The root cap: a short story of life and death

Transcription

1 Journal of Experimental Botany, Vol. 66, No. 19 pp , 2015 doi: /jxb/erv295 Advance Access publication 11 June 2015 DARWIN REVIEW The root cap: a short story of life and death Robert P. Kumpf 1,2 and Moritz K. Nowack 1,2, * 1 Department of Plant Systems Biology, Vlaams Instituut voor Biotechnologie, B-9052 Ghent, Belgium 2 Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Ghent, Belgium * To whom correspondence should be addressed. moritz.nowack@vib.be Received 15 April 2015; Revised 13 May 2015; Accepted 18 May 2015 Editor: Christine Foyer Abstract Over 130 years ago, Charles Darwin recognized that sensory functions in the root tip influence directional root growth. Modern plant biology has unravelled that many of the functions that Darwin attributed to the root tip are actually accomplished by a particular organ the root cap. The root cap surrounds and protects the meristematic stem cells at the growing root tip. Due to this vanguard position, the root cap is predisposed to receive and transmit environmental information to the root proper. In contrast to other plant organs, the root cap shows a rapid turnover of short-lived cells regulated by an intricate balance of cell generation, differentiation, and degeneration. Thanks to these particular features, the root cap is an excellent developmental model system, in which generation, differentiation, and degeneration of cells can be investigated in a conveniently compact spatial and temporal frame. In this review, we give an overview of the current knowledge and concepts of root cap biology, focusing on the model plant Arabidopsis thaliana. Keywords: Arabidopsis, columella, differentiation, development, formative cell division, programmed cell death, root cap. Introduction We believe that there is no structure in plants more wonderful, as far as its functions are concerned, than the tip of the radicle Darwin and Darwin (1880). In one of his last books, The Power of Movement in Plants, Charles Darwin concluded that the course pursued by the growing root must be determined by its tip. Darwin realized that the root tip functions as a sensory organ, and that its role is of paramount importance not only for root growth but also for growth and development of the entire plant. Today, we know that many of the functions that Darwin attributed to the tip of the radicle are actually controlled by a particular plant organ that ensheathes the growing root tip like a thimble: the root cap. The root cap, or calyptra, has the functions of protecting the delicate stem cells within the root tip, and of receiving and transmitting environmental signals to the growing root. In order to fulfil this function, the root cap has to maintain its position at the very tip of the root. As the root proper grows by producing new cells at its tip, root cap cells also need to be continually generated. However, in contrast to the root proper, which grows in an indeterminate fashion, the root cap shows a determinate, constant organ size (Barlow, 2003; Fendrych et al., 2014). In order to restrict the root cap organ to the root tip, the generation of new root cap cells has to be compensated for by the disposal of cells at the edge of the root cap organ. Different plant taxa have come up with various solutions to this problem: some plants, including cereals and legumes, dissolve the cellular connection between the outermost root cap cells and their neighbours, releasing long-lived border cells into the rhizosphere (Hamamoto et al., 2006). Others, including the model species Arabidopsis thaliana, actively kill and degrade the majority of root cap cells on the root surface, while a limited amount of Abbreviations: COL, columella root cap; EPI, epidermis; LRC, lateral root cap; PCD, programmed cell death; QC, quiescent centre; TF, transcription factor. The Author Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please journals.permissions@oup.com

2 5652 Kumpf and Nowack short-lived border-like cells are released into the rhizosphere (Durand et al., 2009; Fendrych et al., 2014). The notion of regulating cell number homeostasis in developing organs via cellular turnover is well established in animals (Penzo-Méndez and Stanger, 2014), but the importance of this concept has not been recognized widely in plant development. Intriguingly, due to its particular mode of development, the root cap unites crucial features of plant development in a compact spatial and temporal context. In Arabidopsis, for instance, formative cell division, transient cell amplification, cellular differentiation, and programmed cell death (PCD) occur within a matter of days, and can be observed simultaneously within a single microscopic frame. Furthermore, the root cap plays an important role in modulating root growth direction and in the elaboration of root system architecture according to environmental cues, which allows the investigation of diverse signal transduction pathways. In this review, we summarize current findings and concepts regarding the genetic and molecular control of root cap generation, differentiation, and degeneration, and the functional signalling processes that co-ordinate root cap development and function. Evolutionary origin of the root cap organ With the colonization of the terrestrial habitat some 500 million years ago, plant species had to adapt to life on land where their bodies were surrounded by air instead of water (Becker and Marin, 2009). In order to obtain water and necessary solutes, plants had to develop organs to forage the soil for these vital resources. Early land plants such as mosses, liverworts, and hornworts developed rhizoids, root hair-like structures to extract water and minerals from the soil and to anchor the plant to its substrate. During land plant evolution, an extensive post-embryonic development of the sporophytic generation and a competition for light led to the formation of larger and higher plant bodies. This development required more complex and capacious below-ground structures to sustain and stabilize the plant body. As evolution followed its course, more advanced land plant taxa such as the club mosses, ferns, and horsetails were among the first to form complex multicellular roots, which are produced by the sustained action of adult stem cells at the root tips, a mechanism adopted by all more advanced land plant taxa (Fig. 1a) (Prigge and Bezanilla, 2010). The evolution of stem cells at the root tip necessitated specific adaptations to protect these delicate meristematic cells from mechanical damage during growth. With a few exceptions, all plants with true multicellular roots form a root cap, which ensheathes and protects the stem cell niche at the root tip (Fig. 1). In ferns and horsetails, a single stem cell, the so-called apical cell, is the ultimate source of all cells of the root, including the root cap (Fig. 1c, d). This apical cell is of tetrahedral shape and undergoes formative divisions in different division