© 2013 Scandinavian Plant Physiology Society, ISSN 0031-9317
Physiologia Plantarum 151: 43–51. 2014
MINIREVIEW
Auxin gradients across wood – instructive or incidental? Rishikesh P. Bhalerao and Urs Fischer∗ ˚ SE-90183, Department of Forest Genetics and Plant Physiology, Umea˚ Plant Science Centre, Swedish University of Agricultural Sciences, Umea, Sweden
Correspondence *Corresponding author, e-mail: [email protected] Received 20 August 2013; revised 18 November 2013 doi:10.1111/ppl.12134
Various aspects of wood formation have been linked to the action of auxin, e.g. cambial activity, dormancy, secondary cell wall deposition and tension wood formation. The presence of a radial auxin concentration gradient across wood-forming tissue has been suggested to regulate cambial activity and differentiation of cambial derivatives by providing positional information to cells within the tissue. Similar patterning mechanisms that depend on the interpretation of auxin thresholds have subsequently been proposed for shoot and root apical meristems. However, direct evidence for the existence of auxin gradients has only been obtained for the cambium of various tree species. While the auxin gradient theory is based on a plethora of descriptive and pharmacological experiments, in recent years, auxin function on wood formation has been underpinned by molecular and functional data. Here, we review the latest progress in understanding the role of auxin in wood formation and discuss how auxin concentration gradients could be established and interpreted in wood-forming tissues.
Introduction In plants, pattern formation not only occurs in embryos but differentiation and spatial organization of meristematic cells into organs happens throughout the entire plant life. Meristematic cells divide indeterminately and parts of their derivatives differentiate into tissues and organs. Cell fate decisions in plants are usually made according to the spatial position of an undifferentiated cell within the meristem. In the absence of cell migration, it seems plausible that lineage-dependent pattern forming processes are of minor importance. The positional dependence of differentiation in plants implies that each cell in an incipient primordium can sense its position. In the shoot apical meristem (SAM), mathematical modeling and auxin reporter gene analyses have revealed local maxima of the plant hormone auxin, which coincide with the position of incipient primordia (de Reuille et al. 2006, Smith et al. 2006), while in the root, auxin
response maxima correlate with the determination of lateral root cell identity (Dubrovsky et al. 2008). The idea of auxin gradients providing positional information has also been proposed as a patterning mechanism for the vascular cambium (Uggla et al. 1996, Sundberg et al. 2000), a lateral meristem that contributes to secondary radial growth of plant organs, e.g. to wood in stems. In fact, auxin gradients were first measured across woodforming tissues and it was speculated that these gradients provide spatial information for patterning (Uggla et al. 1996, 1998, Tuominen et al. 1997). Molecular dissection of the auxin gradient across wood has proven difficult (Tuominen et al. 1997) and the hypothesis that auxin acts as a positional signal during wood formation still lacks strong experimental support. However, in recent years, molecular tools and model systems have emerged that now enable the auxin gradient theory in the cambium to be tested. Here, we review the latest progress in understanding the role of auxin in wood formation and
Abbreviations – ABC, ATP-binding cassette; ARF, auxin response factor; AuxREs, auxin response elements; PIN1, PINFORMED 1; rev, revoluta; SAM, shoot apical meristem; WAT1, WALLS ARE THIN1.
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discuss these findings in the light of an auxin gradient as a patterning mechanism.
What is the mechanism underlying the establishment of an auxin concentration gradient?
Auxin levels along a concentration gradient correlate with distinct zones of differentiation in wood-forming tissues
The major sources of auxin in wood-forming tissues are the shoot apices. Auxin is thought to be transported rootwards in the cambium and distributed radially into xylem and phloem (Sundberg and Uggla 1998). As auxin diffuses only very poorly in the apoplast (Kramer 2006), redistribution by means of PIN-FORMED 1 (PIN1)-like auxin efflux carriers has been suggested as a mechanism of gradient formation in trees (Schrader et al. 2003, Hellgren et al. 2004). In the root tip of Arabidopsis, the auxin gradient has been explained based on the expression and localization of PIN proteins and auxin diffusion (Grieneisen et al. 2007). In order to redistribute polarly transported auxin from the cambium into phloem and xylem, efflux carriers would need to be localized to the lateral membrane of cambial cells and their derivatives. Although PIN proteins have not yet been systematically localized in cambia, PIN1, which is strongly expressed in Arabidopsis inflorescence stems, has been shown to localize not only to basal plasma membranes but also in the lateral plasma membrane at the basal end of parenchymatic xylem cells (G¨alweiler et al. 1998, Sauer et al. 2006). Similarly, PIN3, which is involved in redirecting the auxin flux upon gravistimulation in the root tip, localizes to the inner lateral membrane of the starch sheath of the Arabidopsis inflorescence stem (Friml et al. 2002). Interestingly, the first periclinal cell divisions, from which the interfascicular cambium originates, occur in the starch sheath (Altamura et al. 2001). In both a pin1 and pin3 mutant, the radial extension of derivatives of the interfascicular cambium is reduced (Agusti et al. 2011a). However, it is not known whether this is due to reduced cell division activity, cell growth or expansion, and whether PIN1 and PIN3 have a direct effect on auxin distribution in the cambium and its derivatives. In contrast, application of the polar auxin transport inhibitor 1-N-naphthylphthalamic acid (NPA) does not affect secondary xylem expansion in the Arabidopsis hypocotyl (Ragni et al. 2011). In stems of revoluta (rev ) mutants, which are defective in the formation of interfascicular fibers, PIN3 and PIN4 expression is dramatically reduced (Zhong and Ye 2001). Although in rev inflorescence stems rootward auxin transport has been shown to be strongly impaired, it is not known whether reduced PIN expression translates into alterations in radial auxin distribution. Besides systematic localization of PIN proteins in the cambial zone, analysis of multiple pin mutants will be required in order to reveal the contribution of this family of auxin transport facilitators to the establishment of a radial auxin gradient.
