The role of auxin gradients in plant development

It remained a mystery for years how a simple molecule like auxin can influence the variety of seemingly unrelated developmental processes such as embryonic axis formation, organogenesis, meristem maintenance, tropisms, root and shoot elongation, apical hook formation and others. The availability of molecular tools for visualization and manipulation of PAT components as well as for visualization of auxin and its activity enabled researchers to address more specifically the role of PAT-dependent auxin distribution in each of these developmental processes. These studies convincingly demonstrated that regulated, local auxin acummulation underlies most of auxin-mediated development.

1.5.1 Monitoring of auxin distribution in planta

One of the major obstacles in studies addressing the role of auxin in plant development was the inability to visualize auxin distribution in planta. An important breakthrough was brought through the discovery of genes that are rapidly upregulated by auxin (reviewed in Hagen & Guilfoyle, 2002). The consensus sequence TGTCTC (auxin response element, AuxRE) was identified within promoters of these genes, which confers the auxin responsiveness (Ulmasov et al., 1995). Multiple repeats of the AuxREs yielded synthetic promoters DR5 or DR5rev (another variant with inverse repeats), which are highly responsive to auxin and, therefore, can be used for indirect monitoring of auxin levels. Indeed, multiple strategies were utilized to show the correlation between DR5 activity and auxin accumulation. Firstly, direct measurements of auxin content within Arabidopsis roots demonstrated elevated levels at the tip, where the highest DR5 activity was also detected (Casimiro et al., 2001). Secondly, exogenously supplied auxin was able to induce DR5 activity in all cells and inhibition of PAT changed the DR5 expression pattern, suggesting that the spatially restricted signals in untreated plants visualize differences in auxin levels between cells. Finally, the accumulation of auxin itself (monitored using an anti-IAA antibody) mirrors the DR5 pattern (Plates 1.1J and 1.1K). In summary, despite the theoretical limitations for the use of DR5 and related tools, so far it seems that DR5 activity can be used as a reasonable approximation for auxin levels at least in embryonic and meristematic tissues. Indeed, the DR5::GUS and/or DR5rev::GFP constructs have been instrumental in detecting the spatial pattern of auxin accumulation in many tissues including Arabidopsis embryos (Plate 1.1I), roots (Plate 1.1J) and organ primordia (Plate 1.1H), and have demonstrated a universal role for auxin gradients in plant development (Sabatini et al., 1999; Friml et al, 2002a,b, 2003; Benkova etal., 2003).

