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Ontogeny of the sheathing leaf base in maize (Zea mays) Robyn Johnston, Samuel Leiboff and Michael J. Scanlon Department of Plant Biology, Cornell University, Ithaca, NY 14853, USA
Summary Author for correspondence: Michael J. Scanlon Tel: +1 607 254 1156 Email: [email protected] Received: 10 May 2014 Accepted: 23 July 2014
New Phytologist (2015) 205: 306–315 doi: 10.1111/nph.13010
Key words: auxin, disc of insertion (DOI), founder cells, leaf margin, maize (Zea mays), monocot.
Leaves develop from the shoot apical meristem (SAM) via recruitment of leaf founder cells. Unlike eudicots, most monocot leaves display parallel venation and sheathing bases wherein the margins overlap the stem. Here we utilized computed tomography (CT) imaging, localization of PIN-FORMED1 (PIN1) auxin transport proteins, and in situ hybridization of leaf developmental transcripts to analyze the ontogeny of monocot leaf morphology in maize (Zea mays). CT imaging of whole-mounted shoot apices illustrates the plastochron-specific stages during initiation of the basal sheath margins from the tubular disc of insertion (DOI). PIN1 localizations identify basipetal auxin transport in the SAM L1 layer at the site of leaf initiation, a process that continues reiteratively during later recruitment of lateral leaf domains. Refinement of these auxin transport domains results in multiple, parallel provascular strands within the initiating primordium. By contrast, auxin is transported from the L2 toward the L1 at the developing margins of the leaf sheath. Transcripts involved in organ boundary formation and dorsiventral patterning accumulate within the DOI, preceding the outgrowth of the overlapping margins of the sheathing leaf base. We suggest a model wherein sheathing bases and parallel veins are both patterned via the extended recruitment of lateral maize leaf domains from the SAM.
Introduction Leaves evolved multiple times within the land plants (Townsley & Sinha, 2012). Despite the abundance of leaf morphological diversity found in nature, all leaves develop from founder cells recruited from the periphery of a stem cell niche called the shoot apical meristem (SAM) (Kaplan, 2001). One of the earliest described molecular markers of leaf initiation is the downregulation of KNOTTED1-like homeobox (KNOX) protein accumulation in the founder cells of the incipient leaf (P0), a process that requires transport of the plant hormone auxin (Smith et al., 1992; Scanlon, 2003; Hay et al., 2006). Among the angiosperms, eudicot leaves typically have nonsheathing bases and branched venation. By contrast, most monocot leaves have parallel veins and a sheathing base that surrounds the stem at the node, with a prominent distal lamina or leaf blade (reviewed in Kaplan, 1973). Distinct patterns of KNOX down-regulation and leaf founder cell recruitment in monocot and eudicot SAMs correlate with morphological differences in mature leaves within these clades. In eudicots, local polarized PIN1 expression in the L1 layer of the SAM peripheral zone creates an auxin maximum at the incipient (P0) leaf primordium (Reinhardt et al., 2003b). Subsequent alterations in PIN1 expression and localization at these convergence points redirect the flow of auxin from the L1 to internal cell layers, creating a canalized pattern of auxin flow that will pattern the 306 New Phytologist (2015) 205: 306–315 www.newphytologist.com
central midvein of the new primordium (Reinhardt et al., 2000; Benkova et al., 2003). Auxin-mediated KNOX down-regulation occurs at the leaf initiation site (Hay et al., 2006). In eudicots such as Arabidopsis, the domain of KNOX down-regulation and founder cell recruitment is localized to only a small portion of the SAM (Long et al., 1996), such that a nonsheathing, peg-like leaf primordium eventually emerges from the SAM. These findings have led to models in which the lateral domains comprising the Arabidopsis leaf lamina are initiated after founder cell recruitment from a peg-like primordium, while the nonsheathing leaf base comprises a limited lateral region near the point of insertion at the node (Kaplan, 1973, 2001; Sack & Scoffoni, 2013). In Arabidopsis, PIN-FORMED1 (PIN1)-mediated auxin transport initiates from the margins of the leaf primordium toward the midvein, thereby generating a series of vascular traces connecting the expanding lateral domains of the growing primordium to the midvein (Scarpella et al., 2006). The resulting eudicot leaf has branched venation, and does not form a sheathing leaf base at the insertion point on the node. By contrast, initiation of the sheathing monocot leaf coincides with KNOX down-regulation around the entire circumference of the maize SAM (Smith et al., 1992), such that the lateral domains of the monocot blade and sheathing leaf base are initiated during founder cell recruitment as opposed to during primordial stages. The margin regions of the basal sheath are completely L1-derived in maize. Fate mapping demonstrates that the margins are Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust
New Phytologist derived from overlapping leaf founder cell domains in the disc of insertion (DOI) (Sharman, 1942). Thus, the margins already overlap as they emerge from the DOI (Poethig & Szymkowiak, 1995; Scanlon & Freeling, 1997; Scanlon, 2000). Although initiation of the maize midvein is also correlated with PIN1-mediated auxin transport at the site of leaf initiation (Carraro et al., 2006; Lee et al., 2009), the correlation of PIN localization and parallel venation in the lateral regions of the monocot primordium has not been investigated. Previous studies utilizing inhibitors of polar auxin transport (PAT) have shown that maize shoots cultured in the presence of the auxin transport inhibitor 1-naphthylphthalamic acid (NPA) form tubular bases in which the margins fail to separate and exhibit ectopic KNOX accumulation (Scanlon, 2003). A model was generated in which the sheath margins are recruited from a ring of L1-derived cells at the apex, to form a sheathing monocot leaf base. In this study, computed tomography (CT) imaging illustrates the emergence of the overlapping margins of the sheathing maize leaf from the tubular DOI at the node. When combined with analyses of PIN1 accumulation and transcription of leaf patterning genes during maize leaf initiation, the data suggest a model for the development of the distinctive parallel venation and sheathing bases of monocot leaves.
Materials and Methods Plant material and growth conditions ZmPIN1a-YFP and DR5-RFP transgenic seed were provided by D. Jackson (Cold Spring Harbor Laboratory). Seedlings of Zea mays L. inbred line B73 were used for in situ hybridizations; plants were glasshouse grown and harvested at 14 d. In situ hybridizations and immunolocalizations In situ hybridizations were performed as described previously (Jackson, 1992; Juarez et al., 2004). Immunolocalizations were carried out as described previously using an Arabidopsis PIN1 antiserum diluted 1 : 300 and the Alexa Fluor 488-conjugated secondary antibody (Life-Technologies, Grand Island, NY, USA) (Boutte et al., 2006; Lee et al., 2009). Microscopic imaging Light microscopic imaging of sectioned samples was performed as described previously (Douglas et al., 2010). Confocal imaging was performed as described previously (Shimizu et al., 2009). CT imaging was performed on maize seedlings harvested 14 d after planting. Hand-trimmed apices were fixed overnight in formalin-acetic-alcohol (FAA) then dehydrated to 100% ethanol as described previously (Ruzin, 1999). Apices were stained for 4 d in 1% crystalline iodine dissolved in 100% ethanol. After a brief series of rinses in 100% ethanol, apices were transferred to 100% xylene and then to liquid paraffin as described previously (Ruzin, 1999). Paraffin-embedded samples were utilized for CT imaging. Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust
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Tomographic data sets were acquired using the Xradia (Pleasanton, CA, USA) Versa XRM-500 at one of the following settings: (1) 80 keV, 7 W, 2-s exposures with 2400 projections, 49 binned at 2000-nm pixel resolution; (2) 60 keV, 5 W, 5-s exposures with 1800 projections through the LE1 filter, 49 binned at 1533- or 1496-nm pixel resolution. Data were exported as TIFF-stacks to the image-processing software OSIRIX (Rosset et al., 2004). Using the 3D MPR and 3D volume-rendering tools in OSIRIX v.5.8.1 64-bit, the shoot apex and leaf primordia were examined from longitudinal, lateral, transverse, and paramarginal vantage points. Final micrographs were compiled by volumetric rendering of between 5 and 45 lm of optical data, depending upon the thickness of the plant microstructure that was imaged.
