Toxicity responses of Cu and Cd: the involvement of miRNAs and the transcription factor SPL7
© The Author(s). 2016
Received: 30 March 2016
Accepted: 14 June 2016
Published: 28 June 2016
MicroRNAs are important posttranscriptional regulators of gene expression playing a role in developmental processes as well as in stress responses, including metal stress responses. Despite the identification of several metal-responsive miRNAs, the regulation and the role of these miRNAs and their targets remain to be explored. In this study, miRNAs involved in the response to Cd and Cu excess in Arabidopsis thaliana are identified. In addition, the involvement of the transcription factor SPL7, namely the key regulator of Cu homeostasis, in these metal stress responses is demonstrated by the use of an spl7 knockout mutant. Furthermore, more insight is given in the Cd-induced Cu deficiency response through determining the effects of adding supplemental Cu to Cd-exposed plants.
Thirteen miRNAs were identified in response to Cu and Cd excess in A. thaliana. Several of these miRNAs (miR397a, miR398b/c and miR857) were oppositely affected under Cu and Cd exposure. The induced expression of these miRNAs after Cd exposure was totally abolished in the spl7 mutant (SQUAMOSA promoter binding protein like7), indicating a major role for SPL7 in the Cd response. Plants exposed to Cd showed a higher Cu content in the roots, whereas the Cu content in the leaves of the spl7 mutant was reduced. Furthermore, the Cd-induced Cu deficiency response disappeared when supplemental Cu was added.
Copper- and Cd-responsive miRNAs were identified and several of them are SPL7-dependently regulated. SPL7 seems to be a shared component between both the Cu toxicity and the Cd toxicity response, yet oppositely regulated, that is inactivated after Cu exposure and activated after Cd exposure. Since SPL7 is the key regulator of Cu homeostasis, and Cd affects the Cu homeostasis, we hypothesize that SPL7 is activated in response to Cd possibly due to a Cd-induced Cu deficiency. Since adding additional Cu to Cd-exposed plants resulted in the disappearance of the Cu deficiency response, Cd possibly provokes Cu deficiency, thereby activating SPL7 and inducing subsequently the Cu deficiency response.
Plants require mechanisms to control growth, development and stress responses and therefore an accurate regulation of gene expression is essential. In recent years, the recognition of microRNAs (miRNAs) as important posttranscriptional regulators of gene expression has emerged. MiRNAs are non-protein coding small endogenous RNAs of approximately 21 nucleotides that are complementary with target mRNA thereby causing its translational repression or cleavage .
Over the last decade, cloning and sequencing of small RNAs and computational analysis have revealed many miRNAs. Most of these annotated miRNAs have a role in the regulation of developmental processes, like organ morphogenesis and polarity, meristem boundary, floral patterning, vascular development, lateral root development and stomatal development [2–6]. However, different functional studies also showed the participation of miRNAs in stress responses. Liu et al  identified 14 miRNAs induced by high-salinity, drought and low temperature in Arabidopsis thaliana on a microarray-based analysis, among which miR168, miR171 and miR396 responded to all three stresses. MiR395, negatively regulating ATP sulfurylases and a sulphate transporter, was strongly induced upon sulphate starvation [8–10]. In addition, phosphate starvation induced the expression of miR399, which targets an E2 conjugase PHOSPHATE2, that functions upstream of a subset of phosphate starvation-induced genes [11–13]. Additionally, Pant et al.  identified also other P- and N-starvation responsive miRNAs in A. thaliana. These results demonstrate that miRNAs are affected by a fluctuating nutrient availability and are essential in regulating nutrient homeostasis.
Besides this, miRNAs are also involved in responses to metal stress in diverse plant species . Zhou et al.  identified six miRNAs responsive to Cd, mercury (Hg) and aluminium (Al) in Medicago truncatula. Recent studies also reported altered expression of some miRNAs in Brassica napus [17, 18] and in rice [19, 20] after Cd exposure. It should be noted however that different metals can have different responses upon miRNA expression. On the one hand, the expression of miR398 is downregulated by copper (Cu) and iron (Fe), causing increased copper/zinc superoxide dismutase (CSD) expression [21, 22]. On the other hand, excess cadmium (Cd) induces miR398 and this response is also seen under low Cu availability [22, 23]. The induction of the miR398 expression under Cu deprivation is regulated by the active transcription factor SQUAMOSA promoter binding protein-like7 (SPL7) assumed to be a central regulator in Cu homeostasis .