1.5.2 Embryonic axis formation

Embryogenesis is a process that transforms a single-celled zygote into the embryo containing all basic pattern elements of the future plant. The mature embryo displays an axis of polarity, with the shoot meristem at the apical end and root meristem at the basal end. This remarkably uniform apical-basal pattern has been traced in Arabidopsis back to the earliest stages of embryogenesis. The first manifestation of the apical-basal axis is the asymmetric division of the zygote, which produces a small apical cell and a larger basal cell. The apical cell divides vertically and generates the 'proembryo', which later gives rise to the most regions of the seedling. The basal cell continues to divide horizontally and produces the suspensor - a file of cells that attaches the proembryo to maternal tissue. At the early globular stage, the uppermost cell is specified to become the hypophysis - the founder of the root meristem. At the triangular stage, the early patterning is finished with the initiation of two symmetrically positioned embryonic leaves - cotyledons (Jiirgens, 2001). A role for auxin in embryo patterning has been suspected for some time, not the least because embryo defects can be induced by blocking PAT (Hadfi et al, 1998). In addition, genetic disruption of auxin response in Arabidopsis mutants such as monopteros (mp) and bodenlos (bdl) led to defects in embryonic axis formation. Molecular analysis of these mutants revealed that MP encodes a transcriptional activator - the auxin response factor 5 (ARF5), and BDL encodes the corresponding transcriptional repressor - IAA12, both components of auxin signaling (Hardtke & Berleth, 1998; Hamann et al., 2002). To really pinpoint the role of auxin in embryogenesis, the distribution of auxin and its response was monitored using anti-IAA antibodies and DR5rev::GFP. Immediately after the division of the zygote, when the apical cell is specified, auxin accumulates in this cell and during subsequent development persists in the proembryo. At around the 32-cell stage, when the basal embryo pole is being specified, the gradient of auxin accumulation suddenly reverses and forms a new maximum in the uppermost suspensor cells, including the hypophysis (Plate 1.1I). At later stages of embryogenesis, additional DR5 reporter gene signals appear in the tips of the developing cotyledons (Friml et al., 2003). Both chemical (AEIs) and genetic (gn or multiple pin mutants) inhibition of PAT interfere with this dynamic distribution of auxin during embryogenesis. Furthermore, they cause identical developmental defects, ranging from cup-shaped embryos with misspecified apical structures and a nonfunctional root pole (Plates 1.1F and 1.1G), to ball-shaped embryos without any discernible apical-basal axis. These findings, together with analysis of PIN expression and localization, completed the picture, indicating a role for PIN-dependent auxin distribution in embryo patterning. At early stages (Fig. 1.5A), auxin is actively provided to the apical cell from the adjacent basal cell by the action of apically localized PIN7. This apical-basal auxin gradient is required for the specification of the apical cell. At subsequent stages, the cells of the suspensor continue to localize PIN7 at their apical side, while in the proembryo another protein, PIN1, is expressed without apparent polarity. But after the 32-cell stage (Fig. 1.5B), PIN1 becomes localized to the basal membranes of the provascu-lar cells, suggesting downward transport toward the region of the future root pole. Simultaneously, the asymmetric localization of PIN7 is reversed within the basal cells, mediating auxin transport out of the embryo. Subsequently, PIN4 expression starts at the basal pole of the embryo, supporting the action of both PIN1 and PIN7. As a result of these changes in auxin flow, the auxin gradient reverses, displaying its new maximum in the uppermost suspensor cell, which in response to auxin is specified to become the hypophysis - the founder of the future root meristem. Thus, developmentally regulated changes in polarity of PIN proteins result in the redirection of auxin fluxes for local auxin accumulation, which is then required first for specification of the apical and later for the basal pole of the embryonic apical-basal axis (Friml et al., 2003).

Figure 1.5 Auxin transport and distribution during embryogenesis. Sites of auxin accumulation are shadowed. Arrows indicate routes of auxin efflux mediated by PIN1, PIN4 and PIN7. Also depicted are proteins involved in embryo patterning and related to auxin transport (encircled) or auxin response. (A) Two cell stage embryo - apical cell specification. Auxin accumulates in the proembryo through PIN7-dependent transport via the suspensor. Auxin response (mp, bdl) and transport (gn) mutants show defects in the establishment of the apical cell. (B) Triangular stage - from early globular stage on auxin accumulates, in a PIN1- and PIN4-dependent manner, in the hypophysis, which is specified and is further transported through the suspensor via a PIN7-dependent route. New sites of auxin accumulation emerge at the tips of forming cotyledons. mp, bdl and gn show defects in root pole as well as cotyledon establishment. Adapted from Friml et al. (2003), with permission.

Figure 1.5 Auxin transport and distribution during embryogenesis. Sites of auxin accumulation are shadowed. Arrows indicate routes of auxin efflux mediated by PIN1, PIN4 and PIN7. Also depicted are proteins involved in embryo patterning and related to auxin transport (encircled) or auxin response. (A) Two cell stage embryo - apical cell specification. Auxin accumulates in the proembryo through PIN7-dependent transport via the suspensor. Auxin response (mp, bdl) and transport (gn) mutants show defects in the establishment of the apical cell. (B) Triangular stage - from early globular stage on auxin accumulates, in a PIN1- and PIN4-dependent manner, in the hypophysis, which is specified and is further transported through the suspensor via a PIN7-dependent route. New sites of auxin accumulation emerge at the tips of forming cotyledons. mp, bdl and gn show defects in root pole as well as cotyledon establishment. Adapted from Friml et al. (2003), with permission.

