Functional characterisation of Arabidopsis SPL7 conserved protein domains suggests novel regulatory mechanisms in the Cu deficiency response
© Garcia-Molina et al.; licensee BioMed Central Ltd. 2014
Received: 14 April 2014
Accepted: 18 August 2014
Published: 30 August 2014
The Arabidopsis SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) transcription factor SPL7 reprograms cellular gene expression to adapt plant growth and cellular metabolism to copper (Cu) limited culture conditions. Plant cells require Cu to maintain essential processes, such as photosynthesis, scavenging reactive oxygen species, cell wall lignification and hormone sensing. More specifically, SPL7 activity promotes a high-affinity Cu-uptake system and optimizes Cu (re-)distribution to essential Cu-proteins by means of specific miRNAs targeting mRNA transcripts for those dispensable. However, the functional mechanism underlying SPL7 activation is still to be elucidated. As SPL7 transcript levels are largely non-responsive to Cu availability, post-translational modification seems an obvious possibility. Previously, it was reported that the SPL7 SBP domain does not bind to DNA in vitro in the presence of Cu ions and that SPL7 interacts with a kin17 domain protein to raise SPL7-target gene expression upon Cu deprivation. Here we report how additional conserved SPL7 protein domains may contribute to the Cu deficiency response in Arabidopsis.
Cytological and biochemical approaches confirmed an operative transmembrane domain (TMD) and uncovered a dual localisation of SPL7 between the nucleus and an endomembrane system, most likely the endoplasmic reticulum (ER). This new perspective unveiled a possible link between Cu deficit and ER stress, a metabolic dysfunction found capable of inducing SPL7 targets in an SPL7-dependent manner. Moreover, in vivo protein-protein interaction assays revealed that SPL7 is able to homodimerize, probably mediated by the IRPGC domain. These observations, in combination with the constitutive activation of SPL7 targets, when ectopically expressing the N-terminal part of SPL7 including the SBP domain, shed some light on the mechanisms governing SPL7 function.
Here, we propose a revised model of SPL7 activation and regulation. According to our results, SPL7 would be initially located to endomembranes and activated during ER stress as a result of Cu deficiency. Furthermore, we added the SPL7 dimerization in the presence of Cu ions as an additional regulatory mechanism to modulate the Cu deficiency response.
SQUAMOSA PROMOTER BINDING PROTEINS (SBP) constitute a transcription factor (TF) family exclusively found in green plants. Arabidopsis thaliana (hereinafter Arabidopsis) homologs have been related to developmental and adaptive programmes, such as plastochron determination , leaf morphogenesis , vegetative phase transition , flowering , anther and gynoecium development – or innate immunity  and copper deficiency response ,.
Despite evolutionary divergence between the different family members, the tertiary structure of all SBP proteins encompasses the founding SBP-domain. It consists of a 76 amino acid signature including a functional bipartite nuclear localisation signal (NLS) and a series of 8 conserved cysteine and histidine residues organized in two unconventional zinc fingers (ZF1 and ZF2) –. Structural and functional studies suggested that ZF1 would maintain the SBP folding, while ZF2 would confer selectivity for the DNA sequence to bind ,. Therefore, the SBP domain facilitates nuclear translocation and confers the capability to bind DNA-motifs harbouring a GTAC core sequence ,,.
The SBP genes appear in moderately sized-families. The Arabidopsis genome encodes 16 different SBP-Like (SPL) proteins grouped in 2 classes according to size, sequence similarity and structure and expression patterns of the respective genes. Based on these criteria, the denoted large SPLs (SPL1/7/12/14/16) conform a class representing the most complex and constitutively expressed genes. The other class is constituted by the small SPLs, whose expression is refined by the well-conserved and related microRNAs miR156/7, with SPL8 as a notable exception ,.