Despite these examples, information about other miRNAs involved in the response to Cd and Cu excess in A. thaliana is still rather scarce. In this study, we used an RT-qPCR based pri-miRNA platform  to identify Cd- and Cu-responsive (pri-)miRNAs. The expression of their target genes was measured to unravel the role of these miRNAs in the Cd and Cu stress response. In addition, the involvement of the transcription factor SPL7 in these metal stress responses was demonstrated by the use of an spl7 knockout mutant and more insight was gained in the Cd-induced Cu deficiency response through determining the impact of adding supplemental Cu to Cd-exposed plants.
Plant fresh weight is reduced after exposure to Cu and Cd
Exposure to excess Cu and Cd induces lipid peroxidation
Several miRNAs are Cd and/or Cu responsive
Metal exposure changed the expression of miRNAs already after short exposure time. Three miRNAs had an altered expression at the earliest time-point of 2 h: in roots and leaves an upregulation of pri-miR395c after Cu exposure and of pri-miR398a after Cd and Cu exposure was observed, while a downregulation of pri-miR826 in Cu- and Cd-exposed roots was noticed. However, most of the responsive pri-miRNAs were induced or repressed after 24 h and 72 h of exposure (Table 1). A time-dependent strengthening of the effect only seems to be the case for the induction of pri-miR857 expression in the roots of 5 μM Cd-exposed plants on one hand and for the reduction of pri-miR398b and pri-miR398c expression in the leaves after 0.5 μM Cu exposure on the other hand (Table 1). The induced expression of pri-miR398a in the Cd- and Cu-exposed roots and in the Cu-exposed leaves diminished over time and even disappeared after 72 h (Table 1).
Some pri-miRNAs showed an opposite expression pattern after exposure to Cu or Cd. The expressions in the leaves of pri-miR398b and c were repressed after 24 h and 72 h of Cu exposure and induced after 24 h and 72 h of Cd exposure (Table 1). Moreover, several pri-miRNAs seemed to be Cd-specific in this experimental design, by which is meant that these pri-miRNAs (in roots: pri-miR157a, pri-miR397a and pri-miR857; in leaves: pri-miR167c and pri-miR857) were only expressed after exposure to Cd and not (or very lowly) expressed in control conditions as well as after Cu exposure (Table 1).
Target gene expression levels of Cd- and Cu-responsive miRNAs
The involvement of the transcription factor SPL7 in the Cd and Cu response
The transcription factor SPL7, already known to be important for Cu homeostasis in plants , binds to GTAC motifs in the promoters of several miRNAs (cupro-miRNAs) thereby upregulating their expression . A number of these cupro-miRNAs, namely miR397a, miR398b, miR398c and miR857 , were also Cd responsive (Table 1). Therefore, Cu and Cd responses were further investigated using an spl7 knockout mutant to define a possible role for SPL7 in these responses.
Copper and Cd sensitivity of root growth in wild-type plants and the spl7 mutant
Effect of Cd and excess Cu on fresh weight, lipid peroxidation and metal content
Whereas a significant increase in lipid peroxidation after 24 h exposure to 2 μM Cu was observed in both leaves and roots, 5 μM Cd exposure had no effect on lipid peroxidation. Under all conditions, there was no genotype difference in lipid peroxidation (Additional file 2: Figure S1).