1.5.3 Postembryonic organ formation

Embryo development establishes the basic body plan of both animals and plants. However, the adult form of a plant also depends largely on postembryonic development. Plants, unlike animals, can postembryonically initiate new organs such as leaves, flowers, flower organs, ovules and lateral roots. The regular initiation pattern of leaves and flowers (called phyllotaxis) is the major determinant of adult plant architecture. During organ formation, first a site of primordium initiation is selected and then a new growth axis of the organ primordium is established. It seems that PIN-dependent redirection of auxin flow and local accumulation of auxin play a fundamental role in both of these processes. Exogenous auxin application is sufficient to trigger leaf or flower formation in the shoot apex (Reinhardt et al., 2000) or lateral root initiation (Laskowski et al., 1995), and endogenous accumulation of auxin and its response was detected at the initiation site of incipient organs in shoots and roots (Benkova et al., 2003). On the other hand, interference with PAT (AEIs, pin mutants) or auxin response (mp, solitary root) blocks organ formation (Okada et al., 1991; Przemeck et al, 1996; Fukaki et al., 2002). In the shoot, PIN1 localization in the outermost layer (L1) of the meristem undergoes dynamic rearrangement toward these loci of auxin accumulation (Reinhardt et al., 2003). The pattern of auxin accumulation and PIN localization suggests that auxin is transported toward the meristem through the L1 cell layer. There, auxin becomes absorbed by

Figure 1.6 Auxin transport and distribution during organogenesis. Sites of auxin accumulation are shadowed. Presumptive routes of auxin transport are depicted by arrows. (A) Lateral root primordium: Auxin is provided by PIN1-dependent auxin transport through the primordium interior toward the tip, where it accumulates. From here, part of the auxin is retrieved through the outer layers. (B) Auxin is provided to the primordium tip through the outer layers. From the tip, auxin is drained through a gradually established transport route toward the vasculature. Reproduced from Benkova et al. (2003), with permission.

Figure 1.6 Auxin transport and distribution during organogenesis. Sites of auxin accumulation are shadowed. Presumptive routes of auxin transport are depicted by arrows. (A) Lateral root primordium: Auxin is provided by PIN1-dependent auxin transport through the primordium interior toward the tip, where it accumulates. From here, part of the auxin is retrieved through the outer layers. (B) Auxin is provided to the primordium tip through the outer layers. From the tip, auxin is drained through a gradually established transport route toward the vasculature. Reproduced from Benkova et al. (2003), with permission.

already initiated primordia, which transport auxin into their interior and further toward already differentiated vasculature (Benkova et al., 2003). Therefore, auxin is depleted from the surroundings of the primordia (sink function) and its highest concentration remains at the most distant position, where a new primordium is initiated. Thus, a positive feedback represented by auxin accumulation in combination with lateral inhibition provides a mechanism for reiterativity and stability of the spiral or other types of phylotactic pattern (Reinhardt et al., 2003). In the root, auxin also accumulates by a PAT-dependent mechanism at sites of organ initiation (Benkova et al., 2003), but how it is precisely regulated remains so far unclear.

Once sites of organ initiation are selected, cell division is activated and the organ primordium develops along a new growth axis. In all types of primordia, PIN polar localization reorganizes and the new direction of PIN-mediated auxin transport determines the growth axis of the developing organ, establishing an auxin gradient with its maximum at the tip (Benkova et al., 2003). Interestingly, the main auxin transport routes appear to be reversed, when lateral root primordia are compared with other types of organs. In aerial organs, auxin is supplied through the outer layer and accumulates at the primordium tip (Fig. 1.6B). From the tip, auxin is transported into the interior of the primordium. In the case of lateral roots, auxin is provided to the tip through the inner cells and distributed away through the primordium surface (Fig. 1.6A). Regardless of these differences, a common auxin-gradient-dependent mechanism seems to underlie organ initiation as well as primordium development in all plant organs regardless of their mature morphology or developmental origin.

