MicroRNAs (miRNAs) are an abundant class of small, endogenous non-protein-coding RNAs, approximately 21 nucleotides in length, that modulate the expression of animal and plant target genes at the post-transcriptional level. Recent work has shown that miRNA-based gene regulation plays a crucial role in pathways involved in plant growth and development. However, knowledge about the timing and spatial regulation of plant miRNA expression is still limited. Here we used in situ analysis to demonstrate that miRNAs accumulate spatially and temporally in a highly restricted manner in Nicotiana benthamiana and Arabidopsis thaliana. The presence of the seven investigated miRNAs was characteristic of the developing organs, implying a role in cell-fate establishment, differentiation and cell-cycle progression. Spatial analyses revealed that six of the studied miRNAs were present in vascular bundles, suggesting that mobile miRNAs in the phloem could contribute to the coordination of organogenesis and development. The obvious absence of miR167 in vascular bundles represented an exception to this observation, implying an active process in regulating the presence of miRNAs in the vascular system. Taken together, our results imply that the spatially and temporally organized accumulation of miRNAs plays a pivotal role in fine-tuning of target gene expression in plant development.
The development of multicellular organisms is regulated by spatially and temporally coordinated complex regulatory networks that organize cell division and differentiation. The discovery of a novel gene-regulatory mechanism, based on microRNAs (miRNAs), has had a significant impact on the understanding of developmental processes. miRNAs are approximately 21 nucleotide-long non-coding RNA molecules that are generated by sequential processing of longer precursor molecules (termed pre-miRNA; Baulcombe, 2004; Dugas and Bartel, 2004; Filipowicz et al., 2005; Kidner and Martienssen, 2005). The plant miRNA loci encode capped and polyadenylated transcripts (pri-miRNAs) that are processed to pre-miRNAs in the nucleus, probably by the Dicer-like enzyme DCL1. The genetically defined pre-miRNAs possess a self-complementary fold-back structure that is processed to a double-stranded intermediate comprising the miRNA and the complementary miRNA* strands, respectively. The miRNA–miRNA* duplex is unwound and the miRNA is subsequently loaded in the RISC complex, while the miRNA* strand is eliminated. In plants, the miRNA–target duplex shows near-perfect or perfect base pairing, and the RISC acts via cleavage of the target mRNA. In contrast, most animal miRNAs and their target mRNAs form imperfect duplexes resulting in translational inhibition by RISC. However, there are also exceptions in plants. The near-perfect complementarity of plant miR172 and its target sequence in APETALA2 (AP2) predicts induction of cleavage-based regulation, but instead the translational inhibition of AP2 mRNA was reported (Aukerman and Sakai, 2003; Chen, 2004).
As plant miRNAs usually show a near-perfect match to their target sequences, it is possible to identify new miRNA loci by computational approaches (Adai et al., 2005; Wang et al., 2004). To date, about 92 loci have been identified encoding 27 miRNA species in Arabidopsis thaliana, and a similar number of miRNAs have also been found in Oryza sativa (Griffiths-Jones, 2004). A large proportion of the plant miRNAs regulate genes that play roles in developmental processes, such as control of meristem identity, cell proliferation, developmental timing and patterning (Kidner and Martienssen, 2005). Disruption of miRNA-mediated processes or miRNA–target mRNA interactions usually results in developmental abnormalities (Achard et al., 2004; Chellappan et al., 2005; Chen, 2004; Emery et al., 2003; Guo et al., 2005; Juarez et al., 2004; Kidner and Martienssen, 2004; Kim et al., 2005; Laufs et al., 2004; Mallory et al., 2004; Palatnik et al., 2003; Tang et al., 2003; Vaucheret et al., 2004). It has been proposed that miRNAs act by eliminating target regulatory genes during the cell-fate changes. For example, spatially coordinated expression of miR165/166 regulating PHAVOLUTA and PHABULOSA genes has been demonstrated to be important in adaxial/abaxial determination of leaves (Juarez et al., 2004; Kidner and Martienssen, 2004). The expression profile of miR165 was largely complementary to the expression of its target mRNAs, while mutations in ARGONAUTE1 resulting in distorted miRNA accumulation were associated with organ polarity defects (Kidner and Martienssen, 2004). The expanding expression profile of miR166 during leaf development, together with its accumulation in phloem, suggested that miR166 may act as a signaling molecule (Juarez et al., 2004). The possible role of miRNAs in signalization was also suggested by the presence of various miRNAs in Cucurbita maxima phloem sap (Yoo et al., 2004). In contrast, transgenic experiments showed near-perfect co-localization between miR171 transcription and activity, indicating that miRNAs act at the site of production (Parizotto et al., 2004). These experiments demonstrate that spatial characterization of miRNA expression is essential for a better understanding of miRNA-mediated processes. However, due to their small size, detection of miRNAs by in situ hybridization is technically demanding.
