The Role of PIN Auxin Efflux Carriers in Polar Auxin Transport and Accumulation and Their Effect on Shaping Maize Development

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1 REVIEW ARTICLE The Role of PIN Auxin Efflux Carriers in Polar Auxin Transport and Accumulation and Their Effect on Shaping Maize Development Cristian Forestan 1 and Serena Varotto Department of Environmental Agronomy and Crop Science University of Padova, Viale dell Università 16, Legnaro (PD), Italy ABSTRACT In plants, proper seed development and the continuing post-embryonic organogenesis both require that different cell types are correctly differentiated in response to internal and external stimuli. Among internal stimuli, plant hormones and particularly auxin and its polar transport (PAT) have been shown to regulate a multitude of plant physiological processes during vegetative and reproductive development. Although our current auxin knowledge is almost based on the results from researches on the eudicot Arabidopsis thaliana, during the last few years, many studies tried to transfer this knowledge from model to crop species, maize in particular. Applications of auxin transport inhibitors, mutant characterization, and molecular and cell biology approaches, facilitated by the sequencing of the maize genome, allowed the identification of genes involved in auxin metabolism, signaling, and particularly in polar auxin transport. PIN auxin efflux carriers have been shown to play an essential role in regulating PAT during both seed and post-embryonic development in maize. In this review, we provide a summary of the recent findings on PIN-mediated polar auxin transport during maize development. Similarities and differences between maize and Arabidopsis are analyzed and discussed, also considering that their different plant architecture depends on the differentiation of structures whose development is controlled by auxins. Key words: auxin; PIN-formed; auxin efflux carrier; PAT; Polar Auxin Transport; kernel development; meristem; organogenesis; Zea mays. INTRODUCTION The developmental program of multicellular organisms implies a continuous exchange of molecular information among neighboring cells. Proper seed development requires the coordinated expression of embryo and endosperm genes and relies on the interaction between the two seed compartments and between the seed and maternal tissues (Bommert and Werr, 2001). Similarly, the continuing plant post-embryonic organogenesis, which relies on groups of undifferentiated meristematic cells, requires that different cell types are correctly differentiated, in time and space, inside the developing organism (Benkova and Bielach, 2010; Bohn-Courseau, 2010; De Smet and Beeckman, 2011). This plastic growth and adaptability to the external surrounding environment are mainly mediated by plant hormones and are fundamental for the plant s reproductive success. Auxin, and in particular auxin asymmetric distributions, have been shown to regulate a multitude of physiological and developmental processes during vegetative and reproductive development: at cellular level, auxins are known to regulate basic growth processes such as cell division and cell elongation (Campanoni and Nick, 2005; Perrot-Rechenmann, 2010), while an apparently ever-expanding list of processes in which auxin is involved includes: embryogenesis (Friml et al., 2003; Cheng et al., 2007; De Smet and Jurgens, 2007; Stepanova et al., 2008), all types of organogenesis (Reinhardt et al., 2000; Casimiro et al., 2001; Benkova et al., 2003; Reinhardt et al., 2003; Cheng et al., 2006, 2007; De Smet et al., 2007; Dubrovsky et al., 2008), root meristem maintenance (Sabatini et al., 1999; Kerk et al., 2000; Friml and Palme, 2002; Friml et al., 2002a; Blilou et al., 2005; Cheng et al., 2007; Yamada et al., 2009), vascular tissue differentiation (Mattsson et al., 1999, 2003; Cheng et al., 2006; Scarpella et al., 2006; Stepanova et al., 2008), hypocotyl and root elongation (Jensen et al., 1998; Tao et al., 2008; Yamada et al., 2009), apical hook formation (Lehman et al., 1996; Vandenbussche et al., 2010; Zadnikova et al., 1 To whom correspondence should be addressed. cristian.forestan@ unipd.it, tel , fax ª The Author Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS. doi: /mp/ssr103 Received 3 August 2011; accepted 16 November 2011

2 788 Forestan & Varotto 2010), apical dominance (Leyser, 2005; Stepanova et al., 2008), fruit ripening (Ellis et al., 2005), and growth responses to environmental stimuli such as light or gravity (Marchant et al., 1999; Friml et al., 2002b; Friml, 2003; Blakeslee et al., 2004; Kimura and Kagawa, 2006; Palme et al., 2006). Auxin biological function is carried out by the strict coordination of three complex processes: auxin metabolism, auxin transport, and auxin signaling. Auxin is synthesized in many plant tissues throughout several different pathways (Normanly, 2010; Zhao, 2010): the indole-3-pyruvic acid (IPA) pathway has been very recently completely defined, resulting in the main IAA biosynthetic pathway in Arabidopsis (Mashiguchi et al., 2011; Won et al., 2011). Local auxin measurements together with functional characterization of mutants impaired in auxin synthesis revealed the important role of localized auxin biosynthesis in specific developmental processes in different species (Gallavotti et al., 2008a; Normanly, 2010; Zhao, 2010; Phillips et