1.5.4 Root meristem maintenance

Plants, in contrast to animals, not only can initiate new organs postembryonically, but also possess specialized, permanently dividing and differentiating tissues called meristems, which enable a perpetuation of postembryonic growth. Meristems contain populations of 'stem' cells, which, according to their position, differentiate into different cell types. The Arabidopsis root meristem displays a highly regular and predictable pattern of cell divisions and differentiation and is, therefore, an ideal system for studies on mechanisms of meristem patterning. Manipulation of auxin distribution as well as auxin signaling have demonstrated a role for auxin in regulating the root meristem activity (Kerk & Feldman, 1994; Ruegger et al., 1997; Sabatini et al., 1999). Accumulation of auxin and auxin response was detected in the columella initial and first columella layer cells (Sabatini et al., 1999; Benkova et al., 2003). Interestingly, the PIN4 auxin efflux regulator displays a polar localization pointing toward the same area, suggesting a role of PIN4 in maintenance of this auxin accumulation. In support of this, pin4 mutation or chemical inhibition of PAT disrupts this auxin accumulation, which results in changes in cell fate specification (Friml et al., 2002b). These observations suggest that PIN4 mediates PAT through the central root meristem tissues, thus actively maintaining an auxin gradient with its maximum in the distal root tip (Plate 1.1M). The PIN4 function appears to be also necessary for local auxin turnover, sincepin4 mutant root tips display elevated auxin levels and fail to canalize exogenously applied auxin properly (Friml et al., 2002b). Following the most plausible scenario, auxin from upper tissues is actively concentrated by PIN-dependent transport in the distal root tip, which serves as an 'auxin sink'. There, part is probably rendered inactive by an as yet unknown mechanism and other part is redistributed back by PIN2 action through the outer layers (Plate 1.1M).

1.5.5 Tropisms

Tropisms are growth responses to external stimuli such as light (phototropism) or gravity (gravitropism), resulting from differential elongation rates on either side of a plant organ. The Cholodny-Went hypothesis proposed that differential growth rates result from the asymmetrical distribution of auxin, which subsequently promotes or inhibits cell growth and elongation (Went, 1974). Indeed, differential auxin or auxin response distributions were visualized in various plant organs including gravity stimulated tobacco shoots (Li et al., 1991), light and gravity stimulated Arabidopsis hypocotyls (Friml et al., 2002a) or developing peanut gynophores (Moctezuma, 1999). Because AEIs interfere with the asymmetric distribution of auxin as well as with tropisms, PAT has been implicated as the process underlying asymmetric auxin distribution (Lehman et al., 1996; Friml et al., 2002a) and the existence of a lateral auxin transport in shoots has been proposed. This would facilitate the exchange of auxin between the basipetal stream in vasculature and peripheral regions, where control of elongation occurs (Fig. 1.2). The analysis of localization and function of the auxin efflux regulator PIN3 provided molecular support for this concept (Friml et al., 2002a). Thepin3 mutants are defective in hypocotyl phototropism and gravitropism as well as root gravitropism, although these defects are rather subtle, suggesting functional redundancy. In addition, PIN3 is predominantly localized at the lateral side of shoot endodermis cells, where it is perfectly positioned to regulate lateral auxin flow (Friml et al., 2002a).

In roots, the situation is more complex, since gravity is perceived in the root cap but the growth response occurs in the elongation zone where elevated auxin levels on the lower side inhibit growth, resulting in downward bending (Chen et al., 2002). It seems that following gravity stimulation, auxin is redistributed laterally toward the lower side of the root cap, from where it is transported to the elongation zone (Saba-tini etal., 1999; Rashotte etal., 2000). Localization and mutant analyses suggest that the auxin influx component AUX1 facilitates auxin uptake in the lateral root cap and epidermis region and that PIN2 efflux regulator mediates directional auxin translocation toward the elongation zone (Muller et al., 1998; Swarup et al., 2001). The notion that both auxin influx and efflux are required for root gravitropism is further supported by the experiments with inhibitors of both processes (Parry et al., 2001). But how is gravity perception linked to the initial lateral auxin redistribution in the root cap? Gravity is perceived by sedimentation of starch-containing organelles (statoliths) in the columella root cap cells (Chen et al., 2002). PIN3 is localized in these cells under normal growth conditions without any apparent asymmetry at the cell boundaries. Intriguingly, when roots are gravistimulated, already within 2 min PIN3 changes its position and relocates, presumably to the new lower cell boundary (Plate 1.1M) (Friml et al., 2002a). It is conceivable that the rapid recycling of PIN3 between endosomes and the plasma membrane along the actin cytoskeleton provides a mean for its rapid retargeting in response to the environmental stimulus. However, how the statolith sedimentation and PIN3 relocation are linked remains unclear. It is possible that actin reorganization following the statoliths sedimentation would redirect intracellular traffic of PIN3 along the sedimentation routes and PIN3 would preferentially accumulate at the lower side of the cell. It remains to be demonstrated whether PIN3 relocation also mediates the auxin redistribution in the shoot. However, it is likely, since both statoliths and PIN3 are present in the shoot endodermis (Friml et al., 2002a), which is essential for shoot tropism responses (Fukaki et al., 1998). Also the link between light perception and lateral auxin redistribution during phototropism remains a topic for future investigations.

