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Plant stem cell niches: from signalling to execution
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Plant stem cell niches: from signalling to execution Robert Sablowski The shoot and root meristems contain small populations of stem cells that constantly renew themselves while providing precursor cells to build all other plant tissues and organs. Cell renewal, growth and differentiation in the meristems are coordinated by networks of transcription factors and intercellular signals. The past two years have revealed how auxin and cytokinin signals are integrated with each other and with regulatory genes in the shoot and root meristems. Small RNAs have also emerged as novel intercellular signals. Downstream of meristem regulatory genes, links have been made to cell division control and chromatin function. Protection of genome integrity, partly through programmed cell death after DNA damage, has recently been revealed as a specialised function in plant stem cells. Address Cell and Developmental Biology, John Innes Centre, Norwich NR4 7UH, United Kingdom Corresponding author: Sablowski, Robert ([email protected])
Current Opinion in Plant Biology 2011, 14:4–9 This review comes from a themed issue on Growth and development Edited by Jiayang Li and Nam-Hai Chua Available online 23rd August 2010 1369-5266/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2010.08.001
Introduction Virtually all cells of a mature plant descend from small populations of stem cells that are maintained within the meristems. As in animals, these stem cells are defined by their ability to both renew themselves and contribute daughter cells to produce new tissues. A feature found throughout multicellular organisms is that stem cells are maintained in specialised microenvironments called stem cell niches, where extracellular signals are available that maintain the stem cells. The functional similarities found between stem cells of very different multicellular organisms probably result from convergent evolution [1,2]. The best-studied plant stem cell niches reside in the shoot and root apical meristems (SAM and RAM, respectively). Stem cells are maintained at the summit of the SAM by a signal produced by underlying cells that Current Opinion in Plant Biology 2011, 14:4–9
express WUSCHEL, which encodes a homeodomain transcription factor (TF) (Figure 1A and C) [3]. Descendants of the stem cells are displaced to the periphery of the meristem and recruited into new organs, while the innermost region of the SAM, called the rib meristem, produces the internal tissues of the stem. In the RAM, stem cells are maintained by signals from a small group of cells called the quiescent centre (QC), which rarely divide but also functions as a long-term reserve of stem cells [4] (Figure 1B). The QC, and therefore the root stem cell niche, is positioned where activity of the TFs SCARECROW (SCR) and SHORT ROOT (SHR) overlaps with expression of the PLETHORA (PLE) TFs, which in turn are controlled by an apical–basal gradient of auxin maintained by PIN transporters [2]. Control of the stem cell maintenance signal in the RAM shows similarities to the SAM: the WUS homologue WUS-RELATED HOMEOBOX5 (WOX5) is required in the QC to prevent differentiation of the stem cells of the columella [5]. Both the SAM and RAM maintain stable patterns of gene expression in spite of constant cell division and displacement [6,7]. This requires intense intercellular signalling to assess the position of each cell and correct its gene expression accordingly. These signals also balance the rate of cell division with differentiation and initiate tissue patterning. Key regulatory genes that respond to intercellular signalling (e.g. WUS, PLE, SHR, SCR and WOX5) ultimately control rates of cell growth, division and the decision to enter a differentiation pathway, but the molecular details of how these processes are controlled remain unknown. Here, I review progress made in the past two years on intercellular signalling and on the functions downstream of key transcriptional regulators in the meristems.
Cell signalling in meristem maintenance and differentiation The best understood intercellular signal used in meristem maintenance is the glucosylated dodecapeptide CLAVATA3 (CLV3) [8], which is produced by the shoot stem cells and diffuses to underlying cells to inhibit WUS in a negative feedback loop that regulates the size of the stem cell population [9]. Until recently, it was thought that CLV3 was perceived by a receptor made of CLAVATA1 (CLV1) and CLAVATA2 (CLV2) subunits, but new results suggest instead that CLV3 is perceived in parallel by two different receptors, one including CLV1 and another made of CLV2/CORYNE subunits [10]. It has been suggested that a pathway related to CLV/WUS functions in the RAM, because peptides homologous to www.sciencedirect.com
Plant stem cell niches: from signalling to execution Sablowski 5
Figure 1
Physical structure and regulatory networks in the shoot and root meristems. A, B: three-dimensional reconstructions based on confocal images of shoot meristems (A; IM and FM indicate inflorescence and flower meristem, respectively) and a root apical meristem (B). Key regions of the meristems are marked in false colours: red for the stem cells, blue for the source of stem cell maintenance signals (organising centre in A, quiescent centre in B), yellow for the rib meristem (A) and stele (B); the green line in B surrounds the columella. Size bar: 50 mm. C, D: schematic representations of the meristems shown in A and B, with regulatory interactions indicated. Regions marked in red, blue and yellow correspond to those in A, B; positive and negative inputs are indicated by arrows and blunted lines. In C, the main hub of the regulatory network is WUS (marked in white); the orange region indicates where the cytokinin (CK) concentration is proposed to be sufficient to activate the AHK4 receptor; the dashed green line contains the region where AHK4 is expressed, whose overlap with the orange region defines the WUS-expression domain (blue); a dashed arrow indicates that small RNAs (sRNAs) produced in the developing vasculature are also required to maintain WUS expression. In D, boxes contain three different sets of regulatory interactions: on the top, SHR moves from the stele to the endodermis, where it heterodimerises with SCR to activate expression of miRNAs 165 and 166, which move back to the stele to control xylem differentiation. The middle box shows the integration of cytokinin (CK) and auxin signals through SHY2 to regulate expression of PIN1 and consequently auxin transport to the root tip (block arrow). The lower box represents the signalling network controlling WOX5 expression and differentiation of stem cells (SC) in the columella, which is bounded by the dashed green lines.
CLV3 inhibit root meristem growth [11,12] and because WOX5 function in the RAM resembles WUS in the SAM [5]. The similarity has been reinforced by the finding that CLE40 (the closest homologue of CLV3) promotes differentiation of columella cells, partly by restricting expression of WOX5 [13]. There are, however, important differences www.sciencedirect.com
to the CLV3/WUS interaction. First, the effect of CLE40 is mediated by a receptor kinase unrelated to CLV1/2: ARABIDOPSIS CRINKLY4 (ACR4), which restricts cell division in the columella [14], is required for CLE40 signalling, although it is not known if ACR4 binds to CLE40 or functions downstream of CLE40 signalling. Current Opinion in Plant Biology 2011, 14:4–9
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Second, CLE40 signals from differentiating cells to control WOX5, whereas CLV3 signals from stem cells to inhibit WUS.
in animals [24], loss of the potential to function as a stem cell is a gradual process that can be reversed in differentiating cells.
Other well-studied signals that regulate meristem function are hormones. As mentioned above, auxin has a central role in positioning the root stem cell niche; now cytokinin has been implicated in positioning the stem cell niche in the SAM. It was found that cytokinin responses and WUS activity reinforce each other through multiple feedback loops and reflect the localised expression pattern of cytokinin receptors (ARABIDOPSIS HISTIDINE KINASE 2 and 4: AHK2, 4) in the rib meristem [15]. Combined with data indicating that cytokinin is produced in the apical region of the SAM [16,17], these results support a model in which the WUS-expression domain is continuously specified at the position where a sufficient amount of apically produced cytokinin reaches its receptors in the rib meristem (Figure 1C). It will be interesting to see whether activation of WUS by cytokinin is mediated by the WUS homologue STIMPY, which has been placed upstream of WUS and downstream of cytokinin in the SAM [18,19].
