Role of recently evolved miRNA regulation of sunflower HaWRKY6 in response to temperature damage


Author for correspondence:
Pablo A. Manavella
Tel: +49 7071 601 1405


  • MicroRNAs (miRNAs) are small 21-nucleotide RNAs that post-transcriptionally regulate gene expression. MiR396 controls leaf development by targeting GRF and bHLH transcription factors in Arabidopsis. WRKY transcription factors, unique to plants, have been identified as mediating varied stress responses. The sunflower (Helianthus annuus) HaWRKY6 is a particularly divergent WRKY gene exhibiting a putative target site for the miR396. A possible post-transcriptional regulation of HaWRKY6 by miR396 was investigated.
  • Here, we used expression analyses, performed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and northern blots together with computational approaches to establish the regulatory interaction between HaWRKY6 and the identified sunflower miR396. Arabidopsis plants expressing a mi396-resistant version of HaWRKY6 confirmed the miRNA-dependency of the HaWRKY6 silencing.
  • Sunflower plants exposed to high temperatures or salicylic acid presented opposite expression of HaWRKY6 and miR396. Experiments using the wildtype and miRNA-resistant versions of HaWRKY6 showed altered stress responses. Our results showed a role of the recently evolved miR396 regulation of HaWRKY6 during early responses to high temperature.
  • Our study reveals how a miRNA that normally regulates development has been recruited for high-temperature protection in sunflower, a plant particularly well adapted to this type of stress.


Many external factors, such as drought, extreme temperatures and pathogen infections, influence plant development. As a response to adverse environmental conditions, plants change their protein concentrations, both by transcriptional and/or post-transcriptional regulatory mechanisms. At the transcriptional level, the main players are transcription factors through cis-acting elements present in the regulatory regions. At the post-transcriptional level, small RNAs, and particularly microRNAs (miRNAs), play major roles on gene silencing.

MicroRNAs are small RNA molecules 21 nucleotides in length. They originate from primary longer RNA transcripts that include a self-complementary fold-back, from which the mature miRNAs are excised (Voinnet, 2009). In Arabidopsis thaliana alone, almost 300 miRNAs have been identified. The majority of them are evolutionarily young, not present in other plant species, suggesting that birth and loss of these small RNAs are frequent processes (Fahlgren et al., 2007; Axtell & Bowman, 2008). On the other hand, conserved miRNAs generally act in similar regulatory networks across species, making them master modulators of processes such as plant development, hormone signalling and stress response (Axtell, 2008). The key feature that makes miRNAs fundamental for plant homeostasis is their evolutionary preference to silence transcription factors (Axtell & Bowman, 2008). In Asteraceae, several conserved miRNAs have been predicted based on expressed sequence tag (EST) sequences (Monavar Feshani et al., 2012). However, their biological activity and relevance are largely unknown.

Many gene regulatory networks, including those involving miRNAs, tend to be conserved across large evolutionary distances (Nardmann & Werr, 2007; Zhong et al., 2010). However, the existence of species- and family-specific adaptations implies that the regulatory genes can acquire new characteristics. One example of such innovation has been described for the two A. thaliana MIR396 genes, MIR396A and MIR396B. The miR396 miRNAs silence members of the GROWTH-REGULATING FACTOR (GRF) family, which regulate cell proliferation and leaf development (Rodriguez et al., 2010). The miR396-GRF regulatory network is conserved across plant species (Axtell & Bowman, 2008; Cuperus et al., 2011). However, it has been recently shown that, in Brassicaceae and Cleomaceae, an additional transcription factor target has been recruited to this regulatory network, through acquisition of a miR396 target site. In this way, bHLH74, which also impacts leaf development, has come under miR396 control (Debernardi et al., 2012).

The Asteraceae family contains unique genes compared with model plants. An example is the sunflower gene HaHB4. The encoded transcription factor is a divergent member of the HD-Zip I family, presenting atypical domains outside the conserved HD-Zip (Gago et al., 2002; Arce et al., 2011). This structural divergence means HaHB4 has unique functions, not shared by its closest A. thaliana homologues (Dezar et al., 2005; Manavella et al., 2006, 2008). WRKY transcription factors have been associated with responses to biotic and abiotic stresses (Ulker & Somssich, 2004; Eulgem & Somssich, 2007; Rushton et al., 2010). They are defined by the presence of a 60 amino acid conserved region named WRKY domain, characterized by a WRKY and a zinc-finger-like motifs. At least 97 WRKY genes have been identified in sunflower (Giacomelli et al., 2010), although their biological roles are largely unknown. Moreover, phylogenetic analyses of the WRKY genes revealed the existence of Asteraceae-specific clades (Giacomelli et al., 2010).