In recent years, the Chlamydomonas reinhardtii Copper response regulator Crr1 and its closest Arabidopsis homolog SPL7 attracted attention because of their deeply conserved function as central orchestrators of Cu homeostasis ,,. Cu is an essential micronutrient for virtually all eukaryotes since its redox properties are optimal for essential catalytic functions in protein complexes. Indeed, plant cells rely on Cu-proteins to deal with oxidative stress, energy production, lignification, or pollen tube guidance ,. Furthermore, Cu has also been reported to play a structural role in the ethylene and salicylic acid receptors, as well as in the molybdenum cofactor –. However, an excess of free Cu ions will damage cellular components, e.g. lipids, proteins or nucleic acids, due to the generation of reactive oxygen species (ROS) . To cope with this dual nature of Cu, cells possess a fine-tuned homeostatic network aimed at maintaining Cu levels within a proper range. Although the general features of this network are conserved among all eukaryotes, main evolutionary divergences concern the regulatory mechanisms. During Cu starvation in Arabidopsis, SPL7 directly binds to GTAC motif-containing Cu response elements (CuRE) located in the promoter regions of Cu responsive genes ,. In a first response, a Cu-uptake system based on the Cu-metalloreductases FRO4/5 and the plasma membrane-related Cu transport proteins COPT1/2/6 is promoted ,. Secondly, SPL7 reprograms cellular gene expression for a more efficient Cu usage and (re-)distribution within the plant, thereby prioritizing delivery to essential enzymes. In this way, levels of particular microRNAs, denoted Cu-miRNAs and including miR398 and miR408, are raised to translationally repress production of non-essential Cu-requiring proteins, such as the cytosolic Cu/Zn superoxide dismutase (CSD1), chloroplastic CSD2, plantacyanin or the laccases. Suppression of CSD2 and the promotion of FSD1 represent a coordinated substitution of the chloroplastic superoxide dismutases that facilitates a preferential delivery of Cu to plastocyanin (PC) ,,.
However, the mechanism underlying SPL7 activation is not fully understood, especially with regard of Cu sensing and protein regulation. SPL7 is a constitutively expressed gene detected in all plant tissues regardless of Cu availability. Consequently, a post-translational regulation for this TF has been proposed ,,. Within this context, we recently reported the physical interaction between SPL7 and a kin17-domain encoding protein (KIN17) to stimulate SPL7 targets during Cu starvation . Moreover, the in vitro SBP-DNA binding could be prevented by Cu ions probably replacing complexed Zn ions and thereby changing the conformation of SPL7-like proteins ,. Here, we present a functional characterisation of conserved domains in the SPL7 protein as to come to a better understanding of how its activity may be regulated in response to cellular Cu status in Arabidopsis. Our subcellular and biochemical approaches revealed that the presence of a TMD recruits SPL7 to the microsomal fraction, likely at the ER membrane and suggests a proteolytic cleavage prior to its nuclear translocation. Interestingly, our data indicate that Cu deficiency implicates ER stress and could constitute a driving force to activate SPL7. Moreover, a SPL7 dimerization domain could act in a mechanism to prevent the protein from entering the nucleus.
A conserved transmembrane domain is sufficient to anchor SPL7-like proteins to the plasma membrane
SPLexhibits a dual subcellular localisation and likely requires proteolytic cleavage to become translocated to the nucleus
The above-stated results concerning the presence of a TMD seem to oppose the function of a conserved bipartite NLS within the SBP domain and the rather constitutive nuclear localization reported in our previous observations, as well as for several other SBP-domain proteins ,,. Therefore, we addressed the question whether SPL7 could display a dual subcellular distribution. For this purpose, we generated CaMV 35S promoter-driven transgenes consisting of the entire SPL7 coding region and fused in frame either 5′ or 3′ to GFP to allow the constitutive expression of either N-terminal or C-terminal tagged SPL7 protein (GFP::SPL7 and SPL7::GFP; Figure 1a). Since we failed to reliably detect GFP fluorescent signals in Arabidopsis plants stably transformed with these constructs, we decided to use agro-infiltration of tobacco leaves as a heterologous system to assess the subcellular localization of the encoded protein products. Strikingly, while GFP::SPL7 distributed homogeneously within the nucleoplasma excluding the nucleolus, the C-terminal tagged version located around the nucleus and to filamentous structures in cytoplasmic strands (Figure 1b, 1c and Additional file 2: Figure S2). As the latter pattern suggested possible association with the ER, we co-infiltrated the SPL7::GFP-encoding construct with an ER marker fused to the mCherry fluorescent protein (ER-rk ). This revealed a high degree of co-localization of both fluorescent signals, most intensely around the nucleus (Figure 1d). Moreover, we also subjected total extracts from 35S::GFP::SPL7 and 35S::SPL7::GFP transformed leaves to a biochemical fractionation, as described above. Indeed, GFP::SPL7 protein was detected in the nuclear enriched fraction, whereas SPL7::GFP primarily associated with the microsomal fraction (Figure 2a), thereby corroborating the microscopic observations. Consequently, these data strongly suggest SPL7 to distribute between the nucleus and the endomembrane system.