We have previously reported highly sensitive and specific detection of miRNAs by Northern blot analysis using locked nucleic acid (LNA)-modified oligonucleotides (Valoczi et al., 2004), and have now adapted this method to in situ hybridization of miRNAs in plants. In a recent study, LNA-modified oligonucleotide probes were successfully used to determine the temporal and spatial expression patterns of 115 conserved vertebrate miRNAs in zebrafish embryos by in situ hybridization (Wienholds et al., 2005). Most miRNAs were expressed in a highly tissue-specific manner during segmentation and later stages, but not early in development. Thus, these miRNAs may not be essential for tissue establishment, but rather play crucial roles in the maintenance of tissue identity or in differentiation (Wienholds et al., 2005).
Here, we show that LNA-modified oligonucleotide probes are well suited for specific and sensitive detection of miRNAs in plant tissues by in situ hybridization. Our results revealed that the seven studied plant miRNAs accumulate spatially and temporally in a highly coordinated manner. We furthermore demonstrate that these miRNAs are localized in the cells and organs that show the most active division and differentiation activity. In contrast to the suggested role of miRNAs in animals, our results support the notion that plant miRNAs have a crucial role in cell-fate determination and differentiation. Moreover, the presence or absence of various miRNAs in vascular bundles in developing organs suggests the existence of active regulation based on phloem-mobile miRNAs that could function in the coordination of developmental processes.
Detection of miR171 accumulation by in situ hybridization in Nicotiana benthamiana leaves using LNA-modified oligonucleotide probes
We have previously described a new method for sensitive detection of miRNAs by Northern blot analysis using LNA-modified oligonucleotides (Valoczi et al., 2004). This method is also highly specific, as demonstrated by the use of various single and double mismatched LNA probes. Here we used digoxygenin-labelled LNA-modified oligonucleotide probes complementary to the well-characterized miR171 (Llave et al., 2002b) to assess the sensitivity and specificity of the method in detecting miRNAs by in situ hybridization (Llave et al., 2002b) in the developing tissue of Nicotiana benthamiana (Valoczi et al., 2004). Consecutive sections of paraffin-embedded young developing leaves of N. benthamiana were used for in situ hybridizations with four different probes: miR171LNA (specific for miR171), miR171LNA/MM11 (mismatch probe with a single mismatch at nt position 11 compared to the mature miRNA target sequence), miR171LNA/2MM (mismatch probe with two mismatches compared to the mature miRNA target sequence) and miR124LNA (complementary to mouse miR124, with no predicted homologous target sequences in plants). The in situ hybridization results showed strong hybridization signals in young tobacco leaves when the perfect match miR171LNA was employed (Figure 1a). In contrast, we observed only very weak hybridization signals with miR171LNA/MM11 (Figure 1b) and miR171LNA/2MM (Figure 1c), and no signals were detectable with the mouse miR124LNA probe (Figure 1d). Combined, these results show that the LNA-modified oligonucleotide probes are highly efficient in detection of miRNAs in plant tissues by in situ hybridization, in accordance with recent results obtained with zebrafish miRNAs (Wienholds et al., 2005). Moreover, the LNA detection probes appear to be highly specific, as a single mismatch in the probe significantly reduces the in situ hybridization signal.
Comparison of LNA oligonucleotides with RNA probes in miRNA detection by in situ hybridization
To compare LNA-modified oligonucleotide probes with conventional RNA probes for miRNA in situ detection, we used a tandem repeated DNA clone of miR171 for in vitro RNA synthesis. The sense and antisense digoxygenin-labelled RNA transcripts for miR171 (miR171s and miR171as) were used for in situ hybridization in N. benthamiana sections in parallel with digoxygenin-labelled LNA-modified (miR171LNA) and DNA (miR171DNA) oligonucleotides respectively (Figure 2). The sections showed similar expression patterns with both miR171LNA and miR171as probes, except that the sections hybridized with the LNA probe displayed a dramatically enhanced signal intensity compared to the miR171as RNA probe. In contrast, the DNA oligonucleotide probe did not detect any in situ signals. Taken together, these results indicate that LNA-modified probes are highly effective in detecting plant miRNAs by in situ hybridization.
