting that hemin may work as a signalling molecule independent of ROS signals.
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Fig. 1 ABI4 and CBFA are involved in multiple signalling. a Response of LHCB, CBFA and CBP transcripts and total RNA levels to NF, Sucrose, Linc, Mg-Proto IX, Hemin or ABA treatments in wild-type (Col-0) seedlings or abi4 and cbfA mutants. For RNA quantification, DNA was used as a loading control. b Hemin induces
ROS accumulation only in the high light (HL) conditions. LL low light. Superoxide and H2O2 levels were visually detected by NBT and DAB, respectively. Ctrl, control seedlings without any treatment. All experiments were repeated three times, and typical results are presented. Error bars show standard deviations (n = 3)
ABA double cellular total RNA through ABI4 and CBFA-mediated pathways
gated all four candidate TFs regulated by plastid signals as Strand et al. (2003) identified through a micorarray analysis. Among them, only the CCAAT binding factor CBFA was found to be possibly involved in the multiple signalling to CBP gene (Fig. 2). NF, sucrose, hemin or ABA, all these treatments induced CBFA repression (Fig. 1a and Supplemental Fig. 1). On the other hand, cfbA mutant had a little but significantly higher level of CBP transcripts (Fig. 1a and Supplemental Fig. 1). Thus, we suppose that CBFA might be a negative regulator to CBP expression. After the treatments, ABI4 repressed CBFA expression and then CBFA repressed CBP expression. Thus, ABI4 regulated CBP expression positively as a whole.
It is interesting that ABA not only repressed LHCB expression but also increased CBP expression and total RNA levels (Fig. 1a and Supplemental Fig. 1). ABA signalling to CBP gene was completely abolished in abi4 mutant, but not in gun1 mutant (Fig. 2), implying that ABI4 mediates a greater number of signal transduction outputs than GUN1. Considering that ABI4 is a negative regulator of PhANGs expression (Koussevitzky et al. 2007), there might be some other TFs mediating CBP induction positively. We investi-
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Fig. 2 The roles of putative transcription factors in multiple signaling. gun1, abi4, cbfA, At1g19850, abf2, At4g36990 mutants were investigated. CBP expression levels were detected by quantitative real-time PCR. The levels of the wild-type (Col-0) seedlings without any treatment are normalized to 100 %. Error bars show standard deviations (n = 3). 7 %S, 7 % sucrose; Ctrl, control seedlings without any treatment
ABI4 bound to LHCB and CBFA promoters persistently The persistence of ABI4-mediated signalling was testified by the following experiments: The seedlings were treated with NF, 7 % sucrose, 200 lM hemin or ABA in the low light for 24 h to trigger the signals, and then transferred to water (1/2 Hoagland solution) under the normal light condition (100 lmol m-2 s-1) or HL condition (250 lmol m-2 s-1) for additional 48 and 96 h to enhance LHCB and CFBA transcripts (attempts to reverse the signalling). However, neither diluting out the signalling triggers alone/illuminating with the HL alone (data not shown) nor the combination of these two transfers de-repressed LHCB/CFBA expression (Fig. 3a). ABI4 and GBF interact with a common binding sequence CCACGT (a G-box element), and therefore they are a couple of competitive transcription factors (Koussevitzky et al. 2007; Darieva et al. 2010). This core G-box element was found in promoters of both LHCB gene and CBFA gene. When this box was mutated, CBFA transcript could not be repressed by NF, 7 % sucrose, hemin or ABA treatment (Fig. 3b). When the plants were grown under the normal conditions without any signalling triggers, GBF bound to LHCB and CBFA promoters and almost no ABI4 binding could be detected (Fig. 3c; Supplemental Fig. 2). HL induced GBF expression and subsequently more GBF binding in control seedlings (Figs. 3c, 4; Supplemental Fig. 2). However, after the treatments, the binding of ABI4 to the G-box element could not be changed by the following HL illumination, and the greatly enhanced GBF protein could not pull ABI4 down from the G-box element (Figs. 3c, 4; Supplemental Fig. 2). To acquire further evidence of regulations to TF’s binding, transgenic plants over-expressing ABI4, GBF1, CBFA or LEC1 (discussed below) were constructed. Eight transgenic lines for each gene were confirmed by RT-PCR (Supplemental Fig. 3) and Western blotting (Supplemental Fig. 4), and one line with the highest expression level for