Results and Discussion Computed tomography imaging of the emerging maize leaf base CT permits X-ray imaging of intact biological samples. Optical sections are collated to form 3-dimensional (3D) images of biological structures that can be viewed from any planar orientation. We used CT imaging of fixed, iodine-stained 14-d-old seedlings to observe the successive stages of morphological development during maize leaf ontogeny. CT enables the simultaneous observation of different plastochron (P) stages of leaf margin development, from all possible vantage points and orientations (Fig. 1a–c), in a single study. A plastochron comprises the time period between successive leaf initiations from the vegetative SAM; significant developmental changes occur within each leaf primordium during the length of a single plastochron. Although CT imaging captures a morphological ‘still shot’ of multiple leaf primordia in a single seedling, any given P3 primordium (for example) may be at a slightly different developmental stage than another P3-staged sample within the same plastochron. As shown in Fig. 1(d–g,j), the left and right edges of P2 and early-staged P3 leaf primordia insert into the tubular DOI at the leaf base without overlapping. When considered alongside the data from numerous fate maps of maize leaf development (Poethig, 1984; Poethig & Szymkowiak, 1995; Scanlon & Freeling, 1997), these observations suggest that the entirety of the as yet elaborated leaf primordium observed during these early stages of maize leaf development comprises blade tissue, the margins of which do not overlap the shoot apex. Moreover, the data further suggest that the sheath components of these P2 and early P3 primordia comprise as yet unelaborated initials within the DOI. Beginning in late P3 (Fig. 1h,k) and early P4 (Fig. 1i,i0 ,m–o), the inner and outer sheath margins emerge from the DOI as two separate, overlapping fronts of tissue growth. In this way, a sheathing leaf base is formed in which the right and left margins overlap from their very inception, not simply as a result of postprimordial, lateral growth. Fig. 1(i,i0 ) show paramarginal views of the outer and inner edges of these P4 sheath margins, respectively. Supporting Information Movie S1 shows paramarginal optical sections of the P4 leaf base, revealing the edges of the outer (time-point 00:02) and inner (time-point 00:06) P4 New Phytologist (2015) 205: 306–315 www.newphytologist.com
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Fig. 1 Computed tomography analysis of the ontogeny of the maize sheathing leaf base. (a–c) Median, longitudinal views of maize shoot apical meristems (SAMs). Dotted lines designate the plane of optical sections in succeeding panels. (d) Paramarginal view of the maize shoot apex focusing on the P2 primordium. Distinct, nonoverlapping marginal outgrowths (arrows) from the disc of insertion (DOI) of the P2 primordium reveal that, at this stage of development, all of the as yet emerged leaf primordium comprises blade tissue in which the margins do not overlap the shoot apex. (j) Transverse view of the P2 primordium shown in (d). (e–h) Paramarginal views of four individual P3 primordia show progressive stages of leaf development. In early P3 (e), the margins of the leaf primordium emerge as separate, nonoverlapping tissue (arrows), presumably fated to form blade in the mature leaf. As development continues (f, g), sheath margins emerge from the DOI with overlapping edges (h). (k) Transverse view of the P3 primordium shown in (h) reveals that margins are ‘pre-wrapped’ before their emergence from the DOI, such that one margin emerges on the outside of the other forming unfused, overlapping leaf edges. (i, i0 ) Paramarginal views of the outer margin (blue arrow in (i)) and inner margin (orange arrow in (i0 )) of the P4 leaf. By P4, margins form tightly overlapped, unfused tissue fated to form the sheath in the mature leaf. (l) Transverse view of the primordium shown in (i). (m–o) Transverse, acropetal optical sections of a P4 primordium base reveal that the outer (blue arrow) and inner (orange arrow) margins emerge as pre-wrapped, overlapping outgrowths from the tubular DOI. Note that the DOI shown in (m) forms a ring of tissue that is separate from the outer edge of the stem (highlighted by green arrows). (p, q) Rotational views of a 3D rendering of a maize seedling shoot apex show that the highly overlapped P5 margins insert at separate outer (blue arrow) and inner (orange arrow) regions along the perimeter of the DOI. SAM, shoot apical meristem; MG, marginal axis. (r) 3D rendering of P6 leaf tissue shows the arrangement of parallel leaf veins (asterisks) in the young leaf primordium. N, node; MG, marginal axis. Bars, 50 lm; serial sections in (m–o) are vertically spaced by 5 lm.
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sheath margins. As shown in serial, transverse optical sections (Fig. 1m–o) and at time-point 00:07 in Movie S2, the DOI below the emerging P4 sheath margins forms an uninterrupted ‘tube’ of tissue that is distinct from and surrounds the stem. By contrast, serial sections distal to the DOI reveal that the P4 sheath margins emerge as separate, but already overlapping, sheets of primordial tissue. These multidimensional CT data provide support to the interpretations of previous cell fate analyses (Poethig & Szymkowiak, 1995; Scanlon & Freeling, 1997; Scanlon, 2000), which suggested that the maize sheathing leaf base does not arise as a result of the extended, differential growth of primordial sheath margins. By contrast, the primordial sheath margins overlap the shoot apex from their very inception, thereby forming a sheathing leaf base. Whole-mount 3D reconstructions clearly illustrate the separate insertion points of the overlapping P5 sheath margins (Fig. 1p,q), as does time-point 00:11 in Movie S3. Lastly, the parallel arrangement of the primordial leaf vasculature is clearly illustrated in CT scans of maize leaf primordia, as shown in Fig. 1(r).