al., 2011). In addition to the localized biosynthesis and metabolism, auxin directional transport is crucial in building up the spatial auxin maxima and minima (also called auxin gradients) that provide the positional and directional information required for the coordination of plant development. The contribution of this polar transport, a peculiar property of auxin, in creating the hormone gradients that control the above list of developmental processes has been extensively reviewed in the last years (Tanaka et al., 2006; Vieten et al., 2007; Zazimalova et al., 2007; Petrasek and Friml, 2009; Vanneste and Friml, 2009; Zazimalova et al., 2010; and Peer et al., 2011 represent a short and incomplete list of interesting papers for those who wish to learn more about the field of auxin-mediated development). THE AUXIN FAIRYTALE: MOVING FROM GRASSES TO ARABIDOPSIS AND BACK Although our current knowledge of auxin and its mechanisms of action is almost exclusively based on the results from studies on Arabidopsis thaliana (a dicotyledonous species) during the last 20 years, the auxin story began more than a century ago with the work of Charles and Francis Darwin on tropic response in grasses. They showed that unilateral light is perceived by coleoptile tips of etiolated canary grass seedlings and the asymmetric stimulus is then transmitted to the lower region beneath the tip, which responds with differential cellular growth on the shade side, causing the curvature of the coleoptile. Based on their bending experiments, they hypothesized the existence of a mobile plant growth-regulating substance (auxin, from the Greek verb auxano, which means to grow, to expand ) that mediates phototropism curvature in grass coleoptiles (Darwin and Darwin, 1881). In , Frits Went implemented the Darwin experiments, developing the Avena coleoptiles curvature test later used by Kenneth Thimann to identify the nature of this growth-promoting molecule (Went and Thimann, 1937). Indole-3-acetic acid (IAA), the main natural auxin, was initially identified and purified from fungal cultures (Kögl et al., 1934; Thimann and Koepfli, 1935) and only 10 years later eventually from the first plant, Zea mays (Haagen-Smit et al., 1946). Once identified as the main actor, the IAA script during plant development became the goal of researchers. Based on the IAA chemical properties and on in planta physiological data derived from [14C]-IAA radiolabeling assays, in the mid-1970s, the first model to explain polar auxin transport, the Chemiosmotic Hypothesis, was formulated (Rubery and Sheldrake, 1974; Raven, 1975). Since PAT is unidirectional, saturable, energy- and protein synthesis-dependent, the existence of specific auxin influx and efflux carriers catalyzing the cell-to-cell auxin transport was predicted. Even though no molecular data were available at that time, the model was revealed to be very accurate and has since been validated using specific chemical influx or efflux inhibitors and by the isolation of auxin carriers. Indeed, several classes of genes required for auxin influx and efflux have been identified in Arabidopsis thaliana, mainly by molecular genetics approaches and mutant characterizations. Starting from the Arabidopsis thaliana pin-formed1 (pin1) mutant, which shows defective organ initiation and phyllotaxy, resulting in naked, needle-like inflorescence stems, the first PIN auxin efflux carrier member was identified (Okada et al., 1991; Galweiler et al., 1998). Subsequently, seven other genes similar to PIN1 were identified in Arabidopsis. PIN3, PIN4, and PIN7 are required for tropic growth responses, root meristem patterning, and early embryo development, respectively (Friml et al., 2002a, 2002b, 2003), and a mutation in the PIN2 gene, concurrently published as ETHYLENE INSENSITIVE ROOT 1 (EIR1) and AGRAVITROPIC 1 (AGR1), causes a defect in root gravitropism (Chen et al., 1998; Luschnig et al., 1998; Muller et al., 1998; Utsuno et al., 1998). The above-mentioned PIN genes encode proteins that can be asymmetrically distributed at the cell plasma-membrane and function in auxin redistribution/accumulation, while the ER-localized PIN5 and PIN8 transporters have opposite roles in the regulation of intracellular IAA homeostasis (Mravec et al., 2009). Finally, PIN6, the last member of the Arabidopsis gene family, has not yet been functionally characterized. Similarly, the auxin1 (aux1) mutant allowed the isolation of ageneputativelyencodinganauxininfluxcarrier(bennettetal., 1996). In addition, it was demonstrated that a group of ATPbinding cassette transporters (ABCB) of the multidrug-resistant class (MDR) of P-glycoproteins (PGP) are involved in PAT, directly catalyzing both auxin influx and efflux (Blakeslee et al., 2005a, 2005b; Geisler et al., 2005; Geisler and Murphy, 2006; Yang and Murphy, 2009), and interacting at the plasma membrane with PIN proteins (Blakeslee et al., 2007; Mravec et al., 2008; Titapiwatanakun et al., 2009; Yang and Murphy, 2009). In Arabidopsis, many works during the last few years highlighted the crucial role of PINs in determining the direction of auxin transport and, in turn, in creating the auxin gradients that are required for development. Furthermore, many gene products and molecular mechanisms involved in the regulation of PIN polar targeting to the plasma-membrane were identified (see Grunewald and Friml, 2010; Richter et al., 2010 for reviews).