each gene was selected for the subsequent studies (Supplemental Fig. 3). The possessive binding of ABI4 was affirmed further in gbf1 mutant and GBF1 over-expressing transgenic plants. Neither of them had dramatically changed LHCB or CBP transcripts comparing with the wild-type plants (Fig. 5), indicating that ABI4 binding was independent of GBF levels. Interestingly, over-expressing ABI4 also could not affect the signalling (Fig. 5). The redundant transcription factor CBFA functions irreplaceably in signalling transduction CBFA is one of CCAAT binding factors, which are evolutionary conserved oligomeric transcription factors that contain three non-identical subunits called HAP2, HAP3, and HAP5 (Heme activator protein) that interact to form a complex (Maity and de Crombrugghe 1998). There are 10 HAP3 subunits in Arabidopsis, and could be divided into two classes on the basis of sequence similarity in the B domain: the LEAFY COTYLEDON1 (LEC1)-type, consisting of LEC1 and LEC1-LIKE (L1L), and the nonLEC1-type comprising the remaining subunits, including CBFA (Kwong et al. 2003). LEC1 is a regulator of embryogenesis that controls many different aspects of embryo development and the lec1 mutant shows an embryo lethal phenotype (West et al. 1994). While the other HAP3 s were considered to be redundant ones without importantly biological functions (Lee et al. 2003). The complementary strand of CBP promoter has a CCAAT box at -223 bp. When this box was mutated, CBP transcript could not be increased by NF, 7 % sucrose, hemin or ABA treatment (Fig. 6a). When the plants were grown under the normal conditions without any signalling triggers, both CBFA and LEC1 bound to the CBP promoter (Fig. 6b). HL slightly induced CBFA expression (Fig. 4) and subsequently a little more CBFA binding in the control seedlings (Fig. 6b). However, once the signal transduction fulfilled, CBFA transcripts and its binding persistently
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Fig. 3 Permanent binding makes ABI4-madiated signalling longlasting. a Low light to high light transfer can not reverse the signalling. Wild-type seedlings were pretreated with NF, 7 % sucrose (7 %S), hemin or ABA for 24 h, and then transferred to high-light (HL) treatment for additional 0, 48 or 96 h. General reverse transcription (RT)-PCR results are shown in Fig. 3a. b GUS staining of the Arabidopsis seedlings transiently transformed with 800 bp of the CBFA promoter fused to the GUS reporter gene. On the right, the
CCACGT box in the promoter was mutated to TTACAA. c ChIP assays for ABI4 (the left part) and GBF1 (the right part) binding to the G-box of the CBFA promoter in vivo. The samples were collected from the wild-type plants as (pre)treated in a. No AB shows the control signals without the antibody (AB). IgG shows the control signals of Rabbit control IgG. The data are presented as mean ± SD (n = 3). Ctrl, control seedlings without any treatment. All experiments were repeated three times, and typical results are presented
Fig. 4 The responses of ABI4, GBF1, LEC1 and CBFA genes to signaling triggers and high light treatments. Only the wild-type plants were used. Gene expression levels were detected by quantitative realtime PCR. The levels of the control (Ctrl) seedlings without any
treatment are normalized to 100 %. Error bars show standard deviations (n = 3). 7 %S, 7 % sucrose. The high-light (HL) treatments were performed with 1/2 Hoagland solution without any signaling triggers
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Fig. 5 ABI4, GBF1, CBFA and LEC1 levels regulate LHCB and CBP expression. Wild-type (Col-0) seedlings, gbf1 mutant, and transgenic plants overexpressing ABI4, CBFA, GBF1 and LEC1 genes by the 35S promoter (35S-) were investigated. Gene expression levels were detected by quantitative real-time PCR. The levels of the control (Ctrl) seedlings without any treatment are normalized to 100 %. Error bars show standard deviations (n = 3). 7 %S, 7 % sucrose. The high-light (HL) treatments were performed with 1/2 Hoagland solution without any signaling triggers
