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Nature. Author manuscript; available in PMC May 20, 2011.
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PMCID: PMC2967782
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Cell signalling by microRNA165/6 directs gene dose-dependent root cell fate

Abstract

A key question in developmental biology is how cells exchange positional information for proper patterning during organ development. In plant roots the radial tissue organization is highly conserved with a central vascular cylinder in which two water conducting cell types, protoxylem and metaxylem, are patterned centripetally. We show that this patterning occurs through crosstalk between the vascular cylinder and the surrounding endodermis mediated by cell-to-cell movement of a transcription factor in one direction and microRNAs in the other. SHORT ROOT, produced in the vascular cylinder, moves into the endodermis to activate SCARECROW. Together these transcription factors activate MIR165a and MIR166b. Endodermally produced microRNA165/6 then acts to degrade its target mRNAs encoding class III homeodomain-leucine zipper transcription factors in the endodermis and stele periphery. The resulting differential distribution of target mRNA in the vascular cylinder determines xylem cell types in a dosage-dependent manner.

Organ development involves extensive communication between cells to orchestrate tissue specification and differentiation. This communication is often mediated by mobile molecules such as hormones, mRNAs, proteins and small RNAs1. In plants and animals, transcription factors and/or mRNAs have been shown to move to neighbouring cells and transfer positional information26. Recently, small RNAs including microRNAs (miRNAs), small interfering RNAs (siRNAs) and trans-acting siRNAs (tasiRNAs) have emerged as potential mediators of cell-to-cell communication. Studies on the mobility of small RNAs have established that siRNAs and tasiRNAs are mobile, but evidence for the mobility of miRNAs has remained elusive27.

The plant root is well suited to study cell-to-cell communication in tissue patterning. Along the radial axis, epidermis, cortex and endo-dermis form around the stele, in which the pericycle surrounds the vascular cylinder8 (Fig. 1a). Here, xylem develops in the centre and forms symmetric arcs towards the pericycle. Phloem develops between these arcs, with procambium/cambium, the vascular stem cells separating xylem and phloem. Xylem precursors in the centre differentiate into metaxylem with pitted secondary cell walls, while peripheral precursors differentiate as protoxylem with spiral walls. The strong evolutionary conservation of xylem patterning9,10 suggests the presence of common molecular mechanisms that constrain its organization.

Figure 1
Endodermal SHR and SCR control xylem patterning via PHB

Here we report a novel regulatory pathway (Supplementary Fig. 1) that involves bidirectional cell signalling mediated by miRNA165/6 (miR165/6) and the transcription factors SHORT ROOT (SHR) and SCARECROW (SCR) controlling xylem patterning.

Endodermal SHR controls xylem patterning

SHR is produced in the stele and moves into the endodermis to activate SCR1115. In mutants of SHR or SCR, the asymmetric cell division that forms endodermis and cortex fails to occur and the quiescent centre is not maintained, resulting in short roots with only one ground tissue layer12,16,17. Despite the endodermal-specific expression of SCR genome-wide mRNA profiling of wild-type, shr and scr roots14,15 (Methods) indicated that nearly half of the genes co-regulated by SHR and SCR were expressed at the highest level in the stele (Supplementary Fig. 2). Furthermore, in both scr and shr mutants, metaxylem differentiates ectopically in the place of proto-xylem (Fig. 1e, f, g), indicating that SHR and SCR affect stele development in a non-cell autonomous manner.

Because SHR is normally present in both the stele and the endodermis, we determined where its activity is required for xylem patterning. We first expressed SHR strictly in the stele of shr-2 by introducing a construct in which a non-mobile version of SHR18 containing a nuclear localization signal was driven by the stele-specific promoter of CRE1 (ref. 19). Recovery of root meristem size (Student’s t-test; P < 0.001, α = 0.05) and root growth (P < 0.001, α = 0.05) showed that this version of SHR was functional. Although we observed more immature phloem sieve cells than in shr-2 (Supplementary Fig. 3), protoxylem formation was not rescued (Fig. 1b). We then expressed SHR strictly in the ground tissue by introducing UAS::SHR:YFP into shr-2 harbouring J0571, an enhancer trap line that drives expression specifically in the ground tissue (www.plantsci.cam.ac.uk/Haseloff/; Fig. 1c and Supplementary Fig. 4). In addition to multiple ground tissue cell divisions, protoxylem formation was observed in 73% (24 of 33) of the lines (Fig. 1c), compared with 14% in shr-2 J0571 alone. When SCR was expressed in the ground tissue in the absence of SHR, neither protoxylem nor endodermis was rescued (Fig. 1d). Therefore, xylem patterning requires both SHR and SCR to be present in the endodermis.