planes, allowing the generation of a complex three-dimensional root morphology (Piekarska- Stachowiak and Nakielski, 2013). In ferns, analysis of cell division in the root apex revealed distinct root cap lineages (Piekarska-Stachowiak and Nakielski, 2013). Furthermore, an orthologue of the transcription factor WUSCHEL is expressed in all cells generated by the apical cell, except the root cap cells, indicating a clearly distinct genetic programme for root cap files (Nardmann and Werr, 2012). Club mosses, such as Selaginella (Fig. 1b), as well as gymnosperms, and angiosperms have evolved more complex root meristems with groups of different stem cells in which cell production and root growth are co-ordinated (Barlow, 1994; Prigge and Bezanilla, 2010). In dicotyledonous angiosperms, open and closed meristem structures can be distinguished. Plants with closed meristems, such as Brassica species, form their cell lineages, including the root cap lineage, from specific stem cells (Fig. 1e). In open meristems, like that of pea (Fig. 1f), a large pool of unspecific stem cells gives rise to the different root tissue types in terms of function with regard to their morphological positions (reviewed by Rost, 2011). Although there are a variety of different root cap organizations, the root cap organ as such is a highly conserved structure in land plant evolution. Much research on root cap development and function has been carried out in Arabidopsis, which shows a closed meristem organization. In the remainder of this review, we will focus on the molecular regulation of root cap formation, maintenance, and function in A. thaliana. Stem cell maintenance and cellular differentiation in the root cap In the root cap of Arabidopsis, two tissue types can be distinguished; the central columella root cap (COL) and the peripheral lateral root cap (LRC) (Fig. 2) (Dolan et al., 1993). COL cells are located distally to the quiescent centre (QC), a group of cells near the root apex that divide infrequently. COL cells have large volumes and contain statoliths, starchfilled plastids that sediment according to the gravity vector and enable COL cells to sense gravity (Strohm et al., 2012), as described later. LRC cells are located on the periphery of the COL and extend proximally (shootwards) up to the end of the meristem (Fig. 2). LRC cells do not contain statoliths and are, especially towards the upper part of the meristem, long and slender of shape. COL and LRC cells are generated by distinct groups of stem cells. COL stem cells form a cell plate distal to the QC (Fig. 2 and Fig. 3a) and create, by formative divisions, proximal daughter cells retaining the stem cell fate, and distal differentiating COL daughter cells (Wenzel and Rost, 2001). New LRC cells are formed by the epidermis (EPI)/ LRC initials, a ring of stem cells that is located laterally to the COL stem cells (Fig. 2 and Fig. 3a). In contrast to COL stem cells, EPI/LRC initials undergo not one but two divergent formative divisions. A first anticlinal division produces a distal epidermis daughter cell, while the proximal cell maintains EPI/LRC stem cell identity (Fig. 3b). The second formative division occurs in a periclinal plane, perpendicular to the first one, producing a peripheral LRC daughter cell and a central cell that again maintains EPI/LRC stem cell fate (Fig. 3b) (Wenzel and Rost, 2001; Willemsen et al., 2008).

3 a roots* Developmental biology and functions of the root cap 5653 Angiosperms (Brassica, Pisum) Gymnosperms Monilophytes (Equisetum, Dennstaed a) Lycophytes (Selaginella) Hornworts Mosses Liverworts million years before present b c d e f Selaginella Equisetum Dennstaed a Brassica Pisum (root cap) stem cells differen a ng root cap cells Fig. 1. Root cap morphologies in land plants. (a) Phylogenetic relationships among land plants indicating the evolutionary origin of roots (arrow). The asterisk indicates that roots might have evolved independently in different land plant clades. (b d) The club moss Selaginella forms the root cap from specific stem cells (b), while the horse tail Equisetum (c) and the fern Dennstaedtia (d) derive it from the root apical cell that is the stem cell for all root tissues. (e, f) The angiosperm oilseed (Brassica species) shows a closed meristem structure with defined root cap stem cells and a layered root cap structure (e), while the angiosperm pea (Pisum species) has an open meristem with less clearly defined root cap stem cells (f). (a) Modified after Prigge and Bezanilla (2010); (b d) modified after von Guttenberg (1966); (e) modified after Sitte et al. (1998); (f) modified after Haberlandt (1904). The division of the COL stem cells is co-ordinated with the periclinal formative division of the EPI/LRC stem cells, so that coherent new root cap layers are formed in an iterative fashion (Wenzel and Rost, 2001). As a result, the root cap is organized into multiple well-defined layers of increasing age (Fig. 2). The youngest root cap layer is directly adjacent to the COL and EPI/LRC stem cells, and consists of young, differentiating COL and LRC cells. With every formative division, a new root cap layer is created proximal to the existing one, displacing the next older root cap layer towards the root periphery. While COL cells do not divide further, cells in young LRC layers divide anticlinally, increasing the number of LRC cells in every layer (Wenzel and Rost, 2001). Together with their adjacent epidermal neighbours, LRC cells traverse the meristem, first dividing and then differentiating and elongating (Campilho et al., 2006). When the oldest, outermost LRC cells reach the elongation zone, their epidermal neighbour cells start to expand, while the LRC cells enter a PCD process (Fendrych et al., 2014). The oldest COL cells, however, are sloughed off alive (del Campillo et al., 2004). Both cell death and cell sloughing ensure a continuous turnover of root cap layers at the growing root tip, as will be described later in more detail. Transcriptional control by several related NAC [for NAM (NO APICAL MERISTEM), ATAF (ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR), and CUC (CUP-SHAPED COTYLEDON)] transcription factors (TFs) has been shown to regulate the balance between root cap stem cell maintenance and cellular differentiation. The NAC TF FEZ specifically promotes the formative divisions in the COL and EPI/LRC stem cells (Fig. 4) (Willemsen et al., 2008). While the anticlinal (epidermis-producing) formative divisions in fez mutants occur at wild-type rates, both the formative division in the COL stem cells as well as the periclinal (LRC-producing) formative division in the EPI/ LRC stem cells occur at lower frequencies (Willemsen et al., 2008). The SOMBRERO (SMB) NAC TF has been found to promote the differentiation of young root cap cells. In the smb mutant, there are additional COL and LRC layers, and the youngest layers are delayed in differentiation (Willemsen et al., 2008). A regulatory feedback loop was proposed, in which FEZ activates SMB expression in the differentiating daughter cells after the formative division, and SMB in turn represses FEZ expression to prevent further formative divisions and promotes early root cap differentiation, as well as subsequent LRC maturation (Fig. 4) (Willemsen et al., 2008; Bennett et al., 2010). Two other NAC TFs, BEARSKIN1 (BRN1) and BRN2, have been implicated in the regulation of cell sloughing at the COL (Fig. 4). In brn1 brn2 double mutants, mature COL layers fail to detach from the younger layers, suggesting that BRN genes redundantly control cell cell separation in mature COL layers. In smb brn1 brn2 triple mutants, the cell sloughing phenotype is enhanced and the entire COL appears disorganized, arguing for a redundant role of SMB and BRN genes in controlling root cap maturation (Bennett et al., 2010). Interestingly, the root-cap-expressed CELLULASE3 (CEL3) and CEL5, which have been implicated in sloughing of COL cells (del Campillo et al., 2004), were strongly downregulated in the smb brn1 brn2 mutants, suggesting a transcriptional

4 5654 Kumpf and Nowack Gravity vector and direc on of growth Meristem Elonga on Zone dead LRC cells undergo autolysis LRC cells prepare and undergo PCD LRC proliferate and differen ate COL cells are shed Auxin Flux QC quiescent centre EPI/LRC stem cells COL cells of increasing age LRC cells of increasing age epidermal cells of increasing age Fig. 2. Root tip morphology and auxin fluxes in Arabidopsis. Longitudinal section of a 6-d-old Arabidopsis root. On the left side, the epidermis (EPI) and successive layers of the columella root cap (COL) and the lateral root cap (LRC) are highlighted. Increasing differentiation and age of the individual tissues are indicted by darker shades of yellow, green, and purple, respectively. The backdrop is a root visualized by the WAVE131 plasma membrane marker (Geldner et al., 2009). On the right side, the same root is shown as a diagram in order to visualize the known (solid red lines) and hypothesized (dashed red lines) major trajectories of auxin flux. regulation of these cell-wall-degrading enzymes by NAC TFs (Bennett et al., 2010). The Arabidopsis RETINOBLASTOMA-RELATED (RBR) protein has also been shown to be involved in the control of cell-cycle exit and differentiation of COL stem cell daughters (Fig. 4). Reduction of RBR levels in the root leads to an increase in undifferentiated stem-cell-like COL layers (Wildwater et al., 2005; Cruz-Ramírez et al., 2013). As co-depletion of FEZ and RBR results in a partial rescue of the RBR loss of function, FEZ activity is involved in producing this phenotype, although it is yet unclear whether RBR represses FEZ in COL stem cells (Bennett et al., 2014). Next to RBR, two auxin response factor (ARF) proteins, ARF10 and ARF16, are involved in controlling COL stem cell divisions (Fig. 4). Combined loss of ARF10 and ARF16 function leads to a disorganized root cap with additional COL stem-cell-like cells (Wang et al., 2005). Similar to the situation of RBR depletion, FEZ contributes to the arf10 arf16 phenotype, but as FEZ levels are only slightly upregulated in this double mutant, a regulation of FEZ by these ARFs is unlikely (Bennett et al., 2014). Mutant combinations of SMB, RBR, and ARF10/ARF16 showed additive phenotypes of COL and LRC differentiation, suggesting that RBR and ARFs contribute to the control of root cap differentiation in pathways that are operating largely in parallel with SMB and FEZ (Bennett et al., 2014). Ablation of QC cells has been shown to drive COL stem cells into differentiation, suggesting that QC-derived signals prevent differentiation of these stem cells (van den Berg et al., 1997). The QC-expressed WUSCHEL-RELATED HOMEOBOX5 (WOX5) encodes a TF involved in this signalling process (Fig. 4). It has been proposed that WOX5, similar to WUSCHEL in the shoot meristem, moves to the stem cells to maintain their dedifferentiated state (Sarkar et al., 2007). Recently, it has been shown that WOX5 controls COL stem cell activity via SMB (Bennett et al., 2014). While SMB is ectopically expressed in wox5 mutants, SMB is depleted from all COL cells 24 h after inducible systemic misexpression of WOX5. Remarkably, inducible WOX5 misexpression leads not only to a failure of COL stem cells to differentiate, but also to a de-differentiation and re-entry into mitosis of previously differentiated COL cells. Notably, WOX5 misexpression

5 Developmental biology and functions of the root cap 5655 a b activates ACR4 to restrict WOX5 expression, which in turn allows COL cell differentiation (Stahl and Simon, 2009). In addition to ACR4, the RLK CLAVATA1 (CLV1) has been implicated in COL differentiation (Stahl et al., 2013). Similar to acr4 and cle40 mutants, the clv1 mutant shows accumulation of undifferentiated COL stem cells. ACR4 and CLV1 expression patterns overlap, and the RLKs can physically interact in planta, suggesting a model in which ACR4 and CVL1 can homo- or heterodimerize to different extents at different sites to repress COL stem cell fate and promote COL differentiation (Stahl et al., 2013). Taken together, an intricate cross-talk of hormone responses, signalling processes, and transcriptional control regulates the stemness maintenance of root cap stem cells, and the differentiation and fate of COL and LRC cells (Fig. 4). The tight control of root cap differentiation is not only important for this organ it also has major implications for the life and development of the entire plant. planes of forma ve divisions EPI/LRC stem cells Columella stem cells LRC cells epidermal cells Fig. 3. The stem cell niche of the root cap and the epidermis. (a) Detailed view of the Arabidopsis root stem cell niche. The COL and EPI/LRC stem cells are striated. The plane of the COL formative division is marked with a dotted line. The different cell types are labelled by colour according to Fig. 2 and (b). (b) Two different formative divisions occur in the EPI/ LRC stem cells (from left to right): a periclinal division generates a new LRC layer, while an anticlinal division creates a new epidermis cell and regenerates the EPI/LRC stem cell. only affected SMB expression in COL cells, and not in LRC cells (Bennett et al., 2014). Taken together, WOX5 probably controls COL stem cell fate in a non-cell-autonomous manner, although the actual movement of WOX5 out of the QC remains to be shown. Stem cell maintenance in the root cap also involves receptor-like kinase (RLK) signalling processes, a common theme in spatial differentiation control during plant development (Wierzba and Tax, 2013). From the over 600 RLK genes encoded in the Arabidopsis genome, several have been implicated in root cap development (Fig. 4). The RLK ARABIDOPSIS CRINKLY4 (ACR4) is expressed in COL and LRC stem cells and their direct descendants. acr4 mutants are defective in COL differentiation, which results in additional COL stem-cell-like layers (De Smet et al., 2008). Several putative peptide ligands of RLKs have been identified. A