Northern blot analyses of miRNA accumulation in N. benthamiana and A. thaliana
To study the spatial accumulation of various miRNAs in N. benthamiana, we selected highly conserved miRNAs with roles in plant developmental processes based on the microRNA Registry database (Griffiths-Jones, 2004). LNA-modified oligonucleotide probes (Valoczi et al., 2004) complementary to miR156, miR159, miR160, miR167, miR164 and miR319 were designed and validated by Northern blot analyses of total RNA samples from N. benthamiana and A. thaliana (Figure 3). The Northern blot results showed strong bands for all the selected plant miRNAs in N. benthamiana and A. thaliana, while no plant small RNAs could be detected with the mouse-specific miR124 control probe. These observations imply that our LNA probes for the selected, conserved plant miRNAs would be suitable for in situ hybridization experiments across various plant species.
Spatial accumulation of miR171 in N. benthamiana
SCARECROW (SCR) is a member of the plant-specific GRAS gene family encoding putative transcription factors, and plays a significant role in the radial patterning of both roots and shoots and hormone signalling (Helariutta et al., 2000; Kamiya et al., 2003; Wysocka-Diller et al., 2000). Three members of the Arabidopsis SCR-like putative transcription factors (SCL), SCL6-II, SCL6-III and SCL6-IV, were reported to possess perfectly complementary target sites for miR171 (Llave et al., 2002a; Reinhart et al., 2002). Four SCL genes in Oryza sativa have also been shown to contain the miR171 target site (Reinhart et al., 2002). It has previously been demonstrated that SCL6-III and SCL6-IV are regulated by miR171-mediated cleavage (Llave et al., 2002b).
We investigated the spatial accumulation of miR171 in various organs of N. benthamiana during the developmental process. Analyses of longitudinal sections of the apex revealed that miR171 accumulates to high levels in meristems in both tunica and corpus (Figure 4a), and also in the procambial and early vascular tissues. Longitudinal sections of young flowers reveal high expression levels of miR171 in the ovary, anthers and petals (Figure 4b), while cross-sections of the young stem also showed the presence of miR171 in the vascular system and the epidermal cell layer (Figure 4c). In the leaf primordia and young developing leaves, miR171 accumulates at a very high level, showing gradually decreasing expression levels during development (Figure 4e–g). The presence of miR171 in the veins of young developing leaves is also obvious (Figure 4d). Comparison of miR171 in situ expression pattern with the chemical staining of nuclei (Figure 4n,o) revealed that the nuclei of developing trichomes and the epidermal cells of the developing stem show high signal intensity, indicating that miR171 is present in the nuclei. This is in line with previous findings that Arabidopsis thaliana nuclei contain mature miRNAs (Park et al., 2005). Examination of cross-sections from young developing inflorescence tissues revealed that miR171 accumulates in anthers, ovary and petals in a highly polarized manner (Figure 4h,i,k), which is maintained during the development of anthers and also in petals, suggesting that accumulation of miR171 is spatially and temporally coordinated during development. Pronounced accumulation of miR171 in anthers was observed in the developing pollen sacs, pollens and in the vascular bundles (Figure 4h,i,k), and spatially regulated expression of miR171 was also characteristic for the development of the ovary (Figure 4h,i,k). The presence of miR171 throughout the ovules and the vascular system of the ovary is visible in transverse and longitudinal sections of the ovary with developing ovules (Figure 4k–m). The accumulation of miR171 at high levels in ovules, funiculus, inner epidermis and in the vascular system of the placenta indicates that miR171 could play a role in ovule development. To compare the miR171 expression pattern with that of a housekeeping gene, we applied a glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific RNA probe on the tissue sections. GAPDH-specific signals showed an expression pattern that was largely complementary to that of miR171, indicating that miR171 accumulates mainly in metabolically inactive regions of flower (Figure 4r–u).
Spatial distribution of miRNAs associated with flower development
SQUAMOSA PROMOTER BINDING PROTEIN-box genes (SBP-box genes) encode plant-specific proteins that share a highly conserved DNA binding domain (the SBP domain). In Arabidopsis, SBP box-containing genes constitute a structurally heterogeneous family of 16 members known as SPL genes (Cardon et al., 1999). miR156 is thought to target several members of the SPL gene family (Jones-Rhoades and Bartel, 2004). Ten of the 16 members in the SPL family have been predicted to be targeted by miR156 (Rhoades et al., 2002). Transgenic overexpression of miR156b resulted in delayed flowering, faster initiation of rosette leaves and decreased apical dominance (Schwab et al., 2005).