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Polarized auxin transport is correlated with recruitment of all maize leaf founder cell domains To infer the direction of auxin transport during leaf founder cell recruitment, accumulation of PIN1 auxin transport proteins was examined in ZmPIN1a-YFP transgenic plants (Gallavotti et al., 2008) and by immunolocalization using an Arabidopsis PIN1 antiserum (Boutte et al., 2006; Lee et al., 2009) (Fig. 2). As described previously (Lee et al., 2009), maize PIN1 proteins accumulate in the L1 layer of the SAM directly above the presumptive midrib domain of the incipient leaf (P0) (Figs 2a,c, S1a–c,g–i). PIN1 proteins are predominately localized to the basal membranes of these L1 cells; these data indicate that L1 auxin transport is almost exclusively basipetally at the P0, to generate an inferred auxin maximum at the site of leaf initiation. As described previously (Lee et al., 2009), we occasionally observed L1 cells with apical orientation of PIN1a (specifically one example in > 20 shoot apices examined; Fig. S1d–f). Notably, L1 cells at the P0 midrib with apical PIN1 orientation are located at the
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Fig. 2 Auxin dynamics during maize leaf initiation. (a–e) ZmPIN1a-YFP expression in longitudinal sections of maize shoot apices. (f–i) PIN1 immunolocalization in transverse sections of the maize shoot apices. (c–e) and (g–i) Enlargements of boxed regions in (a–b) and (f), respectively. (a, c) Median longitudinal section along the midrib-to-margin axis. (b, d, e) Median longitudinal section cut perpendicular to the midrib-to-margin axis. PIN1 localization in the L2 layer of the apex marks the provascular strands of the P0 (pv0), P1 (pv1), and P2 (pv2) primordia. (f–i) Transverse section through the shoot apex at the level of P0. Arrows and black arrowheads indicate the direction of auxin transport inferred from PIN1 localization. Longitudinal sections show primarily basipetal transport of auxin in L1 to the site of leaf initiation at the midrib and lateral domains (a–e). Transverse sections show auxin transport from internal (L2) cell layers to L1 and from adjacent cells of L1 converging at the site of leaf initiation (f–i). P1, P2 designate plastochron number. D2 indicates the disc of insertion for phytomer 2. L1 and L2 indicate cell layer. The dashed line in (g) indicates the L1 layer. Individual cells are outlined in bright-field image in (h) (white, L1; blue, L2; black, P2). Asterisks indicate PIN1-depleted areas. Images in (a, b, d, f, g) have bright-field overlay. Bars: (a, b, f) 100 lm; (g–i) 20 lm. Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust
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lower end of the PO, such that auxin transport is inferred to converge at the tip of the incipient leaf (Lee et al., 2009; Fig. S1e–f). Immunolocalization of PIN1 in transverse sections through the shoot apex at the level of the midrib of the incipient leaf (Fig. 2f–i) revealed that auxin is also transported from internal (L2) cell layers of the SAM to the L1 layer, and from adjacent cells of the L1, converging at the site of leaf initiation (Fig. 2g). Subsequently, PIN1 is diverted internally to the L2 layers where it marks the provascular strand of the midrib. PIN1 accumulation is high in the adaxial sides of leaf buttresses and leaf primordia (Fig. 2a). Basal to this is a darker, PIN1-depleted region (denoted by asterisks in Fig. 2a–c); leaf outgrowth occurs along this axis of high versus low PIN1 accumulation. We investigated PIN1 localization during recruitment of lateral and marginal leaf domains, to determine if founder cell recruitment in lateral domains of the SAM is correlated with PIN1-mediated auxin transport. Lateral sections of seedling apices (i.e. longitudinal sections cut perpendicular to the midribmargin axis) show ZmPIN1a-YFP accumulation in the basal membranes of at least three tiers of L1 cells stacked directly above the emerging leaf buttress (Figs 2b,d,e, S1j–l). Thus, recruitment of lateral leaf founder cells in maize correlates with similar patterns of PIN1 accumulation observed during leaf initiation at the midrib domain (modeled in Fig. 5a). However, PIN1 localization in the P0 L2 directing auxin into the L1 (as described in Fig. 2f–i) was not observed in lateral domains. The extended and reiterative localization of PIN1-mediated, basipetal auxin transport in lateral domains about the circumference of the shoot apex is correlated with the formation of multiple, parallel, provascular strands (labeled as ‘pv’ in Fig. 2) in the young primordia. More uniform PIN1 accumulation is noted in younger primordial domains (Scarpella et al., 2006). Beginning at the P0 midrib (Fig. 2a), the canalization of PIN1 into provascular strands proceeds toward the margins as the leaf develops (Fig. 2f). By P3, provascular strands are apparent in lateral domains, while the margins have a more uniform localization of PIN1 (Fig. 3h). PIN1 accumulation becomes restricted to discreet provascular strands, which extend internally at the node (Fig. 2a–c). Provascular strands precede the eventual formation of lateral veins during later plastochrons; this described pattern of PIN1 localization suggests a model for the development of the monocot pattern of parallel leaf venation (modeled in Fig. 5b). A different pattern of PIN1 localization is associated with the recruitment of founder cells that give rise to the sheath margins (Fig. 3). The sheath margins comprise the final lateral domain to be recruited, and are derived from the SAM flank opposite to the midrib domain (Scanlon & Freeling, 1997). Clonal sector analyses show that the extended edges of the maize sheath margins are completely L1-derived (Poethig & Szymkowiak, 1995), and this dermal tissue is likewise devoid of vasculature (Scanlon et al., 1996). At the SAM flanks corresponding to the initiating margins of P1, P2 and P3 leaf primordia, PIN1 accumulates in the outer membranes of L2 cells that are adjacent to L1 cells (Fig. 3c–k). This polarized pattern of PIN1 localization indicates that auxin is transported from L2 cells to the L1 cells that will give rise to the sheath margins. These observations may explain the fused leaf New Phytologist (2015) 205: 306–315 www.newphytologist.com
New Phytologist margin phenotype conditioned by inhibition of auxin transport; shoots cultured in the presence of the auxin transport inhibitor NPA form tubular bases in which the margins fail to separate and exhibit ectopic KNOX accumulation (Scanlon, 2003). In this view, auxin is transported from the L2-derived cells to the L1-derived sheath margins, and is required for margin separation (modeled in Fig. 5a–c). Accumulation of leaf patterning transcripts in the maize leaf base We reasoned that genes functioning in auxin biosynthesis, dorsiventral patterning, and lateral organ boundary specification are good candidates to be involved in patterning the outgrowth of the DOI into two overlapping edges of the sheathing maize leaf base. The auxin biosynthetic YUCCA genes are required for leaf margin development in Arabidopsis (Wang et al., 2011). In situ hybridization of the maize YUCCA homolog SPARSE INFLORESCENCE1 (SPI1) (Gallavotti et al., 2008) reveals SPI1 expression encircling the SAM during the early stages of leaf initiation, and persisting in the margin domain later in primordium development (Fig. 4a). At the P1 stage, SPI1 is detected in the margin flanks of