Xylem patterning requires PHB restriction

In a screen for altered vascular development (Supplementary Fig. 5) we identified a mutant with a short root and frequent differentiation of metaxylem in place of protoxylem (Fig. 1h). This mutant had a point mutation in the miR165/6 target site in the class III homeo-domain leucine zipper (HD-ZIP III) gene PHABULOSA (PHB). Consistent with the phenotype of this new allele, phb-7d, the strong gain-of-function allele phb-1d (ref. 20) invariably formed ectopic metaxylem (Supplementary Fig. 5e).

Comparison of vascular development using cell and tissue markers reinforced the striking similarity of the phb-7d and shr-2 phenotypes (Supplementary Fig. 6 and Supplementary Table 1). To determine if the phb-7d phenotype was caused by reduced SHR activity, we expressed the green fluorescent protein fusion SHR::SHR:GFP13 in phb-7d (Supplementary Fig. 7) but found no deviation from wild-type expression, indicating that this was unlikely.

As expected if the phb-7d mutation renders the PHB transcript resistant to miRNA-mediated degradation, its mRNA levels were elevated in the mutant (Supplementary Fig. 5d). RNA in situ hybridization in wild type showed that PHB mRNA localized primarily to the metaxylem precursors and neighbouring procambial cells, and at a residual level in protoxylem precursors (Fig. 2a). In phb-7d the PHB mRNA domain expanded throughout as well as outside the stele (Fig. 2b), indicating that miR165/6 normally acts to exclude PHB mRNA from the stele periphery and ground tissue.

Figure 2
SHR post-transcriptionally represses PHB

The importance of miRNAs in xylem cell specification was supported further by the ectopic metaxylem phenotype of the mutant of HYL1, which specifically binds the miRNA during miRNA-mediated mRNA degradation21,22 (Supplementary Fig. 8). We did not detect ectopic metaxylem in mutants of several well-characterized genes in the si- and tasiRNA-mediated RNA degradation pathways, indicating that xylem patterning is primarily mediated by miRNAs.

SHR post-transcriptionally represses PHB

Genome-wide expression profiling of shr, scr and wild-type roots indicated that PHB and the other four HD-ZIP III genes targeted by miR165/6 (refs 2325) are upregulated in the shr and scr mutant backgrounds (Supplementary Fig. 9). To characterize further the relationship between SHR and the HD-ZIP III transcription factors we crossed shr-2 with loss-of-function mutants of three HD-ZIP III transcription factors, PHB, PHAVOLUTA (PHV) and REVOLUTA (REV), which are closely related and functionally redundant in leaf development23. The phb-6 phv-5 rev-9 mutant did not show any major deviation in xylem patterning26 from wild-type roots (Supplementary Figs 10 and 19). However, in the phb-6 phv-5 rev-9 shr-2 quadruple mutant, the xylem patterning defect of shr was completely rescued (Supplementary Fig. 10). This provides strong evidence that ectopic metaxylem formation in shr is the result of upregulation of at least one of these HD-ZIP III transcription factors. This quadruple mutant also largely rescued the number of vascular cell files and root length (Supplementary Fig. 10). Analysis of the segregating double mutants showed that phb shr fully recovered protoxylem (in about 80%) and root growth, whereas phv shr, rev shr and phv rev shr did not (Fig. 1i and Supplementary Figs 10 and 11). Loss-of-function mutations in the two other HD-ZIP III genes, ATHB8 and CORONA/ATHB15 (CNA) could not restore the root growth of shr-2, but in both double mutants, athb8-11 shr-2 and cna-2 shr-2, stretches of protoxylem were infrequently observed (Supplementary Fig. 11). Combining mutation in phb with scr also fully recovered protoxylem (Supplementary Fig. 11). Hence, these genetic analyses indicate that SHR/SCR repression of PHB is the primary pathway for xylem patterning.