Copper and cadmium content in A. thaliana wildtype and spl7 knockout plants
7.50 ± 0.53abc
6.53 ± 0.37bc
8.95 ± 0.81abe
9.76 ± 0.66ade
2 μM Cu
12.43 ± 0.32d
11.63 ± 0.44de
16.70 ± 1.33f
16.79 ± 0.74f
5 μM Cd
6.31 ± 0.21bc
4.93 ± 0.74c
6.82 ± 0.17abc
4.64 ± 0.48c
25.00 ± 2.81abc
25.61 ± 1.93abc
18.91 ± 1.18c
23.47 ± 2.24bc
2 μM Cu
1069.45 ± 30.28d
712.47 ± 38.00e
943.04 ± 43.13de
854.71 ± 50.80de
5 μM Cd
29.97 ± 2.50ab
27.38 ± 2.18ab
45.85 ± 1.71f
34.45 ± 2.04af
5 μM Cd
514.45 ± 6.05a
699.38 ± 156.61a
1831.70 ± 53.69b
1549.40 ± 151.09b
5 μM Cd
884.79 ± 116.14a
1282.86 ± 152.62a
2421.08 ± 163.42b
3034.21 ± 113.16c
Gene expression levels of SPL7-regulated Cd- and Cu-responsive (pri-)miRNAs and their targets
The impact of adding additional Cu to Cd-exposed plants
Since SPL7 is involved in the Cd response and Cu homeostasis is disturbed after Cd exposure, knowing the impact of adjustments to external Cu concentrations in combination with Cd exposure will provide more insights in the Cd-induced Cu deficiency responses. Therefore, WT plants were simultaneously exposed to 5 μM Cd and increasing concentrations of Cu (0.5, 1 or 2 μM Cu additionally added to the Hoagland solution) for 72 h whereafter metal content and gene expression analyses were performed.
Additional Cu restores Cu levels and reduces Cd levels in leaves of Cd-exposed plants
The Cd-induced Cu deficiency response disappears with supplemental Cu
Plant growth and development is challenged due to the occurrence of several environmental stressors, including elevated metal concentrations. Therefore, an appropriate regulation of stress responses is of major importance. MiRNAs have a regulatory role in numerous stresses, including metal stress . High-throughput expression profiling of plants exposed to metals have identified several metal-responsive miRNAs [19, 20, 26–30], however the regulation of these miRNAs and the precise role of these miRNAs and their targets in metal responses remain to be explored. Therefore, identifying Cu- and Cd-responsive miRNAs in A. thaliana and their regulation after Cu and Cd exposure represent important research questions.
To identify Cu- and Cd-responsive miRNAs in A. thaliana, we exposed 19-days-old plants for 2, 24 and 72 h to 0.5 μM Cu, 5 μM Cd or grew them under control conditions. The applied metal concentrations in our study are sublethal and based on the concentrations found in pore water of sandy soils in polluted areas of Belgium [31–33]. Whether the applied metal concentrations were toxic was verified at the morphological level [impaired root development (Fig. 1)] and at the cellular level [induced lipid peroxidation (Fig. 2)]. These parameters were previously shown to be affected upon Cd and Cu exposure [22, 34–39].
In order to clarify whether Cu- and Cd-induced responses are dependent on miRNAs, we analyzed the expression profile of 180 pri-miRNAs in Cu- and Cd-exposed roots and leaves. Pant et al.  validated the use of a pri-miRNA profiling platform as a useful screening tool. Although it is recently demonstrated that the level of pri-miRNAs not always reflects the level of its mature miRNA , other studies have shown the correlation between pri-miRNA and mature miRNA levels [11, 14] as was also observed for the majority of the analysed miRNAs in our study (Table 6). Until lately, miR398 was the only miRNA family that was known to be involved in Cu and Cd stress in A. thaliana [21, 22]. But recently Barciszewska-Pacak et al.  identified more than 40 Arabidopsis pri-miRNAs responsive to (among others) Cd and Cu excess using an RT-qPCR mirEX platform. The Cd and Cu responsive pri-miRNAs that we identified in our experimental setup (Table 1), showed no altered expression in their study, except for pri-miR398a/b/c after Cu exposure, which could be due to the different experimental setups. Barciszewska-Pacak et al.  screened the pri-miRNA expression in leaves of plants grown on 0.8 % agar 1/2MS plates exposed for 24 h to 10 μM Cu and 10 μM Cd , while we used both leaf and root samples of plants grown in hydroponic culture exposed during 2, 24 and 72 h. Although fast alterations in miRNA expression levels after 0.5 μM Cu and 5 μM Cd were observed in our study, a negative correlation with the transcript levels of their targets was not always seen, especially not in the Cu-exposed plants (Table 2). However, Cd exposure resulted in the leaves in induced expression levels of miR395, miR397 and miR398 and this led to reduced transcript levels of their targets SULTR2;1, LAC2, LAC4 and LAC17, and CSD1 and CSD2 (Tables 1 and 2). Cuypers et al.  already demonstrated an upregulation of miR398b together with a downregulation of CSD1 and CSD2 in A. thaliana after 24 h exposure to 5 μM Cd. The negative correlations between Cd-responsive miRNAs and their targets show the importance of miRNAs in regulatory networks.