We showed that miR156a accumulates at low levels in anthers and ovary of young flowers (Figure 5a). However, at a later stage of flower development, we could not detect hybridization signals above the technical background (Figure 5b). miR156 was clearly present in young leaves (Figure 5h, compare with mouse miR124 control hybridization), ovules (Figure 5c,d,f) and in meristematic tissues (Figure 5e).
miR164 has been identified as the regulator of NAC-domain genes, including CUP-SHAPED COTYLEDON1 (CUC1), CUC2 and NAC1 (Rhoades et al., 2002). miR164 was implicated in regulation of the boundary domain around developing primordia at the shoot apical and floral meristems, and also in regulation of petal number and lateral root development (Baker et al., 2005; Guo et al., 2005; Laufs et al., 2004; Mallory et al., 2004). We found that miR164 accumulated in developing flower organs, and in this regard it was similar to miR171. miR164 also showed a highly polarized accumulation in the developing pollen sacs and ovary as well as in the poles of developing petals (Figure 5a,b). Ovules and the vascular system of the placenta also displayed abundant expression levels of miR164 (Figure 5c,d,f). The presence of miR164 was very obvious in the vascular bundles of anthers, flowers and leaves (Figure 5b,g,h). In addition, miR164 accumulated to high levels in procambium and meristems (Figure 5e).
miR159-mediated cleavage of mRNAs encoding GAMYB-related proteins was demonstrated to be important in the regulation of short-day photoperiod flowering time and anther development (Achard et al., 2004). miR159 shows a similar polar accumulation in anthers and petals and in young flowers as miR164 and miR171. It is present in vascular bundles and meristematic tissues. We also investigated the accumulation of miR319 (miR-JAW), which shows sequence similarity to miR159 (Griffiths-Jones, 2004). The overexpression of miR319 results in aberrant leaf development and delayed flowering time with greenish petals (Palatnik et al., 2003). miR319 was shown to guide messenger RNA cleavage of several TCP genes controlling leaf development. The accumulation pattern of miR319 obtained by in situ hybridization revealed that it was undetectable or poorly detectable in young flowers (Figure 5a,b) and predominantly accumulated in ovules (Figure 5c,d,f). miR319 was also present in vascular bundles of the flower (Figure 5g) and in young developing leaves (Figure 5h). Longitudinal sections of the apex revealed the accumulation of miR319 in procambium and meristematic tissues (Figure 5e).
The small signaling molecule auxin elicits multiple developmental responses, such as patterning in embryogenesis, apical dominance and cell elongation (Berleth and Sachs, 2001). Genes upregulated by auxin contain auxin-response elements (AuxRE) in their promoters, which bind transcription factors of the auxin response factor (ARF) family (Ulmasov et al., 1997, 1999). There are 23 members in the ARF gene family in Arabidopsis, and some of them have been shown to be able to repress or to activate expression of genes with an AuxRE promoter element (Ulmasov et al., 1999). miR160 has been demonstrated to regulate ARF10, ARF16 and ARF17 via cleavage of the target mRNA (Mallory et al., 2005). Transgenic plants expressing miR160 cleavage-resistant ARF17 show various developmental abnormalities, including reduced petal size, abnormal stamens and sterility (Mallory et al., 2005). miR167 is predicted to target other members of the ARF gene family (ARF6 and ARF8; Jones-Rhoades and Bartel, 2004). Combined, these data demonstrate that at least five members of the ARF gene family are regulated by miRNAs.