decreased. Correspondingly, more LEC1 bound to CBP promoters. Although lec1 mutant is lethal (lec1 seeds don’t completely mature, so the seeds die if not rescued), therefore not testing in this study; LEC1 over-expressed transgenic plants had a little but significantly higher level of CBP transcripts (Fig. 5). Thus, we suppose that LEC1 might be a positive regulator to CBP expression. All these binding can not be reversed by the following HL treatment (Fig. 6b). However, the changes should be attributed to CBFA repression caused by the long-lasting ABI4 binding, but not the assumed persistence of CBFA binding. Actually, signalling-induced CBFA repression could be reversed by over-expressing CBFA gene, which significantly restrained CBP transcripts (Fig. 5). Accordingly, the binding of LEC1 to CBP promoters reduced in CBFA over-expressed transgenic plants (Supplemental Fig. 5). A previous study also showed that over-expressing some HAP2 or HAP3 subunit may impair formation of a HAP2/3/5 complex leading to reduced expression of target genes (Wenkel et al. 2006).
(Fig. 7), which indicated the impaired basal drought-tolerance in these mutants. If the wild-type plants were pretreated with 50 lM NF for 48 h (inducing RNA multiplication and oxidative stress acclimation, Zhang et al. 2011b), their drought-tolerance could be largely enhanced (Fig. 7). However, this NF-stimulated droughttolerance was completely abolished in gun1, cbfA and cbp mutants (Fig. 7). On the contrary, the abi4 mutant showed a great resistance to drought stress (Arenas-Huertero et al. 2000; Cheng et al. 2011), however its drought-tolerance could not affected by the NF pre-treatment (Fig. 7). All the data suggested that the signalling components (including three important TFs) play an irreplaceable role in plant’s adaptation to environmental stresses. Consisting with gene expression regulations, transgenic plants over-expressing ABI4, CBFA, GBF1 or LEC1, did not show dramatically changed drought-tolerance, when comparing with the wild-type plants (Supplemental Fig. 6). Although 35S-CBFA plants and 35S-LEC1 plants showed a little weaker tolerance and a little stronger tolerance respectively (Supplemental Fig. 6).
The physiological roles of ABI4, CBFA and CBP in drought-tolerance Discussion The physiological relevance of the regulatory actions of ABI4 and CBFA has been investigated further. After 14-day drought treatment, the relative water content (RWC) declined from 89 to 52 % in the wild-type plants, while the RWC values for gun1, cbfA and cbp mutants were 43, 45 and 37 % respectively at the 14th day of stress
The signalling roles of the herbicide norflurazon and the accompanying tetrapyrrole Mg-Proto IX NF treatments induce Mg-Proto IX accumulation (Strand et al. 2003). With this herbicide, a series of GUN mutants
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(McCormac and Terry 2002, 2004). The fact that most gun mutants have defects in tetrapyrrole biosynthesis that would affect Mg-Proto IX production suggested that Mg Proto IX or its related intermediates might be the signal molecules. Tetrapyrrole feeding experiments also provided direct and strong evidence that Mg-Proto IX is the putative signaling molecule, not the other tetrapyrroles (Strand et al. 2003; Ankele et al. 2007; Zhang et al. 2011a). However, the role of Mg-Proto IX in plastid signalling has been challenged by two later reports (Mochizuki et al. 2008; Moulin et al. 2008), and the NF treatment generates a much more complex signal other than simple reactive oxygen species (ROS) or simple Mg-proto IX accumulation (Kindgren et al. 2011; Zhang et al. 2011a). Our previous studies showed that ROS eliminator treatments only partly reversed the NF-induced repression of LHCB and suggested that transient accumulation of Mg-Proto IX in plastids transmits a signal to nucleus to represses PhANGs (Zhang et al. 2011a). The signalling role of the tetrapyrrole heme