In this study, we exposed plants to Cu and Cd, two metals that have different physico-chemical properties, i.e. Cu is an essential nutrient and redox-active whereas Cd is nonessential and not redox-active. Striking is the opposite regulation after Cu and Cd exposure of miR398b/c in the leaves and the specific Cd-responsive miRNAs, namely miR157a, miR167c, miR397a and miR857 that were not (or very low) expressed under control conditions or after Cu exposure (Table 1). Some of these miRNAs, more specifically miR397a, miR398b/c and miR857 all have several GTAC motifs in their promoter regions, which are binding places for the transcription factor SQUAMOSA promoter binding protein-like7 (SPL7) . SPL7 is assumed to be the key regulator of Cu homeostasis. Under Cu deficiency, it activates transcription of the so-called cupro-miRNAs (miR397, miR398b/c, miR408 and miR857) targeting Cu-containing proteins, i.e. laccases and Cu/Zn superoxide dismutases . Additionally, GTAC motifs are present in the promoters of several Cu-transporters, such as COPT1, COPT2 and COPT6, and a copper chaperone CCH and the expression of all these genes is increased under Cu deficiency through the action of SPL7 . Because of the SPL7-dependent decrease of non-essential or replaceable Cu-containing proteins and the SPL7-dependent increase of Cu transporters under Cu deficiency, it is suggested that the limited available Cu is preferred for plastocyanin in photosynthesis and Cu uptake in the roots is stimulated . In general, after Cu excess the expression of the cupro-miRNAs (miR397a, miR398b/c and miR857) were decreased or not detectable at all (Table 1). In addition, exposure to Cd increased the transcript levels of these cupro-miRNAs (Table 1). The induced expression of miR398b/c after Cd exposure was already demonstrated in previous studies [22, 41], but this regulation was not yet seen for miR397a and miR857 in A. thaliana (Table 1).
Gayomba et al.  demonstrated that SPL7 is involved in the Cd-induced increase of miR398, as well as in the upregulated expression of FSD1 and COPT1/2/6. Therefore, we wanted to confirm this SPL7 dependency of the Cd-induced Cu deficiency response in our experimental setup with expansion of the data to all cupro-miRNAs and their targets. Furthermore, we were also interested in the possible involvement of SPL7 in the response to Cu excess. To analyse this, we used an spl7 knockout mutant. The Cu and Cd sensitivity of the spl7 mutant was determined based on root growth of plants grown on vertical agar plates and exposed to a range of Cu and Cd concentrations. On one hand, primary root growth (Fig. 3a), lateral root length per unit primary root length (Fig. 3b) and root FW (Fig. 4) was less or not diminished in Cu-exposed spl7 mutants compared to WT plants. On the other hand, exposure to Cd reduced primary root growth and lateral root length (Fig. 3) in both genotypes, however, lateral root length per unit primary root length decreased tremendously more in the spl7 mutant. Therefore we can conclude that the spl7 mutant is less sensitive to excess Cu and more sensitive to Cd toxicity compared to the WT plants.