To assess the role of miRNAs in the regulation of the ARF gene family during development, we hybridized tissue sections representing various developmental stages with miR160- and miR167-specific LNA oligonucleotide probes. In situ hybridization of sections from young developing leaves revealed that, whereas miR167 accumulated to detectable levels, miR160 was undetectable or accumulates at a very low level that does not allow detection using the LNA probe technology (Figure 6l). This was further confirmed by Northern blot analyses, which demonstrated that miR167 was readily detected, while miR160 showed a faint signal in RNA samples from young leaves (Figure 3d,g). Cross-sections and longitudinal sections of young flower buds revealed that neither miR160 nor miR167 accumulate to high levels in developing organs (Figure 6a–c,h). However, both miRNAs were present in the vascular bundles of the anthers, while only miR160 was detectable in vascular bundles of the ovary (Figure 6c). The presence of miR160 in vascular bundles was also evident in sections representing the basal part of the developing flower (Figure 6k). In floral and vegetative meristems, we could detect only a low level of expression for miR160 and miR167 (Figure 6h). The most striking differences between the miR160 and miR167 patterns were detected in the cross-sections and longitudinal sections, respectively, of the developing ovary (Figure 6d–f). miR160 accumulated exclusively throughout the ovules, while no signals were detected elsewhere in the tissue, whereas miR167 showed high expression levels in the ovules and also in vascular bundles of the placenta. Closer examination of the ovules revealed that, in contrast to miR167, miR160 accumulated to high levels in the basal part of ovules, in the funiculus attaching the ovules to the wall of the ovary, in the vascular bundle system of the placenta and in the inner epidermis (Figure 6e,f). Hybridization with an miR167-specific probe revealed that this miRNA accumulates throughout the immature seeds (Figure 6g). In contrast, miR160 disappeared or its accumulation dropped below the detection level in immature seeds (Figure 6g), indicating that expression of these miRNAs is temporally regulated.
Accumulation of miR160 and miR167 in A. thaliana
To demonstrate the utility of LNA-modified oligonucleotide probes in miRNA in situ detection in A. thaliana, we applied miR160- and miR167-specific LNA probes on A. thaliana sections. In contrast to N. benthamiana tissues, which respond well in in situ hybridization experiments, A. thaliana tissues showed weaker in situ signals. The miR160LNA probe detected a signal mainly in the funiculus and the basal part of the ovary, while the miR167LNA probe showed an in situ signal throughout the ovary (Figure 7). These results confirmed the in situ data obtained from N. benthamiana tissue sections. Moreover, we also detected the presence of miR160 in the vascular bundles, comparable to the results in N. benthamiana, while miR167 was not detectable in the veins. Combined, our results indicate that, although technically more demanding, LNA-modified probes can also be used for miRNA in situ detection in A. thaliana.
Although the founding member of the miRNA class, lin-4, was described 12 years ago (Lee et al., 1993), the biological function of these small, regulatory RNAs is still poorly understood. Hundreds of miRNAs have been predicted to be present in higher eukaryotes, regulating multiple target mRNAs, and thus one of the future challenges is to understand the biological roles of miRNAs in various organisms. Reliable and thorough analysis of the spatio-temporal distribution of miRNAs is a prerequisite for understanding the function of individual miRNAs. However, the detection of miRNAs by in situ hybridization has proven difficult due to their small size (21–25 nt) as well as the fact that members of the same miRNA family often differ only by one or two bases. To overcome this problem, we have developed a new method, based on the use of LNA-modified oligonucleotide probes, which significantly enhance the sensitivity of miRNA detection while simultaneously retaining high specificity (Valoczi et al., 2004; Wienholds et al., 2005). Here we used LNA-modified oligonucleotide probes for in situ detection of miRNAs in plant tissues.
In situ hybridization of seven miRNAs using LNA probes complementary to the mature miRNAs in developing N. benthamiana and A. thaliana plants revealed highly restricted temporal and spatial expression patterns during development. Control experiments with LNA probes designed to detect the miR171* strand and the miR171 precursor RNA in A. thaliana did not detect any hybridization signals, implying that the observed in situ hybridization signals originated from the mature miRNA species and not from the precursor molecules (data not shown). The observed temporal regulation of miRNA expression suggests that timing of miRNA accumulation in developing tissues is an important factor for miRNA-mediated gene regulation. For example, miR171 accumulates at high levels in young leaves, decreasing gradually in parallel with the advancing developmental process (Figure 4), suggesting that miR171 is involved in differentiation and development of the leaf tissue. On the other hand, miR160 and 167 do not accumulate, or accumulate below the detection level, at the beginning of ovule differentiation, but at increasing levels during the ovule development (compare Figure 4 and Figure 6), while miR167, but not miR160, is continuously expressed in premature seeds (Figure 6g).