The vascular cambium is organized in radial strands of cells and forms to its inner (centripetal) side xylem and to the outer side phloem. Hence, there are only three primary cell fates a derivative of the cambium can adopt: either it differentiates into secondary xylem or phloem, or it does not differentiate and stays meristematic. Pattern formation in this case is strictly dependent on the position of the derivative in relation to the cambium. Daughter cells facing the inner side can differentiate into xylem, whereas daughter cells facing the outer side can differentiate into phloem. After dividing, cambial daughter cells first expand radially, and then secondary cell wall thickening takes place. This subdivides the xylary side of the cambium into three distinct zones of differentiation, namely the cambium, elongation and maturation zones (Fig. 1). Similar zonation patterning occurs in the phloem. However, the boundaries are structurally less apparent. Accordingly, the degree of differentiation increases with distance from the cambium. In wood-forming tissue, auxin concentrations peak in the cambium and decay rapidly toward the xylem and phloem (Uggla et al. 1996, 1998, Tuominen et al. 1997). High auxin concentrations localize to the cambium, intermediate concentrations to the elongation zone and low auxin concentration correlate with the maturation zone. Thus, it is tempting to speculate that the auxin gradient provides positional information for the division of wood-forming tissue into distinct zones of differentiation. High auxin concentrations may be interpreted as a signal for cell division, intermediate levels may promote cell expansion and low levels may be read out as a signal inducing the deposition of secondary cell walls (Fig. 1; Sundberg et al. 2000, Bhalerao and Bennett 2003). In addition, auxin concentrations could be interpreted for cell fate decisions. In this case, a high concentration of auxin would keep cells meristematic, whereas low levels would lead to differentiation into either xylem or phloem. In Pinus explants lacking an apical auxin source, cambial derivatives are not able to maintain their fusiform shape and differentiate into parenchymatic cells instead of xylem tracheids (Savidge 1983). This suggests that while auxin is required for maintaining the meristematic identity of the cambium, factors other than auxin are likely to contribute to subsequent cell fate decisions. 44
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Fig. 1. Auxin concentration gradient across wood-forming tissue. Auxin concentrations higher than threshold 1 (T1), correspond to the cell division zone, concentrations between T1 and T2 with cell expansion and below T2 with secondary cell wall formation (SCW).
Another candidate transporter that may be involved in actively generating an auxin gradient is the ATPbinding cassette (ABC) transporter ABCB14. It is expressed subapically, and in an abcb14 mutant, both polar auxin transport and the expression of the auxin response reporter DR5 have been shown to be reduced (Kaneda et al. 2011). The abcb14 mutant displays a mild radial cell expansion phenotype in the xylem. For ABCB1/PGP1 and ABCB19/PGP19, paralogous to ABCB14, auxin efflux activity has been demonstrated (Geisler et al. 2005). Localization and determination of transport substrates of ABCB14 could help to clarify whether the observed mutant phenotype is a consequence of a changed auxin gradient. In summary, phenotypes of auxin carrier mutants related to wood formation have either not been reported or are difficult to interpret due to an absence of auxin distribution data in those mutants (Agusti et al. 2011a, Kaneda et al. 2011).
How can the auxin concentration gradient be interpreted? Auxin is perceived by co-receptors comprising a ´ VilTIR1/AFB F-BOX and an AUX/IAA subunit (Calderon lalobos et al. 2012). Upon auxin binding, the AUX/IAA subunit is ubiquitinated and subsequently degraded by the proteasome. The Arabidopsis genome harbors 29 AUX /IAA genes, whose short-lived gene products can form heterodimers with auxin response factors (ARFs). Upon AUX/IAA-ARF heterodimerization, the ARF-mediated auxin response is inactivated, whereas binding of auxin to the co-receptor activates degradation of AUX/IAA and allows the ARFs to bind to promoters containing auxin response elements (AuxREs).
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Interestingly, auxin affinity to the co-receptor is primarily dependent on the AUX/IAA subunit of the co-receptor, and different AUX/IAAs confer differences in receptor ´ affinities of more than an order of magnitude (Calderon Villalobos et al. 2012). This means that by using a combination of different AUX/IAA and TIR1/AFB subunits, a large set of co-receptors with graded affinity can be obtained. Low affinity co-receptors would only lead to AUX/IAA degradation in the presence of high auxin concentration, whereas high affinity receptors could be active at low and high auxin concentrations. Low affinity and high affinity receptor activity could result in the expression or repression of different suites of genes, and this could provide a mechanistic explanation for how the read-out of an auxin gradient could result in discrete, threshold-dependent auxin responses rather than gradual changes. During differentiation, cambial derivatives experience a gradual decline in auxin concentration unless they stay meristematic. Different auxin concentrations are therefore likely to induce different responses, which must not occur simultaneously in a given cell. This means that auxin perception in differentiating wood does not constitute a simple on–off switch that leads to an irreversible, permanent outcome but rather a response that can be overruled by responses to lower concentrations of auxin. If for the different auxin functions in wood formation only a single auxin receptor or family of receptors with the same binding affinity is found to be responsible, this receptor would need to be multi-stable with at least three different steady states rather than being a simple bi-stable on–off switch. Differential expression of various AUX /IAA genes across the secondary xylem suggests co-occurrence of receptors with different affinities for auxin in wood-forming tissues (Moyle et al. 2002). 45
It remains to be tested whether these AUX/IAAs heterodimerize with specific ARFs or whether they interact more broadly with a larger number of ARFs as has been suggested for the SAM (Vernoux et al. 2011). In addition, some ARF genes might be expressed differentially across the wood-forming tissue (Schrader et al. 2004). Such a pre-patterning occurs in the embryo, where different ARF s, expressed in different domains, allow cell-specific auxin responses (Rademacher et al. 2012). Although direct determination of auxin concentration across wood-forming tissue by mass spectrometry has proven to be highly informative, it does not provide sufficient spatial resolution to distinguish auxin concentrations between neighboring cell files, where cells can acquire different cell fates, e.g. xylem vessels or xylem fibers. Resolution at a single cell level is possible by using reporter gene constructs, which have proven useful for measuring auxin response activity in the shoot and root apical meristem of Arabidopsis. These constructs do not report actual auxin concentrations but provide a read-out of auxin signaling. Nevertheless, they help to resolve TIR1/AFB dependent auxin responses at a cellular level. Reporter gene constructs driven by the synthetic promoter DR5, which consists of multiple AuxREs, have often been used for this purpose. In Arabidopsis inflorescence stems, DR5::GUS is expressed at the distal pole of the vascular bundles (Ranocha et al. 2010) and DR5::GFP is expressed in the protoxylem and metaxylem of vascular bundles (Suer et al. 2011, Agusti et al. 2011b). In addition, DR5::GFP has been found in the phloem and cortical cells of stems undergoing secondary growth (Suer et al. 2011). Intriguingly, neither in Arabidopsis nor in poplar (Chen et al. 2013) has DR5-reporter gene activity been detected in the cambium. This surprising finding was further substantiated by the work of Nilsson et al. (2008). The latter authors depleted stem segments of hybrid aspen from endogenous auxin and then added exogenous auxin while monitoring global gene expression. Such time course experiments allowed auxin-regulated genes in stem segments undergoing secondary growth to be identified. Surprisingly, expression patterns of only a very minor fraction of these auxin-regulated genes were found to mirror the auxin distribution across wood-forming tissue. Taken together, these results show that in wood-forming tissues, the auxin concentration maximum does not overlap with the DR5 auxin response maximum. While in the root apical meristem, the site of strongest DR5 promoter activity and highest auxin concentration has been shown to localize to the quiescent center (Petersson et al. 2009), a similar discrepancy between predicted auxin concentration maximum and auxin response has been found in the 46
SAM (de Reuille et al. 2006). Auxin insensitivity of cells at the summit of the SAM has been suggested to be the cause of DR5 promoter inactivity (de Reuille et al. 2006). In the light of this finding, it is interesting to note that the expression of TIR1 in hybrid aspen can be modulated and is reduced by 50% during seasonal cambial inactivity (Baba et al. 2011). As an alternative to auxin insensitivity, the AuxREs in the DR5 promoter may be insufficient to drive gene expression in the cambium. The native soybean GH3 promoter, from which the AuxREs were originally isolated, has been shown to drive GUS expression in the cambium and the developing secondary xylem of poplar (Teichmann et al. 2008). However, this reporter gene exhibits the highest activity in the cortex. Interestingly, cambial activity of GH3::GUS is induced in the cambial zone upon mechanical induction of tension wood (Teichmann et al. 2008). Previously, no changes in cambial auxin concentrations during tension wood formation were recorded (Hellgren et al. 2004). Hence, the induced GH3 auxin response upon mechanical stimulation might be a consequence of increased auxin responsiveness rather than augmented auxin concentrations in the cambium.