We asked whether PHB was repressed by SHR at the transcriptional or post-transcriptional level. For this we analysed transcriptional (PHB::GFP) and translational GFP fusions (PHB::PHB:GFP) driven by the PHB promoter27. Patterns of PHB::GFP indicated that PHB is transcribed throughout the stele and endodermis (ground tissue in shr) in both wild-type and shr root meristems (Fig. 2f), whereas PHB::PHB:GFP was restricted to the central vascular cylinder in wild type (Fig. 2h). When the phb-7d mutation was introduced into the cDNA of the translational fusion, the GFP domain became similar to the PHB transcriptional domain in wild type (Fig. 2i). In shr, PHB::PHB:GFP and PHB mRNA domains expanded throughout the stele, similar to the pattern of PHB::GFP (Fig. 2g, h). These data indicate that SHR restricts PHB at the post-transcriptional level.

These results further implied that the meristematic zone is where PHB determines the root xylem cell types. Supporting this, we found that a miRNA-resistant version of PHB (with a silent mutation) expressed in the meristematic region under the stele-specific CRE1 promoter was sufficient for ectopic metaxylem to form both in wild type and in the phb shr double mutant (Supplementary Fig. 12).

In summary, our data indicate that miR165/6 post-transcriptionally restricts PHB within the root meristem to the stele centre for proper xylem patterning and that SHR regulates this process by promoting miR165/6 activity in the stele periphery and endodermis.

SHR activates miR165/6 in the endodermis

We compared levels of miR165/6 in root tips of shr, scr and wild type. In shr, miR165/6 levels were reduced eightfold, and in scr threefold compared to wild type (Supplementary Fig. 13; Methods). Comparison of gene expression profiles of components related to small RNA pathways in whole roots14,15 showed no statistically significant expression changes in these mutant backgrounds (Supplementary Table 2), indicating that SHR and SCR primarily control miR165/6 activity by regulating MIR165/6 expression.

To determine which of the MIR165/6 genes are controlled by SHR and SCR, expression patterns of transgenic promoter::GFP (endoplasmic reticulum-localized) lines with the complete intergenic region of eight of the MIR165/6 genes (except 165b) were analysed in wild type, shr-2 and scr-1 (Fig. 3a, b and Supplementary Fig. 14). Only MIR165a and 166b promoters drove detectable GFP in distinct patterns in wild-type roots. We note that another study28 suggests that MIR166a is also expressed at low levels in roots. pMIR165a::GFP showed endodermis-specific expression throughout the root (Fig. 3a), whereas pMIR166b::GFP was expressed strongly in the endodermis and quiescent centre and weakly in cortex and epidermis of the meristems of embryonic, primary and lateral roots (Fig. 3b and Supplementary Fig. 14). In shr roots, GFP was not detected from pMIR165a::GFP in any tissue, and only low-level activity was observed in the ground tissue and epidermis from pMIR166b::GFP (Fig. 3b and Supplementary Fig. 14). The constructs behaved similarly in scr, showing marked reduction in expression, with the exception that pMIR165a::GFP was detected in the ground tissue from the late maturation zone (Fig. 3a, b and Supplementary Fig. 14). As in wild type, GFP driven by any of the other six promoters could not be detected in shr roots. Consistent with the GFP data, real time PCR with reverse transcription analysis (RT–PCR) indicated significant reduction of pri-MIR165a and pri-MIR166b in both shr and scr roots (Supplementary Fig. 13).

Figure 3
miR165/6 activated by SHR inthe endodermis is active in the stele

To determine whether SHR is a direct regulator of MIR165/6 we performed chromatin immunoprecipitation (ChIP) followed by real-time quantitative PCR (Methods). We reproducibly found enrichment of fragments approximately 1 kb upstream of the transcription start site of MIR165a, 4.5 kb upstream of MIR166b (Fig. 3c), and 2.5 kb upstream of MIR166a (data not shown). Taken together, our data indicate that SHR activates transcription of MIR165a and 166b in the endodermis directly.

miR165/6 acts non-cell-autonomously

We reported previously that the HD-ZIP III transcriptional domains are largely restricted to the vascular cylinder27. PHB is the only one whose transcriptional domain partially overlaps with the endodermal activity domain of SHR and SCR. Hence, for miR165/6 produced in the endodermis to encounter HD-ZIP III mRNA in the stele, there must be a mobile signal.