The spatial accumulation of miRNAs in developing organs is also highly regulated, underlining the role of miRNAs in coordinating developmental events. Some miRNAs show abundant expression in several organs (miR171, miR164, miR159), implying a general role in the developmental processes. Moreover, these miRNAs appear to accumulate in a highly polarized manner, which is maintained during the development, identifying the most actively dividing and differentiating tissue parts. The expression levels of the miRNAs studied here decrease dramatically when the developmental process had been completed, suggesting that these miRNAs are crucial for tissue-fate establishment and differentiation (see, for example, Figure 4e–g). This observation is further supported by the presence of most of the investigated miRNAs in the developing ovules. Moreover, the development of ovules requires spatially well-defined expression of miRNAs, as depicted by miR160 and miR167, which target the same gene family but show different expression patterns in ovules. Thus, the accumulation of miRNAs targeting different members of the same gene family appears to be differentially coordinated, implying a role in fine-tuning of target gene expression.
Interestingly, we found that six of the seven studied miRNAs were present in the vascular tissues. This was especially pronounced for miR159, miR160, miR164 and miR171 (Figures 4–6). By contrast, miR167 was not detected in the vascular tissues, although it accumulated at high levels in the ovules, suggesting that the presence or absence of a given miRNA in the vascular system could be actively regulated. Although our results do not provide direct evidence as to whether miRNAs are produced or transported in the vascular system, their presence in the veins of the developing organs implies that phloem-mobile miRNAs could play a role in the coordination of developmental processes. For example, miR164 has been demonstrated to be a common regulatory factor controlling the separation of adjacent embryonic, vegetative and floral organs (Mallory et al., 2004). Here, we find that miR164 is highly abundant in the vascular system of developing flowers, which supports the notion that miR164 may provide a circulating signal controlling the formation of various organs. Furthermore, the seven plant miRNAs in this study were predominantly present in young developing organs and showed significantly decreased expression in mature tissues, which suggests a role in cell-fate determination and differentiation. Alternatively, miRNAs may also affect cell-cycle progression. CUC genes in Arabidopsis, targeted by miR164, appear to play an important role in setting up boundaries in the meristems, suggesting their potential as cell-cycle regulators (Jakoby and Schnittger, 2004). A recent analysis of C. maxima phloem sap revealed the presence of various miRNA and small interfering (si) RNA species, in accordance with their role as signalling agents (Yoo et al., 2004). By contrast, another study showed that, in transgenic plants, expression of the miR171 precursor showed a near-perfect overlap with its activity (Parizotto et al., 2004). Our results do not suggest a cell-to-cell spreading mechanism for plant miRNAs, as shown by the highly restricted miRNA expression patterns. However, it is possible that miRNA-mediated signalling is relevant only for the developing organs or parts of the organs, and not valid for whole plants, and furthermore that a fine resolution technology is necessary to detect short-distance signalling events. As our results do not distinguish between transport of mature miRNAs and the local production of miRNAs, a main task for future plant miRNA research would be to develop methods that enable spatio-temporal detection of miRNA precursors.
Nicotiana benthamiana plants grown in soil under normal growth conditions were used for collecting leaves, flowers and terminal buds. Flowers (1 to 10 mm in size) were collected and embedded with shoot apices. Arabidopsis thaliana (Columbia wild-type) plants grown in soil at 25°C were used for collecting flowers.
RNA isolation and Northern blot analysis
Total RNA was extracted from plant and mouse tissues using TRI Reagent (Sigma-Aldrich, St Louis, MO, USA) according to the manufacturer's instructions. The total RNA samples were fractionated on denaturing 12% polyacrylamide gels containing 8 m urea, transferred to Nytran N membrane (Schleicher & Schuell, Dassel, Germany) by capillary method, and fixed by ultraviolet cross-linking. Membranes were probed with 32P-labelled LNA oligonucleotide probes (miRCURY probes, Exiqon, Vedbaek, Denmark, http://www.exiqon.com), complementary to the mature miRNAs. A 10 pmol aliquot of each oligonucleotide probe was end-labelled with [γ32P]ATP using T4 polynucleotide kinase. Pre-hybridization of the filters was carried out in 50% formamide, 0.5% SDS, 5x SSPE, 5x Denhardt's solution, and 20 μg ml−1 sheared, denatured, salmon-sperm DNA, followed by hybridization in the same solution as described previously (Valoczi et al., 2004). Hybridization with miR164 resulted in unspecific background. To eliminate this, the membrane was treated with RNaseA in NTE buffer similar to the in situ hybridization protocol (0.5 m NaCl, 10 mm Tris, pH 7.5, 1 mm EDTA, 20 μg ml−1 RNaseA) at 37°C for 30 min. The membrane was washed again after RNase digestion.