In addition to hormones, mobile macromolecules control cell differentiation in the RAM. For example, SHR moves from the stele (the innermost tissues of the stem and root, corresponding to the developing vasculature in the root tip) to adjacent endodermis cells, where it functions together with SCR to control asymmetric cell division and differentiation [25]. Now miRNAs have also been implicated in communication across the boundary between the endodermis and stele: miRNAs 165/166 are activated by SCR/SHR in the endodermis and move to the developing vasculature to control xylem differentiation [26].
Apart from positioning the stem cell niches, auxin and cytokinin interact to regulate the balance of cell division and differentiation. In the RAM, auxin and cytokinin have antagonistic effects, with auxin promoting cell division, while cytokinin promotes cell differentiation. The regulatory basis for this antagonism has been revealed: the cytokinin-response regulator ARABIDOPSIS RESPONSE REGULATOR 1 (ARR1) activates expression of SHORT HYPOCOTYL 2 (SHY2), which represses auxin signalling and PIN expression; conversely, auxin caused degradation of SHY2, allowing PIN expression and the auxin flow required to maintain the RAM [20] (Figure 1D). The effect of cytokinin on auxin transport has been independently confirmed [21]. In contrast to their opposite role in the RAM, auxin and cytokinin have synergistic effects on shoot organogenesis, and details have also emerged of how auxin and cytokinin signals are integrated in the SAM: auxin-activated MONOPTEROS (MP) directly represses the negative regulators of cytokinin responses ARR7/15, which in turn inhibit meristem function at least partly by activating CLV3 [22] (Figure 1C). Another important new insight is that the control of cell differentiation by hormones appears to be superimposed on their role in niche maintenance. After excision of the root tip, it was seen that missing cell types, such as the columella, were re-established before the stem cell niche had regenerated [23]. This early re-specification of missing cell types required auxin transport, showing that the hormonal control of tissue patterning can be separated from the control of stem cell maintenance and indeterminate growth. This work also confirmed that in plants, as Current Opinion in Plant Biology 2011, 14:4–9
More generally, signalling by small RNAs (sRNAs) is an emerging theme in plant development. Long-distance movement of sRNAs through the vasculature controls epigenetic changes in recipient cells [27]; the actual mobile molecule is probably duplex small RNAs [28]. Although direct evidence for sRNA signalling from the developing stem to the SAM is missing, several recent papers support the idea. First, a mobile small RNA (tasiR-ARF) that controls abaxial–adaxial leaf patterning also moves to the SAM from underlying stem tissues [29]. Second, PINHEAD/ZWILLE (PNH/ZLL), which encodes an ARGONAUTE protein and therefore is implicated in sRNA function, is required in the developing vasculature of the embryo to produce a mobile signal that maintains WUS expression [30]. Third, PNH/ ZLL functions in parallel with the tasiR-ARF pathway to maintain the SAM by reducing the meristematic levels of miRNAs 165/166, which in turn downregulate HD-ZIP transcription factors required for SAM development [31]. In conclusion, recent work has clarified how hormone signals are integrated in the meristems and has revealed sRNAs as potentially important intercellular signals. The results also highlight the role of the developing stele (which in the shoot includes the rib meristem and developing vasculature) in signalling processes that position the stem cell niches and control differentiation in both the SAM and RAM (Figure 1).
Downstream effectors of meristem functions The intercellular signals discussed above constantly adjust the expression of genes that control stem cell functions, including cell division. Accordingly, SHR and SCR have been shown to directly activate a specialised cycD isoform, which in turn regulates an asymmetric stem cell division that gives rise to the precursor cells of the cortex and endodermis of the root [32]. Another link to cell division is that a SUMO E3 ligase functions downstream of PLE genes to maintain cells in the mitotic www.sciencedirect.com
Plant stem cell niches: from signalling to execution Sablowski 7
cycle (rather than entering the endocycle associated with cell differentiation) [33]. Direct targets have also been identified for WUS combining ChIP-chip and transcriptome analysis [34]. The set of WUS targets included CLV1 and was enriched for genes involved in hormone responses and meristem development, in addition to genes involved in pollen and ovule development, reflecting later functions of WUS in reproductive organs. Comparison with a global analysis of gene expression in different regions of the SAM [35] showed that most of the direct targets of WUS are excluded from the WUS domain, suggesting that WUS is primarily a repressor. WUS was confirmed to function mostly as a transcriptional repressor in the meristem, although it also activates the reproductive organ identity gene AGAMOUS [36]. The identification of direct targets of SHR, SCR and WUS complemented work on the cell type-specific transcriptomes in the RAM and SAM. In the SAM, gene expression was profiled in cells marked by CLV3, WUS and FIL reporters (expressed in stem cells, organising centre and initiating organs, respectively) [35]. Gene ontology analysis showed that the stem cells preferentially express genes involved in chromatin modification and DNA repair. Similar insight emerges from a comparison between QC gene expression in Arabidopsis [37] and a more recent analysis in maize [38]. Thus results from both the root and shoot support the idea that dynamic chromatin and stringent protection of genome integrity are important features of plant stem cell niches. The idea that plant stem cells have specialised mechanisms to maintain genome integrity has also been supported by work on how stem cells respond to DNA damage. UV irradiation and DNA double strand breaks preferentially kill stem cells in the root and shoot, a response that requires transduction of DNA damage signals by the kinases ATAXIA-TELANGIECTASIA MUTATED (ATM) and ATM-RELATED (ATR) [39,40] and activation of DNA damage responses by the TF SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1) [41,42]. ATR has also been implicated in the inhibition of RAM activity by aluminium (which among other stresses causes DNA damage) [43]. Another link between DNA damage and stem cell function is that the DNA repair gene BARD1 represses WUS [44]. All the data above indicate that protection of genome integrity is an important function in plant stem cell populations; this could be important to prevent the propagation of mutations to large parts of the plant body, including the germline in the case of the SAM. In summary, gene expression profiling has revealed that cell division and intercellular signalling are key processes targeted by regulators of stem cell function. Global www.sciencedirect.com
analysis of gene expression in the meristems also indicated that chromatin dynamics and maintenance of genome integrity are key functions, although the molecular links between these processes and key meristem regulators remain unclear.
Conclusions and perspectives The past two years have revealed how multiple signals are integrated in the meristems to position and maintain the stem cell niches, balance cell proliferation with differentiation and initiate tissue patterning. Although it is satisfying to have an increasingly unified view, the signalling network has become too complex to understand intuitively. Computer models are already necessary to predict the behaviour of meristem regulatory networks [45], and these models will no doubt be improved by incorporating advances in dynamic, 3D meristem imaging [46] and mechanical modelling [47]. To ultimately understand how the behaviour of signalling and transcriptional networks is translated into meristem functions, the molecular links to cellular functions will also need to be explored further. Links to chromatin functions are particularly important because of the likely role of chromatin structure in pluripotency, which is a defining feature of stem cells [48]. The control of genome integrity in the meristem is a new and mostly unexplored area – we do not know whether repair mechanisms differ in the stem cell niches or what are the molecular pathways linking DNA damage and programmed cell death. To reveal the functional relevance of these mechanisms, it will be necessary to measure the accumulation of mutations in stem cells and in differentiating progeny. No shortage of challenges for the future.
Acknowledgements I am grateful to Lars Østergaard and Silvia Costa for comments. Work in my lab is funded by the Biotechnology and Biological Sciences Research Council and the European Union.