The read-out Auxin is a crucial factor for promoting cell division, e.g. cells of callus cultures do not divide in growth medium lacking auxin. When the auxin response in the cambium is reduced by stabilizing the AUX/IAA protein PttIAA3, cambial cells divide less frequently, while radial extension of the cambial zone increases (Nilsson et al. 2008). Interestingly, PttIAA3 could not be ubiquitinated by extracts from dormant hybrid aspen, whereas extracts from actively growing trees did result in PttIAA3 ubiquitination. This indicates that during active growth PttIAA3 is degraded and auxin signaling can take place, whereas during dormancy, it is stabilized (Baba et al. 2011). Considering that auxin levels in the cambium undergo only subtle seasonal changes (Uggla et al. 2001), these findings are in keeping with the idea that auxin signaling controls cambial activity by modulation of auxin responsiveness. Recent work in Arabidopsis has helped to unravel how auxin can regulate cambial activity. Cell division activity in the cambium is promoted by the release of the phloem derived CLV3 like peptides CLE41 and CLE44, which can bind to the leucine-rich receptor kinase PXY in the cambium. Receptor signaling then promotes cambial activity through expression of the WUSCHELlike homeodomain transcription factor WOX4 in the cambial zone (Etchells and Turner 2010, Hirakawa et al. 2010). Suer et al. (2011) found that WOX4 expression
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can be induced by exogenous auxin and that long-lasting induction requires PXY . These results indicate that auxin acts upstream of PXY and regulates cambial activity through WOX4 induction. Interestingly, DR5 promoter activity only sparsely overlaps with WOX4 expression (Suer et al. 2011). Hence, either the activation of WOX4 by auxin is mediated by a mobile factor different than auxin or alternatively, WOX4 expressing cells are not auxin responsive. In hybrid aspen, expression of stabilized PttIAA3 under the control of the ubiquitous 35S promoter not only decreases cambial activity but also radial cell expansion of xylem fibers and vessels (Nilsson et al. 2008). Similarly, in early and late wood, cell division activity, radial cell expansion and secondary wall formation are tightly coupled processes (Uggla et al. 2001). During early wood formation, cell division activity and cell expansion are high, whereas secondary cell wall deposition is low. In contrast, during late wood formation, cambial activity and cell expansion are low, but secondary cell walls become thicker. These observations can be explained by a narrower auxin peak in the cambium and more rapid decay of the auxin gradient during late wood formation (Uggla et al. 2001). From this angle, it is not surprising that reduction of auxin sensitivity by stabilizing PttIAA3 across wood-forming tissues not only targets a single isolated process of differentiation but affects various, probably interlinked aspects of wood formation. Secondary cell wall formation, the last step in the differentiation of cambial derivatives, corresponds to low auxin concentration in the gradient. Secondary cell wall deposition is controlled by a few members of the NAC transcription factor family. NST1 and NST3/SND1 switch on expression of secondary wall genes in xylem fibers, whereas VND6 and VND7 act on xylem vessel maturation (for a review see Wang and Dixon 2012). In a nst1, nst3/snd1 double mutant, secondary cell wall deposition in interfascicular fibers is completely abolished. A similar phenotype has been found in rev mutants. REV encodes for a class III homeodomain leucine zipper transcription factor, which together with other members of the gene family is involved in organ polarity. REV has been thought to act through PIN efflux carriers on polar auxin transport (Zhong and Ye 2001). However, it is not clear whether interfascicular fiber identity is affected in rev mutants or REV instead acts downstream on secondary cell wall deposition. Whereas RNAi lines against a REV homolog in poplar do not display a phenotype, overexpression of a miRNA resistant REV variant causes interruptions in the cambial cylinder (Robischon et al. 2011). Interestingly, the endings of such a gapped xylem did not join but extended into the
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cortex, resulting in ‘open loops’ with mirrored cambial layers. The positions of these gaps seemed to correlate with the position of lateral organs. Over- and/or ectopic REV expression may hinder the establishment of an interfascicular cambium, and therefore REV may be involved in cell fate determination rather than directly promote secondary cell wall deposition. As observed for rev mutants, mutations in WALLS ARE THIN1 (WAT1), which encodes for a tonoplast localized auxin efflux facilitator, cause a similar reduction in polar auxin transport and misregulation of genes associated with auxin response (Ranocha et al. 2010, 2013). Secondary cell wall deposition in xylary and interfascicular fibers is strongly reduced in wat1 mutants. However, as there is residual lignification of fibers, fiber cell identity seems not to be affected by wat1 mutations. This indicates that WAT1 acts more specifically on late fiber differentiation, whereas REV might be involved in patterning. Its interaction with REV and how WAT1 affects the radial auxin gradient are yet to be resolved. Secondary cell wall deposition also occurs outside of wood-forming tissue, e.g. in highly specialized cells of the silique, in the valve margin. During pod shatter, siliques become dehydrated, and as a consequence, the lignin component in valve margin cells is thought to create tensile forces, which separate the valve from the replum (Spence et al. 1996, Roberts et al. 2002). Like secondary cell wall deposition in xylary fibers, the NAC domain transcription factors, NST1 and NST3/SND1, switch on expression of secondary cell wall genes in the valve margins (Mitsuda and Ohme-Takagi 2008) after cell fate decisions in the abscission zone have taken place. Secondary cell wall deposition is preceded by formation of a local auxin response minimum in the valve margins (Sorefan et al. 2009). Formation of this auxin response minimum is thought to be a consequence of IND induced PIN1 and PIN3 de-localization in the valve margin, which in turn might deplete the valve margin from stigma-derived auxin. In the gynoecium, auxin is transported basipetally, like in the cambium (Cheng et al. 2006, Sohlberg et al. 2006). Interestingly, the auxin response remains high in the replum, the tissue in-between two neighboring valve margins. Cell division and expansion have been observed in the replum (Wu et al. 2006). However, it is not known whether these divisions coincide with the auxin maximum in space and time and whether secondary wall formation in the valve margins is triggered by auxin depletion.