To understand the nature of this non-cell autonomous regulation, we first compared the spatial distribution of miR165/6 activity in wild-type and shr-2 roots using a ‘miRNA-sensor’29 (Fig. 4a; Methods). In this system, high miR165/6 activity is reflected by low GFP expression. Without the miRNA recognition site the GFP levels were uniform in the stele in both wild type and shr. In wild-type roots the sensor GFP with the miRNA recognition site was significantly lower in the ground tissue and stele periphery than in other cell types, consistent with the observed reduction in the PHB mRNA and protein domains in wild type. In shr-2, the sensor GFP expression level was uniform throughout the root.

Figure 4
Non-cell-autonomous action of MIR165a.

Second, we determined the spatial distribution of mature miR165/6 in the root meristem of wild type and shr using in situ hybridization with locked nucleic acid (LNA) probes (Fig. 4b and Supplementary Fig. 15; Methods). In wild-type meristems, mature miR165/6 was detected at a low level in the quiescent centre and surrounding cells, but became progressively higher and ubiquitous throughout the root radius 30–40 μm distal from the quiescent centre. In cells close to the quiescent centre, mature miR165/6 levels were considerably higher in the cortical and epidermal cell layers than in the endodermis and stele cells. Hence, the pattern seemed complementary to the domain of PHB transcription. A complementary pattern of a mature miRNA and its target is consistent with previous observations30, but rather contradictory to our results showing the highest promoter activity of MIR165a and MIR166b in the endodermis. We proposed that the hybridization signal may reflect mainly the distribution of free miR165/6. Consistent with this, we detected less of a differential distribution of miR165/6 in the phb-13 mutant and in several loss-of-function HD-ZIP III mutants where target mRNA is very low or absent. In these backgrounds the miR165/6 signal became high and ubiquitous throughout the root radius, even in tissues close to the quiescent centre (Fig. 4b and Supplementary Fig. 15). In shr-2, the miR165/6 pattern was similar to wild type, but at a markedly lower level, probably reflecting the residual expression of MIR166b (Fig. 4b). Hence, the sensor and in situ hybridization results indicate that the mature miRNA165/6 moves radially both outward and inward from the endodermis.

Third, we drove MIR165a expression by a ground-tissue-specific promoter (shr-2 J0571; UAS::MIR165a) in shr and observed the resulting xylem pattern (Fig. 4c and Supplementary Fig. 4). Five independent segregating T2 lines showed a clear recovery of protoxylem (Fig. 4c) at frequencies ranging from 33% to 88%, accompanied by suppression of mRNA levels of all the HD-ZIP IIIs (Fig. 4d) and restriction of PHB and CNA mRNA domains within the stele (Supplementary Fig. 16). Co-segregation of protoxylem formation with the activator was verified by backcrossing. Similarly, both J0571 [dbl greater-than sign] MIR165a (Fig. 4c) in scr-4 and MIR165a expressed under another ground-tissue-specific promoter, pSCR, in the scr-6 allele, rescued protoxylem formation (Supplementary Fig. 17).

Further support for this hypothesis is that protoxylem recovery observed when SHR was specifically expressed in the ground tissue of shr (Fig. 1c) was accompanied by an increase in MIR165a and 166b levels, and a decrease in mRNA levels of all five HD-ZIP III genes (Supplementary Fig. 18a), whereas SHR exclusively localized to the stele of the shr mutant did not affect either miR165/6 or HD-ZIP III mRNA levels (Fig. 1d and Supplementary Fig. 18b). Taken together, our results strongly support that miR165/6 in the endodermis mediates the non-cell-autonomous action of SHR and SCR in xylem patterning, by moving into the stele to restrict the HD-ZIP III mRNA domains.