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activation of ARR1 and SHY2, cytokinin repressed expression of the auxin transporters PIN1, 3, 7, consequently changing auxin distribution and promoting cell differentiation. 21. Ruzycka K, Simaskova M, Duclercq J, Petrasek J, Zazimalova E, Simon S, Friml J, Van Montagu MCE, Benkova E: Cytokinin regulates root meristem activity via modulation of the polar auxin transport. Proc Natl Acad Sci U S A 2009, 106:4284-4289. 22. Zhao Z, Andersen SU, Ljung K, Dolezal K, Miotk A, Schultheiss SJ, Lohmann JU: Hormonal control of the shoot stem-cell niche. Nature 2010, 465:1089-1092. This work showed how cytokinin and auxin inputs are integrated in the shoot meristem. Inhibition of the negative cytokinin-response regulators ARR7/15 caused increased meristem size, partly by inhibiting CLV3, and caused phylotaxis defects, suggesting a link to auxin responses. Of several auxin response regulators tested, MP was found to directly repress ARR7/ 15. Thus auxin inhibits a repressor of cytokinin responses, explaining why auxin and cytokinin have synergistic effects in shoot development. 23. Sena G, Wang XN, Liu HY, Hofhuis H, Birnbaum KD: Organ regeneration does not require a functional stem cell niche in plants. Nature 2009, 457:1150-U1110. Sena et al. asked whether re-establishment of root cell types after excision of the root tip required first the recovery of the root stem cell niche. On the basis of marker genes, starch accumulation and gravitropic response, recovery of columella cells preceded re-establishment of the QC and the stem cell niche. Mutants that are unable to maintain the stem cell niche ( plt1, plt2 and scr) still re-established columella cells and overall root tip morphology after excision. Thus the stem cell niche is required for indeterminate growth, but not for the re-direction of cell differentiation during organ regeneration. 24. Nakagawa T, Sharma M, Nabeshima Y, Braun RE, Yoshida S: Functional hierarchy and reversibility within the murine spermatogenic stem cell compartment. Science 2010, 328:62-67. 25. Nakajima K, Sena G, Nawy T, Benfey P: Intercellular movement of the putative transcription factor SHR in root patterning. Nature 2001, 413:307-311. 26. Carlsbecker A, Lee J-Y, Roberts CJ, Dettmer J, Lehesranta S, Zhou J, Lindgren O, Moreno-Risueno MA, Vaten A, Thitamadee S et al.: Cell signalling by microRNA165/6 directs gene dosedependent root cell fate. Nature 2010, 465:316-321. This paper shows that mobile miRNAs regulate cell differentiation in the root stele. In the endodermis, SCR and SHR activated miRNAs 166/165, which degrade HD-ZIP mRNAs. The miRNAs not only functioned in the endodermis, but also moved to adjacent cells, as shown by the discrepancy between miRNA gene expression and the pattern of mature miRNAs detected by in situ hybridisation and by a miRNA sensor construct. Graded degradation of HD-ZIP mRNAs by miRNAs 165/6 in the stele was required for the differentiation of different xylem cell types. 27. Molnar A, Melnyk CW, Bassett A, Hardcastle TJ, Dunn R, Baulcombe DC: Small silencing RNAs in plants are mobile and direct epigenetic modification in recipient cells. Science 2010, 328:872-875. This paper demonstrated long-distance movement of functional small RNAs. Wild-type Arabidopsis shoots were grafted onto roots of mutants deficient in small RNA production; libraries of small RNAs extracted from these roots showed that a large number of sRNAs are mobile. Grafting experiments also confirmed that mobile 24 nt sRNAs, which have been implicated in chromatin silencing, caused methylation of matching loci in the recipient roots. The authors speculated that sRNAs could also move to the shoot meristem to control development or reinforce transposon silencing. 28. Dunoyer P, Schott G, Himber C, Meyer D, Takeda A, Carrington JC, Voinnet O: Small RNA duplexes function as mobile silencing signals between plant cells. Science 2010, 328:912-916. Using vascular-specific expression of genes that participate in sRNA synthesis or that sequester sRNAs, this work shows that sRNAs move from the leaf vasculature to target cells in the Arabidopsis leaf mesophill. Experiments using particle bombardment to directly introduce GFP sRNAs into cells of sRNA-deficient mutants confirmed that GFP silencing was non-cell autonomous and provided evidence that double-stranded sRNAs function as the mobile signal. 29. Chitwood DH, Nogueira FTS, Howell MD, Montgomery TA, Carrington JC, Timmermans MCP: Pattern formation via small RNA mobility. Genes Dev 2009, 23:549-554. www.sciencedirect.com
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This work revealed that an endogenous small RNA (tasiR-ARF) that controls development of the leaf abaxial–adaxial axis is a mobile patterning signal. In situ hybridisation showed a gradient of tasiR-ARF from the adaxial to the abaxial side of developing leaves, contrasting with the localised expression of AGO7 and TAS3A (which are required for tasiRARF synthesis) in the adaxial side of developing leaves. Similarly, discrepancy between the vascular expression of components required to produce tasiR-ARF and the SAM accumulation of tasiR-ARF indicated movement from stem tissues to the SAM. 30. Tucker MR, Hinze A, Tucker EJ, Takada S, Ju¨rgens G, Laux T: Vascular signalling mediated by ZWILLE potentiates WUSCHEL function during shoot meristem stem cell development in the Arabidopsis embryo. Development 2008, 135:2839-2843. 31. Liu Q, Yao X, Pi L, Wang H, Cui X, Huang H: The ARGONAUTE10 gene modulates shoot apical meristem maintenance and establishment of leaf polarity by repressing miR165/166 in Arabidopsis. Plant J 2009, 58:27-40. Liu et al. showed that AGO10 (also known as PNH and ZLL) maintains the embryonic SAM by repressing miRNAs 166/165, which antagonise HDZIP genes required for SAM development (see also [26]). Increased miRNA166/165 levels were detected in the embryonic SAM of the ago10 mutant. Meristem loss in ago10 was enhanced by loss of HDZIP function and suppressed by dominant, miRNA-resistant HD-ZIP or in mutants with decreased miRNA function (e.g. dcl1). The tasiR-ARF pathway (see [29]) functioned in parallel with AGO10 to reduce miRNA166/ 165 levels. 32. Sozzani R, Cui H, Moreno-Risueno MA, Busch W, Van Norman JM, Vernoux T, Brady SM, Dewitte W, Murray JAH, Benfey PN: Spatiotemporal regulation of cell-cycle genes by SHORTROOT links patterning and growth. Nature 2010, 466:128-132. Sozzani et al. discovered that SHR directly activates a specific isoform of cyclin D to regulate asymmetric division of the root stem cells that give rise to the cortex and endodermis. CycD6,1 responded rapidly to activation of SHR or SCR, coinciding with the asymmetric division. Chromatin immunoprecipitation revealed that cycD6,1 is a direct target of both SHR and SCR. Asymmetric division was delayed in the cycD6,1 mutant and rescued by forced expression of CYCD6,1 in the ground tissue of the shr mutant. Mutation and ectopic expression of other cyclin D isoforms did not affect asymmetric division of the cortex/endodermis initials, showing that cyclin D isoforms have specialised developmental roles. 33. Ishida T, Fujiwara S, Miura K, Stacey N, Yoshimura M, Schneider K, Adachi S, Minamisawa K, Umeda M, Sugimoto K: SUMO E3 ligase HIGH PLOIDY2 regulates endocycle onset and meristem maintenance in Arabidopsis. Plant Cell 2009, 21:2284-2297. 34. Busch W, Miotk A, Ariel FD, Zhao Z, Forner J, Daum G, Suzaki T, Schuster C, Schultheiss SJ, Leibfried A et al.: Transcriptional control of a plant stem cell niche. Dev Cell 2010, 18:841-853. WUS target genes were identified by transcriptional profiling after induction of WUS or CLV3 overexpression, combined with chromatin immunoprecipitation. Target genes have known or predicted roles in sugar metabolism, meristem development and hormone responses. Characterised targets include CLAVATA1 and several members of the TOPLESS family encoding transcriptional co-repressors. The set of direct WUS targets also includes genes that are probably controlled by WUS during reproductive organ development (e.g. TETRASPORE, DYAD and genes encoding S-locus kinases). 35. Yadav RK, Girke T, Pasala S, Xie M, Reddy GV: Gene expression map of the Arabidopsis shoot apical meristem stem cell niche. Proc Natl Acad Sci U S A 2009, 106:4941. Yadav et al. compared the trancriptomes of flow cytometry-purified SAM cells expressing CLV3, WUS and FIL markers. The possible overlaps of expression in three different markers defined a set of seven different meristem regions. The dataset correctly assigned the expression pattern of numerous well-known meristem genes and successfully predicted the expression pattern revealed by in situ hybridisation for many novel genes. Gene ontology analysis of genes preferentially expressed in the shoot stem cells suggested that DNA repair and chromatin remodelling are particularly active in shoot stem cells. 36. Ikeda M, Mitsuda N, Ohme-Takagi M: Arabidopsis WUSCHEL is a bifunctional transcription factor that acts as a repressor in stem cell regulation and as an activator in floral patterning. Plant Cell 2009, 21:3493-3505.