The morphogen analogy As early as the first description of an auxin concentration gradient spanning wood-forming tissue, an analogy 47
to morphogen gradients was recognized (Uggla et al. 1996). The idea that auxin is able to form similar gradients as a morphogen has swiftly been adopted in mathematical models of auxin distribution across the SAM and root tip (Smith et al. 2006, Grieneisen et al. 2012). Most metazoan morphogen models require a localized source of morphogen secretion, diffusion or transport and decay of the morphogen (Wartlick et al. 2009). All cells along a morphogen gradient should be able to sense the morphogen and to interpret its concentration in a threshold-dependent manner, i.e. cells can determine their position in relation to the source. Although various mathematical auxin distribution models have been suggested, auxin concentrations at the shoot and root tip have still not been determined in intact meristems with sufficient spatial resolution to separate different morphologic zones (i.e. auxin measurements from 500 μm thick sections in the root tip, Fischer et al. 2006, Petersson et al. 2009, vs 30 μm in the cambium, Sundberg et al. 2000), and model validation has mainly been based on consideration of model robustness and DR5 promoter activity. Moreover, differences in experimentally determined auxin concentrations along the main growth axis or between various cell types of the root are well within an order of a magnitude (Fischer et al. 2006, Petersson et al. 2009), whereas the modeled dynamic range of auxin concentration spans several orders of magnitude (Grieneisen et al. 2012). While Arabidopsis shoot and root apices are superior model systems for functional analysis and protein localization of auxin transporters, wood-forming tissues are easily accessible to spatially highly resolved auxin concentration measurements. In both the root tip and SAM, formation of auxin gradients has been explained by the localization and activity of PIN proteins (Smith et al. 2006, Grieneisen et al. 2007). However, little attention has been paid to other mechanisms that do not rely on directed transport but still could form rapidly decaying auxin gradients in the direction of expanding growth axes. Given that radially redirected auxin in wood-forming tissues originates mainly from polarly transported auxin from shoot apices and that auxin is most likely transported rootwards in cambial cells, a mechanism based on cell division and expansion, which dilutes auxin, seems to be plausible. In such a scenario, some cambial derivatives would be disconnected from the polar auxin flux and their inherited auxin content would gradually be diluted by cell growth and expansion, and auxin catabolism (Fig. 2). Intriguingly, the decay of auxin concentration is most rapid in the cell expansion zone of the xylem, and auxin levels are barely detectable at the beginning of the maturation zone where cell expansion 48
Fig. 2. Clonal dilution model. Auxin dilution by cell growth and expansion. Auxin is provided to the cambium by polar auxin transport (PAT) via auxin efflux carriers. S, cambial stem cell; T1, threshold 1; T2, threshold 2 (Fig. 1).
has ceased (Sundberg et al. 2000). A similar model has been proposed for the formation of certain morphogen gradients in animals. For example, the distribution of fgf8 mRNA in the mouse embryo seems to be a consequence of cell division and expansion (Dubrulle and Pourqui´e 2004). The plausibility of such a model, based on the inheritance of a morphogenetically active, nonor poorly-secreted substance from its source, has been ˜ et al. 2006, shown by mathematical modeling (Ibanes Wartlick et al. 2009). A mechanism which acts through clonal dilution of auxin could also explain cell fate decisions in the cambium. Slightly asymmetrical cambial cell division would lead to unequal distribution of auxin between the two daughter cells. After cell growth, these stochastic irregularities could result in a different auxin concentration, and if as a consequence of asymmetric cell division and cell growth the auxin concentration
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drops below a certain threshold, the cambial derivate could undergo differentiation. In the case where the daughter cell with lower auxin concentration is located at the inner side of the cambium, it would be expected to differentiate into xylem. It would be interesting to determine whether the simple ‘cell lineage transport’ model can explain the observed auxin gradients across wood-forming tissues and to what extent auxin dilution by cell expansion contributes to the formation or robustness of the proposed auxin gradients in the root tip and SAM. For wood-forming tissues, meta-analyses of available data from tangential sectioning and thorough examination of auxin transport mutants in Arabidopsis should enable this hypothesis to be tested. ¨ Acknowledgements – We thank Bjorn Sundberg for critically reading this manuscript, the Berzelii Centre and Bio4Energy for financial support.
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Kramer EM (2006) How far can a molecule of weak acid travel in the apoplast or xylem? Plant Physiol 141: 1233–1236 Mitsuda N, Ohme-Takagi M (2008) NAC transcription factors NST1 and NST3 regulate pod shattering in a partially redundant manner by promoting secondary wall formation after the establishment of tissue identity. Plant J 56: 768–778 Moyle R, Schrader J, Stenberg A, Olsson O, Saxena S, Sandberg G, Bhalerao RP (2002) Environmental and auxin regulation of wood formation involves members of the Aux/IAA gene family in hybrid aspen. Plant J 31: 675–685 Nilsson J, Karlberg A, Antti H, Lopez-Vernaza M, Mellerowicz E, Perrot-Rechenmann C, Sandberg G, Bhalerao RP (2008) Dissecting the molecular basis of the regulation of wood formation by auxin in hybrid aspen. Plant Cell 20: 843–855 Petersson SV, Johansson AI, Kowalczyk M, Makoveychuk A, Wang JY, Moritz T, Grebe M, Benfey PN, Sandberg G, Ljung K (2009) An auxin gradient and maximum in the Arabidopsis root apex shown by high-resolution cell-specific analysis of IAA distribution and synthesis. Plant Cell 21: 1659–1668 Rademacher EH, Lokerse AS, Schlereth A, Llavata-Peris CI, Bayer M, Kientz M, Freire Rios A, Borst JW, Lukowitz W, Jurgens G, Weijers D (2012) Different auxin response machineries control distinct cell fates in the early plant embryo. Dev Cell 22: 211–222 Ragni L, Nieminen K, Pacheco-Villalobos D, Sibout R, Schwechheimer C, Hardtke CS (2011) Mobile gibberellin directly stimulates Arabidopsis hypocotyl xylem expansion. Plant Cell 23: 1322–1336 Ranocha P, Denance N, Vanholme R, Freydier A, Martinez Y, Hoffmann L, Koehler L, Pouzet C, Renou J-P, Sundberg B, Boerjan W, Goffner D (2010) Walls are thin 1 (WAT1), an Arabidopsis homolog of Medicago truncatula NODULIN21, is a tonoplast-localized protein required for secondary wall formation in fibers. Plant J 63: 469–483 Ranocha P, Dima O, Nagy R, Felten J, Corratge-Faillie C, Novak O, Morreel K, Lacombe B, Martinez Y, Pfrunder S, Jin X, Renou JP, Thibaud JB, Ljung K, Fischer U, Martinoia E, Boerjan W, Goffner D (2013) Arabidopsis WAT1 is a vacuolar auxin transport facilitator required for auxin homoeostasis. Nat Commun 4: 2625 de Reuille PB, Bohn-Courseau I, Ljung K, Morin H, Carraro N, Godin C, Traas J (2006) Computer simulations reveal properties of the cell-cell signaling network at the shoot apex in Arabidopsis. Proc Natl Acad Sci USA 103: 1627–1632 Roberts JA, Elliott KA, Gonzalez-Carranza ZH (2002) Abscission, dehiscence, and other cell separation processes. Annu Rev Plant Biol 53: 131–158