3 Forestan & Varotto 789 Undoubtedly, the introduction of Arabidopsis thaliana as a model and reference organism and the subsequent availability of many molecular and cell biology tools have concentrated auxin research on this species for many years. However, during the last decade, the exigency to move from model organisms to crop species gave a new impulse to auxin research in monocots, maize in particular. Mutant characterization, molecular and cell biology approaches, together with the sequencing of the maize genome, allowed the identification of genes involved in auxin metabolism, signaling, and particularly in polar auxin transport. In this review, we will provide a summary of the recent findings on PIN-mediated polar auxin transport in maize during pre- and post-embryonic development. POLAR AUXIN TRANSPORT IN MAIZE: PHYSIOLOGICAL AND BIOCHEMICAL EVIDENCE The first indications of the role of polar auxin transport in controlling maize (and more in general monocots) development derived from mutant characterizations and applications of PAT inhibitors such as 1-N-Naphthylphthalamic Acid (NPA; Katekar and Geissler, 1980; Rubery, 1990; Morris, 2000; Petrasek et al., 2003). The semaphore1 (sem1) maize mutant shows a significant reduction in basipetal auxin transport in the shoot that is accompanied by a reduction in plant height, defects in embryo, endosperm, leaves, lateral roots, and pollen development (Scanlon et al., 2002). A similar correlation between decrease of basipetal auxin transport and dwarf phenotype can be observed in brachytic2 (br2) mutant plants, which also show vasculature defects (Multani et al., 2003; Pilu et al., 2007), while, in the defective endosperm B-18 (de18) mutantplants,alackof IAA accumulation causes a reduction of dry matter accumulation in kernels (Torti et al., 1986). Similar defects in embryo and endosperm development (Forestan et al., 2010) and in leaf initiation and growth (Tsiantis et al., 1999; Scanlon, 2003) have been reported after treating plants with NPA. Application of PAT inhibitors totally blocks axillary meristem initiation in maize inflorescences (Wu and McSteen, 2007), producing a phenotype very similar to the needle-like inflorescence stem of Arabidopsis pin1 mutant (Okada et al., 1991) and confirming the conservation of the pivotal role of PAT in controlling axillary meristem and organ initiation in di- and monocots. Similar pin1-like phenotypes also characterize the inflorescences of barren inflorescence 2 (bif2), barren stalk 1 (ba1), vanishing tassel 2 (vt2), sparse inflorescence 1 (sp1), and developmental disaster 1 (dvd1) mutants that will be further discussed in the following sections. In maize, a transposon tagging approach allowed cloning of the BR2 gene that encodes for type B adenosine triphosphate (ATP)-binding cassette transporters (ABCB) of the multidrugresistant (MDR) class of P-glycoproteins (PGPs), responsible for IAA transport in the stem intercalary meristems and in root apexes and considered the ortholog of the Arabidopsis gene ABCB1/PGP1 (Multani et al., 2003; Knoller et al., 2010; McLamore et al., 2010). However, unlike in Arabidopsis, forward genetics approaches have been so far unsuccessful in identifying efflux and influx transporters orthologous to PIN and AUX carriers in maize. THE MAIZE PIN FAMILY OF AUXIN EFFLUX CARRIERS The first monocot gene belonging to the PIN family (Rice EIR1 Homolog; OsREH1) was identified in rice by database searching for sequences homologous to the EIR1/AtPIN2 gene (Luschnig et al., 1998). However, later works and phylogenetic analysis revealed that this gene represents one of the four PIN1 rice orthologs (Paponov et al., 2005; Xu et al., 2005; Wang et al., 2009), indicating that the monocot PIN family is wider and more divergent than the dicot one. Rice and sorghum PIN proteins that do not cluster with any dicot sequence have also been identified, suggesting also the presence of monocot-specific PIN proteins (Paponov et al., 2005; Xu et al., 2005; Wang et al., 2009; Shen et al., 2010). Similarly, three maize PIN1 genes (called ZmPIN1a, ZmPIN1b, and ZmPIN1c) have so far been characterized (Carraro et al., 2006; Forestan et al., 2010), while a fourth has recently been identified (ZmPIN1d, Forestan et al., unpublished results). The amino acid identity among the putative maize proteins is about 85%, while the AtPIN1 protein exhibits an amino acid identity of about 70% with maize PIN1s. Complementation assays demonstrate that ZmPIN1a can partially rescue the Arabidopsis pin1 mutant, suggesting that it may play a role in PAT (Gallavotti et al., 2008b). To better understand the role of PIN-mediated auxin transport during maize development, we are now isolating all the members of the maize PIN family. In addition to the four PIN1s, a gene closely related to AtPIN2 (ZmPIN2), three putative orthologs of AtPIN5 (ZmPIN5a, ZmPIN5b, and ZmPIN5c) and the ortholog of AtPIN8 (ZmPIN8) have been isolated. The identification in maize, as in rice and sorghum, of ZmPIN9, ZmPIN10a, and ZmPIN10b, three monocot-specific genes (Forestan et al., unpublished results), confirms the widening of the monocot PIN family in comparison with the dicot one. ROLE OF ZmPIN1 DURING MAIZE KERNEL DEVELOPMENT Polar auxin transport, as previously described, plays a major role in pre- and post-embryonic development in Arabidopsis thaliana. Development, anatomy, and morphology of monocots and dicots differ considerably (McSteen et al., 2000; Chandler et al., 2008; McSteen, 2010): could differences in IAA polar transport mechanisms, regulation, and function account for the peculiarities of monocot patterning (see Figure 1, discussed also in McSteen, 2010)? ZmPIN1s and Maize Embryogenesis In Arabidopsis embryo, the coordination of PIN1, PIN3, PIN4, and PIN7 expression and cell membrane polarization creates auxin gradients that are fundamental for the correct