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37. Nawy T, Lee JY, Colinas J, Wang JY, Thongrod SC, Malamy JE, Birnbaum K, Benfey PN: Transcriptional profile of the Arabidopsis root quiescent center. Plant Cell 2005, 17:1908-1925. 38. Jiang KN, Zhu T, Diao ZY, Huang HY, Feldman LJ: The maize root stem cell niche: a partnership between two sister cell populations. Planta 2010, 231:411-424. 39. Curtis MJ, Hays JB: Tolerance of dividing cells to replication stress in UVB-irradiated Arabidopsis roots: requirements for DNA translesion polymerases eta and zeta. DNA Repair 2007, 6:1341-1358. 40. Fulcher N, Sablowski R: Hypersensitivity to DNA damage in plant stem cell niches. Proc Natl Acad Sci U S A 2009, 106:20984-20988. This paper showed that root and shoot stem cells are particularly sensitive to DNA double strand breaks (DSBs) and respond by activating programmed cell death. Low levels of DSBs caused by zeocin, X-rays or by mutations that disrupt DSB repair caused death of stem cells and daughter cells, while the quiescent centre and rapidly dividing meristem cells survived. Cell death showed features of autophagy and required transduction of DNA damage signals, so it was not a consequence of DNA damage itself. Selective cell death may help to prevent the accumulation of mutations in plant stem cell populations, which can be exposed to DNA-damaging environments. 41. Furukawa T, Curtis MJ, Tominey C, Duong YH, Wilcox BWL, Aggoune D, Hays JB, Britt AB: A shared DNA-damage-response pathway for induction of stem-cell death by UV-B and by gamma irradiation. DNA Repair 2010, In Press, doi:10.1016/ j.dnarep.2010.06.006. 42. Yoshiyama K, Conklin PA, Huefner ND, Britt AB: Suppressor of gamma response 1 (SOG1) encodes a putative transcription factor governing multiple responses to DNA damage. Proc Natl Acad Sci U S A 2009, 106:12843-12848. This work identifies a central regulator of DNA damage responses in plants, functionally comparable to p53 in animals. SOG1, which encodes a plant-specific NAC transcription factor, was identified in a screen for mutants that suppress the sensitivity to gamma rays in a DNA repair mutant. SOG1 is required for the majority of the plant’s responses to DNA damage, including death of stem cells in response to DNA damage (see [41]). 43. Rounds MA, Larsen PB: Aluminum-dependent root-growth inhibition in Arabidopsis results from AtATR-regulated cellcycle arrest. Curr Biol 2008, 18:1495-1500. This paper showed that the root meristem is a key target of aluminium toxicity, which inhibits cell division and QC function via ATR, a kinase that transduces replication stress and DNA damage signals. Dominant negative ATR mutations restored root meristem activity in mutants hypersensitive to aluminium and in wild-type plants exposed to toxic aluminium levels. 44. Han P, Li Q, Zhu Y-X: Mutation of Arabidopsis BARD1 causes meristem defects by failing to confine WUSCHEL expression to the organizing center. Plant Cell 2008, 20:1482-1493. 45. Jonsson H, Krupinski P: Modeling plant growth and pattern formation. Curr Opin Plant Biol 2010, 13:5-11. 46. Fernandez R, Das P, Mirabet V, Moscardi E, Traas J, Verdeil J-L, Malandain G, Godin C: Imaging plant growth in 4D: robust tissue reconstruction and lineaging at cell resolution. Nat Methods 2010, 7:547-553. 47. Hamant O, Heisler MG, Jonsson H, Krupinski P, Uyttewaal M, Bokov P, Corson F, Sahlin P, Boudaoud A, Meyerowitz EM et al.: Developmental patterning by mechanical signals in Arabidopsis. Science 2008, 322:1650-1655. Microtubules are known to be required for directional growth; this work showed that the microtubule cytoskeleton also responds to physical stresses resulting from growth. The observed orientation of microtubules matched the pattern of physical stresses predicted by computer models during normal growth and after perturbations such as cell ablation or inhibition of organ emergence. Thus microtubules function in a feedback loop between cellular activities and the physical properties of meristem tissues. 48. Sang Y, Wu M-F, Wagner D: The stem cell-chromatin connection. Semin Cell Dev Biol 2009, 20:1143-1148.
Current Opinion in Plant Biology 2011, 14:4–9
Plant stem cell niches: from signalling to execution Robert Sablowski The shoot and root meristems contain small populations of stem cells that constantly renew themselves while providing precursor cells to build all other plant tissues and organs. Cell renewal, growth and differentiation in the meristems are coordinated by networks of transcription factors and intercellular signals. The past two years have revealed how auxin and cytokinin signals are integrated with each other and with regulatory genes in the shoot and root meristems. Small RNAs have also emerged as novel intercellular signals. Downstream of meristem regulatory genes, links have been made to cell division control and chromatin function. Protection of genome integrity, partly through programmed cell death after DNA damage, has recently been revealed as a specialised function in plant stem cells. Address Cell and Developmental Biology, John Innes Centre, Norwich NR4 7UH, United Kingdom Corresponding author: Sablowski, Robert ([email protected])
Current Opinion in Plant Biology 2011, 14:4–9 This review comes from a themed issue on Growth and development Edited by Jiayang Li and Nam-Hai Chua Available online 23rd August 2010 1369-5266/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2010.08.001
Introduction Virtually all cells of a mature plant descend from small populations of stem cells that are maintained within the meristems. As in animals, these stem cells are defined by their ability to both renew themselves and contribute daughter cells to produce new tissues. A feature found throughout multicellular organisms is that stem cells are maintained in specialised microenvironments called stem cell niches, where extracellular signals are available that maintain the stem cells. The functional similarities found between stem cells of very different multicellular organisms probably result from convergent evolution [1,2]. The best-studied plant stem cell niches reside in the shoot and root apical meristems (SAM and RAM, respectively). Stem cells are maintained at the summit of the SAM by a signal produced by underlying cells that Current Opinion in Plant Biology 2011, 14:4–9
express WUSCHEL, which encodes a homeodomain transcription factor (TF) (Figure 1A and C) [3]. Descendants of the stem cells are displaced to the periphery of the meristem and recruited into new organs, while the innermost region of the SAM, called the rib meristem, produces the internal tissues of the stem. In the RAM, stem cells are maintained by signals from a small group of cells called the quiescent centre (QC), which rarely divide but also functions as a long-term reserve of stem cells [4] (Figure 1B). The QC, and therefore the root stem cell niche, is positioned where activity of the TFs SCARECROW (SCR) and SHORT ROOT (SHR) overlaps with expression of the PLETHORA (PLE) TFs, which in turn are controlled by an apical–basal gradient of auxin maintained by PIN transporters [2]. Control of the stem cell maintenance signal in the RAM shows similarities to the SAM: the WUS homologue WUS-RELATED HOMEOBOX5 (WOX5) is required in the QC to prevent differentiation of the stem cells of the columella [5]. Both the SAM and RAM maintain stable patterns of gene expression in spite of constant cell division and displacement [6,7]. This requires intense intercellular signalling to assess the position of each cell and correct its gene expression accordingly. These signals also balance the rate of cell division with differentiation and initiate tissue patterning. Key regulatory genes that respond to intercellular signalling (e.g. WUS, PLE, SHR, SCR and WOX5) ultimately control rates of cell growth, division and the decision to enter a differentiation pathway, but the molecular details of how these processes are controlled remain unknown. Here, I review progress made in the past two years on intercellular signalling and on the functions downstream of key transcriptional regulators in the meristems.