Interestingly, although estimating that GFP would contribute ~23 KDa and SPL7 ~ 90 KDa, the observed apparent molecular weight of both GFP-tagged SPL7 versions seemed more consistent with ~63 KDa (Figure 2a). Since membrane-anchored proteins must be somehow released prior to their translocation to the nucleus and exert their function, we investigated if these observed bands could correspond to cleaved SPL7 products. To this end, total protein extracts from transformed tobacco leaves expressing either GFP::SPL7 or SPL7::GFP were also analysed by Western blot. A pattern including two specific bands was obtained regardless of the position of the tag (Figure 2b). We considered that the upper band (~125 KDa) could correspond to the full-sized SPL7 while the presence of the second lower band (~63 KDa) in both cases might be explained if SPL7 would have been cleaved in the middle (Figure 2a). This processing would thus render a derived polypeptide fitting the observed size (~45 KDa from half of SPL7 + ~23 KDa from GFP; Figure 2a,b).
These results are consistent with an arranged pattern where the N-terminal half of SPL7 translocates to the nucleus following proteolytic cleavage, whereas the C-terminal half would remain attached to some endomembrane, such as the ER.
Cu deficiency generates endoplasmic reticulum stress, a metabolic perturbation that promotes SPLtarget activity
SPLis able to homodimerize in vivo
Arabidopsis transgenic lines expressing the SPL7-SBP domain exhibit constitutive activation of SPL7 targets
All together, our data indicate that neither the stability of the SBP domain nor its function is severely affected by Cu ions in planta. Based on these results, we suggest that protein domains outside the SBP-domain of SPL7 are likely to have a more profound effect on SPL7 activity in response to Cu availability.
In this sense, ER-membrane tethered TFs (ER-MTTFs) might provide an illustrative example to infer the SPL7 mechanism since they exhibit a similar behaviour. This class of TFs display an initial latent form when attached to membranes and require some sort of processing to be released and eventually translocated to the nucleus . ER-MTTFs nuclear-localised versions are generated as a result of two main strategies, namely mRNA processing and proteolytic cleavage. Although alternative mRNA splicing has been reported to produce a non-anchored version of the bZIP60 ER stress transducer ,, this mechanism would not be expected for SPL7 because its known or predicted splicing variants (AT5G18830.2 and -.3; TAIR10 genome release; www.arabidopsis.org) do not disrupt the TMD domain. More often, specific proteolytic activities, such as the regulated intramembrane proteolysis (RIP) and the rhomboid proteases, produce a cleavage at the vicinity of the TMD ,. However, because the apparent molecular weights of both SPL7 nuclear and ER-attached fragments correspond approximately to half of that of the predicted full-size protein, a proteolytic cleavage in the middle is suggested as the strategy to release and activate SPL7 (Figure 8). Thus, regulated ubiquitin/proteasome-dependent processing (RUP) and the so-called receptor-activated proteolysis (RAP) would be more conceivable for this case ,. Nevertheless, we envisage a relative rapid-acting mechanism as the presence of the full-sized GFP::SPL7 was barely detectable in co-immunoprecipitation experiments and remained even undetectable following biochemical fractionation. Consequently, it would be particularly interesting to identify the responsible protease(s) and the cleavage site(s) in SPL7, as it will shed more light on the precise mechanism activating SPL7 and contribute to relate its function to additional biological responses.