In order to verify whether the targets of the SPL7-regulated miRNAs are also under the control of SPL7 after Cu and Cd exposure, target gene expression levels were measured. Clear up- or downregulation of the cupro-miRNAs (Table 4) is not always leading to alterations in target expression levels (Table 5), since the observed target gene transcript levels are a result of both, transcriptional activity regulated by other transcription factors and the SPL7-dependent miRNA-mediated post-transcriptional breakdown (Fig. 8). Multiple studies describe the reduced expression of miR398b/c and induction of CSD transcript levels after exposure to excess Cu [21, 22]. This is in accordance with our findings where a downregulation of miR398b/c and a maintained or small upregulation of CSDs were seen (Tables 4 and 5). In addition, it has been shown that excess Cu leads to an elevated production of reactive oxygen species (ROS) to which plants have to defend themselves [22, 34, 42]. Therefore, the putatively SPL7-dependent miR398-mediated maintenance of CSD transcript levels can be a first-line defence against Cu-induced oxidative stress. Nevertheless, Cd also induces oxidative stress ( and references therein), but compared to the Cu toxicity response, CSD transcript levels were decreased in our study (Table 2). However, alternatively Cd induced FSD1 expression (Table 4), also regulated by SPL7, to maintain the SOD capacity (Fig. 8).
It is known that Cd competes with other essential elements for cellular uptake sites and for binding sites in metalloenzymes, thereby disturbing the homeostasis of these elements [42, 44]. For example, Yoshihara et al.  and Xu et al.  demonstrated the disturbance of the iron homeostasis after Cd exposure at morphological and molecular level. In this regard, Cd possibly also interferes with Cu homeostasis (Fig. 8) as shown by the higher Cu content in the roots after exposure to Cd (Table 3 and Fig. 6c) and by the induction of several Cu transporters (Table 6 and Fig. 8) . This disturbance in Cu homeostasis, causing Cu deficiency, possibly can result in the activation of SPL7, the major regulator for Cu homeostasis in A. thaliana. This subsequently results into upregulations of the Cu transporters and cupro-miRNAs and hence downregulations of their targets as seen in this study (Tables 4,5,6 and Fig. 8). In addition, Gayomba et al.  stated that this SPL7-dependent Cu deficiency response, i.e. Cu uptake and reallocation, is required for basal Cd tolerance in A. thaliana.
However, how SPL7 is activated and how Cd induces the Cu deficiency response is still unknown. Recently, a working model for the regulation of SPL7 function was reported  as well as several scenarios for the link between Cd and SPL7 activation proposed . On one hand it is proposed that Cd could directly interact with SPL7 or on the other hand could alter the cellular Cu availability and hence affect SPL7 activation . Therefore, in a subsequent experiment, extra Cu was supplemented (0.5, 1 and 2 μM Cu) to Cd-exposed plants and the Cd-induced Cu deficiency responses were analysed. The decreased Cu content in leaves of Cd-exposed plants (Fig. 6a)  restored to control level when extra Cu was added, while the opposite was seen for Cd (Fig. 6b). In addition, at the transcript level, the observed Cu deficiency response after Cd exposure disappeared when extra Cu was added (Table 6). Taken together, these data support our hypothesis that adding extra Cu to Cd-exposed plants restores Cu levels in leaves resulting in the disappearance of the Cu deficiency response. Thus, Cd possibly provokes Cu deficiency, thereby activating SPL7 and inducing subsequently the Cu deficiency response (Fig. 8). However, whether the disappearance of the Cd-induced Cu deficiency response after adding extra Cu is due to restored Cu levels or due to reduced Cd levels should be further investigated.
In this study, we identified several miRNAs that are involved in responses to Cu and Cd excess. Copper and Cd are physico-chemically different metals and have an opposite effect on the expression of some Cu transporters, miRNAs (miR397a, miR398b/c, miR857) and the gene FSD1 (Fig. 8), that all have GTAC binding sites for the transcription factor SPL7 in their promoter. SPL7 seems to be a shared component between both the Cu toxicity and the Cd toxicity response, yet oppositely regulated, that is inactivated after Cu exposure and activated after Cd exposure (Fig. 8). Since SPL7 is the key regulator of Cu homeostasis, and Cd affects the Cu content in the roots and the Cu translocation to the shoots, we hypothesize that the involvement of SPL7 in the Cd response is possibly due to a Cd-induced Cu deficiency (Fig. 8). How Cd induces Cu deficiency and how SPL7 is activated remain interesting research topics that require further investigation.
