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- Materials and Methods
Vascular plants comprise the lycophytes and euphyllophytes, with the latter including monilophytes, gymnosperms and angiosperms. Evidence from fossils and phylogenetic analyses indicates that lycophyte and euphyllophyte lineages probably diverged over 400 million yr ago and that the common ancestor comprised leafless branching axes (Kenrick & Crane, 1997; Pryer et al., 2001). As such, indeterminate apical growth probably evolved before the lycophyte–euphyllophyte divergence, but complex shoot architecture evolved independently in each lineage. To date, comparative studies have suggested that, although some developmental pathways evolved in parallel in both lycophytes and euphyllophytes (Harrison et al., 2005; Sanders et al., 2010), others play distinct roles in each group (Floyd & Bowman, 2006).
In angiosperms, shoot architecture is regulated, in part, by the modulation of auxin transport and homeostasis. For example, vascular tissue is patterned along polar auxin transport (PAT) routes in stems and leaves (reviewed in Berleth et al., 2000), and branching is controlled by patterns of auxin flow (reviewed in Müller & Leyser, 2011). A simplified model of leaf initiation describes how auxin flux between developing primordia and the shoot apex creates local auxin maxima that establish leaf initiation sites and thus determine phyllotactic pattern (Reinhardt et al., 2000, 2003). In Arabidopsis and tomato, loss of PAT results in a loss of leaf production (Reinhardt et al., 2000) and, in maize, aberrant PAT disrupts leaf placement and hence phyllotactic pattern at the apex (Lee et al., 2009). Similar effects are seen in gymnosperms, in that disrupted PAT causes a loss of cotyledon boundaries and abnormal meristem development in spruce embryos (Larsson et al., 2008). In combination, these observations suggest that the role of auxin transport in shoot patterning is conserved across seed plants.
In angiosperms, auxin efflux is inhibited by 1-n-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA) (Depta & Rubery, 1984). These compounds have therefore been used to examine the role of PAT in the regulation of plant growth (e.g. Mattsson et al., 1999; Rashotte et al., 2000; Reinhardt et al., 2000; DeMason & Chawla, 2004) and to infer the underlying mechanisms (Poli et al., 2003). In Arabidopsis, morphological perturbations induced by auxin efflux inhibitors are accompanied by perturbed function of PIN-FORMED (PIN1) and P-GLYCOPROTEIN (PGP) auxin efflux carriers (Geldner et al., 2001; Geisler et al., 2005). Disruption of PIN1 function leads to similar morphological perturbations, including the loss of leaf formation and disrupted phyllotaxy (Gälweiler et al., 1998; Reinhardt et al., 2003; Blilou et al., 2005; Lee et al., 2009). Although the disruption of PAT in the moss Physcomitrella patens results in morphological aberrations (Fujita et al., 2008), the role of auxin in the regulation of moss development is not well understood. In the lycophyte Selaginella, previous work has suggested that auxin transport is bidirectional (Wochok & Sussex, 1973), but the lack of controls in that study prevented conclusive interpretations of the data. To ascertain more precisely how auxin and PAT regulate developmental processes in lycophytes, we have characterized the morphological consequences of perturbed PAT in the lycophyte Selaginella kraussiana. The data obtained provide a comparative framework to infer how the role of auxin has changed during land plant evolution and, specifically, to compare the mechanisms that regulate shoot architecture in lycophytes and euphyllophytes.
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- Materials and Methods
Auxin transport mechanisms are conserved in lycophytes and euphyllophytes. To provide a comparative framework for the assessment of the role of auxin in the development of early diverging vascular plants and seed plants, we investigated the mechanisms of auxin transport in the lycophyte Selaginella kraussiana and characterized the morphological consequences of the perturbation of transport processes. We first showed that, as in seed plants, PAT is exclusively basipetal and is sensitive to known PAT inhibitors (Fig. 1). The finding that PAT is exclusively basipetal in Selaginella contrasts with previous reports which concluded a 2 : 1 basipetal to acropetal ratio in Selaginella shoots (Wochok & Sussex, 1973). However, given that these earlier studies did not demonstrate the extent of diffusion using auxin efflux inhibitors or other organic acids (see Lewis & Muday, 2009 for a discussion of such methods), the results may not be reliable. Notably, the quantity of auxin taken up into Selaginella shoots was c. 100 times lower than that in angiosperms, such as maize or Arabidopsis (Fig. 1; e.g. Rashotte et al., 2000; Poli et al., 2003; Lewis & Muday, 2009). It is unclear whether the smaller quantities reflect differences in the experimental method, size of the tissue, amount of vascular tissue or reduced efficiency of auxin transport systems within Selaginella. However, the data suggest that the molecular mechanisms underlying PAT are likely to be conserved across all vascular plants. Consistent with this suggestion, eight PIN1-like sequences are present in the Selaginella genome (Banks et al., 2011).