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Robischon M, Du J, Miura E, Groover A (2011) The Populus class III HD ZIP, popREVOLUTA, influences cambium initiation and patterning of woody stems. Plant Physiol 155: 1214–1225 Sauer M, Balla J, Luschnig C, Wisniewska J, Reinohl V, Friml J, Benkova E (2006) Canalization of auxin flow by Aux/IAA-ARF-dependent feedback regulation of PIN polarity. Genes Dev 20: 2902–2911 Savidge RA (1983) The role of plant hormones in higher plant cellular differentiation. 2. Experiments with the vascular cambium and sclerid and tracheid differentiation in the pine, Pinus contorta. Histochem J 15: 447–466 Schrader J, Baba K, May ST, Palme K, Bennett M, Bhalerao RP, Sandberg G (2003) Polar auxin transport in the wood-forming tissues of hybrid aspen is under simultaneous control of developmental and environmental signals. Proc Natl Acad Sci USA 100: 10096–10101 Schrader J, Moyle R, Bhalerao R, Hertzberg M, Lundeberg J, Nilsson P, Bhalerao RP (2004) Cambial meristem dormancy in trees involves extensive remodelling of the transcriptome. Plant J 40: 173–187 Smith RS, Guyomarc’h S, Mandel T, Reinhardt D, Kuhlemeier C, Prusinkiewicz P (2006) A plausible model of phyllotaxis. Proc Natl Acad Sci USA 103: 1301–1306 Sohlberg JJ, Myrenas M, Kuusk S, Lagercrantz U, Kowalczyk M, Sandberg G, Sundberg E (2006) STY1 regulates auxin homeostasis and affects apical-basal patterning of the Arabidopsis gynoecium. Plant J 47: 112–123 Sorefan K, Girin T, Liljegren SJ, Ljung K, Robles P, Galvan-Ampudia CS, Offringa R, Friml J, Yanofsky MF, Ostergaard L (2009) A regulated auxin minimum is required for seed dispersal in Arabidopsis. Nature 459: 583–586 Spence J, Vercher Y, Gates P, Harris N (1996) ’Pod shatter’ in Arabidopsis thaliana, Brassica napus and B. juncea. J Microsc 181: 195–203 Suer S, Agusti J, Sanchez P, Schwarz M, Greb T (2011) WOX4 imparts auxin responsiveness to cambium cells in Arabidopsis. Plant Cell 23: 3247–3259 Sundberg B, Uggla C (1998) Origin and dynamics of indoleacetic acid under polar transport in Pinus sylvestris. Physiol Plant 104: 22–29 Sundberg B, Uggla C, Tuominen H (2000) Cambial growth and auxin gradients. In: Savidge R, Barnett J, Napier R (eds) Cell and Molecular Biology of Wood Formation. BIOS, Oxford, pp 169–188 Teichmann T, Bolu-Arianto WH, Olbrich A, Langenfeld-Heyser R, Goebel C, Grzeganek P, Feussner I, Haensch R, Polle A (2008) GH3 :: GUS reflects cell-specific developmental patterns and stress-induced changes in wood anatomy in the poplar stem. Tree Physiol 28: 1305–1315
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Edited by K. Ljung
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Physiologia Plantarum 151: 43–51. 2014
MINIREVIEW
Auxin gradients across wood – instructive or incidental? Rishikesh P. Bhalerao and Urs Fischer∗ ˚ SE-90183, Department of Forest Genetics and Plant Physiology, Umea˚ Plant Science Centre, Swedish University of Agricultural Sciences, Umea, Sweden
Correspondence *Corresponding author, e-mail: [email protected] Received 20 August 2013; revised 18 November 2013 doi:10.1111/ppl.12134
Various aspects of wood formation have been linked to the action of auxin, e.g. cambial activity, dormancy, secondary cell wall deposition and tension wood formation. The presence of a radial auxin concentration gradient across wood-forming tissue has been suggested to regulate cambial activity and differentiation of cambial derivatives by providing positional information to cells within the tissue. Similar patterning mechanisms that depend on the interpretation of auxin thresholds have subsequently been proposed for shoot and root apical meristems. However, direct evidence for the existence of auxin gradients has only been obtained for the cambium of various tree species. While the auxin gradient theory is based on a plethora of descriptive and pharmacological experiments, in recent years, auxin function on wood formation has been underpinned by molecular and functional data. Here, we review the latest progress in understanding the role of auxin in wood formation and discuss how auxin concentration gradients could be established and interpreted in wood-forming tissues.
Introduction In plants, pattern formation not only occurs in embryos but differentiation and spatial organization of meristematic cells into organs happens throughout the entire plant life. Meristematic cells divide indeterminately and parts of their derivatives differentiate into tissues and organs. Cell fate decisions in plants are usually made according to the spatial position of an undifferentiated cell within the meristem. In the absence of cell migration, it seems plausible that lineage-dependent pattern forming processes are of minor importance. The positional dependence of differentiation in plants implies that each cell in an incipient primordium can sense its position. In the shoot apical meristem (SAM), mathematical modeling and auxin reporter gene analyses have revealed local maxima of the plant hormone auxin, which coincide with the position of incipient primordia (de Reuille et al. 2006, Smith et al. 2006), while in the root, auxin
response maxima correlate with the determination of lateral root cell identity (Dubrovsky et al. 2008). The idea of auxin gradients providing positional information has also been proposed as a patterning mechanism for the vascular cambium (Uggla et al. 1996, Sundberg et al. 2000), a lateral meristem that contributes to secondary radial growth of plant organs, e.g. to wood in stems. In fact, auxin gradients were first measured across woodforming tissues and it was speculated that these gradients provide spatial information for patterning (Uggla et al. 1996, 1998, Tuominen et al. 1997). Molecular dissection of the auxin gradient across wood has proven difficult (Tuominen et al. 1997) and the hypothesis that auxin acts as a positional signal during wood formation still lacks strong experimental support. However, in recent years, molecular tools and model systems have emerged that now enable the auxin gradient theory in the cambium to be tested. Here, we review the latest progress in understanding the role of auxin in wood formation and
Abbreviations – ABC, ATP-binding cassette; ARF, auxin response factor; AuxREs, auxin response elements; PIN1, PINFORMED 1; rev, revoluta; SAM, shoot apical meristem; WAT1, WALLS ARE THIN1.
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discuss these findings in the light of an auxin gradient as a patterning mechanism.
What is the mechanism underlying the establishment of an auxin concentration gradient?