Cell signalling in meristem maintenance and differentiation The best understood intercellular signal used in meristem maintenance is the glucosylated dodecapeptide CLAVATA3 (CLV3) [8], which is produced by the shoot stem cells and diffuses to underlying cells to inhibit WUS in a negative feedback loop that regulates the size of the stem cell population [9]. Until recently, it was thought that CLV3 was perceived by a receptor made of CLAVATA1 (CLV1) and CLAVATA2 (CLV2) subunits, but new results suggest instead that CLV3 is perceived in parallel by two different receptors, one including CLV1 and another made of CLV2/CORYNE subunits [10]. It has been suggested that a pathway related to CLV/WUS functions in the RAM, because peptides homologous to www.sciencedirect.com
Plant stem cell niches: from signalling to execution Sablowski 5
Figure 1
Physical structure and regulatory networks in the shoot and root meristems. A, B: three-dimensional reconstructions based on confocal images of shoot meristems (A; IM and FM indicate inflorescence and flower meristem, respectively) and a root apical meristem (B). Key regions of the meristems are marked in false colours: red for the stem cells, blue for the source of stem cell maintenance signals (organising centre in A, quiescent centre in B), yellow for the rib meristem (A) and stele (B); the green line in B surrounds the columella. Size bar: 50 mm. C, D: schematic representations of the meristems shown in A and B, with regulatory interactions indicated. Regions marked in red, blue and yellow correspond to those in A, B; positive and negative inputs are indicated by arrows and blunted lines. In C, the main hub of the regulatory network is WUS (marked in white); the orange region indicates where the cytokinin (CK) concentration is proposed to be sufficient to activate the AHK4 receptor; the dashed green line contains the region where AHK4 is expressed, whose overlap with the orange region defines the WUS-expression domain (blue); a dashed arrow indicates that small RNAs (sRNAs) produced in the developing vasculature are also required to maintain WUS expression. In D, boxes contain three different sets of regulatory interactions: on the top, SHR moves from the stele to the endodermis, where it heterodimerises with SCR to activate expression of miRNAs 165 and 166, which move back to the stele to control xylem differentiation. The middle box shows the integration of cytokinin (CK) and auxin signals through SHY2 to regulate expression of PIN1 and consequently auxin transport to the root tip (block arrow). The lower box represents the signalling network controlling WOX5 expression and differentiation of stem cells (SC) in the columella, which is bounded by the dashed green lines.
CLV3 inhibit root meristem growth [11,12] and because WOX5 function in the RAM resembles WUS in the SAM [5]. The similarity has been reinforced by the finding that CLE40 (the closest homologue of CLV3) promotes differentiation of columella cells, partly by restricting expression of WOX5 [13]. There are, however, important differences www.sciencedirect.com
to the CLV3/WUS interaction. First, the effect of CLE40 is mediated by a receptor kinase unrelated to CLV1/2: ARABIDOPSIS CRINKLY4 (ACR4), which restricts cell division in the columella [14], is required for CLE40 signalling, although it is not known if ACR4 binds to CLE40 or functions downstream of CLE40 signalling. Current Opinion in Plant Biology 2011, 14:4–9
6 Growth and development
Second, CLE40 signals from differentiating cells to control WOX5, whereas CLV3 signals from stem cells to inhibit WUS.
in animals [24], loss of the potential to function as a stem cell is a gradual process that can be reversed in differentiating cells.
Other well-studied signals that regulate meristem function are hormones. As mentioned above, auxin has a central role in positioning the root stem cell niche; now cytokinin has been implicated in positioning the stem cell niche in the SAM. It was found that cytokinin responses and WUS activity reinforce each other through multiple feedback loops and reflect the localised expression pattern of cytokinin receptors (ARABIDOPSIS HISTIDINE KINASE 2 and 4: AHK2, 4) in the rib meristem [15]. Combined with data indicating that cytokinin is produced in the apical region of the SAM [16,17], these results support a model in which the WUS-expression domain is continuously specified at the position where a sufficient amount of apically produced cytokinin reaches its receptors in the rib meristem (Figure 1C). It will be interesting to see whether activation of WUS by cytokinin is mediated by the WUS homologue STIMPY, which has been placed upstream of WUS and downstream of cytokinin in the SAM [18,19].
In addition to hormones, mobile macromolecules control cell differentiation in the RAM. For example, SHR moves from the stele (the innermost tissues of the stem and root, corresponding to the developing vasculature in the root tip) to adjacent endodermis cells, where it functions together with SCR to control asymmetric cell division and differentiation [25]. Now miRNAs have also been implicated in communication across the boundary between the endodermis and stele: miRNAs 165/166 are activated by SCR/SHR in the endodermis and move to the developing vasculature to control xylem differentiation [26].
Apart from positioning the stem cell niches, auxin and cytokinin interact to regulate the balance of cell division and differentiation. In the RAM, auxin and cytokinin have antagonistic effects, with auxin promoting cell division, while cytokinin promotes cell differentiation. The regulatory basis for this antagonism has been revealed: the cytokinin-response regulator ARABIDOPSIS RESPONSE REGULATOR 1 (ARR1) activates expression of SHORT HYPOCOTYL 2 (SHY2), which represses auxin signalling and PIN expression; conversely, auxin caused degradation of SHY2, allowing PIN expression and the auxin flow required to maintain the RAM [20] (Figure 1D). The effect of cytokinin on auxin transport has been independently confirmed [21]. In contrast to their opposite role in the RAM, auxin and cytokinin have synergistic effects on shoot organogenesis, and details have also emerged of how auxin and cytokinin signals are integrated in the SAM: auxin-activated MONOPTEROS (MP) directly represses the negative regulators of cytokinin responses ARR7/15, which in turn inhibit meristem function at least partly by activating CLV3 [22] (Figure 1C). Another important new insight is that the control of cell differentiation by hormones appears to be superimposed on their role in niche maintenance. After excision of the root tip, it was seen that missing cell types, such as the columella, were re-established before the stem cell niche had regenerated [23]. This early re-specification of missing cell types required auxin transport, showing that the hormonal control of tissue patterning can be separated from the control of stem cell maintenance and indeterminate growth. This work also confirmed that in plants, as Current Opinion in Plant Biology 2011, 14:4–9
More generally, signalling by small RNAs (sRNAs) is an emerging theme in plant development. Long-distance movement of sRNAs through the vasculature controls epigenetic changes in recipient cells [27]; the actual mobile molecule is probably duplex small RNAs [28]. Although direct evidence for sRNA signalling from the developing stem to the SAM is missing, several recent papers support the idea. First, a mobile small RNA (tasiR-ARF) that controls abaxial–adaxial leaf patterning also moves to the SAM from underlying stem tissues [29]. Second, PINHEAD/ZWILLE (PNH/ZLL), which encodes an ARGONAUTE protein and therefore is implicated in sRNA function, is required in the developing vasculature of the embryo to produce a mobile signal that maintains WUS expression [30]. Third, PNH/ ZLL functions in parallel with the tasiR-ARF pathway to maintain the SAM by reducing the meristematic levels of miRNAs 165/166, which in turn downregulate HD-ZIP transcription factors required for SAM development [31]. In conclusion, recent work has clarified how hormone signals are integrated in the meristems and has revealed sRNAs as potentially important intercellular signals. The results also highlight the role of the developing stele (which in the shoot includes the rib meristem and developing vasculature) in signalling processes that position the stem cell niches and control differentiation in both the SAM and RAM (Figure 1).