mutant in the CLE40 peptide, a member of the CLAVATA3/EMBRYO SURROUNDING REGION (CLE) family, phenocopies acr4 mutants, while root treatment with CLE40 induces premature differentiation of COL stem cells (Stahl et al., 2009). The insensitivity of acr4 mutants to CLE40 application, and the expansion of WOX5 expression in acr4 and cle40 mutants suggest a model in which CLE40 Role of the root cap in regulation of directional root growth While the root grows through the soil, the root cap is the first part of the plant to face the environmental conditions that will later surround the mature root. Since the trajectory of the growing root tip predetermines the location of the mature root, the control of root growth direction is of utmost importance for the entire plant. Aside from its function to protect the meristem, the root cap has the ability to sense stimuli from its environment and to trigger different tropisms in the root proper, controlling the direction of growth towards or away from diverse environmental cues. One major tropism acting on root growth is gravitropism. The perception of gravity takes place in the root cap, and ablation of root cap cells leads to defects in the plant s gravity response (Tsugeki and Fedoroff, 1999). Numerous authors have discussed the involvement of the plant hormone auxin in root gravitropism (see Sato et al., 2015, for a detailed review). In a nutshell, in roots that are growing straight down, a symmetric auxin distribution in the root cap and the root meristem is achieved by co-ordinated cellular influx and efflux of auxin (Band et al., 2014). PIN- FORMED (PIN) proteins promote the auxin efflux towards a specific neighbouring cell by polar localization at the plasma membrane on one side of the cell. In this fashion, auxin is actively transported from the upper parts of the plant via stele cells with basally localized PIN1 (Band et al., 2014). At the root tip, COL cells expressing the auxin influx carrier AUX1 act as an auxin sink and redistribution centre (Swarup et al., 2005). From the COL cells, auxin is transported symmetrically towards the peripheral LRC cells by PIN3 and PIN7 proteins, and from there towards the elongation zone through PIN2-expressing LRC and epidermis cells (Swarup et al., 2005). In the transition zone, the auxin flux is thought to be directed to the inner tissues of the stele (Grieneisen et al., 2007), from where it is again transported towards the root tip (Fig. 2).

6 5656 Kumpf and Nowack When the growing root axis is deviated out of the gravity vector, for instance by an obstacle in the soil, statoliths within COL cells change their intracellular position in a gravity-dependent fashion. According to the starch statolith hypothesis, changes in statolith sedimentation create a cellular signal that triggers the actual root growth modulation in the root elongation zone (Haberlandt, 1900). How exactly the physical gravistimulus is perceived in the cell is not yet entirely understood, although endoplasmic reticulum-localized mechanosensitive ion channels, the actin cytoskeleton, and hydrostatic pressure have been implicated in gravity perception (Sato et al., 2015). Auxin has been identified as the long-range signal that translates gravity stimulation into a growth response in the root elongation zone (Friml et al., 2002). Different auxin response reporters have shown an asymmetric distribution of auxin in the upper and lower flanks of the gravistimulated root tip (Ottenschläger et al., 2003; Band et al., 2012). This asymmetric auxin flux is caused by polarization of the auxin efflux carriers PIN3 and PIN7 to the lower side of the COL cells (Kleine-Vehn et al., 2010). From here, PIN2 transports auxin towards the elongation zone, creating an auxin maximum at the lower flank of the elongation zone. As high auxin concentrations interfere with cell elongation, an asymmetry in root elongation results in a downward bending of the root (Band et al., 2012). A major function of roots is to acquire water and dissolved micro- and macronutrients from the soil. Hydrotropism directs growing root tips to available water, and is competing with gravitropism for directional growth of the root tip (for a recent review, see Moriwaki et al., 2013). Several molecular regulators of hydrotropism have been identified so far. MIZU-KUSSEI1 (MIZ1), a root-cap-localized protein of unknown function, has been shown to be a positive regulator of hydrotropism but not gravitropism. Hydrotropism is impaired in miz1 loss-of-function mutants, while it is accentuated in MIZ1 overexpressors (Kobayashi et al., 2007; Moriwaki et al., 2011). In MIZ1 overexpressors, auxin concentration and response are attenuated, but to what extent auxin is involved in regulating hydrotropism remains unclear (Moriwaki et al., 2013). Another gene involved in hydrotropism regulation is GNOM, encoding a guanine nucleotide exchange factor of the ADP-ribosylation factor GTPase. By controlling vesicle trafficking, GNOM regulates the polar localization of PIN proteins (Friml et al., 2003). As polar (re-)localization of PIN proteins is important for gravitropism, gnom partial loss-of-function mutants are agravitropic (Geldner et al., 2004). A particular GNOM allele, GNOM miz2, has been found to specifically affect the hydrotropic but not the gravitropic response by a still unknown function of GNOM other than PIN trafficking (Moriwaki et al., 2014). In summary, controlling growth directionality of the root tip is of decisive importance for plant growth. However, a plant root system is determined not only by the growth direction of the primary root but also by the formation of lateral roots in a controlled manner. Role of the root cap in influencing root system architecture With increasing plant age, increasingly complex root systems are formed, and factors influencing the patterning of lateral root initiation, and their emergence and growth determine many functional parameters of root system architecture (for a recent review, see Robbins and Dinneny, 2015). Although the root cap is located at the very tip of the root, far away from emerging lateral roots, root-cap-derived processes have been shown to regulate lateral root formation in several pathways. The Arabidopsis root-cap-expressed IBR3 gene encodes a protein involved in the conversion of the auxin precursor indole-3-butyric acid (IBA) into auxin. IBR3 and other Arabidopsis IBR paralogues show overlapping expression patterns in the LRC cells, suggesting that conversion of IBA into auxin acid takes place preferentially in the LRC cells, and that this local auxin might play a role in marking the sites of future lateral root emergence (De Rybel et al., 2012). Interestingly, oscillatory fluctuations of the auxin response in the root proper adjacent to the LRC cells are involved in pre-branch-site determination and lateral root patterning (Moreno-Risueno et al., 2010). Not all pre-branch sites become lateral roots, but the amplitude of the oscillation determines the frequency and