Auxin levels along a concentration gradient correlate with distinct zones of differentiation in wood-forming tissues
The major sources of auxin in wood-forming tissues are the shoot apices. Auxin is thought to be transported rootwards in the cambium and distributed radially into xylem and phloem (Sundberg and Uggla 1998). As auxin diffuses only very poorly in the apoplast (Kramer 2006), redistribution by means of PIN-FORMED 1 (PIN1)-like auxin efflux carriers has been suggested as a mechanism of gradient formation in trees (Schrader et al. 2003, Hellgren et al. 2004). In the root tip of Arabidopsis, the auxin gradient has been explained based on the expression and localization of PIN proteins and auxin diffusion (Grieneisen et al. 2007). In order to redistribute polarly transported auxin from the cambium into phloem and xylem, efflux carriers would need to be localized to the lateral membrane of cambial cells and their derivatives. Although PIN proteins have not yet been systematically localized in cambia, PIN1, which is strongly expressed in Arabidopsis inflorescence stems, has been shown to localize not only to basal plasma membranes but also in the lateral plasma membrane at the basal end of parenchymatic xylem cells (G¨alweiler et al. 1998, Sauer et al. 2006). Similarly, PIN3, which is involved in redirecting the auxin flux upon gravistimulation in the root tip, localizes to the inner lateral membrane of the starch sheath of the Arabidopsis inflorescence stem (Friml et al. 2002). Interestingly, the first periclinal cell divisions, from which the interfascicular cambium originates, occur in the starch sheath (Altamura et al. 2001). In both a pin1 and pin3 mutant, the radial extension of derivatives of the interfascicular cambium is reduced (Agusti et al. 2011a). However, it is not known whether this is due to reduced cell division activity, cell growth or expansion, and whether PIN1 and PIN3 have a direct effect on auxin distribution in the cambium and its derivatives. In contrast, application of the polar auxin transport inhibitor 1-N-naphthylphthalamic acid (NPA) does not affect secondary xylem expansion in the Arabidopsis hypocotyl (Ragni et al. 2011). In stems of revoluta (rev ) mutants, which are defective in the formation of interfascicular fibers, PIN3 and PIN4 expression is dramatically reduced (Zhong and Ye 2001). Although in rev inflorescence stems rootward auxin transport has been shown to be strongly impaired, it is not known whether reduced PIN expression translates into alterations in radial auxin distribution. Besides systematic localization of PIN proteins in the cambial zone, analysis of multiple pin mutants will be required in order to reveal the contribution of this family of auxin transport facilitators to the establishment of a radial auxin gradient.
The vascular cambium is organized in radial strands of cells and forms to its inner (centripetal) side xylem and to the outer side phloem. Hence, there are only three primary cell fates a derivative of the cambium can adopt: either it differentiates into secondary xylem or phloem, or it does not differentiate and stays meristematic. Pattern formation in this case is strictly dependent on the position of the derivative in relation to the cambium. Daughter cells facing the inner side can differentiate into xylem, whereas daughter cells facing the outer side can differentiate into phloem. After dividing, cambial daughter cells first expand radially, and then secondary cell wall thickening takes place. This subdivides the xylary side of the cambium into three distinct zones of differentiation, namely the cambium, elongation and maturation zones (Fig. 1). Similar zonation patterning occurs in the phloem. However, the boundaries are structurally less apparent. Accordingly, the degree of differentiation increases with distance from the cambium. In wood-forming tissue, auxin concentrations peak in the cambium and decay rapidly toward the xylem and phloem (Uggla et al. 1996, 1998, Tuominen et al. 1997). High auxin concentrations localize to the cambium, intermediate concentrations to the elongation zone and low auxin concentration correlate with the maturation zone. Thus, it is tempting to speculate that the auxin gradient provides positional information for the division of wood-forming tissue into distinct zones of differentiation. High auxin concentrations may be interpreted as a signal for cell division, intermediate levels may promote cell expansion and low levels may be read out as a signal inducing the deposition of secondary cell walls (Fig. 1; Sundberg et al. 2000, Bhalerao and Bennett 2003). In addition, auxin concentrations could be interpreted for cell fate decisions. In this case, a high concentration of auxin would keep cells meristematic, whereas low levels would lead to differentiation into either xylem or phloem. In Pinus explants lacking an apical auxin source, cambial derivatives are not able to maintain their fusiform shape and differentiate into parenchymatic cells instead of xylem tracheids (Savidge 1983). This suggests that while auxin is required for maintaining the meristematic identity of the cambium, factors other than auxin are likely to contribute to subsequent cell fate decisions. 44
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Fig. 1. Auxin concentration gradient across wood-forming tissue. Auxin concentrations higher than threshold 1 (T1), correspond to the cell division zone, concentrations between T1 and T2 with cell expansion and below T2 with secondary cell wall formation (SCW).
Another candidate transporter that may be involved in actively generating an auxin gradient is the ATPbinding cassette (ABC) transporter ABCB14. It is expressed subapically, and in an abcb14 mutant, both polar auxin transport and the expression of the auxin response reporter DR5 have been shown to be reduced (Kaneda et al. 2011). The abcb14 mutant displays a mild radial cell expansion phenotype in the xylem. For ABCB1/PGP1 and ABCB19/PGP19, paralogous to ABCB14, auxin efflux activity has been demonstrated (Geisler et al. 2005). Localization and determination of transport substrates of ABCB14 could help to clarify whether the observed mutant phenotype is a consequence of a changed auxin gradient. In summary, phenotypes of auxin carrier mutants related to wood formation have either not been reported or are difficult to interpret due to an absence of auxin distribution data in those mutants (Agusti et al. 2011a, Kaneda et al. 2011).
How can the auxin concentration gradient be interpreted? Auxin is perceived by co-receptors comprising a ´ VilTIR1/AFB F-BOX and an AUX/IAA subunit (Calderon lalobos et al. 2012). Upon auxin binding, the AUX/IAA subunit is ubiquitinated and subsequently degraded by the proteasome. The Arabidopsis genome harbors 29 AUX /IAA genes, whose short-lived gene products can form heterodimers with auxin response factors (ARFs). Upon AUX/IAA-ARF heterodimerization, the ARF-mediated auxin response is inactivated, whereas binding of auxin to the co-receptor activates degradation of AUX/IAA and allows the ARFs to bind to promoters containing auxin response elements (AuxREs).