Here we show that HaWRKY6 is a particularly divergent sunflower WRKY gene with a recently evolved miR396 target site. There is a direct correlation between miR396 accumulation and HaWRKY6 silencing. This regulation is critical for the response to high temperatures.

Materials and Methods

Plant material and growth conditions

Arabidopsis thaliana (L.) Heynh. accession Col-0, Nicotiana benthamiana Domin and Helianthus annuus L. cv HA89 genotype were grown in a growth chamber at 22–24°C, under long-day photoperiods. A. thaliana plants were grown in Petri dishes containing 0.8% agar–Murashige and Skoog (MS) medium or in soil pots (837 cm3). N. benthamiana plants were grown in soil pots. Sunflowers were grown in 30 × 45 cm soil pots until reaching the desired developmental stage (Schneiter & Miller, 1981).

RNA isolation and real-time reverse transcription polymerase chain reaction (RT-PCR)

Total RNA was extracted using Trizol® reagent (Life Technologies, Carlsbad, CA, USA). Quantitative RT-PCRs were performed as previously described (Manavella et al., 2006). Specific oligonucleotides are specified in Supporting Information, Table S1. Three biological replicates, tested by triplicate, were used to calculate standard deviation. Differences were considered significant when the P-values were < 0.05 (Student’s t-test). MiRNA-guided 3′ cleavage products were detected using RNA ligase-mediated 5′ RACE (rapid amplification of cDNA ends; Llave et al., 2002).

Northern blots

Total RNA (1–5 μg) was resolved on 17% polyacrylamide gels under denaturing conditions (7 M urea). The gel was transferred to HyBond-N+ membranes (Amersham) by semi-dry electroblotting, and hybridized to DNA probes labelled with digoxigenin (DIG) using the second-generation DIG oligonucleotide 3′-end labelling kit (Roche). Probe sequences are listed in Table S1.

Protein analysis

Proteins were isolated from 300 mg of ground tissue with 300 μl of extraction buffer (50 mM Tris, pH 7.5; 150 mM NaCl; 1 mM EDTA; 10% (v/v) glycerol; 1 mM dithiothreitol (DTT); 1 mM Pefabloc (Roche) and one tablet of complete protease inhibitor cocktail (Roche)) vortexing for 10 s. Tissue debris was eliminated by centrifugation at 12 000 g at 4°C for 20 min. Protein concentration was measured using a commercial Bradford kit assay (Bio-Rad). Twenty micrograms of crude protein extract per sample were resolved on an 8% polyacrylamide gel. Anti-HA (Hemagglutinin) high-affinity monoclonal antibody (Roche) was used to detect HA-tagged proteins.

Plant treatments

Sunflower V2 plants, grown in hydroponics, were transferred to plates containing 1× liquid MS medium to which 1 mM salicylic acid (SA) was added. Seedlings were incubated for the period of time specified on the figures and frozen in liquid nitrogen before RNA extraction. For heat-shock treatments, 2-week-old plants were placed during 2 h in a culture chamber maintained at 45°C. Then, the plants were irrigated and placed in normal growth conditions.

Constructs and plant transformation

The HaWRKY6 wildtype complete coding region (786 bp) was isolated by RT-PCR and cloned behind the constitutive CaMV 35S promoter in the pBI121 vector. The sequence of HaWRKY6 EST can be found in the EMBL/GenBank under the accession number BU024714. The miR396-resistant form of HaWRKY6 was generated by PCR-directed mutagenesis. The precursor of the miR396 was isolated from HA89 seedlings, cloned in the pCR8/GW/TOPO plasmid (Life Technologies) and recombined behind a 35S promoter into a pGREEN-IIS based vector (Hellens et al., 2000). Primers used for cloning are listed in Table S1. A. thaliana plants were transformed by the floral dip procedure (Clough & Bent, 1998). Fifteen homozygous independent lines for each construct were used to analyse phenotypes and expression levels. Transient transformation of sunflower leaf discs and tobacco leaves was carried out as previously described (Manavella & Chan, 2009; de Felippes & Weigel, 2010).