the developing primordium. At P2 a discernible bump demarcates the DOI (D2 in Fig. 4a). SPI1 transcripts accumulate in the DOI, from which the margins will initiate in later plastochrons. By late P3, sheath margins have begun to emerge from the leaf base. Although no SPI1 transcript is detected in the already elaborated P3 leaf margin, SPI1 transcript accumulates in the P3 DOI, from which as yet unelaborated sheath margin will continue to form. SPI1 transcript accumulates most strongly in the L1 layer of incipient maize leaf margins, although weaker signal is detected in L2 cells (Fig. 4a). When considered with regard to the leaf margin ZmPIN1 localization data described previously (Fig. 3), these data suggest that polarized PIN1 expression in the L2 cells combined with localized L1-specific auxin biosynthesis in the incipient leaf margins may act to concentrate auxin in the L1 cells that give rise to the sheath margins. Intriguingly, whereas the synthetic auxin response reporter DR5-RFP generally co-localizes with ZmPIN1a-YFP maxima in incipient leaves, in the tips of leaf primordia, and in vascular traces, no DR5-RFP signal is correlated with ZmPIN1a-YFP or SPI1 transcript localization near maize sheath margins (Fig. 3a, b). Taken together, these data suggest that auxin maxima generated by auxin transport and/or localized biosynthesis are correlated with recruitment of the midrib, lateral, and marginal leaf founder cell domains (modeled in Fig. 5a,b), although domainspecific differences in the tissue layer localization of PIN1a and of the auxin response reporter DR5 are observed and SPI1 transcript accumulation is primarily detected in the lateral and marginal domains. The accumulation of AUXIN RESPONSE FACTOR3a (ARF3A) transcripts in abaxial domains of young maize leaf primordia and in developing vasculature has been described previously (Douglas et al., 2010). ARF3a expression in the margin domain is first detected as a patch of transcript accumulation in Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust
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Fig. 3 Auxin dynamics during leaf margin initiation. (a) Co-localization of ZmPIN1a YFP and DR5-RFP (an auxin response reporter containing a synthetic auxin promoter) in the maize seedling shoot apex. (b) DR5-RFP expression in same sample as (a). (c) Enlargement of the boxed region in (a), which includes the base of P1 and the disc of insertion (DOI) of P2. Note, DR5-RFP is not detected in the P2 margin domain. (d–k) PIN1 immunolocalization in maize shoot apices. (a–g) Median longitudinal sections through the midrib-to-margin axis. (h–k) Transverse sections through the shoot apex at the level of P2. (e–g, i–k) Enlargements of boxed regions in (d) and (h), respectively. P1, P2 and P3 designate plastochron number; white arrowheads indicate P2 margin domain. Arrows and black arrowheads indicate the direction of auxin transport inferred from PIN1 localization. In the margin domain, the direction of auxin transport is from the inner cell layers (L2) to the outer cell layer (L1). Dashed lines in (e) and (i) demarcate the L1 layer. Individual cells are outlined in brightfield images (f, j: white, L1; blue, L2; black, P3). Images in (a–e, h, i) have bright-field overlay. Bars: (a, b) 100 lm; (c, e–g, i–k) 300 lm; (d, h) 50 lm.
the DOI of the P2 primordium (marked as D2 in Fig. 4b). By P3, the DOI forms a prominent bulge just above where it inserts into the node (D3). Two stripes of ARF3a expression in the DOI appear to mark the abaxial domains of the as yet uninitiated inner and outer sheath margins, and predict the emergence of these margin domains. In P4 leaves, ARF3a transcripts are abaxialized in the elaborated margins of the primordial blade and sheath, although a stripe of ARF3a expression persists in the P4 DOI. Our previous analyses of NPA-treated maize shoots demonstrated that the P4-staged DOI still contains pre-marginal domains that have not yet emerged from the node (Scanlon, 2003). We suggest that this stripe of ARF3a transcript accumulation in the P4 DOI Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust
demarcates the abaxial domain of the as yet unelaborated outer leaf margin. HOMEODOMAIN-LEUCINE ZIPPERIII (HD-ZIPIII) genes have well-described functions during adaxial patterning and vascular development (McConnell & Barton, 1998; McConnell et al., 2001; Emery et al., 2003; Juarez et al., 2004; Itoh et al., 2008). Transcript localization of the maize genes ROLLED1 (RLD1) (Juarez et al., 2004) and ZmPHABULOSA1 (ZmPHAB) (Juarez et al., 2004; Zhang et al., 2012) were re-examined by in situ hybridization. Both RLD1 and ZmPHAB1 are expressed in adaxial domains of leaf primordia and in developing vascular bundles, as described previously (Fig. 4c,d); however, transcripts New Phytologist (2015) 205: 306–315 www.newphytologist.com
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also accumulate in the DOI of P2 and P3 leaf primordia, a pattern that has not been described previously. Bands of RLD1 and ZmPHAB transcript accumulation are detected on the distal side of the P2 DOI. An equivalent patch of ZmPHAB1 expression is also observed in the P3 DOI of the seedling sample shown in Fig. 4(d), which has still not elaborated P3 marginal domains from this flank of the SAM. By contrast, the slightly older P3 primordium in Fig. 4(c) has begun elaboration of the inner sheath margin, and shows corresponding adaxial RLD1 expression (green arrowhead). The unelaborated outer margin is marked by a stripe of RLD1 expression in the DOI (green arrow in Fig. 4c). These results suggest that dorsiventrality of sheath margin founder cells is specified within the DOI, before their outgrowth. The CUP-SHAPED COTYLEDON (CUC) gene family performs evolutionarily conserved functions during SAM development, organ separation, and leaf margin dissection/serration (Olsen et al., 2005; Aida & Tasaka, 2006). First identified in fused cotyledon and SAM maintenance mutants of Arabidopsis and Petunia hybrida (Souer et al., 1996; Aida et al., 1997), CUC genes are expressed at lateral organ boundaries and several CUC genes are targeted for post-transcriptional regulation by microRNA164 (miR164) (Rhoades et al., 2002; Mallory et al., 2004). Fig. 4(e) presents an in situ hybridization of a maize CUC gene, a putative homolog of the Arabidopsis gene CUC2 (Aida & Tasaka, 2006). In the maize SAM, CUC2 accumulates above the incipient (P0) leaf primordium (Fig. 4e). In elaborated primordia, CUC2 accumulates in notches marking the boundaries between leaf primordia and the SAM (in P1), or between leaf primordia and the stem (for older leaf primordia). CUC2 transcripts are likewise detected above the incipient DOI of the P1, and in the distal boundaries between the P2 and P3 discs of insertion and the stem. Intriguingly, a patch of CUC2 accumulation is New Phytologist (2015) 205: 306–315 www.newphytologist.com
Fig. 4 Accumulation of leaf patterning transcripts in maize shoot apices. In situ hybridizations of the accumulation of maize gene transcripts (a) SPARSE INFLORESCENCE1 (SPI), (b) AUXIN RESPONSE FACTOR3a (ARF3a), (c) ROLLED LEAF1 (RLD1), (d) PHABULOSA1 (PHB1), and (e) CUP-SHAPED COTYLEDON2 (CUC2) are shown in 14-d-old seedlings. Color-coded stages of leaf development denote plastochron stages 0 (yellow), 1 (blue), 2 (red), 3 (green) and 4 (dark blue). D, disc of insertion; arrowheads, elaborated margins; arrows, specific examples of expression within the DOI. Bar, 100 lm.