HD-ZIP III levels determine xylem type

Our results indicated that PHB was the primary determinant, but other HD-ZIP IIIs may have a role in metaxylem specification as well. To investigate further the role of all five HD-ZIP III genes in root vascular patterning, we analysed their expression and assessed their loss-of-function phenotypes. Similar to PHB, CNA and ATHB8 were expressed in xylem precursor cells (Fig. 2c, d): CNA in the metaxylem domain and neighbouring procambial cells and ATHB8 specifically in the xylem axis including the protoxylem precursors. REV was broadly expressed in the vascular tissue just above the quiescent centre (Fig. 2e), but disappeared from the metaxylem domain farther away from the quiescent centre. PHV mRNA was not detected. In summary, the HD-ZIP III mRNA levels in the root meristem appear highest in the centre of the xylem axis and become lower towards the stele periphery.

In contrast with ectopic PHB expression, which causes central metaxylem fate in the stele periphery, the loss of combinations of HD-ZIP III genes resulted in protoxylem differentiating in the central metaxylem positions or even abolished xylem differentiation entirely (Fig. 5 and Supplementary Fig. 19). This is consistent with previous studies showing that HD-ZIP III transcription factors may direct xylem development23,25,3133. All single and most double mutants displayed normal xylem patterning. However, the athb8-11 phb-13 and various triple mutants had ectopic protoxylem partly replacing metaxylem. When four of the five genes were mutated no metaxylem was observed in any of the mutant combinations examined. Consistent with changes in xylem cell fates, low or no expression of the metaxylem marker gene ACL5 (ref. 34) and ectopic expression in the centre of the xylem axis of the protoxylem marker AHP6 (ref. 35) were observed in athb8-11 cna-2 phb-13 phv-11 (Supplementary Fig. 20). This mutant also generated more vascular cells and, in contrast to the invariably diarch vascular arrangement in wild type, often formed a tri- or tetrarch arrangement, indicating that the HD-ZIP III genes redundantly restrict vascular cell proliferation (Supplementary Fig. 19). Surprisingly, loss of all five HD-ZIP III transcription factors failed to form any xylem (Fig. 5b). Partial failure in xylem differentiation was also detected in certain quadruple mutants (Supplementary Fig. 19). These results show that expression levels of HD-ZIP III transcription factors determine not only xylem cell types, but also de novo formation of xylem.

Figure 5
HD-ZIP III levels determine xylem type

Finally, we increased the level of miR165 throughout the stele in wild type and shr-2. In both backgrounds an increased number of stele cells was observed (not shown) and all xylem precursors acquired peripheral fate differentiating exclusively as protoxylem (Fig. 5c, d), similar to the phenotypes of several multiple loss-of-function HD-ZIP III mutants. Thus, xylem patterning requires suppression of HD-ZIP III mRNA in the stele periphery through the activity of miR165/6.

Discussion

Our study highlights a novel regulatory pathway that integrates transcriptional and post-transcriptional regulation and bidirectional cell-to-cell communication to drive tissue patterning in the Arabidopsis root (Supplementary Fig. 1). Formation of vascular tissue with a surrounding endodermal layer was a key milestone in the evolution of land plants17,18. Our study shows that its underlying regulation involves evolutionarily conserved SHR/SCR and HD-ZIP III transcription factors, and miR165/6 (refs 36, 37), implying that this regulatory mechanism might underlie the evolutionary adaptation to terrestrial growth.

The mobility of miR165/6 in the shoot apical meristem has been suggested previously26. Our study indicates that miR165/6 is mobile in the root and its mobility over a short distance is critical for dosage-dependent regulation of HD-ZIP III transcription factors in xylem patterning. A recent modelling study indicates that a mobile small RNA can sharpen the boundary of the activity domain of its target38. Our study shows that this may be the role for the endodermally produced miR165/6 as it moves into the vascular cylinder to encounter its target mRNA, thereby communicating radial positional information between cells of the root meristem.

METHODS SUMMARY

For anatomical and histological analyses, primary roots of vertically grown 4 to 5-day-old seedlings were used. Plastic sectioning, basic fuchsin staining and confocal imaging were performed as described19.

For quantification of mRNA and miRNA, root tips from 6- to 7-day-old seedlings were collected and total RNA including small RNAs was extracted with an miRNeasy Mini kit (Qiagen). To measure the expression level of pri-miRNA and other mRNAs, cDNA was synthesized using SuperScript III first strand synthesis system for RT–PCR (Invitrogen). For mature miRNA, a polyadenylation reaction was performed before the reverse transcription following the method of ref. 39. Differences in gene expression were measured by real time PCR using an ABI 7900HT (Applied Biosystems).