Sequence comparison and phylogenetic analyses

Phylogenetic trees where generated using the maximum likelihood method, with 100 replicates. Branches corresponding to partitions reproduced in < 50% bootstrap replicates were collapsed. Initial tree(s) for the heuristic search were obtained automatically as follows. When the number of common sites was < 100 or less than < 25% of the total number of sites, the maximum parsimony method was used; otherwise the BIONJ method with the MCL distance matrix was used. A discrete gamma distribution was used to model evolutionary rate differences among sites. MiR396 secondary structure was predicted using the Vienna RNA Webservers (

Results and Discussion

A miR396 target site in HaWRKY6

Sunflower WRKY factors potentially involved in stress responses have recently been identified (Giacomelli et al., 2010). As a standard approach to characterize such sunflower transcription factors, we generated transgenic A. thaliana plants overexpressing their cDNAs. One of the most interesting WRKY transcription factors to study was HaWRKY6, because of its apparent evolutionary divergence. Surprisingly, all the obtained HaWRKY6 transgenic plants exhibited very low expression levels of the transgene compared with other sunflower genes encoding transcription factors driven by the 35S promoter (Fig. 1a). Thus we were tempted to hypothesize that the expression of this WRKY gene was somehow specifically silenced in A. thaliana plants.

Figure 1.

A potential miR396 target site in HaWRKY6. (a) Expression levels of sunflower (Helianthus annuus) genes driven by the 35S promoter in transgenic Arabidposis thaliana lines relative to RNA levels in plants transformed with an empty expression vector. Values are means ± SD (n = 3). (b) Sequence comparison of sunflower and A. thaliana miR396 sequences. The relative location of the miR396 target site on HaWRKY6 is represented by a magnification of the corresponding sequence. Regions encoding the WRKY and leucine zipper (LZ) domains are noted. (c) Sequence comparison of miR396 from Asteraceae and A. thaliana. (d) Predicted secondary structure of the miR396 precursor. The blue line marks the region corresponding to the miRNA while the green line is the predicted miRNA*. Colour code indicates base-pairing probabilities.

Using established rules for conventional miRNAs-target binding (Ossowski et al., 2008) we found a putative target site for ath-miR396 located between the sequences encoding the WRKY and the leucine zipper (LZ) domains of HaWRKY6 (Fig. 1b). The finding of such a target site in HaWRKY6 may explain its low expression in A. thaliana transgenic plants, but lacks biological relevance if a sunflower miR396 homologue does not exist. As a first approach to identify a conserved miR396 regulatory network in sunflower, we searched for ath-miR396 homologues in databases containing sequences from Asteraceae spp. ESTs closely matching the A. thaliana miR396 were found in sunflower and in the related species, lettuce (Lactuca sativa), safflower (Carthamus tinctorius) and chicory (Cichorium intybus) (Fig. 1c). Using the EST information, the miR396 precursor was isolated and its secondary structure calculated (Figs 1d, S1). Most conserved miRNAs are encoded by multiple loci, two for the ath-miR396. Despite the identification and cloning of this mir396 precursor, in the absence of a complete genome sequence, we cannot exclude the presence of additional loci encoding miR396 homologues.

Evolutionary divergence of miR396-WRKY6 interaction

MiR396 is an ancient miRNA, found in all land plants, including mosses and lycopods (Axtell et al., 2007). In A. thaliana, miR396 regulates a series of GRF transcription factors, modulating cell elongation and leaf development (Liu et al., 2009; Rodriguez et al., 2010; Wang et al., 2011). Consistent with the conserved mir396-GRF regulation, genes encoding GRFs in Asteraceae exhibit conserved target sequences for miR396 (Fig. 2a). Opposite, the presence of a miR396 target site in WRKY6 seems to be unique in Asteraceae plants. Computational analysis indicated that none of the A. thaliana WRKY genes has any recognizable miR396 target sequence. On the other hand, all the Asteraceae WRKY genes clustering with HaWRKY6 (Figs 2b, S2) shared the identified miR396 target sequence (Fig. 2c). No conservation for the miR396 target site sequence was observed in Asteraceae WRKYs belonging to different subclades (Fig. 2c). The presence of the miR396 target sequence solely in the Asteraceae WRKY6 subclade suggests a recent evolutionary gain of it. Recent evolutionary events generating new miRNA-mediated gene regulation have been described (Merchan et al., 2009). In general, such events involve young miRNAs appearing in plants to target conserved transcription factors. This is the case with the Brassicaceae-specific miR824, which arose after a tandem duplication of AGAMOUS-LIKE 16 (AGL16). The miR824-AGL16 interaction is important in stomata development in A. thaliana leaves (Kutter et al., 2007). Different from young miRNAs evolving to target conserved genes, here we present an opposite situation. A divergent transcription factor has acquired a target site for a conserved miRNA. Intriguingly, a different transcription factor, bHLH74, has come under miR396 control in A. thaliana (Debernardi et al., 2012).