Downstream effectors of meristem functions The intercellular signals discussed above constantly adjust the expression of genes that control stem cell functions, including cell division. Accordingly, SHR and SCR have been shown to directly activate a specialised cycD isoform, which in turn regulates an asymmetric stem cell division that gives rise to the precursor cells of the cortex and endodermis of the root [32]. Another link to cell division is that a SUMO E3 ligase functions downstream of PLE genes to maintain cells in the mitotic www.sciencedirect.com
Plant stem cell niches: from signalling to execution Sablowski 7
cycle (rather than entering the endocycle associated with cell differentiation) [33]. Direct targets have also been identified for WUS combining ChIP-chip and transcriptome analysis [34]. The set of WUS targets included CLV1 and was enriched for genes involved in hormone responses and meristem development, in addition to genes involved in pollen and ovule development, reflecting later functions of WUS in reproductive organs. Comparison with a global analysis of gene expression in different regions of the SAM [35] showed that most of the direct targets of WUS are excluded from the WUS domain, suggesting that WUS is primarily a repressor. WUS was confirmed to function mostly as a transcriptional repressor in the meristem, although it also activates the reproductive organ identity gene AGAMOUS [36]. The identification of direct targets of SHR, SCR and WUS complemented work on the cell type-specific transcriptomes in the RAM and SAM. In the SAM, gene expression was profiled in cells marked by CLV3, WUS and FIL reporters (expressed in stem cells, organising centre and initiating organs, respectively) [35]. Gene ontology analysis showed that the stem cells preferentially express genes involved in chromatin modification and DNA repair. Similar insight emerges from a comparison between QC gene expression in Arabidopsis [37] and a more recent analysis in maize [38]. Thus results from both the root and shoot support the idea that dynamic chromatin and stringent protection of genome integrity are important features of plant stem cell niches. The idea that plant stem cells have specialised mechanisms to maintain genome integrity has also been supported by work on how stem cells respond to DNA damage. UV irradiation and DNA double strand breaks preferentially kill stem cells in the root and shoot, a response that requires transduction of DNA damage signals by the kinases ATAXIA-TELANGIECTASIA MUTATED (ATM) and ATM-RELATED (ATR) [39,40] and activation of DNA damage responses by the TF SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1) [41,42]. ATR has also been implicated in the inhibition of RAM activity by aluminium (which among other stresses causes DNA damage) [43]. Another link between DNA damage and stem cell function is that the DNA repair gene BARD1 represses WUS [44]. All the data above indicate that protection of genome integrity is an important function in plant stem cell populations; this could be important to prevent the propagation of mutations to large parts of the plant body, including the germline in the case of the SAM. In summary, gene expression profiling has revealed that cell division and intercellular signalling are key processes targeted by regulators of stem cell function. Global www.sciencedirect.com
analysis of gene expression in the meristems also indicated that chromatin dynamics and maintenance of genome integrity are key functions, although the molecular links between these processes and key meristem regulators remain unclear.
Conclusions and perspectives The past two years have revealed how multiple signals are integrated in the meristems to position and maintain the stem cell niches, balance cell proliferation with differentiation and initiate tissue patterning. Although it is satisfying to have an increasingly unified view, the signalling network has become too complex to understand intuitively. Computer models are already necessary to predict the behaviour of meristem regulatory networks [45], and these models will no doubt be improved by incorporating advances in dynamic, 3D meristem imaging [46] and mechanical modelling [47]. To ultimately understand how the behaviour of signalling and transcriptional networks is translated into meristem functions, the molecular links to cellular functions will also need to be explored further. Links to chromatin functions are particularly important because of the likely role of chromatin structure in pluripotency, which is a defining feature of stem cells [48]. The control of genome integrity in the meristem is a new and mostly unexplored area – we do not know whether repair mechanisms differ in the stem cell niches or what are the molecular pathways linking DNA damage and programmed cell death. To reveal the functional relevance of these mechanisms, it will be necessary to measure the accumulation of mutations in stem cells and in differentiating progeny. No shortage of challenges for the future.
Acknowledgements I am grateful to Lars Østergaard and Silvia Costa for comments. Work in my lab is funded by the Biotechnology and Biological Sciences Research Council and the European Union.