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Interestingly, auxin affinity to the co-receptor is primarily dependent on the AUX/IAA subunit of the co-receptor, and different AUX/IAAs confer differences in receptor ´ affinities of more than an order of magnitude (Calderon Villalobos et al. 2012). This means that by using a combination of different AUX/IAA and TIR1/AFB subunits, a large set of co-receptors with graded affinity can be obtained. Low affinity co-receptors would only lead to AUX/IAA degradation in the presence of high auxin concentration, whereas high affinity receptors could be active at low and high auxin concentrations. Low affinity and high affinity receptor activity could result in the expression or repression of different suites of genes, and this could provide a mechanistic explanation for how the read-out of an auxin gradient could result in discrete, threshold-dependent auxin responses rather than gradual changes. During differentiation, cambial derivatives experience a gradual decline in auxin concentration unless they stay meristematic. Different auxin concentrations are therefore likely to induce different responses, which must not occur simultaneously in a given cell. This means that auxin perception in differentiating wood does not constitute a simple on–off switch that leads to an irreversible, permanent outcome but rather a response that can be overruled by responses to lower concentrations of auxin. If for the different auxin functions in wood formation only a single auxin receptor or family of receptors with the same binding affinity is found to be responsible, this receptor would need to be multi-stable with at least three different steady states rather than being a simple bi-stable on–off switch. Differential expression of various AUX /IAA genes across the secondary xylem suggests co-occurrence of receptors with different affinities for auxin in wood-forming tissues (Moyle et al. 2002). 45
It remains to be tested whether these AUX/IAAs heterodimerize with specific ARFs or whether they interact more broadly with a larger number of ARFs as has been suggested for the SAM (Vernoux et al. 2011). In addition, some ARF genes might be expressed differentially across the wood-forming tissue (Schrader et al. 2004). Such a pre-patterning occurs in the embryo, where different ARF s, expressed in different domains, allow cell-specific auxin responses (Rademacher et al. 2012). Although direct determination of auxin concentration across wood-forming tissue by mass spectrometry has proven to be highly informative, it does not provide sufficient spatial resolution to distinguish auxin concentrations between neighboring cell files, where cells can acquire different cell fates, e.g. xylem vessels or xylem fibers. Resolution at a single cell level is possible by using reporter gene constructs, which have proven useful for measuring auxin response activity in the shoot and root apical meristem of Arabidopsis. These constructs do not report actual auxin concentrations but provide a read-out of auxin signaling. Nevertheless, they help to resolve TIR1/AFB dependent auxin responses at a cellular level. Reporter gene constructs driven by the synthetic promoter DR5, which consists of multiple AuxREs, have often been used for this purpose. In Arabidopsis inflorescence stems, DR5::GUS is expressed at the distal pole of the vascular bundles (Ranocha et al. 2010) and DR5::GFP is expressed in the protoxylem and metaxylem of vascular bundles (Suer et al. 2011, Agusti et al. 2011b). In addition, DR5::GFP has been found in the phloem and cortical cells of stems undergoing secondary growth (Suer et al. 2011). Intriguingly, neither in Arabidopsis nor in poplar (Chen et al. 2013) has DR5-reporter gene activity been detected in the cambium. This surprising finding was further substantiated by the work of Nilsson et al. (2008). The latter authors depleted stem segments of hybrid aspen from endogenous auxin and then added exogenous auxin while monitoring global gene expression. Such time course experiments allowed auxin-regulated genes in stem segments undergoing secondary growth to be identified. Surprisingly, expression patterns of only a very minor fraction of these auxin-regulated genes were found to mirror the auxin distribution across wood-forming tissue. Taken together, these results show that in wood-forming tissues, the auxin concentration maximum does not overlap with the DR5 auxin response maximum. While in the root apical meristem, the site of strongest DR5 promoter activity and highest auxin concentration has been shown to localize to the quiescent center (Petersson et al. 2009), a similar discrepancy between predicted auxin concentration maximum and auxin response has been found in the 46
SAM (de Reuille et al. 2006). Auxin insensitivity of cells at the summit of the SAM has been suggested to be the cause of DR5 promoter inactivity (de Reuille et al. 2006). In the light of this finding, it is interesting to note that the expression of TIR1 in hybrid aspen can be modulated and is reduced by 50% during seasonal cambial inactivity (Baba et al. 2011). As an alternative to auxin insensitivity, the AuxREs in the DR5 promoter may be insufficient to drive gene expression in the cambium. The native soybean GH3 promoter, from which the AuxREs were originally isolated, has been shown to drive GUS expression in the cambium and the developing secondary xylem of poplar (Teichmann et al. 2008). However, this reporter gene exhibits the highest activity in the cortex. Interestingly, cambial activity of GH3::GUS is induced in the cambial zone upon mechanical induction of tension wood (Teichmann et al. 2008). Previously, no changes in cambial auxin concentrations during tension wood formation were recorded (Hellgren et al. 2004). Hence, the induced GH3 auxin response upon mechanical stimulation might be a consequence of increased auxin responsiveness rather than augmented auxin concentrations in the cambium.
The read-out Auxin is a crucial factor for promoting cell division, e.g. cells of callus cultures do not divide in growth medium lacking auxin. When the auxin response in the cambium is reduced by stabilizing the AUX/IAA protein PttIAA3, cambial cells divide less frequently, while radial extension of the cambial zone increases (Nilsson et al. 2008). Interestingly, PttIAA3 could not be ubiquitinated by extracts from dormant hybrid aspen, whereas extracts from actively growing trees did result in PttIAA3 ubiquitination. This indicates that during active growth PttIAA3 is degraded and auxin signaling can take place, whereas during dormancy, it is stabilized (Baba et al. 2011). Considering that auxin levels in the cambium undergo only subtle seasonal changes (Uggla et al. 2001), these findings are in keeping with the idea that auxin signaling controls cambial activity by modulation of auxin responsiveness. Recent work in Arabidopsis has helped to unravel how auxin can regulate cambial activity. Cell division activity in the cambium is promoted by the release of the phloem derived CLV3 like peptides CLE41 and CLE44, which can bind to the leucine-rich receptor kinase PXY in the cambium. Receptor signaling then promotes cambial activity through expression of the WUSCHELlike homeodomain transcription factor WOX4 in the cambial zone (Etchells and Turner 2010, Hirakawa et al. 2010). Suer et al. (2011) found that WOX4 expression
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can be induced by exogenous auxin and that long-lasting induction requires PXY . These results indicate that auxin acts upstream of PXY and regulates cambial activity through WOX4 induction. Interestingly, DR5 promoter activity only sparsely overlaps with WOX4 expression (Suer et al. 2011). Hence, either the activation of WOX4 by auxin is mediated by a mobile factor different than auxin or alternatively, WOX4 expressing cells are not auxin responsive. In hybrid aspen, expression of stabilized PttIAA3 under the control of the ubiquitous 35S promoter not only decreases cambial activity but also radial cell expansion of xylem fibers and vessels (Nilsson et al. 2008). Similarly, in early and late wood, cell division activity, radial cell expansion and secondary wall formation are tightly coupled processes (Uggla et al. 2001). During early wood formation, cell division activity and cell expansion are high, whereas secondary cell wall deposition is low. In contrast, during late wood formation, cambial activity and cell expansion are low, but secondary cell walls become thicker. These observations can be explained by a narrower auxin peak in the cambium and more rapid decay of the auxin gradient during late wood formation (Uggla et al. 2001). From this angle, it is not surprising that reduction of auxin sensitivity by stabilizing PttIAA3 across wood-forming tissues not only targets a single isolated process of differentiation but affects various, probably interlinked aspects of wood formation. Secondary cell wall formation, the last step in the differentiation of cambial derivatives, corresponds to low auxin concentration in the gradient. Secondary cell wall deposition is controlled by a few members of the NAC transcription factor family. NST1 and NST3/SND1 switch on expression of secondary wall genes in xylem fibers, whereas VND6 and VND7 act on xylem vessel maturation (for a review see Wang and Dixon 2012). In a nst1, nst3/snd1 double mutant, secondary cell wall deposition in interfascicular fibers is completely abolished. A similar phenotype has been found in rev mutants. REV encodes for a class III homeodomain leucine zipper transcription factor, which together with other members of the gene family is involved in organ polarity. REV has been thought to act through PIN efflux carriers on polar auxin transport (Zhong and Ye 2001). However, it is not clear whether interfascicular fiber identity is affected in rev mutants or REV instead acts downstream on secondary cell wall deposition. Whereas RNAi lines against a REV homolog in poplar do not display a phenotype, overexpression of a miRNA resistant REV variant causes interruptions in the cambial cylinder (Robischon et al. 2011). Interestingly, the endings of such a gapped xylem did not join but extended into the