spacing of potential sites of lateral root emergence (Xuan et al., 2015). Under laboratory conditions, it has been observed that lateral roots are formed predominantly on the side of a root that is in contact with a moist surface, a phenomenon referred to as hydropatterning (Robbins and Dinneny, 2015). The underlying mechanisms are not yet understood in detail, but recently, local auxin biosynthesis via the enzyme TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 (TAA1) has been implicated in this process (Bao et al., 2014). TAA1 is expressed in the LRC cells and the epidermis, and a translational TAA1 reporter revealed a clear upregulation of TAA1 protein abundance in LRC cells in contact with a water film. taa1 loss-of-function mutants showed a significant reduction in hydropatterning, suggesting that TAA1-mediated auxin biosynthesis is necessary to induce lateral root formation towards a wet surface. However, as ubiquitous TAA1 expression was able to restore hydropatterning defects in taa1 mutants, it seems that the specific localization of TAA1- mediated auxin synthesis is not required for this response (Bao et al., 2014). As well as moisture, the availability of soluble macronutrients such as phosphate has also been shown to influence root system architecture. Soil phosphate deficiency is known to lead to a reduction in primary root growth concomitant with an increase in the number of lateral roots (Williamson et al., 2001). LOW PHOSPHATE ROOT1 (LPR1) has been identified as a major quantitative trait locus explaining the natural variation of Arabidopsis ecotypes in response to differential phosphate availability (Reymond et al., 2006). LPR1 was mapped to a gene encoding a protein with multicopper oxidase activity, and the quantitative trait locus could be explained by differential expression of LPR1 in the root caps of different Arabidopsis ecotypes. Together with its paralogue,

7 Developmental biology and functions of the root cap 5657 LPR2, LPR1 is responsible for the regular soil phosphate deficiency phenotype (Svistoonoff et al., 2007), suggesting a role for the root cap in sensing, or reacting to, phosphate deficiency. When a growing primary root senses low phosphate concentrations, LPR1 and LRP2 are responsible for a root growth arrest. To date, we are only beginning to understand the molecular mechanisms underlying the root s response to low phosphate, which depends on several processes and involves many different gene functions (for an exhaustive review, see Péret et al., 2014). A role of the root cap as the first interface between root and soil is emerging, enabling the plant to acquire the heterogeneously distributed water and nutrients by an optimally adapted root architecture. Root cap sloughing and PCD In order to fulfil its diverse functions at the root tip, the root cap follows a developmental pattern that is very particular among plant organs. Throughout plant development, organ growth generally follows two opposing developmental principles determinate or indeterminate growth. The indeterminate meristems of roots and shoots are persistently producing new cells, continually increasing shoot and root organ size. By contrast, many lateral organs such as leaves and floral organs are produced by groups of cells with limited proliferation activity, leading to organs of predetermined size (Tsukaya, 2003). The root cap, however, follows neither of these principles. While new root cap cells are constantly produced by root cap stem cells in an indeterminate fashion, the size and cell number of the root cap are determinate (Barlow, 2003). Hence, similar to many animal organs, the root cap shows a cell number and organ size homeostasis in which the disposal of old cells is balanced by the generation of new cells. This particular mode of cellular turnover is beneficial for the root cap, since its cells can be exposed to harsh mechanical stresses while the growing root pushes through the soil. Yet, what is more, the continuous generation of short-lived root cap cells is necessary to facilitate organ size maintenance and the root cap s position at the tip of the growing root: whenever an EPI/LRC stem cell divides periclinically, a new epidermis module and an adjacent LRC module are created (Wenzel and Rost, 2001). As plant cells are connected to their neighbours by a common cell wall and cannot migrate, the root cap stem cells have to continuously create new root cap cells in co-ordination with the stem cells of the root proper. However, in contrast to the root epidermis, which covers the entire root, the root cap only covers the meristematic zone. Therefore, while epidermis cells persist after expansion and maturation, root cap cells have to be disposed of to avoid the extension of the root cap beyond the meristematic region (Fig. 4). Different plant species have come up with different solutions for this problem. Many species, including pea, cereals, and cucumber, dissolve the cell wall connections of root cap cells with their neighbours, causing a release of individual living border cells into the rhizosphere (Driouich et al., 2007). Detached border cells can range from dozens to tens of thousands of cells per root tip, depending on the species and age of the root (Hamamoto et al., 2006). In other species, as in Arabidopsis, LRC cells undergo cell death and rapid autolysis on the root surface as soon as they reach the edge of the elongation zone (Fig. 5) (Fendrych et al., 2014). This cell death process proceeds cell by cell towards the more proximally located root cap cells, until it reaches the LRC cells that are close to the COL cells. Here, a repeated shedding of the dying LRC cells and their still-living neighbouring COL cells occurs. In contrast to true border cells, these borderlike cells are shed as entire packets of cells and do not survive prolonged periods in the rhizosphere (Vicré et al., 2005). Thus, Arabidopsis employs both cell death and cell sloughing to regulate the disposal of root cap layers; while the majority of the LRC cells of a root cap layer on the root surface are disposed of by cell death, the remaining COL cells are sloughed off alive. Between COL and LRC, there is a zone of gradual transition in which packets of dead, dying, and living LRC and COL cells are shed at intervals (Fig. 5) (Fendrych QC WOX5 Epidermis prolifera on Epidermis TRN1 TRN2 EPI/LRC stem cell LRC cells FEZ RBR ARF10/16 SMB BRN1/BRN2 Columella ACR4 stem cell CLE40 COL cells Root Cap Non-/ Root Hair Development PCD PASPA3 BFN1 CEL5 COL sloughing QUA1 Fig. 4. Summarizing model for mechanisms governing stem cell maintenance and differentiation of the root cap. Co-ordinated by the QC, COL and EPI/ LRC stem cells produce COL, LRC, and epidermal cells, respectively, which then follow their individual differentiation pathways. Transcriptional control, as well as receptor-ligand signalling, have been involved in the balance between stem cell maintenance and daughter cell differentiation. See text for further details.