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10. Bleckmann A, Weidtkamp-Peters S, Seidel CAM, Simon R: Stem cell signaling in Arabidopsis requires CRN to localize CLV2 to the plasma membrane. Plant Physiol 2010, 152:166-176. 11. Casamitjana-Martinez E: Root-specific CLE19 overexpression and the sol1/2 suppressors implicate a CLV-like pathway in the control of Arabidopsis root meristem maintenance. Curr Biol 2003, 13:1435. 12. Fiers M, Hause G, Boutilier K, Casamitjana-Martinez E, Weijers D, Offringa R, van der Geest L, Campagne MV, Liu CM: Misexpression of the CLV3/ESR-like gene CLE19 in Arabidopsis leads to a consumption of root meristem. Gene 2004, 327:37-49. 13. Stahl Y, Wink RH, Ingram GC, Simon Rd: A signaling module controlling the stem cell niche in Arabidopsis root meristems. Curr Biol 2009, 19:909-914. This paper showed that in the root, as in the shoot meristem, a CLV3-like peptide regulates the source of stem cell maintenance signals. Mutant analysis and application of synthetic CLE40 revealed that this CLV3 homologue restricts WOX5 expression and promotes differentiation of columella cells. The effects of CLE40 were mediated by the ACR4 receptor (see below). 14. De Smet I, Vassileva V, De Rybel B, Levesque MP, Grunewald W, Van Damme D, Van Noorden G, Naudts M, Van Isterdael G, De Clercq R et al.: Receptor-like kinase ACR4 restricts formative cell divisions in the Arabidopsis root. Science 2008, 322:594-597. Transcriptional profiling of pericycle cells that were synchronously initiating lateral roots revealed ACR4 as a receptor kinase that regulates the asymmetric division that initiates lateral root development. Mutant analysis showed that ACR4 and close homologues also repress division of neighbouring cells to limit the number and location of lateral root primordia. In addition, ACR4 is expressed in columella, epidermal and lateral root cap initials, where it regulates division of daughter cells. 15. Gordon S, Chickarmane V, Ohno C, Meyerowitz E: Multiple feedback loops through cytokinin signaling control stem cell number within the Arabidopsis shoot meristem. Proc Natl Acad Sci U S A 2009. Using live imaging and computer modelling, this paper provides evidence that mutually reinforcing feedback loops between WUS and cytokinin response, superimposed on the expression pattern of cytokinin receptors (AHK2, 4), continuously delimit the domain of WUS expression within the meristem and therefore the position of the shoot stem cell niche. 16. Kurakawa T, Ueda N, Maekawa M, Kobayashi K, Kojima M, Nagato Y, Sakakibara H, Kyozuka J: Direct control of shoot meristem activity by a cytokinin-activating enzyme. Nature 2007, 445:652-655. 17. Veit B: Hormone mediated regulation of the shoot apical meristem. Plant Mol Biol 2009, 69:397-408. 18. Wu X, Dabi T, Weigel D: Requirement of homeobox gene STIMPY/WOX9 for Arabidopsis meristem growth and maintenance. Curr Biol 2005, 15:436. 19. Skylar A, Hong F, Chory J, Weigel D, Wu X: STIMPY mediates cytokinin signaling during shoot meristem establishment in Arabidopsis seedlings. Development 2010, 137:541-549. 20. Dello Ioio R, Nakamura K, Moubayidin L, Perilli S, Taniguchi M, Morita MT, Aoyama T, Costantino P, Sabatini S: A genetic framework for the control of cell division and differentiation in the root meristem. Science 2008, 322:1380-1384. This paper reveals the regulatory basis for the antagonism between auxin and cytokinin in the root meristem. The cytokinin-response regulator ARR1 functions in the root stele to directly activate SHY2, which represses auxin signalling but is degraded in response to auxin. Through Current Opinion in Plant Biology 2011, 14:4–9
activation of ARR1 and SHY2, cytokinin repressed expression of the auxin transporters PIN1, 3, 7, consequently changing auxin distribution and promoting cell differentiation. 21. Ruzycka K, Simaskova M, Duclercq J, Petrasek J, Zazimalova E, Simon S, Friml J, Van Montagu MCE, Benkova E: Cytokinin regulates root meristem activity via modulation of the polar auxin transport. Proc Natl Acad Sci U S A 2009, 106:4284-4289. 22. Zhao Z, Andersen SU, Ljung K, Dolezal K, Miotk A, Schultheiss SJ, Lohmann JU: Hormonal control of the shoot stem-cell niche. Nature 2010, 465:1089-1092. This work showed how cytokinin and auxin inputs are integrated in the shoot meristem. Inhibition of the negative cytokinin-response regulators ARR7/15 caused increased meristem size, partly by inhibiting CLV3, and caused phylotaxis defects, suggesting a link to auxin responses. Of several auxin response regulators tested, MP was found to directly repress ARR7/ 15. Thus auxin inhibits a repressor of cytokinin responses, explaining why auxin and cytokinin have synergistic effects in shoot development. 23. Sena G, Wang XN, Liu HY, Hofhuis H, Birnbaum KD: Organ regeneration does not require a functional stem cell niche in plants. Nature 2009, 457:1150-U1110. Sena et al. asked whether re-establishment of root cell types after excision of the root tip required first the recovery of the root stem cell niche. On the basis of marker genes, starch accumulation and gravitropic response, recovery of columella cells preceded re-establishment of the QC and the stem cell niche. Mutants that are unable to maintain the stem cell niche ( plt1, plt2 and scr) still re-established columella cells and overall root tip morphology after excision. Thus the stem cell niche is required for indeterminate growth, but not for the re-direction of cell differentiation during organ regeneration. 24. Nakagawa T, Sharma M, Nabeshima Y, Braun RE, Yoshida S: Functional hierarchy and reversibility within the murine spermatogenic stem cell compartment. Science 2010, 328:62-67. 25. Nakajima K, Sena G, Nawy T, Benfey P: Intercellular movement of the putative transcription factor SHR in root patterning. Nature 2001, 413:307-311. 26. Carlsbecker A, Lee J-Y, Roberts CJ, Dettmer J, Lehesranta S, Zhou J, Lindgren O, Moreno-Risueno MA, Vaten A, Thitamadee S et al.: Cell signalling by microRNA165/6 directs gene dosedependent root cell fate. Nature 2010, 465:316-321. This paper shows that mobile miRNAs regulate cell differentiation in the root stele. In the endodermis, SCR and SHR activated miRNAs 166/165, which degrade HD-ZIP mRNAs. The miRNAs not only functioned in the endodermis, but also moved to adjacent cells, as shown by the discrepancy between miRNA gene expression and the pattern of mature miRNAs detected by in situ hybridisation and by a miRNA sensor construct. Graded degradation of HD-ZIP mRNAs by miRNAs 165/6 in the stele was required for the differentiation of different xylem cell types. 27. Molnar A, Melnyk CW, Bassett A, Hardcastle TJ, Dunn R, Baulcombe DC: Small silencing RNAs in plants are mobile and direct epigenetic modification in recipient cells. Science 2010, 328:872-875. This paper demonstrated long-distance movement of functional small RNAs. Wild-type Arabidopsis shoots were grafted onto roots of mutants deficient in small RNA production; libraries of small RNAs extracted from these roots showed that a large number of sRNAs are mobile. Grafting experiments also confirmed that mobile 24 nt sRNAs, which have been implicated in chromatin silencing, caused methylation of matching loci in the recipient roots. The authors speculated that sRNAs could also move to the shoot meristem to control development or reinforce transposon silencing. 28. Dunoyer P, Schott G, Himber C, Meyer D, Takeda A, Carrington JC, Voinnet O: Small RNA duplexes function as mobile silencing signals between plant cells. Science 2010, 328:912-916. Using vascular-specific expression of genes that participate in sRNA synthesis or that sequester sRNAs, this work shows that sRNAs move from the leaf vasculature to target cells in the Arabidopsis leaf mesophill. Experiments using particle bombardment to directly introduce GFP sRNAs into cells of sRNA-deficient mutants confirmed that GFP silencing was non-cell autonomous and provided evidence that double-stranded sRNAs function as the mobile signal. 29. Chitwood DH, Nogueira FTS, Howell MD, Montgomery TA, Carrington JC, Timmermans MCP: Pattern formation via small RNA mobility. Genes Dev 2009, 23:549-554. www.sciencedirect.com