4 790 Forestan & Varotto Figure 1. Diagrammatic Overview of Maize Endosperm, Embryo, and Inflorescence Development with Relative Models for ZmPIN1- Mediated Auxin Transport. Pictures are not drawn to scale. (A) A diagrammatic representation of a developing maize kernel in longitudinal section showing the embryo and the different cellular domains of the cereal endosperm. ZmPIN1 transcripts and proteins are expressed in both Basal Endosperm Transfer Layer (BETL) and Embryo Surrounding Region (ESR) cells, while auxin also accumulates in the aleurone layer cells. (B) Schematic representation showing medial longitudinal sections of a proembryo, transition, coleoptilar, and L1 and L2 stage maize embryos. Arrows indicate the polar auxin fluxes deduced by Forestan et al. (2010). First zygote asymmetrical cell division results in a small apical and a large basal cell. At the pro-embryo stage, the basal cell forms the suspensor (su), which later degenerates, while a series of cell divisions of the small apical cell, in unpredictable planes, gives rise to the embryo proper (ep). During the transition stage, adaxial/abaxial polarity is established by the outgrowth of the scutellum (scu) at the abaxial side of the embryo and, at the late transition stage, the shoot apical meristem (SAM) is evident as a group of cytoplasm-rich cells on the adaxial side. Later development, at the coleoptilar stage, the coleoptile (col) becomes histologically evident above the SAM, which starts its morphogenetic phase differentiating five to six leaf primordia (P1 P6) before seed dormancy. The root apical meristem (RAM) is protected by the coleorhiza and the shoot root axis forms an oblique angle relative to the apical/basal polarity of the proembryo. (C) Schematic representation of a maize male inflorescence. The inflorescence meristem (IM) initiates files of axillary meristems (Spikelet Pair Meristem, SPM), depicted as small domes. Each SPM produces two Spikelet Meristems (SM); later, each SM will branch to form two floral meristems for a total of four flowers from a single axillary meristem. ZmPIN1-mediated auxin fluxes hypothesized by Carraro et al. (2006) are depicted with arrows. patterning (Friml et al., 2003; De Smet and Jurgens, 2007). Recent work demonstrated that, despite some differences, ZmPIN1-mediated PAT also plays a fundamental role in seed development in maize (Forestan et al., 2010), in which embryos develop with a more elaborate architecture and endosperm persists at later stages of seed development (Figure 1B; see Bommert and Werr (2001) for a review of kernel development in maize). ZmPIN1a, ZmPIN1b, and ZmPIN1c are indeed upregulated after maize embryosac double fertilization, showing overlapping expression domains in different stages of kernel development. Protein localization studies, auxin accumulation patterns, and the effect of auxin polar transport inhibition indicated that ZmPIN1-mediated auxin transport is associated with cell and tissue differentiation during maize embryogenesis and endosperm development (Forestan et al., 2010). During embryogenesis, PIN1 protein localization and the consequent auxin gradients correlate with apical/basal organization of the pro-embryo and suspensor, with protoderm differentiation, and with the shift from radial to bilateral symmetry following the differentiation of the scutellum on the adaxial side and the shoot apical meristem on the abaxial one. Furthermore, while a switch from an apical to a basal polarization of ZmPIN1 proteins marks the differentiation of the scutellum, the maize single cotyledon, the establishment of two continuous basipetal auxin fluxes from the scutellum and the SAM, respectively, allows the differentiation of the embryonic root. Finally, during the SAM morphogenetic phase, ZmPIN1 proteins localize in the central sub-apical region of the meristem marking the incipient leaf primordia and the differentiation site of inner vascular tissues (Forestan et al., 2010). The involvement of PAT in these developmental processes is confirmed by the effects of NPA treatments on developing kernels: since primordia differentiation and scutellum development requires PIN1-mediated auxin transport, these processes are strongly impaired by NPA applications, which result in the blocking of leaf primordia initiation and in the complete