The initial location of a likely dormant SPL7 at the ER provides a new perspective on the regulation of Cu homeostasis and requires a re-evaluation of the role of the so-called secretory pathway in Cu sensing. As represented in Figure 8, Cu+ imported by the selective Cu-transport proteins CTR/COPT is bound and further distributed by Cu-specific soluble factors or metallochaperones (for a comprehensive description see Burkhead and collaborators ). Among them, ATX1-like metallochaperones interact with the PB-ATPase Ccc2 in Saccharomyces cereviseae, or RAN1/HMA7 in plants, in order to supply Cu-proteins en route,. Whereas Ccc2 resides in the Golgi apparatus of yeast, the exact subcellular localization of Arabidopsis RAN1 has not yet been determined. However, since the ER-located ethylene receptors (ETRs) are largely dependent on Cu supply by RAN1, an ER location has been proposed –. Thus, unlike storage organelles as chloroplasts, mitochondria or vacuoles, the ER could act as a more reliable indicator of the steady-state Cu availability in the cell.
Notably, several studies have recently reported a central role of the ER in sensing/transducing cellular stresses ,. In an attempt to identify ER perturbations that activate SPL7, our initial data suggest an impact in the ER protein-folding capacity during Cu starvation and how ER stress treatments slightly induced selected SPL7-targets in mild Cu-sufficient seedlings. Whether the initial tethering of SPL7 to the ER-membrane could be a cellular strategy to sense Cu limitation through the stress it imposes to the ER needs to be further investigated. Within this context, it is also worth mentioning that the growth inhibitory effect of fumonisin B1 (FB1) was found attenuated in the fbr6 mutant, representing the SPL7-related SPL14 TF . The apoptotic effect of the mycotoxin FB1 is related to a reduction in the cellular ceramide levels, a likely signal for ER-stress –. Given the conservation of the putative TMD among the large SPLs including SPL14, it would also be interesting to address if the strategy proposed for SPL7 could be extended to this class of TFs.
On the other hand, given that the SPL7 orthologs in single-celled algae lack a TMD, this domain could represent an innovation in the evolution of land plants . The positive selection of the TMD may be related to the multicellular and more complex nature of land plants, where many different cell types likely differ in their requirements for Cu and their demand probably even changes with growth and development. Thus, anchoring SPL7-like proteins to membranes could play a role in fine-tuning their activities in a more cell-autonomous context. However, although further comparative studies between Crr1 and SPL7-like proteins are required to provide a more thorough answer, the existence of additional regulatory levels for these TFs in higher plants seems likely.
Based on our data, we also propose SPL7 homodimerization as another checkpoint in the regulation of SPL7 activity. Indeed, independent in vivo approaches indicated that the SPL7 N-terminal half is prone to self-dimerization. Accordingly, only SPL7 protein fragments encompassing at least the conserved signature RXSXKLX4PX3PX2LX7LX7EX3RXGCX3T denoted the IRPGC domain (albeit extended compared to previous reports ,), were isolated in a Y2H screen using SPL7 as bait. Consequently, this signature could be considered to represent a dimerization domain. Homodimer formation involving this domain in the N-terminal half of SPL7 would also explain our observations on co-immunoprecipitated N-terminal SPL7 fragments, most likely generated through post-translational processing as discussed above. Similarly, only split YFP fragments fused as N-terminal tags to SPL7 were successful in BiFC assays. Furthermore, the reconstituted YFP fluorophore signal for N-terminal fusions illuminated the nuclear surroundings and cytoplasmic filaments, in an ER-like distribution. However, these results seem to contradict observations on GFP-tagged SPL7-like proteins clearly located in the nucleus when overexpressed in heterologous systems (our results and ,). Therefore, it is tempting to speculate that SPL7 preferentially enters the nucleus as a monomer. Exclusion of the dimer may be the result of the large size of the protein complex formed or of masking the NLS (Figure 8). In addition, rapid degradation or instability of SPL7 dimers cannot be ruled out as GFP::SPL7 was not easily detectable outside the nucleus neither in fluorescence microscopy nor in biochemical approaches. Hence dimerization, likely promoted by increasing amounts of released SPL7 protein, may be part of a negative feedback mechanism to attenuate the homeostatic Cu deficiency response and eventually avoid spurious effects. Interestingly, given the conservation of the IRPGC signature not only in SPL7 orthologous proteins but also in closely related large SPLs in Arabidopsis , homodimerization, or even heterodimerization, may represent a more general regulatory feature of this type of SBP-domain TFs.