noted in the upper region of the P4 DOI, extending to the boundary between the inner and outer P4 sheath margins. Finally, a stripe of CUC2 expression near the basal region of the P4 DOI presumably marks the boundary between the unelaborated P4 outer sheath margin and the incipient axillary meristem. Intriguingly, CUC2 expression in the SAM peripheral zone and in the DOI of initiating leaf primordia appears to be complementary to that of ARF3a, in keeping with the proposed model wherein auxin negatively regulates CUC2 (Bilsborough et al., 2011). A model for patterning of the sheathing leaf base in maize Our CT imaging data, PIN1a localizations, and in situ hybridizations of leaf patterning transcripts suggest a model for patterning the distinctive parallel venation and sheathing leaf bases that characterize the monocot maize leaf (Fig. 5). In keeping with previous fate map analyses demonstrating the basipetal pattern of maize leaf development (Poethig, 1984; Poethig & Szymkowiak, 1995; Scanlon & Freeling, 1997), our CT analyses illustrate that the distal blade domain of the maize leaf emerges first from the SAM and its margins do not overlap the shoot apex (Fig. 1; modeled in Fig. 5a). Beginning in later stages of plastochron 3, the sheath margins begin to emerge from the DOI as two separate, overlapping fronts of tissue growth to give rise to the wrapped, left and right edges of the sheathing leaf base (Fig. 1; modeled in Fig. 5a). This front of growth continues in plastochron 4 as the remaining portions of the sheath margins emerge. Analyses of polar auxin transportinhibited maize seedlings reveal that complete emergence of the basal-most sheath margins is not completed until plastochron 4 (Scanlon, 2003). Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust
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Fig. 5 Models for ontogeny of the sheathing maize leaf base. (a) Models portray two angled vantage points of three successive stages in the development of a single maize leaf. Leaves are green; hatched edges delineate imaginary lines of dissection of apical regions of leaf primordia; purple delineates expression of CUP-SHAPED COTYLEDON2 (CUC2), an organ-boundary marker; disc of insertion (DOI) is light blue. At the P2 stage the emerged primordium is entirely composed of leaf blade, whose margins do not overlap. In late P3, the left and right sheath margins emerge from the DOI as two fronts of tissue growth, which are already overlapping upon emergence. Pre-wrapped sheath tissue continues to develop from the DOI as late as P4. (b, c) Schematic models of longitudinal (b) and transverse (c) sections of the maize shoot apical meristem (SAM) showing PIN1-mediated basipetal auxin transport within the L1 layer of the SAM toward the site of leaf initiation during recruitment of leaf founder cells in the maize leaves. At the site of leaf initiation at the midrib, PIN1 then directs transport of auxin internally, to generate a provascular strand that will form the midvein (large blue bar in (b); large blue dot in (c)). Subsequent and reiterative PIN1-mediated auxin transport in successive lateral domains generates a series of additional provascular strands (smaller blue bars in b; smaller dots in c) that will eventually give rise to a pattern of parallel lateral veins in the young primordium. Note that the full complement of provascular strands is not presented in this schematic. Finally, and in contrast to the recruitment of midrib and lateral leaf domains, the recruitment of margin founder cells (stippled in b and c) correlates with PIN1-mediated auxin transport from the L2 layer toward L1.
Different patterns of founder cell recruitment in eudicots and monocots result in differences in leaf morphology and venation (Kaplan, 1973, 2001; Sack & Scoffoni, 2013). In eudicots, each auxin maximum generated by polarized PIN1 in the L1 is restricted to a small part of the SAM and redirection of auxin to internal cell layers specifies the midvein (Reinhardt et al., 2000, 2003b; Benkova et al., 2003; Scarpella et al., 2006). In maize, the generation of each auxin maximum extends around the circumference of the SAM (Figs 2, 3) and the primordium emerges as an encircling ridge, rather than a localized bump. PIN1 Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust
relocalization within the ground tissue of the encircling primordium canalizes auxin flow to generate distinct provascular strands. This PIN1 relocalization is observed in transverse shoot apical sections (Figs 2f, 3h), wherein PIN1 localization is uniformly distributed within the ground tissue of more recently recruited margin domains of P2–P3 primordia, but is reorganized into punctate provascular strands nearer to the midrib domains of the same primordia. In this way, a series of parallel veins is patterned within the primordium (modeled in Fig. 5b,c). Thus, the parallel leaf venation that characterizes monocot leaves arises as a consequence of the extended initialization of lateral leaf domains during recruitment of maize leaf founder cells, as opposed to the post-primordial formation of eudicot lateral leaf domains. A different pattern of PIN1a localization characterizes the recruitment of the DOI that gives rise to the sheathing margins of the monocot leaf base. Unlike the L1-localized PIN1a accumulation during recruitment of midrib and lateral leaf domains, recruitment of the maize sheath margins correlates with PIN1amediated auxin transport from the L2 internal layers toward the L1 layer of the marginal meristem flank (Fig. 3; modeled in Fig. 5b,c). In addition, the basipetal relocalization of PIN1 to generate provascular strands is not observed in this sheath marginal region (Fig. 3). Consequently, the extended, overlapping margin domains of the maize sheath are devoid of lateral veins (Scanlon et al., 1996). Our PIN1 localization data indicate that PIN1 acts to create auxin maxima in the L1 layer during all stages of maize leaf initiation, although the direction of transport differs in different leaf domains. In addition, Spi1 transcript accumulation is associated with leaf initiation around the periphery of the SAM, and persists in the DOI until initiation of the leaf margins at P4. The observation that Spi1 is strongest in L1, combined with PIN1 localization data, suggests that Spi1 and PIN1 act synergistically to concentrate auxin within specific regions of L1, and are consistent with previous studies showing that auxin transport and localized auxin biosynthesis act synergistically to promote lateral organ initiation in maize (Gallavotti et al., 2008). These findings also support the idea that L1 plays a crucial role in controlling the initiation of lateral organs in maize, as has been shown in eudicots (Reinhardt et al., 2003a). Analyses of adaxial- and abaxial-specific transcript accumulation further indicate that maize dorsiventral leaf identities are specified during primordium initialization, and that dorsiventral leaf identities of the basal sheath margins are assigned within the pre-primordial DOI, beginning as early as P2 (Fig. 4b–d). The abaxializing transcript ARF3a marks the abaxial sides of the incipient sheath margins, whereas Rld1 and PHB transcripts accumulate on the adaxial sides. All three transcripts maintain these polarized expression patterns as the margins emerge from the DOI. CUC2 functions to repress growth of boundary cells in eudicots Arabidopsis and petunia (Aida et al., 1997; Douglas et al., 2010). Accumulated transcripts of a putative maize ortholog of CUC2 delineate boundaries between leaf primordia and the SAM (Fig. 4e; modeled in Fig. 5). CUC2 also accumulates in the DOI New Phytologist (2015) 205: 306–315 www.newphytologist.com
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Ontogeny of the sheathing leaf base in maize (Zea mays) Robyn Johnston, Samuel Leiboff and Michael J. Scanlon Department of Plant Biology, Cornell University, Ithaca, NY 14853, USA
Summary Author for correspondence: Michael J. Scanlon Tel: +1 607 254 1156 Email: [email protected] Received: 10 May 2014 Accepted: 23 July 2014
New Phytologist (2015) 205: 306–315 doi: 10.1111/nph.13010
Key words: auxin, disc of insertion (DOI), founder cells, leaf margin, maize (Zea mays), monocot.