5 Forestan & Varotto 791 loss of normal scutellum morphology and symmetry (Forestan et al., 2010). These results are also in agreement with previous studies on the distribution of radio-labeled IAA and NPA application in wheat embryos (Fischer-Iglesias et al., 2001). Indeed, the authors demonstrated that the shift from the globular to transition-stage embryos was correlated with a redistribution of auxin in the embryo. Observations of photolytically fixed [ 3 H],5-N 3 IAA and NPA treatments showed that globular embryos are characterized by a NPA-insensitive not polarized auxin redistribution, while, during the transition stage, a polar, NPA-sensitive auxin transport towards the scutellum was inferred (Fischer-Iglesias et al., 2001). Although this approach was not able to reveal the reverse auxin flux from scutellum tip to the root pole that was highlighted in maize, NPA application causes aberrations and loss of symmetry in wheat scutellum like those observed in maize (Fischer-Iglesias et al., 2001; Forestan et al., 2010). The comparison of the first results obtained in maize with the findings from Arabidopsis indicates that the PIN1- mediated auxin transport and accumulation during embryogenesis shares conserved features between mono- and eudicots. However, in comparison to Arabidopsis in which a single PIN1 efflux carrier is expressed, the more complex architecture of the maize embryo is accompanied by the expression of multiple PIN1-like sequences showing more elaborate localization patterns at both transcript and protein levels. Alternatively, since no PIN3, PIN4, PIN7 genes are present in maize genome (Forestan et al., unpublished results), the three maize PIN1 genes might have acquired a certain degree of subfunctionalization that makes them do the work that PIN3, PIN4, and PIN7 proteins do in Arabidopsis embryos. ZmPIN1s and Maize Endosperm Development Concomitantly, ZmPIN1 genes are expressed in maize endosperm, being involved in the early events of endosperm domain differentiation (Forestan et al., 2010). ZmPIN1 proteins initially define the formation of anticlinal membranes during the cellularization phase and later in development mark two of the four domains: the basal endosperm layer (BETL) and embryo surrounding region (ESR; Figure 1A). In these cellular domains, ZmPIN1 proteins show opposite sub-cellular localization: in BETL cells, ZmPIN1 proteins are localized in all the cell membranes, without any detectable polarization, while, in the ESR domain, the proteins are exclusively localized in intracellular vesicles and in endomembrane systems. It is remarkable that the cells of both these domains show high auxin accumulation. This indicates that these two opposite localization patterns, together with the possible role of additional not yet characterized auxin transporters, result in auxin accumulation. Interestingly, NPA applications do not alter BETL or ESR differentiation, while defects have been observed in the aleurone layer. Auxin also accumulates in the aleurone layer (but not in the starchy endosperm), where PIN1 is not expressed. It is also interesting that NPA treatments cause the formation of a pluristratified aleurone with ectopic PIN1 expression (Forestan et al., 2010). Altogether, the results on ZmPIN1s localization during endosperm development indicate that the ZmPIN1-mediated transport of auxin is related to cellular differentiation and open the doors to a series of new questions on the peculiar localization and biological function of ZmPIN1 proteins in ESR cells and in the definition of the aleurone layer identity. While the subcellular localization patterns of PIN proteins in maize will be discussed in Regulation of ZmPIN1 Subcellular Localization, below, it is worth speculating here about how two adjacent cells of the endosperm can differentiate into an aleurone cell and a starchy endosperm cell, respectively. Does auxin accumulation have a function in these neighborly relations? The aleurone or the starchy endosperm cell fates are continuously specified and maintained by positional cues throughout endosperm development (Becraft and Asuncion-Crabb, 2000; Jin et al., 2000; Becraft, 2001) and, despite the positional cue(s) having not yet been identified, the auxin accumulation that splits aleurone and starchy endosperm definitely points to the fundamental role of IAA also in this developmental process. Similarly, auxin accumulation is essential for correct transfer layer differentiation and functioning. In fact, defective endosperm-b18 (de18) mutant kernels present defective transfer cells that, failing to express ZmPIN1 and accumulate IAA, are less active in nutrient import into the developing endosperm and so accumulate less dry matter in kernels (Torti et al., 1986; Forestan et al., 2010). Since IAA accumulation in the aleurone layer does not involve ZmPIN1 proteins, characterization of the maize PIN family and identification of AUX/LAX and ABCB families of auxin carriers are necessary to clearly unravel PAT functions in regulating and coordinating kernel development. ROLE OF ZmPIN1 DURING MAIZE POST-EMBRYONIC DEVELOPMENT The previously described auxin transport-defective mutants br2 and sem1 show as common features a dwarf phenotype and vasculature defects, while NPA applications cause the blocking of leaf and axillary meristem initiation (Scanlon et al., 2002; Multani et al., 2003; Scanlon, 2003; Wu and McSteen, 2007): interestingly, these are the developmental processes marked by ZmPIN1 expression (Carraro et al., 2006; Gallavotti et al., 2008b). ZmPIN1 and Maize Vegetative Development In maize seedlings and young plants, ZmPIN1 transcripts and proteins are detectable in the coleoptile, in the inner central region of the SAM, in the lateral outgrowing primordia, in the tips of young leaves, and in vascular bundles of the apex and leaves (Carraro et al., 2006; Gallavotti et al., 2008b; Nishimura et al., 2009). After germination, IAA polar transport mediated by basally localized ZmPIN1 proteins in coleoptile cells, together with the localized IAA biosynthesis in the coleoptile tip, are responsible for coleoptile elongation and tropism, as hypothesized more than a century ago by the Darwin experiments