Plant culture, exposure and harvest
The spl7 knockout mutant A. thaliana line (Col-0 background; SALK_125385C; Alonso et al. ) was obtained from the European Arabidopsis Stock Centre (uNASC) and was confirmed by PCR to be a homozygous T-DNA insertion line (Additional file 3: Figure S2).
Wild-type (WT) and mutant seeds were surface sterilized and grown in hydroponic culture  in the same conditions as described by Keunen et al. . After 19 days, Hoagland solution was supplemented with 0.5 μM CuSO4, 2 μM CuSO4, 5 μM CdSO4 or 5 μM CdSO4 + extra CuSO4 (0.5, 1 or 2 μM CuSO4) and plants were harvested after an exposure time of 2, 24 and 72 h, to examine immediate responses, responses after 1 full circadian day and responses at metabolic homeostasis respectively. Copper deficient plants were grown from germination on in adapted Cu deficient Hoagland solution (0.5 nM Cu). Root and leaf (entire rosette) samples were taken, snap frozen in liquid nitrogen and stored at -70 °C prior to lipid peroxidation analysis and gene expression analysis. Samples for element analysis were dried prior to extraction.
For root growth analysis, seeds were surface sterilized and grown on vertical agar plates. The preparation of the germination plates was performed as described by Remans et al.  with an adjusted Cu concentration (final concentration of 100 nM) and after 7 days of growth, seedlings were transferred to treatment plates and exposed for another 7 days to 1, 3 and 10 μM CuSO4 or 0.5, 1.5 and 5 μM CdSO4. Plates were scanned on a flatbed scanner at 600 dpi and root growth was analysed using the Optimas6 Image Analysis Software (Media Cybernetics).
Fresh root samples (175–400 mg FW) were washed for 15 min with 10 mM Pb(NO3)2 at 4 °C and rinsed with distilled water, while leaf samples (400–1000 mg FW) were only rinsed with distilled water. Samples were oven-dried (60 °C for 3 weeks), weighed, digested with 70 % HNO3 in a heat block and dissolved in 5 ml of 2 % HCl. Copper and Cd concentrations were measured via inductively coupled plasma-atomic emission spectrometry (ICP-AES, Perkin-Elmer, 1100B, USA). Blanks (only HNO3) and standard references (NIST Spinach 1570a) were included.
Lipid peroxidation analysis
The amount of thiobarbituric acid reactive metabolites (TBArm), as a measure of lipid peroxidation, was determined spectrophotometrically. Roots and leaves (50–100 mg FW) were homogenized in 1 ml of 0.1 % trichloroacetic acid (TCA), centrifuged (10 min, 20000 g, 4 °C) and 3.5 times diluted in 0.5 % TBA. After heating to 95 °C for 30 min, samples were put on ice for 5 min and centrifuged. The absorbance was measured at 532 nm and corrected for unspecific absorbance at 600 nm. The amount of TBA reactive metabolites was calculated using the law of Beer-Lambert (ε = 155 mM-1 cm-1).
Gene expression analysis
Frozen tissues (50–75 mg FW) were disrupted using two stainless steel beads and the Retsch Mixer Mill MM2000 (Retsch, Haan, Germany) under frozen conditions. Total RNA was extracted using the RNAqueous Kit (Life Technologies, Carlsbad, CA, USA) according to manufacturer’s instructions. RNA concentration and purity was determined spectrophotometrically on the NanoDrop ND-1000 (ThermoScientific, Wilmington, DE, USA). The cDNA for the pri-miRNA screening experiment had an RNA input of 3.2 μg that was DNase treated (Turbo DNA-freeTM kit, Life Technologies) and reverse transcribed using SuperScriptTM III Reverse Transcriptase (Life Technologies) in a 30 μl reaction according to manufacturer’s instructions. The cDNA for the experiment using the spl7 knockout mutant, has an RNA input of 1 μg that was DNase treated (Turbo DNA-freeTM kit, Life Technologies) and reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies) according to manufacturer’s instructions. A 10-fold dilution of all the cDNA of both experiments was made using 1/10 diluted TE buffer (1 mM Tris-HCL, 0.1 mM EDTA, pH 8.0, Sigma-Aldrich, Belgium) and stored at -20 °C.