Leaves develop from the shoot apical meristem (SAM) via recruitment of leaf founder cells. Unlike eudicots, most monocot leaves display parallel venation and sheathing bases wherein the margins overlap the stem. Here we utilized computed tomography (CT) imaging, localization of PIN-FORMED1 (PIN1) auxin transport proteins, and in situ hybridization of leaf developmental transcripts to analyze the ontogeny of monocot leaf morphology in maize (Zea mays). CT imaging of whole-mounted shoot apices illustrates the plastochron-specific stages during initiation of the basal sheath margins from the tubular disc of insertion (DOI). PIN1 localizations identify basipetal auxin transport in the SAM L1 layer at the site of leaf initiation, a process that continues reiteratively during later recruitment of lateral leaf domains. Refinement of these auxin transport domains results in multiple, parallel provascular strands within the initiating primordium. By contrast, auxin is transported from the L2 toward the L1 at the developing margins of the leaf sheath. Transcripts involved in organ boundary formation and dorsiventral patterning accumulate within the DOI, preceding the outgrowth of the overlapping margins of the sheathing leaf base. We suggest a model wherein sheathing bases and parallel veins are both patterned via the extended recruitment of lateral maize leaf domains from the SAM.
Introduction Leaves evolved multiple times within the land plants (Townsley & Sinha, 2012). Despite the abundance of leaf morphological diversity found in nature, all leaves develop from founder cells recruited from the periphery of a stem cell niche called the shoot apical meristem (SAM) (Kaplan, 2001). One of the earliest described molecular markers of leaf initiation is the downregulation of KNOTTED1-like homeobox (KNOX) protein accumulation in the founder cells of the incipient leaf (P0), a process that requires transport of the plant hormone auxin (Smith et al., 1992; Scanlon, 2003; Hay et al., 2006). Among the angiosperms, eudicot leaves typically have nonsheathing bases and branched venation. By contrast, most monocot leaves have parallel veins and a sheathing base that surrounds the stem at the node, with a prominent distal lamina or leaf blade (reviewed in Kaplan, 1973). Distinct patterns of KNOX down-regulation and leaf founder cell recruitment in monocot and eudicot SAMs correlate with morphological differences in mature leaves within these clades. In eudicots, local polarized PIN1 expression in the L1 layer of the SAM peripheral zone creates an auxin maximum at the incipient (P0) leaf primordium (Reinhardt et al., 2003b). Subsequent alterations in PIN1 expression and localization at these convergence points redirect the flow of auxin from the L1 to internal cell layers, creating a canalized pattern of auxin flow that will pattern the 306 New Phytologist (2015) 205: 306–315 www.newphytologist.com
central midvein of the new primordium (Reinhardt et al., 2000; Benkova et al., 2003). Auxin-mediated KNOX down-regulation occurs at the leaf initiation site (Hay et al., 2006). In eudicots such as Arabidopsis, the domain of KNOX down-regulation and founder cell recruitment is localized to only a small portion of the SAM (Long et al., 1996), such that a nonsheathing, peg-like leaf primordium eventually emerges from the SAM. These findings have led to models in which the lateral domains comprising the Arabidopsis leaf lamina are initiated after founder cell recruitment from a peg-like primordium, while the nonsheathing leaf base comprises a limited lateral region near the point of insertion at the node (Kaplan, 1973, 2001; Sack & Scoffoni, 2013). In Arabidopsis, PIN-FORMED1 (PIN1)-mediated auxin transport initiates from the margins of the leaf primordium toward the midvein, thereby generating a series of vascular traces connecting the expanding lateral domains of the growing primordium to the midvein (Scarpella et al., 2006). The resulting eudicot leaf has branched venation, and does not form a sheathing leaf base at the insertion point on the node. By contrast, initiation of the sheathing monocot leaf coincides with KNOX down-regulation around the entire circumference of the maize SAM (Smith et al., 1992), such that the lateral domains of the monocot blade and sheathing leaf base are initiated during founder cell recruitment as opposed to during primordial stages. The margin regions of the basal sheath are completely L1-derived in maize. Fate mapping demonstrates that the margins are Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust
New Phytologist Scanlon MJ, Schneeberger RG, Freeling M. 1996. The maize mutant narrow sheath fails to establish leaf margin identity in a meristematic domain. Development 122: 1683–1691. Scarpella E, Marcos D, Friml J, Berleth T. 2006. Control of leaf vascular patterning by polar auxin transport. Genes & Development 20: 1015–1027. Sharman BC. 1942. Developmental anatomy of the shoot of Zea mays L. Annals of Botany 6: 245–282. Shimizu R, Ji J, Kelsey E, Ohtsu K, Schnable PS, Scanlon MJ. 2009. Tissue specificity and evolution of meristematic WOX3 function. Plant Physiology 149: 841–850. Smith LG, Greene B, Veit B, Hake S. 1992. A dominant mutation in the maize homeobox gene, Knotted-1, causes its ectopic expression in leaf-cells with altered fates. Development 116: 21–30. Souer E, van Houwelingen A, Kloos D, Mol J, Koes R. 1996. The no apical meristem gene of Petunia is required for pattern formation in embryos and flowers and is expressed at meristem and primordia boundaries. Cell 85: 159–170. Townsley BT, Sinha NR. 2012. A new development: evolving concepts in leaf ontogeny. Annual Review of Plant Biology 63: 535–562. Wang W, Xu B, Wang H, Li J, Huang H, Xu L. 2011. YUCCA genes are expressed in response to leaf adaxial-abaxial juxtaposition and are required for leaf margin development. Plant Physiology 157: 1805–1819. Zhang X, Douglas RN, Strable J, Lee M, Buckner B, Janick-Buckner D, Schnable PS, Timmermans MC, Scanlon MJ. 2012. Punctate vascular expression1 is a novel maize gene required for leaf pattern formation that functions downstream of the trans-acting small interfering RNA pathway. Plant Physiology 159: 1453–1462.
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Supporting Information Additional supporting information may be found in the online version of this article. Fig. S1 Additional images showing PIN1 localization associated with leaf initiation. Movie S1 Computated tomography video of a paramarginal view of the maize seedling shoot. Movie S2 Computated tomography video of a serial transverse optical section of the maize shoot. Movie S3 Computated tomography three-dimensional reconstructed video of a maize seedling apex. Please note: Wiley Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.
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Ontogeny of the sheathing leaf base in maize (Zea mays) Robyn Johnston, Samuel Leiboff and Michael J. Scanlon Department of Plant Biology, Cornell University, Ithaca, NY 14853, USA
Summary Author for correspondence: Michael J. Scanlon Tel: +1 607 254 1156 Email: [email protected] Received: 10 May 2014 Accepted: 23 July 2014
New Phytologist (2015) 205: 306–315 doi: 10.1111/nph.13010
Key words: auxin, disc of insertion (DOI), founder cells, leaf margin, maize (Zea mays), monocot.
Leaves develop from the shoot apical meristem (SAM) via recruitment of leaf founder cells. Unlike eudicots, most monocot leaves display parallel venation and sheathing bases wherein the margins overlap the stem. Here we utilized computed tomography (CT) imaging, localization of PIN-FORMED1 (PIN1) auxin transport proteins, and in situ hybridization of leaf developmental transcripts to analyze the ontogeny of monocot leaf morphology in maize (Zea mays). CT imaging of whole-mounted shoot apices illustrates the plastochron-specific stages during initiation of the basal sheath margins from the tubular disc of insertion (DOI). PIN1 localizations identify basipetal auxin transport in the SAM L1 layer at the site of leaf initiation, a process that continues reiteratively during later recruitment of lateral leaf domains. Refinement of these auxin transport domains results in multiple, parallel provascular strands within the initiating primordium. By contrast, auxin is transported from the L2 toward the L1 at the developing margins of the leaf sheath. Transcripts involved in organ boundary formation and dorsiventral patterning accumulate within the DOI, preceding the outgrowth of the overlapping margins of the sheathing leaf base. We suggest a model wherein sheathing bases and parallel veins are both patterned via the extended recruitment of lateral maize leaf domains from the SAM.