6 792 Forestan & Varotto (Nishimura et al., 2009; Nishimura and Koshiba, 2010). In the central region of the SAM corpus, proteins are localized at the basal cell plasma-membrane, although cells with ZmPIN1 proteins localized on apical or lateral membranes are also detectable. In provascular tissues of outgrowing primordia and of newly formed leaves, PIN1 proteins are clearly localized on basal cell membranes, suggesting basipetal auxin transport and the subsequent canalization that allows vascular tissues differentiation (Carraro et al., 2006). Localization of the ZmPIN1s proteins appears more controversial in the L1 outer layer of the SAM: using a monoclonal antibody against AtPIN1 that recognizes (at least) three maize PIN1s (ZmPIN1a, ZmPIN1b, and ZmPIN1c), proteins are not detectable in the SAM L1 layer, except where a new leaf primordium is initiated (Carraro et al., 2006). However, a pzmpin1a:zm- PIN1a-YFP reporter line shows the expression in the L1 layer of the shoot apical meristem and in the epidermis of developing primordia (Gallavotti et al., 2008b). It is worth noting that in situ hybridization carried out with probes specific for ZmPIN1a, ZmPIN1b, and ZmPIN1c, respectively, does not show any hybridization signal in the external epidermal layer of the shoot meristem (Carraro et al., 2006; Forestan et al., 2010). Since similar controversial results have been observed in male and female inflorescence meristems, the implication of ZmPIN1 presence/ absence in L1 will be discussed in the next section. ZmPIN1 and Maize Reproductive Development: A Controversial Model After the transition stage, maize SAM differentiates into a branched male inflorescence, the tassel, while the female inflorescences develop at the leaf axils, usually six to seven nodes below the tassel. In both male and female inflorescences, several axillary meristems are formed prior to flower formation (Figure 1C; see McSteen et al. (2000) for a review of maize inflorescence development). As in the vegetative SAM, ZmPIN1 transcripts and proteins mark the central region of the inflorescence meristems, lateral developing primordia, and provascular tissues (Carraro et al., 2006; Gallavotti et al., 2008b). The antibody-based approach shows ZmPIN1 in the corpus of the inflorescence meristem, while the L1/epidermal outer layer is labeled exclusively in the sites where a new organ (branch meristem, spikelet pair meristem, floral meristem, glume, or stamen) starts to differentiate (Carraro et al., 2006). Here, ZmPIN1 proteins are also localized in the cells corresponding to the pro-vascular tissues in the central axis of the outgrowing primordia. Auxin efflux carriers are localized in both lateral and basal membranes of the external cell layer, while, in the inner tissues, only basal membranes are labeled, suggesting basipetal auxin transport towards the organ or stem base (Carraro et al., 2006). According to the canalization hypothesis (Rolland-Lagan and Prusinkiewicz, 2005), this tip-to-base auxin transport allows auxin-mediated vascular tissue patterning. Although this basal ZmPIN1 localization on provascular cells resembles the pattern observed in Arabidopsis, the absence of the PIN1 apical localization on the L1 layer seems to suggest that incipient lateral organs might be sites of auxin biosynthesis since early developmental stages. In Arabidopsis, lateral organ position is determined by an auxin maximum created by acropetal PIN1-mediated auxin transport through the epidermis and the outermost L1 meristem cell layer (Benkova et al., 2003; Reinhardt et al., 2003; Heisler et al., 2005). In maize, ZmPIN1s localization patterns in the meristem corpus seem to point to the importance of internal tissues in auxin redistribution (Carraro et al., 2006; Forestan et al., 2010). As previously reported, different results have been obtained with the ZmPIN1a YFP reporter line developed by Dave Jackson and co-workers (Gallavotti et al., 2008b). Indeed, the fusion protein is detectable in the L1 layer of every meristem and in the epidermis of developing primordia, while no fluorescent signal was observed in the meristem sub-apical region, suggesting that, in maize, the formation of all axillary meristems and lateral organs conforms to the accepted model based on observations from Arabidopsis (Gallavotti et al., 2008b). In situ hybridization assays and the comparison of L1 layer versus corpus cells transcriptome by laser capture micro-dissection coupled with microarray analysis showed that ZmPIN1s transcripts are excluded from the meristem L1 outer layer (Carraro et al., 2006; Ohtsu et al., 2007; Forestan et al., 2010; truman.edu/). Further experiments are therefore necessary to shed light on the auxin-mediated patterning in maize, taking into account that ZmPIN1 proteins are only the tip of the iceberg of the highly regulated network regulating auxin-mediated morphogenesis and that many pieces of the puzzle are still missing. Only when all the maize auxin carriers have been identified and their expression analyzed in detail will it be possible to formulate a comprehensive and solid model, highlighting the auxin redistribution mechanisms conserved or diversified between monocots and dicots. Organ Initiation and Maize Branching: A Multiplayer Game Many mutants affected in inflorescence branching have been