The evolution of PAT mechanisms
The relevance of auxin transport mechanisms before the evolution of vascular plants is uncertain. Current evidence suggests that basipetal PAT occurs in moss seta, but studies reporting the presence or absence of acropetal transport are conflicting (Poli et al., 2003; Fujita et al., 2008). Notably, genes orthologous to PIN1 and to auxin response genes are found in the genome of Physcomitrella patens (Křeček et al., 2009; Paponov et al., 2009), indicating that at least some of the molecular components mediating auxin responses are present in nonvascular plants. Furthermore, NPA causes the disruption of moss embryo development, indicating that PAT in moss is sensitive to auxin efflux inhibitors (Fujita et al., 2008). Poli et al. (2003) suggested that both liverworts and mosses show partial sensitivity to auxin efflux inhibitors, but without diffusion controls their results are hard to interpret. Table 1 summarizes what is known of the morphological effects of reduced auxin transport. Although information is lacking or incomplete for groups other than angiosperms, evidence suggests that PAT plays a ubiquitous role in patterning sporophyte architecture and in regulating vascular tissue development. Intriguingly, a recent study has demonstrated that NPA-sensitive PAT occurs in single cells of Chara corralina at a rate higher than would be expected from diffusion alone (Boot et al., 2012). Putative PIN-like gene sequences have also been identified in streptophyte algae (De Smet et al., 2011). In combination with the data presented here, these findings suggest that basipetal auxin transport may have been co-opted from algal ancestors during the early evolution of the multicellular sporophyte, and that mechanisms were elaborated coincident with the evolution of complex branching systems.
Table 1. Comparison of the effects of polar auxin transport (PAT) inhibitors on plant structure in each of the major groups of land plants
|Shoot architecture||Gametophyte shoot??||No effect on Physcomitrella patens gametophyte leafy shoot|| ||Leaves are produced, meristem arrest in Selaginella||?||?||Leafless shoots, increased branching, meristem continues growth in Arabidopsis|
|Leaf morphology||–||–||–||Decreased length to width ratio in Selaginella||?||?||Shorter petiole, decreased length to width ratio in Arabidopsis. Loss of leaflet initiation in pea and Cardamine hirsuta|
|Root architecture||–||–||–||Callus formation on root tips of Selaginella||Decreased growth rate but no effect on root production in Ceratopteris richardii||?||Inhibition of lateral root formation in Arabidopsis|
|Vascular tissue||–||–||–||Disrupted vascular development and leaf venation, larger tracheids in Selaginella||?||?||Disrupted vascular development and leaf venation, increased number of stem vascular strands in Arabidopsis|
|Other||Embryo??||Pertubations in diploid embryo of P. patens||Gametophyte, embryo??||Rhizophores redetermined to shoots||Disrupted gametophyte development – extra meristem formation in Ceratopteris richardii||Abnormal embryo development in Picea abies||Abnormal embryo development in Arabidopsis|
Auxin mediates dichotomous branching in roots but not shoots of Selaginella
As with all lycophytes, Selaginella shoots and roots branch dichotomously, a trait probably inherited from ancestral vascular plants (Kenrick & Crane, 1997). By contrast, seed plant shoots branch through axillary buds at nodes, and roots branch endogenously from the pericycle. Auxin is a key regulator of branching in both roots and shoots of seed plants. In shoots, auxin is produced in leaves and is transported basipetally, repressing axillary bud outgrowth and maintaining apical dominance (reviewed in Müller & Leyser, 2011). Auxin transport thus provides a ‘top-down’ control of axillary branching in flowering plants. By contrast, the loss of regular branching patterns in excised shoots of Selaginella (Fig. 6) hints at a ‘bottom-up’ control of dichotomous branching in this species. Given that auxin transport is exclusively basipetal (Fig. 1b) and that exogenous auxin cannot ameliorate the excision-mediated disruption of shoot branching (Fig. 6a–c), it seems unlikely that auxin is a regulator of dichotomous shoot branching.
In Arabidopsis, lateral root development is promoted by auxin and inhibited by cytokinin (Laplaze et al., 2007). Similarly, exogenous auxin promotes and cytokinin inhibits the dichotomous branching of Selaginella roots (Fig. 6c,e). From a developmental perspective, the dichotomous branching of Selaginella root tips is fundamentally different from endogenous lateral root formation in angiosperms, yet these data suggest that antagonistic roles of auxin and cytokinin may underpin both mechanisms.