Figure 2.

Conservation of miR396 target sites in GRF, but not WRKY6 genes. (a) Nucleotide sequence alignment of miR396 target motif in GRF genes. (b) Phylogenetic tree of the WRKY6 clade. Bootstrap values for 100 resampled replicates are indicated in each branch. A more detailed tree is shown in Fig S2. (c) Comparison of miR396 target sites in HaWRKY6 and its homologues.

Opposite expression patterns of HaWRKY6 and miR396

Our results suggested a role for miR396 regulation of HaWRKY6 homologues. However, the biological relevance, if any, of WRKY6 silencing by miR396 was unknown. Small RNA blots with different sunflower samples were used to confirm the expression of miR396. MiR396 was abundant in most analysed tissues, with the exception of seedlings and young cotyledons (Fig. 3a). Leaves of Col-0 wildtype plants and the miRNA-biogenesis deficient dcl1-100 mutant were used as positive and negative controls, respectively. In A. thaliana, miR396 has been reported to increase with leaf age (Rodriguez et al., 2010). In agreement, we observed increased amounts of miR396 in older sunflower leaves (Fig. 3b). Interestingly, a weaker miR396 signal was detected in the distal region of sunflower leaves, which is the opposite behaviour to that of its homologue in A. thaliana (Rodriguez et al., 2010). This opposite expression gradient was probably a consequence of the late developmental stage of the assayed leaves. Alternatively, it could also be a genus-specific miR396 expression pattern.

Figure 3.

Expression profiles of HaWRKY6 and miR396. (a) RNA blot analysis of different sunflower (Helianthus annuus) tissues. U6 was used as loading control. (b) RNA blot of sunflower leaves sampled at different developmental stages, according to leaf length (in cm). (c) Expression of HaWRKY6 measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and miR396 measured by RNA blots in sunflower plants subjected to temperature stress. Band intensities of RNA blots were quantified using ImageJ. (d) Expression of HaWRKY6 and miRNAs 156, 159, 160 and 396 in sunflower plants treated with salicylic acid measured as in (c). (e) Expression of HaWRKY6 quantified by qRT-PCR in transiently transformed Nicotiana benthamiana leaves. Leaves were coinfiltrated with wildtype HaWRKY6 (W6) or a miR396 resistant form of it (W6R) in combination with an A. thaliana or sunflower MIR396. The viral protein P19 was coinfiltrated to show miRNA dependency of silencing (grey bars). Values in (c–e) are means ± SD (n = 3). (f) Expression of hemagglutinin (HA)-tagged HaWRKY6 in transiently transformed tobacco leaves. (g) MiR396 cleavage products of HaWRKY6 mRNA. Upper panel: ethidium bromide-stained gels of the first (A) and nested (B) PCR steps of the 5′ rapid amplification of cDNA ends (RACE). In the lower panel, the fraction of clones with the expected 5′ end (among all the sequenced products) is indicated.

Aiming to gain information on the possible crosstalk between HaWRKY6 and miR396, we measured, by quantitative RT-PCR and northern blot, expression levels of both genes under stress conditions. As previously described for A. thaliana (Liu et al., 2008; Laubinger et al., 2010), we found miR396 to be repressed and induced by high and low temperatures, respectively (Fig. 3c). Additionally, we found miR396 to be strongly reduced when sunflower seedlings were treated with SA (Fig. 3d). SA could be potentially toxic to the cells and, therefore, the effect on the accumulation of miR396 could be an indirect consequence. However, the analysis of other nonrelated miRNAs under the same treatment indicated a miR396 specific effect (Fig. 3d). Consistent with miR396 regulation of HaWRKY6, its expression level and that of miR396 were opposite in these conditions (Fig. 3c,d).