identified and some of them also functionally characterized. barren inflorescences2 (bif2) mutants present extremely reduced branching of the male inflorescences (McSteen and Hake, 2001), a phenotype very similar to that caused by NPA applications (Wu and McSteen, 2007), suggesting an involvement of BIF2 in PAT, even though vegetative development is normal in bif2 mutant plants. The BIF2 gene encodes a coortholog of the Arabidopsis PINOID (PID) protein (McSteen et al., 2007), a Ser/Thr protein kinase that regulates the subcellular localization of PIN proteins by direct phosphorylation of specific residues (Friml et al., 2004; Michniewicz et al., 2007). BIF2 phosphorylates in vitro ZmPIN1a and ZmPIN1 proteins localization is altered in bif2 mutants, suggesting that, as in Arabidopsis, correct PIN1 polarization is required for the initiation of axillary meristems and lateral organs (Carraro et al., 2006; McSteen et al., 2007; Skirpan et al., 2009). However, the nuclear localization of BIF2, compared to the cell periphery localization of PID1, and its interaction with the nuclear localized

7 Forestan & Varotto 793 basic helix-loop-helix transcription factor BARREN STALK1 (BA1) implicate additional roles in development for BIF2. ba1 mutant plants do not produce axillary meristem during inflorescence development and, although the role of BA1 is not fully understood, it is likely to act either upstream or downstream of PAT in auxin-mediated axillary meristem initiation (Ritter et al., 2002; Gallavotti et al., 2004, 2008b). Upstream of the auxin transport machinery, auxin biosynthesis likely plays a fundamental role in axillary meristem and organ initiation in maize. The maize sparse inflorescence1 (spi1) mutant has defects in the initiation of axillary meristems and lateral organs during vegetative and inflorescence development in maize (Gallavotti et al., 2008a). Positional cloning showed that spi1 encodes a flavin monooxygenase similar to the YUCCA (YUC) genes of Arabidopsis, which are involved in local auxin biosynthesis in various plant tissues. SPI1 transcripts are transiently expressed in a few cells of the two outermost meristem layers proximal to all newly emerging axillary meristems and lateral primordia at each stage of inflorescence development (Gallavotti et al., 2008a). The mutant phenotype and the expression patterns suggest that SPI1-mediated auxin biosynthesis is essential for regular maize inflorescence development, confirming the suggestion that auxin synthesis begins early in the incipient lateral organ. This also allows it to be speculated that local IAA accumulation at the level of incipient axillary meristems, shown by the DR5 auxin responsive promoter (Gallavotti et al., 2008b), can be due to auxin biosynthesis instead of the polar transport. Of course, the contribution of localized auxin biosynthesis in organ initiation is not restricted to inflorescence development: the vanishing tassel2 (vt2) mutant plants, which present a mutation in a tryptophan aminotransferase gene involved in auxin biosynthesis, show strong vegetative and reproductive defects, ranging from the reduced plant height due to the production of fewer leaves to a lack of axillary meristem initiation in the inflorescences (Phillips et al., 2011). Concluding, the available data indicate that auxin biosynthesis and transport function synergistically to regulate the formation of axillary meristems and lateral organs in maize. Only the complete elucidation of the role of several genes controlling branching in maize inflorescences, such as BA1, developmental disaster1 (Phillips et al., 2009), ramosa1 (Vollbrecht et al., 2005), ramosa2 (Bortiri et al., 2006), ramosa3 (Satoh-Nagasawa et al., 2006; Gallavotti et al., 2010), barren stalk fastigiate1 (Gallavotti et al., 2011), and many others (Veit et al., 1993) can add new details on the auxin network operating during branching and organ initiation in maize. REGULATION OF ZmPIN1 SUBCELLULAR LOCALIZATION The different ZmPIN1 protein sub-cellular localization patterns observed raise some questions about how the ZmPIN1 proteins are directed to distinct sides of the same scutellum cells or why the carriers are not targeted to the cell membrane in the ESR cellular domain, as usually occurs in other cell types. Furthermore, is this ZmPIN1s accumulation in the internal cellular compartments related to particular aspects of their function (Forestan and Varotto, 2010; Forestan et al., 2010)? Of course, understanding the molecular mechanisms controlling the subcellular dynamics of the auxin transport machinery in response to developmental and environmental stimuli is of outstanding importance for both auxin transport studies and the entire subject of cell and tissue polarity in plants. In Arabidopsis, the polarity of PIN localization is also controlled by direct phosphorylation of specific PIN residues: the serine/threonine protein kinase PINOID (PID) directly phosphorylates PIN proteins, marking them as apical cargo, while PIN basal localization is regulated by the dephosphorylation catalyzed by the trimeric serine threonine protein phosphatase 2A (PP2A; Friml et al., 2004; Michniewicz et al., 2007; Gao et al., 2008). Furthermore, Arabidopsis PIN proteins have been shown to constitutively cycle between the plasma membrane and internal pools (Robinson et al., 1999) and this cycling is controlled by GNOM (for PIN1 and PIN3) or AtSNX1 (for PIN2) proteins (Geldner