1.5.6 Downstream of auxin gradients

Recent studies on a role of PAT in plant development revealed that local gradients in auxin accumulation underlie the developmental responses to auxin. So far, this mechanism could be demonstrated for embryonic axis and postembryonic organ formation, meristem pattern maintenance and tropisms. However, how can the accumulation of a structurally rather simple molecule as IAA lead to such a wide variety of different responses? Since it is clear that different cells respond to auxin by activation of different developmental programs, part of the answer lies downstream of auxin gradients. The ability of auxin to bring about diverse responses appears to result from the existence of several independent mechanisms for auxin perception and from a complex transcriptional network at the lower end of the auxin signaling pathway.

So far, we know little about auxin perception, since, despite the fact that several auxin-binding proteins (most prominent among them ABP1) have been isolated from various plant species, their role as auxin receptors have not been demonstrated unequivocally (for overview, see Timpte, 2001). The elucidation of auxin signal transduction pathway has been more successful for very downstream events. Auxin can induce the expression of a variety of primary response genes including members of AUX/IAA, GH3 and SAUR gene families. The TGTCTC auxin response element (AuxRE) that confers auxin inducibility was identified in the promoters of these genes. Transcription factors of the ARF family bind specifically to AuxREs (Hagen & Guilfoyle, 2002). There are at least 22 ARF genes in Arabidopsis, which contain an N-terminal DNA-binding domain (Guilfoyle & Hagen, 2001). Results obtained in assays with transfected protoplasts demonstrated that some ARFs function as activators, while others are repressors (Tiwari et al., 2003). ARFs contain a C-terminal dimerization domain, which is related in amino acid sequence to motifs III and IV that have been found in short-lived nuclear proteins of the AUX/IAA family (Guilfoyle & Hagen, 2001; Liscum & Reed, 2002). There are 24 genes in Arabidopsis that are predicted to encode Aux/IAA proteins, and these contain four conserved motifs (which are referred to as domains I through IV). Domain I is an active repression domain that is transferable and dominant over activation domains (Tiwari et al., 2004). Domain II and domains III and IV play roles in protein stability and dimerization, respectively. Yeast two-hybrid interaction assays suggest that ARF C-terminal dimerization domains and AUX/IAA domains III and IV can homo- and heterodimerize (Kim et al., 1997; Ouellet et al., 2001; Tiwari et al., 2004). Thus, the multitude of mutually competing interactions between transcription factors and their repressors represent a network for activating different sets of genes in response to auxin. But how does an auxin-derived signal enter and activate this transcriptional network? Genetic studies in Arabidopsis implied that ubiquitin-mediated protein degradation is required for auxin response. Mutations in AXR1 and TIR1, both components of the ubiquitination pathway, confer reduced auxin response. TIR1 encodes an F-box protein that interacts with the cullin AtCUL1 and a SKP1-like protein (ASK1 or ASK2) to form an SCF ubiquitin protein ligase (E3) (Ruegger et al., 1998). AXR1, on the other hand, encodes a part of the enzyme that regulates activity of SCF ubiquitin ligases by RUB1 conjugation of the AtCUL1. SCFTIR1 physically interacts with domain II of AUX/IAA proteins and mutations affecting the SCFTIR1 complex increase stability of AUX/IAA proteins (Leyser et al., 1993). Auxin treatment stimulates the interaction between SCFTIR1 and AUX/IAA proteins and promotes their degradation. In support of this, mutations within domain II abolish the interaction between AUX/IAAs and SCFTIR1 and stabilize AUX/IAA repressors in presence of auxin (Gray et al., 2001). Stabilized AUX/IAA protein variants such as bodenlos (bdl, stabilized IAA12) or short hypocotyl 2 (shy2, stabilized IAA3) confer dominant effects on various aspects of auxin-dependent development (Tian & Reed, 1999; Hamann et al., 2002). By integrating all these findings, a current model on how auxin regulates gene expression emerged (Fig. 1.7). When auxin concentrations are below a certain threshold, early auxin response genes are actively repressed, because AUX/IAA repres-sors are dimerized to ARF transcriptional activators, preventing gene transcription

Auxin

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