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cortex, resulting in ‘open loops’ with mirrored cambial layers. The positions of these gaps seemed to correlate with the position of lateral organs. Over- and/or ectopic REV expression may hinder the establishment of an interfascicular cambium, and therefore REV may be involved in cell fate determination rather than directly promote secondary cell wall deposition. As observed for rev mutants, mutations in WALLS ARE THIN1 (WAT1), which encodes for a tonoplast localized auxin efflux facilitator, cause a similar reduction in polar auxin transport and misregulation of genes associated with auxin response (Ranocha et al. 2010, 2013). Secondary cell wall deposition in xylary and interfascicular fibers is strongly reduced in wat1 mutants. However, as there is residual lignification of fibers, fiber cell identity seems not to be affected by wat1 mutations. This indicates that WAT1 acts more specifically on late fiber differentiation, whereas REV might be involved in patterning. Its interaction with REV and how WAT1 affects the radial auxin gradient are yet to be resolved. Secondary cell wall deposition also occurs outside of wood-forming tissue, e.g. in highly specialized cells of the silique, in the valve margin. During pod shatter, siliques become dehydrated, and as a consequence, the lignin component in valve margin cells is thought to create tensile forces, which separate the valve from the replum (Spence et al. 1996, Roberts et al. 2002). Like secondary cell wall deposition in xylary fibers, the NAC domain transcription factors, NST1 and NST3/SND1, switch on expression of secondary cell wall genes in the valve margins (Mitsuda and Ohme-Takagi 2008) after cell fate decisions in the abscission zone have taken place. Secondary cell wall deposition is preceded by formation of a local auxin response minimum in the valve margins (Sorefan et al. 2009). Formation of this auxin response minimum is thought to be a consequence of IND induced PIN1 and PIN3 de-localization in the valve margin, which in turn might deplete the valve margin from stigma-derived auxin. In the gynoecium, auxin is transported basipetally, like in the cambium (Cheng et al. 2006, Sohlberg et al. 2006). Interestingly, the auxin response remains high in the replum, the tissue in-between two neighboring valve margins. Cell division and expansion have been observed in the replum (Wu et al. 2006). However, it is not known whether these divisions coincide with the auxin maximum in space and time and whether secondary wall formation in the valve margins is triggered by auxin depletion.
The morphogen analogy As early as the first description of an auxin concentration gradient spanning wood-forming tissue, an analogy 47
to morphogen gradients was recognized (Uggla et al. 1996). The idea that auxin is able to form similar gradients as a morphogen has swiftly been adopted in mathematical models of auxin distribution across the SAM and root tip (Smith et al. 2006, Grieneisen et al. 2012). Most metazoan morphogen models require a localized source of morphogen secretion, diffusion or transport and decay of the morphogen (Wartlick et al. 2009). All cells along a morphogen gradient should be able to sense the morphogen and to interpret its concentration in a threshold-dependent manner, i.e. cells can determine their position in relation to the source. Although various mathematical auxin distribution models have been suggested, auxin concentrations at the shoot and root tip have still not been determined in intact meristems with sufficient spatial resolution to separate different morphologic zones (i.e. auxin measurements from 500 μm thick sections in the root tip, Fischer et al. 2006, Petersson et al. 2009, vs 30 μm in the cambium, Sundberg et al. 2000), and model validation has mainly been based on consideration of model robustness and DR5 promoter activity. Moreover, differences in experimentally determined auxin concentrations along the main growth axis or between various cell types of the root are well within an order of a magnitude (Fischer et al. 2006, Petersson et al. 2009), whereas the modeled dynamic range of auxin concentration spans several orders of magnitude (Grieneisen et al. 2012). While Arabidopsis shoot and root apices are superior model systems for functional analysis and protein localization of auxin transporters, wood-forming tissues are easily accessible to spatially highly resolved auxin concentration measurements. In both the root tip and SAM, formation of auxin gradients has been explained by the localization and activity of PIN proteins (Smith et al. 2006, Grieneisen et al. 2007). However, little attention has been paid to other mechanisms that do not rely on directed transport but still could form rapidly decaying auxin gradients in the direction of expanding growth axes. Given that radially redirected auxin in wood-forming tissues originates mainly from polarly transported auxin from shoot apices and that auxin is most likely transported rootwards in cambial cells, a mechanism based on cell division and expansion, which dilutes auxin, seems to be plausible. In such a scenario, some cambial derivatives would be disconnected from the polar auxin flux and their inherited auxin content would gradually be diluted by cell growth and expansion, and auxin catabolism (Fig. 2). Intriguingly, the decay of auxin concentration is most rapid in the cell expansion zone of the xylem, and auxin levels are barely detectable at the beginning of the maturation zone where cell expansion 48
Fig. 2. Clonal dilution model. Auxin dilution by cell growth and expansion. Auxin is provided to the cambium by polar auxin transport (PAT) via auxin efflux carriers. S, cambial stem cell; T1, threshold 1; T2, threshold 2 (Fig. 1).
has ceased (Sundberg et al. 2000). A similar model has been proposed for the formation of certain morphogen gradients in animals. For example, the distribution of fgf8 mRNA in the mouse embryo seems to be a consequence of cell division and expansion (Dubrulle and Pourqui´e 2004). The plausibility of such a model, based on the inheritance of a morphogenetically active, nonor poorly-secreted substance from its source, has been ˜ et al. 2006, shown by mathematical modeling (Ibanes Wartlick et al. 2009). A mechanism which acts through clonal dilution of auxin could also explain cell fate decisions in the cambium. Slightly asymmetrical cambial cell division would lead to unequal distribution of auxin between the two daughter cells. After cell growth, these stochastic irregularities could result in a different auxin concentration, and if as a consequence of asymmetric cell division and cell growth the auxin concentration
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drops below a certain threshold, the cambial derivate could undergo differentiation. In the case where the daughter cell with lower auxin concentration is located at the inner side of the cambium, it would be expected to differentiate into xylem. It would be interesting to determine whether the simple ‘cell lineage transport’ model can explain the observed auxin gradients across wood-forming tissues and to what extent auxin dilution by cell expansion contributes to the formation or robustness of the proposed auxin gradients in the root tip and SAM. For wood-forming tissues, meta-analyses of available data from tangential sectioning and thorough examination of auxin transport mutants in Arabidopsis should enable this hypothesis to be tested. ¨ Acknowledgements – We thank Bjorn Sundberg for critically reading this manuscript, the Berzelii Centre and Bio4Energy for financial support.
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Edited by K. Ljung
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