8 5658 Kumpf and Nowack et al., 2014). Intriguingly, in order to maintain a constant root cap organ size with a given number of root cap layers, the generation of new layers and the disposal of mature layers must be balanced precisely. To determine how root cap cell birth, death, and sloughing are co-ordinated will be a challenging task to be addressed in the coming years. As already mentioned, shedding of COL cells has been shown to be controlled by the TFs BRN1 and BRN2, which control the expression of several genes coding for enzymes involved in cell wall modification. CEL5 encodes a root-capexpressed endo-β-1,4-glucanase, and a cel5 loss-of-function mutant shows a slightly reduced efficiency in root cap sloughing (del Campillo et al., 2004). In a complementary approach, mutants defective in the biosynthesis of cell wall xyloglucan, cellulose, or pectin were investigated for sloughing defects. However, only mutants in the homogalacturonan-deficient glycosyltransferase QUASIMODO1 (QUA1) and the putative methyltransferase QUA2 showed an altered border-like cell sloughing. Instead of packets of cells, individual cells were released in qua1 and qua2 mutants (Durand et al., 2009). As COL cell shedding is a conspicuous process, it was often conceived as the only process eliminating Arabidopsis root cap cells. Although several authors suggested the occurrence of a cell death process in the Arabidopsis root cap (Truernit and Haseloff, 2008; Rost, 2011), it has only recently been shown that a developmentally controlled, genetically encoded PCD process takes place in the LRC cells (Fig. 5) (Fendrych et al., 2014). A first symptom of PCD preparation is the expression of genes coding for hydrolytic enzymes, including the BIFUNCTIONAL NUCLEASE 1 (BFN1) and the saposin-like aspartyl protease-encoding PASPA3. The activation of these genes also occurs in other established types of developmental PCD, for instance in the anther tapetum cells and in xylem vessels (Farage-Barhom et al., 2008; Böllhoner et al., 2012; Fendrych et al., 2014). The actual loss of vital functions occurs in a rapid but orderly succession of cellular events. The first conspicuous cellular feature preceding cell death consists of an abrupt cytoplasmic acidification, followed only minutes later by plasma membrane disintegration, and finally the collapse of the large central vacuole (Fendrych et al., 2014). Thus, LRC PCD shows similarities with the PCD type that has been characterized as vacuolar cell death (van Doorn et al., 2011). Interestingly, however, cytoplasmic acidification clearly precedes disintegration of both the plasma membrane and the tonoplast, and was shown to be necessary and sufficient for the effective execution of these subsequent events (Fendrych et al., 2014). This stands in contrast to the prevalent concept that vacuolar collapse causes cytoplasmic acidification during the terminal phase of plant PCD (Hara-Nishimura and Hatsugai, 2011; van Doorn et al., 2011). An initial drop in cytoplasmic ph can trigger several processes that might contribute to cell death initiation. How exactly this happens is unclear to date, but activation of proteases, such as the plant cell-death-associated Arabidopsis cellular degrada on * PCD ini a on and execu on * LRC differen a on and PCD prepara on * COL and LRC cell sloughing * Fig. 5. PCD and cell sloughing regulate root cap turnover. Left, median longitudinal section of a 6-d-old Arabidopsis root expressing the ubiquitous plasma membrane marker WAVE131 (Geldner et al., 2009), with living cells indicate in red/yellow (asterisks), and propidium iodide staining indicate dead and dying cells in blue/grey (arrows). Right, maximum projection showing the surface of a 6-d-old root expressing a tonoplast integrity marker under the control the PASPA3 promoter (Fendrych et al., 2014). In living cells preparing for PCD, cytoplasmic GFP (green) and vacuolar targeted RFP (red) are separated (asterisks), while both fluorescent proteins co-localize after vacuolar collapse, indicating cell death (arrows).