Conserved role of PAT in boundary formation at the shoot apex
Apices of Selaginella are bilaterally symmetrical with the long edge of the apex parallel to the dorsiventral axis. A pair of apical initials ultimately gives rise to pairs of dorsal and ventral leaves that are normally produced on either side of this axis (Harrison et al., 2007). In plants treated with NPA, the shoot apex loses this morphology and leaves are produced from broad regions of the apex, crossing the former dorsiventral axis. Each of these large leaves encompasses the zone normally occupied by one dorsal and one ventral leaf, plus the associated stem tissue. Boundaries defining dorsiventral patterning and leaf phyllotaxy, therefore, fail to be delimited when PAT is inhibited (Fig. 3c). Similar effects have been reported in seed plants, in which multicellular, layered meristems pattern phyllotaxy. For example, spruce embryos treated with NPA grow with fused cotyledons and perturbed meristem patterning (Larsson et al., 2008), and Brassica embryos treated in a similar way phenotypically resemble the embryos of Arabidopsis pin-1 mutants which have fused cotyledons (Liu et al., 1993). In combination, these observations suggest that the role of auxin in maintaining the boundaries between meristem and leaf zones in the apex is conserved between lycophytes and seed plants.
Distinct roles for auxin in the initiation of leaves in lycophytes and euphyllophytes
Arabidopsis inflorescences deficient in the auxin efflux carrier protein PIN1, and tomato shoots cultured on auxin efflux inhibitors, produce shoots lacking leaves (Reinhardt et al., 2000, 2003). Thus, PAT is essential for leaf initiation in angiosperms, but not for shoot meristem maintenance. By contrast, leaves are formed in NPA-treated Selaginella plants, but PAT inhibition ultimately terminates shoot growth. PAT is thus required for proliferation of the two apical initials in the Selaginella apex, but not for leaf initiation, suggesting that different mechanisms regulate shoot growth in lycophytes.
In angiosperms, the failure to initiate leaves when PAT is inhibited can be overcome by the addition of high concentrations of auxin together with NPA. In these circumstances, a greater number of cells than normal are recruited to form leaves, consistent with the idea that auxin maxima at the apex determine leaf positions (Reinhardt et al., 2000). Paradoxically, treatments with NPA and NAA in Selaginella reduce the number of cells recruited into leaves and hence restore normal dorsiventral patterning and leaf positioning (Fig. 3). As such, we suggest that the disruption of phyllotaxis in NPA-treated Selaginella results from perturbed boundary formation, as opposed to a failure to initiate leaves per se (Reinhardt et al., 2003; Lee et al., 2009). By extension, these results imply that auxin does not play a direct role in leaf initiation in Selaginella.
Although the addition of auxin negates the effects of inhibited PAT on dorsiventrality in the shoot of Selaginella, it does not prevent the termination of shoot growth. The morphology of the terminated shoot, however, differs in the presence and absence of auxin. In NPA alone, cells in the apex are all recruited into leaves, whereas, in NPA plus NAA, apices retain normal morphology, but the apical initials stop proliferating. To explain these results, we propose that meristematic activity is dependent on a differential gradient of auxin between the apical cells and their derivatives. This gradient can be perturbed when auxin is either too high (NPA and NAA) or too low (NPA) in the apical cells. As such, both treatments lead to meristematic arrest.
Conserved role for PAT in vascular differentiation, but not in venation patterns
As in angiosperms, the inhibition of PAT in Selaginella results in disrupted leaf venation (Fig. 2; Mattsson et al., 1999; Sieburth, 1999; Scarpella et al., 2006). In Arabidopsis and other angiosperms, the addition of PAT inhibitors results in thicker veins, isolated tracheids and leaf traces that do not connect with the stem vasculature (Mattsson et al., 1999; Sieburth, 1999). In a similar manner, PAT inhibition in Selaginella results in a thicker vein comprising an increased number of tracheids (Fig. 2, Table 1). However, although NPA-treated Arabidopsis leaves display an increase in venation at leaf margins and a reduction in venation more basally (Mattsson et al., 1999; Sieburth, 1999), the single vein in Selaginella leaves is thicker at the leaf base and does not reach the margin at the leaf tip. Mattsson et al. (1999) noted that the perturbed venation pattern in Arabidopsis suggests that a vascular inducing signal emanates from the apex of the leaf, a suggestion supported by the tracking of auxin responses in NPA-treated leaves (Scarpella et al., 2006). In this context, despite basipetal auxin transport through the stem, leaf venation in Selaginella may be patterned by transport from the stem towards the leaf apex. This scenario predicts that, with NPA treatment, auxin would become increasingly confined to the stem vasculature and would, in turn, promote cell expansion in the procambium. Consistent with this hypothesis, tracheid diameters are larger in NPA-treated stems and leaves than in untreated or DMSO-treated plants, and leaf veins do not extend to the leaf tip (Fig. 2). Taken together, these data suggest that vascular tissue development, but not venation patterning, are similarly regulated by PAT in lycophytes and euphyllophytes.