The participation of additional SPL7-interacting proteins in the SPL7 post-translational regulation mechanism cannot be excluded (Figure 8). Indeed, KIN17 associates with SPL7 in order to stimulate SPL7-targets and counteract the oxidative stress under Cu deprivation . Nevertheless, we are not aware of mutants for other genes with a similar or even close impact on the global response to Cu deficiency as spl7 mutants have. Therefore we assume that the likely SPL7-interactome consists of largely functionally redundant components that probably contribute more to refine SPL7 function, rather than to its activation.
Importantly, SPL7 is expected to undergo a high turnover because different tagged full-sized SPL7-like proteins could not be clearly detected in stable transgenics, despite their functionality (our observations and ,,) and reasonable transgene transcript levels (Additional file 5: Figure S5). We also did not succeed to trace SPL7 in planta by observing different tissues at different time-points or using different tags, growth conditions or protein degradation inhibitors (data not shown). However, we demonstrated that expression of an N-terminal GFP-tagged SPL7 fragment including the SBP-domain but lacking the downstream IRPGC domain could be detected and resulted in a constitutive SPL7 function-related response irrespective of the Cu availability. A similar behaviour has been reported for other ER-attached proteins. A constitutive ethylene triple response is achieved by expressing putative C-terminal EIN2-cleaved fragments . Similarly, the anac017-2 mutant, rendering a truncated version of ANAC017 without the TMD, induces its target ALTERNATIVE OXIDASE1 (AOX1), even in non-H2O2-treated plants . Remarkably, the constitutive transcriptional activity of SPL7 targets in SPL7-SBP transgenic lines, even during non-physiological Cu excess, seem to contradict previous data showing that Cu ions negatively interfere the DNA-binding capacity of the SPL7 SBP-domain in vitro. One should take into account that Cu ions cannot move freely within cells due to the efficient Cu-chelating capacity of cells . Moreover, a direct interaction between SPL7 and free Cu ions seems unlikely because Cu is mostly stored in organelles like the chloroplasts and mitochondria, whereas SPL7 distributes between endomembranes and nucleus. Nevertheless, a slight decrease in SPL7-targets could be even noticed in the SPL7-SBP plants during the transition from Cu deficiency to sufficiency. We, therefore, propose that the effect of Cu ions on the functionality of SPL7 is mediated by some interacting factor(s), such as specific metallochaperones (Figure 8). The respective interacting SPL7 domain(s) is then most likely C-terminal of the SBP-domain. An overlap with the IRPGC domain, as the main conserved signature within this region, cannot be excluded. Whether the dimerization through this domain constitutes a possible regulatory mechanism promoting SPL7 turnover needs to be further addressed.
Altogether, our data provide novel insights into the molecular mechanisms underlying the role of the SPL7 TF in orchestrating Cu homeostasis in plants. Additionally, the mechanism of action we have reported here for SPL7 may possibly be extrapolated to other large SBP-domain proteins because a conservation of particular structural features is suggested on the basis of amino acid sequence similarities.
Plant growth and manipulation
The wild-type line used in all the experiments corresponded to the Arabidopsis thaliana ecotype Columbia (Col-0). The spl7-2 mutant has been previously described by Bernal and colleagues . Seeds were stratified at 4°C for 2 days prior to be sown. For in vitro culture, seeds were surface sterilized with sequential washes in ethanol 70% (5 min), bleach (5 min), water (2× 2 min), resuspended in agar 0.1% (w/v) and sown on half-strength MS medium plates (½ MS; Sigma) supplemented with sucrose 1% (w/v) and CuSO4 as indicated. Cu-deficient growth conditions were achieved by adding the specific Cu chelator bathocuproine disulphonate (BCS; Sigma-Aldrich) to the medium. In all cases, long day conditions (16 h light, 20-23°C/8 h darkness, 16°C) were applied. To generate stable transgenic lines, constructs were introduced in wild-type and spl7-2 mutant plants using Agrobacterium tumefaciens GV3101 (pMP90RK) in the floral-dip method ,.