9 Developmental biology and functions of the root cap 5659 METACASPASE9 (MC9) and the mammalian ICE-like protease, have been shown to occur under acidic conditions (Furlong et al., 1997; Watanabe and Lam, 2011). The nuclease BFN1 has been a long-standing candidate for plant cell death execution since it was shown to be associated with senescence (Pérez-Amador et al., 2000) and cell death processes in plants (Farage-Barhom et al., 2008). Although a bfn1 loss-of-function mutant showed no deviation in the execution time point of cell death in LRC cells, Fendrych et al. (2014) described a conspicuous corpse autolysis phenotype: nuclear and cytoplasmic material was degraded significantly slower in bfn1 mutants than in the wild type. This record represents the only phenotype so far reported for a bfn1 mutant in Arabidopsis, although it is in accordance with a nuclear degradation phenotype in xylogenic Zinnia elegans cell cultures depleted of the BFN1 homologue ZEN1 (Ito and Fukuda, 2002). BFN1 and PASPA3 expression are dependent on the root cap differentiation-promoting TF SMB, suggesting that their expression and the ensuing PCD process are the ultimate steps of root cap differentiation (Fendrych et al., 2014). smb loss-of-function mutants show a prolonged root cap division activity and a delayed root cap differentiation (Bennett et al., 2010). As a consequence, preparation for PCD is delayed and smb mutant LRC cells that enter the root elongation zone are unable to induce regular PCD. The loss of SMB function has severe consequences for LRC cells: viable smb mutant LRC cells can reach three times the average length of wild-type root cap cells. The combination of delayed death and increased cell size in the smb mutant LRC cells causes a doubling of root cap organ length. Interestingly, smb mutant meristem size is not increased, representing, to the best of our knowledge, the first example of an uncoupling of meristem size and root cap size control (Fendrych et al., 2014). Eventually, smb mutant LRC cells do die, but they do so in a strikingly aberrant way: BFN1 and PASPA3 are not expressed, and smb LRC cells do not degrade after death. As a consequence, unprocessed LRC cell corpses litter the entire root surface (Fendrych et al., 2014). The smb mutant LRC cell degradation phenotype is much stronger than that observed in bfn1 mutants, suggesting that a number of different hydrolytic enzymes orchestrate cell corpse removal. Interestingly, the cause of LRC cell death in smb mutants appears to be fundamentally different from that in the wild type: treatments with a cell-expansion-reducing drug do not affect PCD in the wild type but lead to a significantly higher rate of LRC cell survival in the smb mutant (Fendrych et al., 2014). This observation suggests that smb LRC cell death is the passive consequence of physical stress exerted by expanding epidermis cells in the elongation zone, rather than an actively controlled PCD process. These results indicate that SMB-conferred LRC differentiation is necessary to prepare PCD induction and execution. Conversely, LRC fate is also sufficient to induce PCD processes ectopically. In tornado (trn) mutants defective in tetraspannin-like membrane proteins, individual epidermal cell files adopt LRC fate (Cnops et al., 2000). Ectopic LRC cell fate in these epidermal cells is sufficient to trigger PASPA3 expression followed by a cell death process similar to the one observed in true LRC cells (Fendrych et al., 2014). The timely execution of LRC PCD and cell autolysis appears to be crucial to facilitate root growth. Both epidermal cell length and root length are reduced by over 20% in smb mutants. This growth reduction might be produced by the living LRC cells or their unprocessed cell corpses present in the elongation zone, which might obstruct the expansion of the root in this zone (Fendrych et al., 2014). Hence, the PCD-regulated organ size control of the LRC is important for optimal root growth, and conceivably for root system architecture. Furthermore, divergent fates and lifespans of root cap cells in different species, i.e. whether root cap cells undergo PCD and degrade on the root surface or live on after being sloughed off, might have important implications for root soil communication in the rhizosphere. Root cap cell fate in the rhizosphere The rhizosphere is the narrow zone of soil that surrounds a plant root and is actively influenced by the growing plant root. The physiological and secretory activities of root cells have direct or indirect impact on the biotic and abiotic conditions in the rhizosphere. The root cap has a prominent role in the influence of the rhizosphere, as it is the first interface of a growing root with the soil. Root cap cells are active secretory cells that produce and exude large amounts of polysaccharide-based mucilage (Wen et al., 2007; Cai et al., 2013). Mucilage secretion is believed to provide a lubricating film that facilitates the soil penetration of the root tip (McKenzie et al., 2013). As well as mucilage, other exudates, secreted proteins, cellular components, and entire cells are also released by the growing root (Philippot et al., 2013). In grasses, rhizodeposits have been shown to contain about 25% of the carbon allocated to the roots (Jones et al., 2009). Next to primary metabolites, diverse secondary metabolites are given off into the rhizosphere, influencing the diverse community of both pathogenic and symbiotic microbiota (Bais et al., 2006). Root cap border cells in pea have been shown to be involved in the local defence against an oomycete pathogen by producing antimicrobial phenolic compounds (Cannesan et al., 2011). Furthermore, after blocking border cell protein secretion with brefeldin A, or after enzymatic degradation of extracellular proteins, pea root tip infection rates by the pathogenic fungus Nectria haematococca dramatically increased (Wen et al., 2007). Similarly, the degradation of border-cellderived extracellular DNA by DNase I increased the infection rates in this pathosystem (Wen et al., 2009). However, the abiotic characteristics, as well as the biotic characteristics, of the rhizosphere are also influenced by the plant root. Soil ph and nutrient availability are modified by ions and chelators that are released by the root (Hinsinger et al., 2009; Tsednee et al., 2012; Li et al., 2015). To sum up, whether living root cap border cells are sloughed off, or whether root cap cells undergo PCD and are degraded on the root surface, might have direct consequences for the rhizosphere and plant life style. Based on the observation