Sectioning, preparation of riboprobes and in situ hybridization were performed as described19. LNA probes with complementary sequences to miR165 and miR166 were synthesized, 5′ digoxigenin-labelled (Exiqon), and then hybridized at 50 °C.

Most transgenic constructs were generated using the modified multisite gateway system27 and all were introduced into plants using the floral dip method40.

To identify in vivo binding activities of SHR to the promoters of MIR165/6, plants expressing SHR::SHR:GFP in shr-2 were used. Roots were collected 6 days after germination and processed for ChIP14. SHR binding affinity was compared between the ChIP DNA treated with and without GFP antibody by measuring the differential enrichment of DNA fragments using real-time PCR.

Supplementary Material

SOM

Acknowledgments

We thank K. Kainulainen, M. Herpola, G.-B. Berglund and J. Jung for technical assistance; M. Prigge, S. Clark, C. Bellini, N. Sauer, J. Colinas, T. Vernoux, K. Gallagher, A. P. Mahonen, A. Bishopp, M. Bonke, N. Fedoroff, J. C. Fletcher, B. J. Reinhart, I. Pekker, ABRC and NASC for materials, and E. Richards and M. Harrison for comments on the manuscript; M. Tsiantis for sharing results before publication. This work was supported by the Boyce Thompson Institute and NSF IOS0818071 to J.-Y.L., Cornell Presidential Life Science Fellowship to J.Z., grants from the NIH (RO1-GM043778) and from the NSF ARABIDOPSIS 2010 programme to P.N.B., a fellowship from the MICINN, Spanish Government to M.A.M.-R., grants by the Academy of Finland, Tekes and ESF to Y.H., S.L. and A.V., European Molecular Biology Organisation (EMBO, ALTF 450-2007) to J.D., Estonian funding agencies (ETF7361 and SF0180071s07) to O.L., Nilsson-Ehle Foundation to C.J.R, and FORMAS and Carl Trygger’s Foundation for Scientific Research to A.C. Imaging at Boyce Thompson Institute was supported by NSF (NSF DBI-0618969) and Triad Foundation.

Footnotes

Supplementary Information is linked to the online version of the paper at www.nature.com/nature.

Author Contributions A.C. and J.-Y.L. contributed equally to this work, C.J.R., J.D., S.L. and J.Z. contributed equally to this work, O.L., M.A.M.-R. and A.V. contributed equally to this work, Y.H. and P.N.B. contributed equally to this work, and the name order was determined by raffle. A.C. designed and performed experiments to characterize HD-ZIP III transcription factors and miR165/6 in vascular patterning, J.-Y.L. identified and characterized the regulatory network of SHR, SCR and miR165/6 in the xylem patterning, C.J.R. analysed the miRNA expression by in situ hybridization and participated in HD-ZIP III mutant characterization. J.D. participated in the analysis of expression of PHB (including mutant forms) and other HD-ZIP III and generated pCRE1::MIR165a as well as participated in the generation of J0571 lines to rescue scr and shr. S.L. participated in the PHB and HD-ZIP III expression analysis, phb-7d mutant characterization and generated the pSCR::MIR165a to rescue scr. J.Z. developed and characterized the xylem patterning led by non-mobile SHR and PHB-m. O.L. participated in the characterization of various HD-ZIP III mutant lines. M.A.M.-R. showed the non-cell autonomous action of non-mobile SHR. A.V. participated in positional cloning of phb-7d and establishment of the J0571 lines to rescue shr. S.T. identified the phb-7d mutant. A.C. identified the scr-6 allele. J.S. characterized shr/scr and HD-ZIP III double mutants and embryo expression patterns in GFP lines. J.L.B. shared informative non-published materials. Y.H. and P.N.B. participated in experimental design. A.C., J.-Y.L., Y.H. and P.N.B. wrote the manuscript. A.C. and J.-Y.L. are co-corresponding authors. All authors discussed the results and commented on the manuscript.

Reprints and permissions information is available at www.nature.com/reprints.

The authors declare no competing financial interests.

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