cDNA fragments corresponding to the entire coding sequence or selected regions of SPL7 (AT5G18830.1) were amplified with specific oligonucleotides (Additional file 6: Table S1) and cloned into pDONR207 by means of the Gateway BP clonase II (Invitrogen). The generated entry clones were further recombined into the pMDC43 or pMDC201 vectors with LR clonase II (Invitrogen) to add a GFP tag at either the amino- or carboxi-terminus, respectively . Similarly, the pALLIGATOR2 vector was chosen to add a 3xHA tag to the N-terminus of full-sized SPL7 (HA::SPL7) . For BiFC, full-sized SPL7 in pDONR207 was LR-recombined into both the pYFN43 and pYFC43 destiny vectors providing N-terminal the two halves of YFP . The ER marker fused to mCherry (ER-rk) used for subcellular co-localizations was described in Nelson and colleagues .
The Y2H assay was performed by Hybrigenics Services SAS using a fragment of SPL7 (aa residues 133 to 762) as bait to screen a random-primed cDNA prey library prepared from 1-week-old Arabidopsis seedlings.
Subcellular localization and bimolecular fluorescence complementation assay on tobacco leaves
To determine the subcellular localization of truncated SPL7 protein versions, Nicotiana benthamiana (tobacco) young leaves were co-infiltrated with diluted cultures of A. tumefaciens harbouring CaMV 35S promoter-driven transgene constructs expressing fluorescent-tagged proteins of interest together with the p19 plasmid  in infiltration buffer [D-glucose 0.5% (w/v); MES 10 mM; MgCl2 10 mM; acetosyringone 0.1 mM] at an OD600 0.2. Small pieces of leaves were excised 2–3 days after infiltration and examined in a confocal laser scanning microscope (Zeiss LSM700) using filters to select for the GFP and chlorophyll signal. For BiFC assays, the pYFN43::SPL7 (nYFP::SPL7) and pYFC43::SPL7 (cYFP::SPL7) destiny vectors were individually or co-expressed in leaf epidermal tobacco cells as indicated above, and examined using widefield epifluorescence microscopy (Olympus BX61) and confocal laser scanning microscopy to asses the restoration of the YFP signal.
Biochemical fractionation was carried out according to Sáez and collaborators  with modifications: 3 g of transiently transformed tobacco leaves were homogenised in 3 volumes of extraction buffer [Tris–HCl 20 mM pH 7.4; glycerol 25% (v/v); KCl 20 mM; MgCl2 2.5 mM; EDTA 2 mM; sucrose 250 mM; Pefabloc 1 mM; cOmplete Protease Inhibitor Cocktail (Roche) 1X], filtered through 2 Miracloth layers and centrifuged at 1000 g for 10 min at 4°C to pellet nuclei. Pellets were gently rinsed with 2 mL of Nuclei Wash Buffer [Tris–HCl 20 mM pH 7.4; glycerol 25% (v/v); MgCl2 2.5 mM; Triton X-100 0.5% (v/v)]. After centrifugation at 1000 g for 30 s pellets were resuspended in 5 volumes of Medium Salt Buffer [Tris–HCl 20 mM pH 7.4; glycerol 5%; NaCl 0.4 M; β-mercaptoethanol 1 mM; EDTA 1 mM; Pefabloc 0.5 mM; cOmplete Protease Inhibitor Cocktail 1X and stored frozen. Samples were thawed on ice, stirred for 15 min and centrifuged at 10000 g for 10 min to recover the supernatant, which was considered as the nuclear enriched fraction. To obtain the microsomal fraction, the initial supernatant was submitted to ultracentrifugation at 100000 g for 1 h in a SW-44 Ti rotor (Beckman) and the sediment was resuspended in extraction buffer. The remaining supernatant was used as the cytosolic fraction. All fractions were concentrated by means of Microcon Centrifugal Filter Devices (Merck Millipore) columns and equal amounts of proteins loaded on 10% NuPAGE precasted gels (Life Technologies). Antibodies used for Western blot were: anti-GFP (1:1000; clones 7.1 and 13.1 Roche), anti-TPR7 (1:1000 ), anti-PEPC (1:15000; Rockland) and anti-H3 (1:10000; Abcam).