Together these results support the miR396-mediated HaWRKY6 silencing scenario. However, the evidence was inconclusive and we could not exclude the possibility that the observed regulation was miRNA independent. In order to test the miR396 dependency of HaWRKY6 repression, we infiltrated N. benthamiana leaves with constructs expressing an HA-tagged version of HaWRKY6, the miR396 and ath-miR396. Three days after coinfiltration, RNA was extracted and HaWRKY6 transcript and protein quantified. Confirming the silencing hypothesis, both HaWRKY6 mRNA and protein concentrations were reduced compared with the miRNA-resistant version of the gene (Fig. 3e,f). Consistent with this regulatory scenario, neither the mRNA nor the protein concentrations in the miR396-resistant HaWRKY6 mutant were affected by the miRNAs. The miR396-resistant version of HaWRKY6 (HaWRKY6R) was generated using synonymous mutations to remove the miRNA target site of the gene, making it insensitive to its action. Aiming to reinforce these results, we made use of a viral silencing suppressor to confirm the miRNA dependency of the HaWRKY6 silencing. The viral protein P19, known to be an inhibitor of the action of small RNAs (Lakatos et al., 2006), was used for this purpose. In agreement with our hypothesis, the coexpression of P19 was sufficient to suppress the effect of miR396 on HaWRKY6 accumulation (Fig. 3e). Specific miR396-mediated HaWRKY6 mRNA cleavage was demonstrated by 5′ RACE (Fig. 3g). The identification of such degradation products in sunflower confirmed the miRNA-mediated silencing of this transcription factor.

Phenotypic effects of miRNA-resistant HaWRKY6 in A. thaliana

Recently evolved miRNA-regulatory interactions are not always biologically relevant, even when they trigger silencing of targeted mRNAs (Axtell, 2008; Felippes et al., 2008). To elucidate the specific effect of the miR396-dependent HaWRKY6 regulation in vivo, we generated transgenic plants expressing the miR396-resistant form of HaWRKY6. Because stable transformation of sunflower is difficult, we evaluated the effect of HaWRKY6 misregulation in a heterologous system, A. thaliana. Transcript abundances of the resistant form of HaWRKY6 were notably higher than those of the wildtype HaWRKY6 (Fig. 4a). Plants expressing miR396-resistant HaWRKY6 were smaller and accumulated more anthocyanin than nontransgenic plants or ones expressing the miR396-targetable form (Fig. 4b). To evaluate stress response, 2-week-old plants were heat-shocked. The treatment caused lethal damage to the plants expressing the miR396-resistant HaWRKY6 form, but not to the control plants (Fig. 4c). Interestingly, the misregulation of HaWRK6 produced an increased sensibility to temperature damage despite being naturally deregulated in such a condition (Fig. 3c). This observation implies that HaWRKY6 is involved in a fine modulation in response to high temperatures. In this way, the constitutive expression of the resistant gene version may produce temporal or spatial deleterious effects. Alternatively, it could be possible that HaWRKY6 acts as a dominant negative factor modulating the effect of other members of the family in the late temperature response. Therefore, its constitutive expression may block processes required for the initial response to high temperatures.

Figure 4.

Arabidopsis thaliana plants expressing miR396-resistant HaWRKY6. (a) HaWRKY6 expression in transgenic lines expressing the resistant (HaWRKY6R) and wildtype (HaWRKY6) versions. Values are means ± SD (n = 3). (b) Rosettes of wildtype and HaWRKY6 or HaWRKY6R plants grown in standard conditions. (c) Plants subjected to a heat shock (2 h at 45°C), immediately after treatment (left panels) and 1 wk later.


We have investigated the biological relevance of the evolutionarily recent gain of miR396-mediated HaWRKY6 regulation in the Asteraceae. The identification of a young and divergent transcription factor coming under the negative control of a highly conserved regulatory element indicates that caution is needed when making inferences about the role of known miRNAs in nonmodel plants. Experiments with A. thaliana as the heterologous system confirmed that WRKY6, and especially the loss of its miRNA-mediated regulation, can affect conserved plant responses to temperature changes. This is particularly meaningful because sunflower and other Asteraceae are well adapted to growth in environments that are often hot. We have found that in addition to temperature, the defence hormone SA regulates WKRY6 activity, which may point to an integration of temperature and biotic stress responses.


This work was supported by ANPCyT (PICT-PAE 37100), UNL (CAI+D 2009) and the Max Planck Society. P.A.M. is a long-term fellow of the Human Frontier Science Program, and R.L.C. is a member of CONICET; J.I.G. is a fellow of the same institution. We thank the DAAD for a short-term fellowship to J.I.G.