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This work revealed that an endogenous small RNA (tasiR-ARF) that controls development of the leaf abaxial–adaxial axis is a mobile patterning signal. In situ hybridisation showed a gradient of tasiR-ARF from the adaxial to the abaxial side of developing leaves, contrasting with the localised expression of AGO7 and TAS3A (which are required for tasiRARF synthesis) in the adaxial side of developing leaves. Similarly, discrepancy between the vascular expression of components required to produce tasiR-ARF and the SAM accumulation of tasiR-ARF indicated movement from stem tissues to the SAM. 30. Tucker MR, Hinze A, Tucker EJ, Takada S, Ju¨rgens G, Laux T: Vascular signalling mediated by ZWILLE potentiates WUSCHEL function during shoot meristem stem cell development in the Arabidopsis embryo. Development 2008, 135:2839-2843. 31. Liu Q, Yao X, Pi L, Wang H, Cui X, Huang H: The ARGONAUTE10 gene modulates shoot apical meristem maintenance and establishment of leaf polarity by repressing miR165/166 in Arabidopsis. Plant J 2009, 58:27-40. Liu et al. showed that AGO10 (also known as PNH and ZLL) maintains the embryonic SAM by repressing miRNAs 166/165, which antagonise HDZIP genes required for SAM development (see also [26]). Increased miRNA166/165 levels were detected in the embryonic SAM of the ago10 mutant. Meristem loss in ago10 was enhanced by loss of HDZIP function and suppressed by dominant, miRNA-resistant HD-ZIP or in mutants with decreased miRNA function (e.g. dcl1). The tasiR-ARF pathway (see [29]) functioned in parallel with AGO10 to reduce miRNA166/ 165 levels. 32. Sozzani R, Cui H, Moreno-Risueno MA, Busch W, Van Norman JM, Vernoux T, Brady SM, Dewitte W, Murray JAH, Benfey PN: Spatiotemporal regulation of cell-cycle genes by SHORTROOT links patterning and growth. Nature 2010, 466:128-132. Sozzani et al. discovered that SHR directly activates a specific isoform of cyclin D to regulate asymmetric division of the root stem cells that give rise to the cortex and endodermis. CycD6,1 responded rapidly to activation of SHR or SCR, coinciding with the asymmetric division. Chromatin immunoprecipitation revealed that cycD6,1 is a direct target of both SHR and SCR. Asymmetric division was delayed in the cycD6,1 mutant and rescued by forced expression of CYCD6,1 in the ground tissue of the shr mutant. Mutation and ectopic expression of other cyclin D isoforms did not affect asymmetric division of the cortex/endodermis initials, showing that cyclin D isoforms have specialised developmental roles. 33. Ishida T, Fujiwara S, Miura K, Stacey N, Yoshimura M, Schneider K, Adachi S, Minamisawa K, Umeda M, Sugimoto K: SUMO E3 ligase HIGH PLOIDY2 regulates endocycle onset and meristem maintenance in Arabidopsis. Plant Cell 2009, 21:2284-2297. 34. Busch W, Miotk A, Ariel FD, Zhao Z, Forner J, Daum G, Suzaki T, Schuster C, Schultheiss SJ, Leibfried A et al.: Transcriptional control of a plant stem cell niche. Dev Cell 2010, 18:841-853. WUS target genes were identified by transcriptional profiling after induction of WUS or CLV3 overexpression, combined with chromatin immunoprecipitation. Target genes have known or predicted roles in sugar metabolism, meristem development and hormone responses. Characterised targets include CLAVATA1 and several members of the TOPLESS family encoding transcriptional co-repressors. The set of direct WUS targets also includes genes that are probably controlled by WUS during reproductive organ development (e.g. TETRASPORE, DYAD and genes encoding S-locus kinases). 35. Yadav RK, Girke T, Pasala S, Xie M, Reddy GV: Gene expression map of the Arabidopsis shoot apical meristem stem cell niche. Proc Natl Acad Sci U S A 2009, 106:4941. Yadav et al. compared the trancriptomes of flow cytometry-purified SAM cells expressing CLV3, WUS and FIL markers. The possible overlaps of expression in three different markers defined a set of seven different meristem regions. The dataset correctly assigned the expression pattern of numerous well-known meristem genes and successfully predicted the expression pattern revealed by in situ hybridisation for many novel genes. Gene ontology analysis of genes preferentially expressed in the shoot stem cells suggested that DNA repair and chromatin remodelling are particularly active in shoot stem cells. 36. Ikeda M, Mitsuda N, Ohme-Takagi M: Arabidopsis WUSCHEL is a bifunctional transcription factor that acts as a repressor in stem cell regulation and as an activator in floral patterning. Plant Cell 2009, 21:3493-3505.
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37. Nawy T, Lee JY, Colinas J, Wang JY, Thongrod SC, Malamy JE, Birnbaum K, Benfey PN: Transcriptional profile of the Arabidopsis root quiescent center. Plant Cell 2005, 17:1908-1925. 38. Jiang KN, Zhu T, Diao ZY, Huang HY, Feldman LJ: The maize root stem cell niche: a partnership between two sister cell populations. Planta 2010, 231:411-424. 39. Curtis MJ, Hays JB: Tolerance of dividing cells to replication stress in UVB-irradiated Arabidopsis roots: requirements for DNA translesion polymerases eta and zeta. DNA Repair 2007, 6:1341-1358. 40. Fulcher N, Sablowski R: Hypersensitivity to DNA damage in plant stem cell niches. Proc Natl Acad Sci U S A 2009, 106:20984-20988. This paper showed that root and shoot stem cells are particularly sensitive to DNA double strand breaks (DSBs) and respond by activating programmed cell death. Low levels of DSBs caused by zeocin, X-rays or by mutations that disrupt DSB repair caused death of stem cells and daughter cells, while the quiescent centre and rapidly dividing meristem cells survived. Cell death showed features of autophagy and required transduction of DNA damage signals, so it was not a consequence of DNA damage itself. Selective cell death may help to prevent the accumulation of mutations in plant stem cell populations, which can be exposed to DNA-damaging environments. 41. Furukawa T, Curtis MJ, Tominey C, Duong YH, Wilcox BWL, Aggoune D, Hays JB, Britt AB: A shared DNA-damage-response pathway for induction of stem-cell death by UV-B and by gamma irradiation. DNA Repair 2010, In Press, doi:10.1016/ j.dnarep.2010.06.006. 42. Yoshiyama K, Conklin PA, Huefner ND, Britt AB: Suppressor of gamma response 1 (SOG1) encodes a putative transcription factor governing multiple responses to DNA damage. Proc Natl Acad Sci U S A 2009, 106:12843-12848. This work identifies a central regulator of DNA damage responses in plants, functionally comparable to p53 in animals. SOG1, which encodes a plant-specific NAC transcription factor, was identified in a screen for mutants that suppress the sensitivity to gamma rays in a DNA repair mutant. SOG1 is required for the majority of the plant’s responses to DNA damage, including death of stem cells in response to DNA damage (see [41]). 43. Rounds MA, Larsen PB: Aluminum-dependent root-growth inhibition in Arabidopsis results from AtATR-regulated cellcycle arrest. Curr Biol 2008, 18:1495-1500. This paper showed that the root meristem is a key target of aluminium toxicity, which inhibits cell division and QC function via ATR, a kinase that transduces replication stress and DNA damage signals. Dominant negative ATR mutations restored root meristem activity in mutants hypersensitive to aluminium and in wild-type plants exposed to toxic aluminium levels. 44. Han P, Li Q, Zhu Y-X: Mutation of Arabidopsis BARD1 causes meristem defects by failing to confine WUSCHEL expression to the organizing center. Plant Cell 2008, 20:1482-1493. 45. Jonsson H, Krupinski P: Modeling plant growth and pattern formation. Curr Opin Plant Biol 2010, 13:5-11. 46. Fernandez R, Das P, Mirabet V, Moscardi E, Traas J, Verdeil J-L, Malandain G, Godin C: Imaging plant growth in 4D: robust tissue reconstruction and lineaging at cell resolution. Nat Methods 2010, 7:547-553. 47. Hamant O, Heisler MG, Jonsson H, Krupinski P, Uyttewaal M, Bokov P, Corson F, Sahlin P, Boudaoud A, Meyerowitz EM et al.: Developmental patterning by mechanical signals in Arabidopsis. Science 2008, 322:1650-1655. Microtubules are known to be required for directional growth; this work showed that the microtubule cytoskeleton also responds to physical stresses resulting from growth. The observed orientation of microtubules matched the pattern of physical stresses predicted by computer models during normal growth and after perturbations such as cell ablation or inhibition of organ emergence. Thus microtubules function in a feedback loop between cellular activities and the physical properties of meristem tissues. 48. Sang Y, Wu M-F, Wagner D: The stem cell-chromatin connection. Semin Cell Dev Biol 2009, 20:1143-1148.
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