Fig. 6 CBFA-madiated signalling and its binding are also longlasting in wild-type plants. a GUS staining of the Arabidopsis seedlings transiently transformed with 800 bp of the CBP promoter fused to the GUS reporter gene. On the lower half, the ATTGG sequence (CCAAT box on the complementary strand) in the promoter was mutated to ACCTT. b ChIP assays for CBFA (the upper half) and LEC1 (the lower half) binding to the G-box of the CBP promoter in vivo. Wild-type seedlings were pretreated with NF, 7 % sucrose (7 %S), hemin or ABA for 24 h, and then transferred to high-light (HL) treatment for additional 0, 48 or 96 h. No AB shows the control signals without the antibody (AB). IgG shows the control signals of Rabbit control IgG. The data are presented as mean ± SD (n = 3). Ctrl, control seedlings without any treatment. All experiments were repeated three times, and typical results are presented
has been defined (Nott et al. 2006). Defects in earlier steps in the tetrapyrrole synthesis pathway before Mg-chelatase (Strand et al. 2003), and overexpression of protochlorophyllide oxidoreductase (POR) that presumably reduces the Mg-Proto IX level, also result in a GUN phenotype
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Previous studies proposed that heme serves as a plastid signal that regulates the expression of nuclear genes in Chlamydomonas (von Gromoff et al. 2008). However, no evidence shows that it also works in higher plants before the year of 2010 (Rockwell et al. 2006; Zhang et al. 2011a). Free heme is highly cytotoxic, most probably due to the Fe atom contained within its protoporphyrin IX ring, which can undergo Fenton’s action and thus leading to free radicals production (Rockwell et al. 2006). Therefore, heme may work as ROS propagators but not as a signalling molecular directly. However, 50 lM hemin feeding under a low light doubled cellular total RNA without generating any ROS (Zhang et al. 2011a). In this study, we further show that 200 lM hemin under a low light repressed LHCB expression. Thus, heme may work as a signalling molecule to control PhANGs expression independent of ROS. A recent report suggested that heme synthesized through plastid ferrochelatase I (but not ferrochelatase II) regulated PhANGs, also indicating a putatively signalling role of heme (Woodson et al. 2011). Their results demonstrated that increased flux through the heme branch of the plastid tetrapyrrole biosynthetic pathway increases PhANGs expression. This observation seems contradictory to our results. The level of exogenous heme treated might be the reason. Woodson et al. (2011) also found that that ALA feeding to seedlings at high concentrations (over 0.8 mM) has a negative effect on PhANGs expression, might suggesting the dual effects of heme on PhANGs expression. Similarly, Enami et al. (2011) also suggested that exogenous heme, but not Mg-Proto IX, might be involved in plastid-to-nucleus signaling to negatively
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Fig. 7 Drought tolerance of wild-type plants and the signalling mutants with or without NF pre-treatment. Some plants were pretreated with (the bottom line) or without NF (50 lM) for 48 h, and then subjected to 14-day drought treatment. gun1, abi4, cbfA and cbp mutants were investigated. Relative water content (RWC) for each sample is shown (mean ± SD, n = 3, at the lower-right corner). All experiments were repeated three times, and typical results are presented
regulate the expression of nucleus-encoded starch biosynthesis genes, although the signalling role of heme and its signalling pathways need further investigations. The physiological role of the signalling transcriptional network in response to environmental stresses This fine-regulated transcriptional network means a lot to plant’s adaptation to the environment. Mg-Proto IXderived signalling has been reported to be involved in stress response in plants (Kindgren et al. 2011) and cell cycle coordination in algae (Kobayashi et al. 2009, 2011). Enami et al. (2011) also suggested that plastid-to-nucleus retrograde signals are essential for the expression of nuclear starch biosynthesis genes during amyloplast