Introduction Leaves evolved multiple times within the land plants (Townsley & Sinha, 2012). Despite the abundance of leaf morphological diversity found in nature, all leaves develop from founder cells recruited from the periphery of a stem cell niche called the shoot apical meristem (SAM) (Kaplan, 2001). One of the earliest described molecular markers of leaf initiation is the downregulation of KNOTTED1-like homeobox (KNOX) protein accumulation in the founder cells of the incipient leaf (P0), a process that requires transport of the plant hormone auxin (Smith et al., 1992; Scanlon, 2003; Hay et al., 2006). Among the angiosperms, eudicot leaves typically have nonsheathing bases and branched venation. By contrast, most monocot leaves have parallel veins and a sheathing base that surrounds the stem at the node, with a prominent distal lamina or leaf blade (reviewed in Kaplan, 1973). Distinct patterns of KNOX down-regulation and leaf founder cell recruitment in monocot and eudicot SAMs correlate with morphological differences in mature leaves within these clades. In eudicots, local polarized PIN1 expression in the L1 layer of the SAM peripheral zone creates an auxin maximum at the incipient (P0) leaf primordium (Reinhardt et al., 2003b). Subsequent alterations in PIN1 expression and localization at these convergence points redirect the flow of auxin from the L1 to internal cell layers, creating a canalized pattern of auxin flow that will pattern the 306 New Phytologist (2015) 205: 306–315 www.newphytologist.com
central midvein of the new primordium (Reinhardt et al., 2000; Benkova et al., 2003). Auxin-mediated KNOX down-regulation occurs at the leaf initiation site (Hay et al., 2006). In eudicots such as Arabidopsis, the domain of KNOX down-regulation and founder cell recruitment is localized to only a small portion of the SAM (Long et al., 1996), such that a nonsheathing, peg-like leaf primordium eventually emerges from the SAM. These findings have led to models in which the lateral domains comprising the Arabidopsis leaf lamina are initiated after founder cell recruitment from a peg-like primordium, while the nonsheathing leaf base comprises a limited lateral region near the point of insertion at the node (Kaplan, 1973, 2001; Sack & Scoffoni, 2013). In Arabidopsis, PIN-FORMED1 (PIN1)-mediated auxin transport initiates from the margins of the leaf primordium toward the midvein, thereby generating a series of vascular traces connecting the expanding lateral domains of the growing primordium to the midvein (Scarpella et al., 2006). The resulting eudicot leaf has branched venation, and does not form a sheathing leaf base at the insertion point on the node. By contrast, initiation of the sheathing monocot leaf coincides with KNOX down-regulation around the entire circumference of the maize SAM (Smith et al., 1992), such that the lateral domains of the monocot blade and sheathing leaf base are initiated during founder cell recruitment as opposed to during primordial stages. The margin regions of the basal sheath are completely L1-derived in maize. Fate mapping demonstrates that the margins are Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust
New Phytologist derived from overlapping leaf founder cell domains in the disc of insertion (DOI) (Sharman, 1942). Thus, the margins already overlap as they emerge from the DOI (Poethig & Szymkowiak, 1995; Scanlon & Freeling, 1997; Scanlon, 2000). Although initiation of the maize midvein is also correlated with PIN1-mediated auxin transport at the site of leaf initiation (Carraro et al., 2006; Lee et al., 2009), the correlation of PIN localization and parallel venation in the lateral regions of the monocot primordium has not been investigated. Previous studies utilizing inhibitors of polar auxin transport (PAT) have shown that maize shoots cultured in the presence of the auxin transport inhibitor 1-naphthylphthalamic acid (NPA) form tubular bases in which the margins fail to separate and exhibit ectopic KNOX accumulation (Scanlon, 2003). A model was generated in which the sheath margins are recruited from a ring of L1-derived cells at the apex, to form a sheathing monocot leaf base. In this study, computed tomography (CT) imaging illustrates the emergence of the overlapping margins of the sheathing maize leaf from the tubular DOI at the node. When combined with analyses of PIN1 accumulation and transcription of leaf patterning genes during maize leaf initiation, the data suggest a model for the development of the distinctive parallel venation and sheathing bases of monocot leaves.
Materials and Methods Plant material and growth conditions ZmPIN1a-YFP and DR5-RFP transgenic seed were provided by D. Jackson (Cold Spring Harbor Laboratory). Seedlings of Zea mays L. inbred line B73 were used for in situ hybridizations; plants were glasshouse grown and harvested at 14 d. In situ hybridizations and immunolocalizations In situ hybridizations were performed as described previously (Jackson, 1992; Juarez et al., 2004). Immunolocalizations were carried out as described previously using an Arabidopsis PIN1 antiserum diluted 1 : 300 and the Alexa Fluor 488-conjugated secondary antibody (Life-Technologies, Grand Island, NY, USA) (Boutte et al., 2006; Lee et al., 2009). Microscopic imaging Light microscopic imaging of sectioned samples was performed as described previously (Douglas et al., 2010). Confocal imaging was performed as described previously (Shimizu et al., 2009). CT imaging was performed on maize seedlings harvested 14 d after planting. Hand-trimmed apices were fixed overnight in formalin-acetic-alcohol (FAA) then dehydrated to 100% ethanol as described previously (Ruzin, 1999). Apices were stained for 4 d in 1% crystalline iodine dissolved in 100% ethanol. After a brief series of rinses in 100% ethanol, apices were transferred to 100% xylene and then to liquid paraffin as described previously (Ruzin, 1999). Paraffin-embedded samples were utilized for CT imaging. Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust
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Tomographic data sets were acquired using the Xradia (Pleasanton, CA, USA) Versa XRM-500 at one of the following settings: (1) 80 keV, 7 W, 2-s exposures with 2400 projections, 49 binned at 2000-nm pixel resolution; (2) 60 keV, 5 W, 5-s exposures with 1800 projections through the LE1 filter, 49 binned at 1533- or 1496-nm pixel resolution. Data were exported as TIFF-stacks to the image-processing software OSIRIX (Rosset et al., 2004). Using the 3D MPR and 3D volume-rendering tools in OSIRIX v.5.8.1 64-bit, the shoot apex and leaf primordia were examined from longitudinal, lateral, transverse, and paramarginal vantage points. Final micrographs were compiled by volumetric rendering of between 5 and 45 lm of optical data, depending upon the thickness of the plant microstructure that was imaged.