Quantitative PCR analysis was performed with the 7900HT Fast Real-Time PCR System (Life Technologies). For the pri-miRNA screening, the pri-miRNA platform  was used and reactions contained Fast SYBR Green Master Mix (Life Technologies), 500 nM of a gene-specific forward and reverse primer, and 1 μl of the diluted cDNA in a final volume of 10 μl. For target genes, primers were designed using primer express 2.0 and a BLAST was performed (http://www.arabidopsis.org/Blast/index.jsp) to check specificity (Additional file 4: Table S2). A two-fold dilution series of a pooled sample (all cDNAs of the experiment) was used to create a standard curve for the evaluation of primer efficiencies that were accepted when they were within a range between 80 and 100 % (measured over 6 dilution points). PCR amplifications were performed at universal cycling conditions and contained Fast SYBR Green Master Mix, 300 nm of a gene-specific forward and reverse primer, and 2.5 μl of the diluted cDNA in a final volume of 10 μl.
For the gene expression analysis of mature miRNAs, total RNA was extracted using the mirVana miRNA isolation kit (Life Technologies, Carlsbad, CA, USA) according to manufacturer’s instructions. RNA concentration and purity was determined spectrophotometrically on the NanoDrop ND-1000. Multiplex reverse transcription and Real-time qPCR were performed using Taqman microRNA assays (Life Technologies, Carlsbad, CA, USA).
All relative expression levels were calculated as 2-ΔCq and normalized by the geometric average of 2-ΔCq values of minimum three reference genes selected by the GrayNorm algorithm . Data of treatment effects are expressed relative to the control of its own genotype set at 1.00. For some genes (pri-miR167c, pri-miR397a, pri-miR857) expression was not detected after 40 cycli, neither after 60 cycli under control conditions. In that case, cyclic threshold was set at 40 for the control samples and thereby treatment effects could be calculated and expressed relative to the control. The calculated and represented treatment effects are therefore minimal fold changes when expression was undetermined after 40 cycli under control conditions. All details of the workflow according to the Minimum Information for publication of Quantitative real-time PCR Experiments (MIQE) guidelines as described by Bustin et al.  are shown in Additional file 5: Table S3.
Statistical analysis was performed using R (version 2.15.1; R Foundation for Statistical Computing, Vienna, Austria). Normal distribution of the datasets was tested using the Shapiro-Wilk test and homoscedasticity was evaluated with the Bartlett’s test. If necessary, transformations of the datasets were applied. Gene expression data were always log transformed. Significant differences were determined using ANOVA test and Tukey correction. If the assumption of normality was not fulfilled, a non-parametrical ANOVA test (Kruskal-Wallis) and correction with pairwise Wilcoxon rank sum test was applied.
We thank Carine Put and Ann Wijgaerts for their skilful technical assistance and we are grateful to the Max Planck Institute of Molecular Plant Physiology (Potsdam-Golm, Germany) to give the opportunity to use the pri-miRNA platform.
This work was supported by Hasselt University by a PhD grant for Heidi Gielen and through the Methusalem project [08M03VGRJ].
Availability of data and materials
The data sets supporting the results of this article are included within the article and its additional files.
HG performed most of the experiments under the supervision of TR, JV and AC. HG, TR and AC participated in the design of experiments and work progress discussions. HG wrote the manuscript and TR and AC assisted with writing the manuscript. All authors revised the manuscript critically for important intellectual content, and read and approved the final manuscript.
The authors declare that they have no competing interests.
Ethics approval and consent to participate
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