Induction of ER stress in Arabidopsis seedlings
ER stress was induced as described by Li and colleagues . Thereto, wild-type and spl7-2 seedlings were grown on ½ MS supplemented with sucrose 1% (w/v) and CuSO4 0.5 μM for 5 days. Subsequently, they were cultured in liquid ½ MS treated with tunicamycin (5 μg/mL) or DTT (2 mM) during 3 h with gentle shaking. Material was harvested and used for gene expression assays by qPCR.
Gene expression analysis by quantitative real-time PCR
For gene expression assays, total RNA was prepared with the Spectrum Plant Total RNA Kit (Sigma-Aldrich) according to manufacturer's instructions. The RNA integrity was visualized in ethidium bromide-agarose gels. RNA samples were treated with DNase I recombinant RNase-free (Roche) and reverse transcribed to cDNA with the Superscript II Reverse Transcriptase (Invitrogen). qPCR analysis were carried out in an iQ5 Real-Time PCR Detection System (Bio-Rad) with EvaGreen (Biotium) and specific primers (see Additional file 7: Table S2) using an initial cycle at 95°C for 3 min and 40 cycles consisting in 95°C for 10 s, 58°C for 20 s and 72°C for 20 s. ACT2 and EF1 were used to normalize gene expression values. Statistical analysis of at least three independent biological samples was performed using Excel (Microsoft Corporation). Student's t-test was used to determine statistically significant differences with a p < 0.05 or p < 0.01 as level of significance.
Total protein extracts from tobacco leaves transiently co-expressing HA::SPL7 and GFP::SPL7 were prepared by grinding frozen material in co-immunoprecipitation (CoIP) buffer [PIPES-KOH 10 mM pH 7; NaCl 50 mM; EDTA 0.5 mM pH 8.0; Triton X-100 0.5% (v/v); cOmplete Protease Inhibitor Cocktail 1X] and crosslinked to a limited extent with formaldehyde 1% (v/v). Samples were centrifuged at maximum speed at 4°C for 5 min and 0.4 volumes 2 M glycine were added to the supernatants in order to stop the crosslinking. Next, HA::SPL7 was pulled-down by incubating 1 mL of protein extract with 1.5 μg of anti-HA high affinity antibody (3 F10 clone, Roche) on rotation at 4°C for 2 h. Then, 50 μL of equilibrated Protein G Mag Sepharose Xtra (GE Healthcare) magnetic beads were added to extracts and rotated overnight at 4°C. Finally, beads were recovered, washed three times with 1 mL CoIP Buffer supplemented with NaCl 300 mM, Triton X-100 0.1% (v/v) and cOmplete Protease Inhibitor Cocktail 1X and boiled at 95°C for 10 min with 50 μL SDS-PAGE load buffer 4X. GFP::SPL7 co-immunopreciptitation was assessed by Western blot with anti-GFP-HRP (1:1000; Molecular Probes) and anti-HA-HRP (1:1000; Roche) antibodies.
Availability of supporting data
All the supporting data are included as additional files.
We thank Elmon Schmelzer (CeMic-MPIPZ, Cologne) for his advice and instructions on confocal microscopy, and Arne Grande, Susanne Höhmann and Rita Berndtgen (MPIPZ, Cologne) for their excellent technical support. Antibodies against H3 and PEPC were kindly provided by George Coupland's and Jane Parker's groups (MPIPZ, Cologne), respectively. François Parcy (CEA-CNRS; Grenoble) and Alejandro Ferrando (IBMCP-CSIC; València) are acknowledged for kindly providing the pALLIGATOR2 and pYFN43 and pYFC43 vectors, respectively.
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