10 5660 Kumpf and Nowack that plants without border cells do not associate with major classes of symbiotic fungi or bacteria, some authors speculate that there is a correlation between border cell production and the ability to form mycorrhizal associations or nitrogen-fixing nodules (Hamamoto et al., 2006). Conclusions Although only short lived, root cap cells fulfil crucial functions for root growth, root system architecture, and plant life in general. As part of Darwin s wonderful structure, the root cap has much potential for research from a 21st-century perspective. Due to its exposed and accessible situation, a plethora of plant biological processes, including hormonal regulation, stem cell maintenance, cellular differentiation, signal transduction, cell wall modification, and PCD, can be conveniently studied in the root cap. Taking the important functions of the root cap for plant growth and nutrition into account, it is high time to put the small root cap organ in the spotlight of scientific attention. Acknowledgements We gratefully acknowledge support for our research by the OMICS@ VIB post-doctoral programme of the Flemish Institute for Biotechnology (VIB) to RPK. We further thank to Tom Beeckman and all members of the Programmed Cell Death Laboratory at the VIB Plant Systems Biology department for critical discussion and helpful comments on the manuscript. Finally, we want to thank Annick Bleys for her help with preparing this manuscript. References Bais HP, Weir TL, Perry LG, Gilroy S, Vivanco JM The role of root exudates in rhizosphere interactions with plants and other organisms. Annual Review of Plant Biology 57, Band LR, Wells DM, Fozard JA, et al Systems analysis of auxin transport in the Arabidopsis root apex. The Plant Cell 26, Band LR, Wells DM, Larrieu A, et al Root gravitropism is regulated by a transient lateral auxin gradient controlled by a tipping-point mechanism. Proceedings of the National Academy of Sciences, USA 109, Bao Y, Aggarwal P, Robbins NE, II, et al Plant roots use a patterning mechanism to position lateral root branches toward available water. Proceedings of the National Academy of Sciences, USA 111, Barlow PW Evolution of structural initial cells in apical meristems of plants. Journal of Theoretical Biology 169, Barlow PW The root cap: cell dynamics, cell differentiation and cap function. Journal of Plant Growth Regulation 21, Becker B, Marin B Streptophyte algae and the origin of embryophytes. Annals of Botany 103, Bennett T, van den Toorn A, Sanchez-Perez GF, Campilho A, Willemsen V, Snel B, Scheres B SOMBRERO, BEARSKIN1, and BEARSKIN2 regulate root cap maturation in Arabidopsis. The Plant Cell 22, Bennett T, van den Toorn A, Willemsen V, Scheres B Precise control of plant stem cell activity through parallel regulatory inputs. Development 141, Böllhoner B, Prestele J, Tuominen H Xylem cell death: emerging understanding of regulation and function. Journal of Experimental Botany 63, Cai M, Wang N, Xing C, Wang F, Wu K, Du X Immobilization of aluminum with mucilage secreted by root cap and root border cells is related to aluminum resistance in Glycine max L. Environmental Science and Pollution Research International 20, Campilho A, Garcia B, van den Toorn H, van Wijk H, Campilho A, Scheres B Time-lapse analysis of stem-cell divisions in the Arabidopsis thaliana root meristem. The Plant Journal 48, Cannesan MA, Gangneux C, Lanoue A, Giron D, Laval K, Hawes M, Driouich A, Vicré-Gibouin M Association between border cell responses and localized root infection by pathogenic Aphanomyces euteiches. Annals of Botany 108, Cnops G, Wang X, Linstead P, Van Montagu M, Van Lijsebettens M, Dolan L TORNADO1 and TORNADO2 are required for the specification of radial and circumferential pattern in the Arabidopsis root. Development 127, Cruz-Ramírez A, Díaz-Triviño S, Wachsman G, et al A SCARECROW-RETINOBLASTOMA protein network controls protective quiescence in the Arabidopsis root stem cell organizer. PLoS Biology 11, e Darwin C, Darwin F The power of movement in plants. London: John Murray. De Rybel B, Audenaert D, Xuan W, et al A role for the root cap in root branching revealed by the non-auxin probe naxillin. Nature Chemical Biology 8, De Smet I, Vassileva V, De Rybel B, et al Receptor-like kinase ACR4 restricts formative cell divisions in the Arabidopsis root. Science 322, del Campillo E, Abdel-Aziz A, Crawford D, Patterson SE Root cap specific expression of an endo-β-1,4-d-glucanase (cellulase): a new marker to study root development in Arabidopsis. Plant Molecular Biology 56, Dolan L, Janmaat K, Willemsen V, Linstead P, Poethig S, Roberts K, Scheres B Cellular organisation of the Arabidopsis thaliana root. Development 119, Driouich A, Durand C, Vicré-Gibouin M Formation and separation of root border cells. Trends in Plant Science 12, Durand C, Vicré-Gibouin M, Follet-Gueye ML, Duponchel L, Moreau M, Lerouge P, Driouich A The organization pattern of root borderlike cells of Arabidopsis is dependent on cell wall homogalacturonan. Plant Physiology 150, Farage-Barhom S, Burd S, Sonego L, Perl-Treves R, Lers A Expression analysis of the BFN1 nuclease gene promoter during senescence, abscission, and programmed cell death-related processes. Journal of Experimental Botany 59, Fendrych M, Van Hautegem T, Van Durme M, et al Programmed cell death controlled by ANAC033/SOMBRERO determines root cap organ size in Arabidopsis. Current Biology 24, Friml J, Vieten A, Sauer M, Weijers D, Schwarz H, Hamann T, Offringa R, Jürgens G Efflux-dependent auxin gradients establish the apical-basal axis of Arabidopsis. Nature 426, Friml J, Wiśniewska J, Benková E, Mendgen K, Palme K Lateral relocation of auxin efflux regulator PIN3 mediates tropism in Arabidopsis. Nature 415, Furlong IJ, Ascaso R, Lopez Rivas A, Collins MKL Intracellular acidification induces apoptosis by stimulating ICE-like protease activity. Journal of Cell Science 110, Geldner N, Dénervaud-Tendon V, Hyman DL, Mayer U, Stierhof Y-D, Chory J Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set. The Plant Journal 59, Geldner N, Richter S, Vieten A, Marquardt S, Torres-Ruiz RA, Mayer U, Jürgens G Partial loss-of-function alleles reveal a role for GNOM in auxin transport-related, post-embryonic development of Arabidopsis. Development 131, Grieneisen VA, Xu J, Marée AFM, Hogeweg P, Scheres B Auxin transport is sufficient to generate a maximum and gradient guiding root growth. Nature 449, Haberlandt G Ueber die Perzeption des geotropischen Reizes. Berichte der Deutschen Botanischen Gesellschaft 18, Haberlandt G Physiologische Pflanzenanatomie. Leipzig: Wilhelm Engelmann. Hamamoto L, Hawes MC, Rost TL The production and release of living root cap border cells is a function of root apical meristem type in dicotyledonous angiosperm plants. Annals of Botany 97,