differentiation in tobacco BY-2 cultured cells. Miller et al. (2007) showed that gun1, gun5, and abi4 mutants had impaired basal thermo-tolerance. gun1, gun3, gun5, and abi4 mutants suffered from more oxidative damages than the wild-type plants under water stress and water stress ? NF co-treatment (Cheng et al. 2011). Here we also show fully or partly hampered drought-tolerance in gun1, cbfA, cbp, and abi4 mutants (Fig. 7). Accumulation of Protoporphyrin IX, protochlorophyllide, Mg-Proto IX may and heme may help plants to better tolerate drought stress (Phung et al. 2011). Plastid-signal-mediated nuclear-generegulation is also important for effectively cellular response to oxidative stress, such as high-light and herbicide costress conditions, where the plastid-signalling defective mutant gun1 showed an apparent photobleaching phenotype (Zhang et al. 2011a). Besides regulations to cell cycles, amyloplast differentiation and stress adaption, multiple plastid-signals also regulate anthocyanin accumulation and
early anthocyanin biosynthesis gene expression in newlygerminated seedlings (Ruckle and Larkin 2009; Cottage et al. 2010) and mature Arabidopsis rosettes (Cheng et al. 2012). Both GUN1 and ABI4 are required for this induction (Ruckle and Larkin 2009; Cottage et al. 2010; Cheng et al. 2012). The delicate communication between plastid and nucleus makes the cell live vividly and flexibly, who is awaiting our more deep-going explorations. ABI4, GBF, CBFA, LEC1 and CBP make up a complex transcription regulatory network ABI4 and LEC1 (discussed below) transcripts are usually expressed early during seedling establishment and may be undetectable afterwards (by Northern Blotting, So¨derman et al. 2000). However these two transcripts in mature Arabidopsis seedlings still could be quantified with the method of RT-PCR by increasing PCR amplification cycles. Quantitative PCR Ct values for ABI4, LEC1 and ACTIN1 genes were 32.2, 33.8 and 24.3 respectively. As shown in Fig. 4, NF and 7 % sucrose significantly induced ABI4 expression, while ABA and high-light treatments only slightly increased ABI4 transcripts, which are consistent with the previous reports (Arroyo et al. 2003; Avonce et al. 2004; Bossi et al. 2009; Sun et al. 2011). Nevertheless, inducing rates of ABI4 expression were much less than the increasing rates of ABI4 binding, indicating some other regulatory mechanism besides transcriptional enhancement. Koussevitzky et al. (2007) showed a similar result that, in wild-type seedlings, expression of ABI4 did not change after lincomycin treatment. We presume that the master switch ABI4 may transmit the signals (bind to the target promoters) through a conformational change
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(a post-transcriptional regulation, Sibe´ril et al. 2001) and this change is highly likely irreversible, which requires more structural studies in the future. ABI4 is a master transcription factor, who charges ABA, sugar and multiple plastid signals, and never concedes his position to other TFs after his binding. While CBFA is another TF working for a large transcription complex. When emergency occurs (such as herbicide treatments and environmental stresses followed by ABA and ROS accumulation), the master TF ABI4 down-regulates some TFs, like CBFA, and then some other TF subunits enter the transcription complex and transcriptional efficiency of HAP is improved instantaneously (Supplemental Fig. 7). These findings broaden our knowledge of gene expression regulation response to stress conditions, cellular signalling and avail of redundant transcription factors. Acknowledgments We thank Prof. Joanne Chory (The Salk Institute, La Jolla, USA) for gun1-9 seeds. We thank Dr. Xiao-Chao Xu (College of Bioindustry, Chengdu University, China) for technical assistance with ChIP assay and antibody preparation. This work was supported by the National Nature Science Foundation of China (31070210, 91017004 and 30970214), the National Key Basic Research ‘973’ Program of China (2009CB118500), and the Doctoral Foundation of the Ministry of Education (20110181110059).
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