et al., 2003; Jaillais et al., 2006; Kleine-Vehn et al., 2008; Richter et al., 2010). Mutagenesis experiments on the Arabidopsis PIN2 sequence showed that a mutation that changes the conserved Serine-97 to Glycine (pin2gly97) leads to the localization of pin2gly97 in intracellular compartments in plant and yeast cells, causing IAA intracellular accumulation (Petrasek et al., 2006). While the activity of BIF2, the maize PID ortholog, in phosphorylating ZmPIN1proteins and controlling their subcellular localization has been clearly shown (Carraro et al., 2006; McSteen et al., 2007), nothing is known about the endosome/plasmamembrane cycling regulatory network of maize PINs. Are the above-described mechanisms also conserved in maize and could they account for the cytoplasmic localization of ZmPIN1 proteins in the ESR? In maize, only the PIN1b protein presents the conserved Serine-97, and transient expressions of ZmPIN1::GFP fusion constructs in maize and tobacco protoplasts reveal that, while ZmPIN1b::GFP and ZmPIN1c::GFP fusion proteins are normally targeted to the plasma-membrane, ZmPIN1a::GFP proteins localize both at the plasma-membrane and in cytoplasmatic vesicles (Forestan and Varotto, 2010). Thus, these differences in ZmPIN1s amino acid sequences could result in different plasma membrane insertion abilities of the three proteins or, more likely, ZmPIN1a, ZmPIN1b, and ZmPIN1c could respond in different ways to a tissue-specific localization signal, resulting in different protein sub-cellular localizations. CONCLUDING REMARKS AND FUTURE PERSPECTIVES The combination of different approaches and methods has demonstrated the essential role of polar auxin transport in regulating growth and development in Zea mays. Although the biological functions of IAA are conserved in monocots, some class-specific differences and peculiarities have also been

8 794 Forestan & Varotto shown, particularly in the number of players and in the complexity of their regulatory network. Genes homologous to the Arabidopsis PIN are present in genomes throughout the plant kingdom, from the model moss Physcomitrella patens to all vascular plants (Paponov et al., 2005; Zazimalova et al., 2007; Krecek et al., 2009; Wang et al., 2009; Shen et al., 2010). Moreover, the relatively high amino acid identity among PIN proteins suggests that all the PIN genes diverged from a single ancestral sequence and at least four independent phylogenetic studies revealed that the monocot PIN family is wider and more divergent than the dicot one, with three or four genes homologous to one single Arabidopsis PIN gene along with the presence of monocot-specific PIN genes (Paponov et al., 2005; Krecek et al., 2009; Wang et al., 2009; Shen et al., 2010). Although only a few PIN sequences have so far been characterized in maize, the identification of four ZmPIN1 genes and the discovery that three loci encoding for putative ZmPIN5 sequences are present in the maize genome (Forestan et al., unpublished results) seems to corroborate this hypothesis. By now, it is widely accepted that maize genome undergoes a whole-genome duplication (Helentjaris et al., 1988; Gaut and Doebley, 1997; Schnable et al., 2009) and several maize genes have been found in duplicated chromosomal regions (Bodeau and Walbot, 1996; Nardmann et al., 2004; Nardmann and Werr, 2006). Further efforts are therefore necessary to understand the evolution of the maize PIN family, and the availability of the complete annotation of sequenced genomes of many mono- and dicot species will be very helpful for these investigations. In addition, only the identification and the expression analysis of all the PAT players (influx carriers and MDR/PGP transporters included) will make it possible to construct a complete road map of auxin fluxes and gradients during maize development. There are still many black holes, ranging from the PAT role in mediating the development and architecture of the complex maize root system, which includes primary root, seminal roots, crown roots, prop roots, and lateral roots, each type with different developmental programs and with different genetic regulation but all of them with auxin as common denominator (Li et al., 2011), to the presence of synergistic interactions between different classes of auxin carriers, as demonstrated in Arabidopsis for PIN and ABCB/MDR-PGP proteins (Blakeslee et al., 2007; Mravec et al., 2008). In addition to the ZmABCB1/BR2 gene described above, three orthologs of AtABCB19 (ZmABCB2, ZmABCB10_1, and ZmABCB10_2) have recently been identified (Knoller et al., 2010). Although the developmental role of these new auxin carriers has not yet been established, they might represent a good starting point to determine the possible interaction between maize ABCB and PIN proteins, unraveling the complex auxin transport network that mediates local asymmetric auxin distribution and triggers different cellular responses. Finally, these studies would be facilitated by the identification of the molecular mechanisms controlling PIN protein polar targeting and, of course, by the isolation/production of specific loss of function mutants. These mutants could provide the functional validation of the proposed model, confirming the initial speculation that the more elaborate pattern of maize development in comparison to Arabidopsis is accompanied by a more complex auxin regulatory network. 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