In situ hybridization
Nicotiana benthamiana and Arabidopsis thaliana samples were collected, fixed in 4% formaldehyde and processed for paraffin embedding as described previously (Jackson, 1992). Cross- and longitudinal sections (10 μm) were taken and applied to microscope slides. The GAPDH probe for mRNA in situ hybridization was obtained by RT -PCR of N. benthamiana total RNA. The cDNA was amplified by PCR using the following oligonucleotides: GAPDHNb-s: 5′-TCTCAAATATGACTCCACCC-3′; GAPDHNb-as: 5′-ACCCCATTCATTGTCATACC-3′; The gel-purified PCR products were ligated into the pGEM-T Easy Vector (Promega, Madison, WI, USA) according to the manufacturer's instructions. The mRNA in situ probes were prepared as described previously (Havelda and Maule, 2000). The sense and antisense probes were prepared after linearizing the pGEM-T Easy vectors containing the target sequence with ApaI or SpeI and transcribing with SP6 or T7 polymerase in the presence of digoxygenin-11-UTP (Roche, Mannheim, Germany).
To produce RNA probe specific to miR171a, DNA oligonucleotides were designed containing the miR171 sequence in triplicate as tandem repeats both in sense and antisense orientations. The two oligonucleotides were annealed and ligated into pBluescript II KS vector (Stratagene, Heidelberg, Germany). To avoid the production of double-stranded RNA from short templates by both RNA polymerases, the sense and antisense probes were prepared after linearizing the pBKS vectors containing the target sequence with PvuII or BglI. Transcribing these templates with T7 or T3 RNA polymerase in the presence of digoxygenin-11-UTP (Boehringer Mannheim) resulted in transcripts of about 400 bases. These were treated with DNase and after precipitation hydrolyzed with carbonate buffer for 10 min at 60°C (Jackson, 1992).
The LNA oligonucleotides were labelled with the DIG oligonucleotide 3′-End Labelling Kit (Roche) according to the manufacturer's instructions. The probes were not further purified after labelling. The resulting 20 μl probe solutions were diluted by addition of 20 μl formamide; 2–6 μl of formamide-containing probe solution was used per slide in 200 μl hybridization solution.
The following LNA-modified oligonucleotide probes were used: miR156LNA oligonucleotide to detect A. thaliana miR156a, 5′-GTGCTCACTCTCTTCTGTCA-3′; miR159LNA to detect A. thaliana miR159a, 5′-TAGAGCTCCCTTCAATCCAAA-3′; miR160LNA to detect A. thaliana miR160, 5′-TGGCATACAGGGAGCCAGGCA-3′; miR164LNA to detect A. thaliana miR164a, 5′-TGCACGTGCCCTGCTTCTCCA-3′; miR167LNA to detect A. thaliana miR167a, 5′-TAGATCATCGTGGCAGCTTCA-3′; miR171LNA to detect A. thaliana miR171a, 5′-GATATTGGCGCGGCTCAATCA-3′; miR319LNA to detect A. thaliana miR319a, 5′-GGGAGCTCCCTTCAGTCCAA-3′; miR124LNA to detect Mus musculus miR124a, 5′-TGGCATTCACCGCGTGCCTTAA-3′.
The hybridization was performed at 50°C with each probe, and slides were washed in 1x SSC/50% formamide at 50°C. During the washing process, the slides were RNaseA-treated to eliminate non-specific background. The colour reactions were stopped depending on the signal intensity (a few hours to two days). The slides were further washed through an ethanol dilution series to eliminate non-specific signals and counter-stained with Alcian Blue (Fluka, Buchs, Switzerland).
Nuclear staining was performed using haematoxylin solution according to Mayer (Fluka). Embedded 10 μm sections of tissues were prepared for haematoxylin staining as well as for in situ hybridization. Tissues were stained for 15 min with haematoxylin solution and washed with water.
We wish to thank Marianne Bonde Mogensen and Mette Bjørn at Exiqon for excellent technical assistance. This research was supported by a grant from the Hungarian Scientific Research Fund (OTKA K61461) and a grant from the European Commission as part of the RIBOREG EU FP6 project (LSHG-CT-2003503022). ZH is the recipient of a Bolyai Janos Fellowship. We thank Exiqon (Vedbaek, Denmark) for providing LNA-modified oligonucleotide probes.