Results and Discussion Computed tomography imaging of the emerging maize leaf base CT permits X-ray imaging of intact biological samples. Optical sections are collated to form 3-dimensional (3D) images of biological structures that can be viewed from any planar orientation. We used CT imaging of fixed, iodine-stained 14-d-old seedlings to observe the successive stages of morphological development during maize leaf ontogeny. CT enables the simultaneous observation of different plastochron (P) stages of leaf margin development, from all possible vantage points and orientations (Fig. 1a–c), in a single study. A plastochron comprises the time period between successive leaf initiations from the vegetative SAM; significant developmental changes occur within each leaf primordium during the length of a single plastochron. Although CT imaging captures a morphological ‘still shot’ of multiple leaf primordia in a single seedling, any given P3 primordium (for example) may be at a slightly different developmental stage than another P3-staged sample within the same plastochron. As shown in Fig. 1(d–g,j), the left and right edges of P2 and early-staged P3 leaf primordia insert into the tubular DOI at the leaf base without overlapping. When considered alongside the data from numerous fate maps of maize leaf development (Poethig, 1984; Poethig & Szymkowiak, 1995; Scanlon & Freeling, 1997), these observations suggest that the entirety of the as yet elaborated leaf primordium observed during these early stages of maize leaf development comprises blade tissue, the margins of which do not overlap the shoot apex. Moreover, the data further suggest that the sheath components of these P2 and early P3 primordia comprise as yet unelaborated initials within the DOI. Beginning in late P3 (Fig. 1h,k) and early P4 (Fig. 1i,i0 ,m–o), the inner and outer sheath margins emerge from the DOI as two separate, overlapping fronts of tissue growth. In this way, a sheathing leaf base is formed in which the right and left margins overlap from their very inception, not simply as a result of postprimordial, lateral growth. Fig. 1(i,i0 ) show paramarginal views of the outer and inner edges of these P4 sheath margins, respectively. Supporting Information Movie S1 shows paramarginal optical sections of the P4 leaf base, revealing the edges of the outer (time-point 00:02) and inner (time-point 00:06) P4 New Phytologist (2015) 205: 306–315 www.newphytologist.com
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Fig. 1 Computed tomography analysis of the ontogeny of the maize sheathing leaf base. (a–c) Median, longitudinal views of maize shoot apical meristems (SAMs). Dotted lines designate the plane of optical sections in succeeding panels. (d) Paramarginal view of the maize shoot apex focusing on the P2 primordium. Distinct, nonoverlapping marginal outgrowths (arrows) from the disc of insertion (DOI) of the P2 primordium reveal that, at this stage of development, all of the as yet emerged leaf primordium comprises blade tissue in which the margins do not overlap the shoot apex. (j) Transverse view of the P2 primordium shown in (d). (e–h) Paramarginal views of four individual P3 primordia show progressive stages of leaf development. In early P3 (e), the margins of the leaf primordium emerge as separate, nonoverlapping tissue (arrows), presumably fated to form blade in the mature leaf. As development continues (f, g), sheath margins emerge from the DOI with overlapping edges (h). (k) Transverse view of the P3 primordium shown in (h) reveals that margins are ‘pre-wrapped’ before their emergence from the DOI, such that one margin emerges on the outside of the other forming unfused, overlapping leaf edges. (i, i0 ) Paramarginal views of the outer margin (blue arrow in (i)) and inner margin (orange arrow in (i0 )) of the P4 leaf. By P4, margins form tightly overlapped, unfused tissue fated to form the sheath in the mature leaf. (l) Transverse view of the primordium shown in (i). (m–o) Transverse, acropetal optical sections of a P4 primordium base reveal that the outer (blue arrow) and inner (orange arrow) margins emerge as pre-wrapped, overlapping outgrowths from the tubular DOI. Note that the DOI shown in (m) forms a ring of tissue that is separate from the outer edge of the stem (highlighted by green arrows). (p, q) Rotational views of a 3D rendering of a maize seedling shoot apex show that the highly overlapped P5 margins insert at separate outer (blue arrow) and inner (orange arrow) regions along the perimeter of the DOI. SAM, shoot apical meristem; MG, marginal axis. (r) 3D rendering of P6 leaf tissue shows the arrangement of parallel leaf veins (asterisks) in the young leaf primordium. N, node; MG, marginal axis. Bars, 50 lm; serial sections in (m–o) are vertically spaced by 5 lm.
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Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust
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sheath margins. As shown in serial, transverse optical sections (Fig. 1m–o) and at time-point 00:07 in Movie S2, the DOI below the emerging P4 sheath margins forms an uninterrupted ‘tube’ of tissue that is distinct from and surrounds the stem. By contrast, serial sections distal to the DOI reveal that the P4 sheath margins emerge as separate, but already overlapping, sheets of primordial tissue. These multidimensional CT data provide support to the interpretations of previous cell fate analyses (Poethig & Szymkowiak, 1995; Scanlon & Freeling, 1997; Scanlon, 2000), which suggested that the maize sheathing leaf base does not arise as a result of the extended, differential growth of primordial sheath margins. By contrast, the primordial sheath margins overlap the shoot apex from their very inception, thereby forming a sheathing leaf base. Whole-mount 3D reconstructions clearly illustrate the separate insertion points of the overlapping P5 sheath margins (Fig. 1p,q), as does time-point 00:11 in Movie S3. Lastly, the parallel arrangement of the primordial leaf vasculature is clearly illustrated in CT scans of maize leaf primordia, as shown in Fig. 1(r).
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Polarized auxin transport is correlated with recruitment of all maize leaf founder cell domains To infer the direction of auxin transport during leaf founder cell recruitment, accumulation of PIN1 auxin transport proteins was examined in ZmPIN1a-YFP transgenic plants (Gallavotti et al., 2008) and by immunolocalization using an Arabidopsis PIN1 antiserum (Boutte et al., 2006; Lee et al., 2009) (Fig. 2). As described previously (Lee et al., 2009), maize PIN1 proteins accumulate in the L1 layer of the SAM directly above the presumptive midrib domain of the incipient leaf (P0) (Figs 2a,c, S1a–c,g–i). PIN1 proteins are predominately localized to the basal membranes of these L1 cells; these data indicate that L1 auxin transport is almost exclusively basipetally at the P0, to generate an inferred auxin maximum at the site of leaf initiation. As described previously (Lee et al., 2009), we occasionally observed L1 cells with apical orientation of PIN1a (specifically one example in > 20 shoot apices examined; Fig. S1d–f). Notably, L1 cells at the P0 midrib with apical PIN1 orientation are located at the
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Fig. 2 Auxin dynamics during maize leaf initiation. (a–e) ZmPIN1a-YFP expression in longitudinal sections of maize shoot apices. (f–i) PIN1 immunolocalization in transverse sections of the maize shoot apices. (c–e) and (g–i) Enlargements of boxed regions in (a–b) and (f), respectively. (a, c) Median longitudinal section along the midrib-to-margin axis. (b, d, e) Median longitudinal section cut perpendicular to the midrib-to-margin axis. PIN1 localization in the L2 layer of the apex marks the provascular strands of the P0 (pv0), P1 (pv1), and P2 (pv2) primordia. (f–i) Transverse section through the shoot apex at the level of P0. Arrows and black arrowheads indicate the direction of auxin transport inferred from PIN1 localization. Longitudinal sections show primarily basipetal transport of auxin in L1 to the site of leaf initiation at the midrib and lateral domains (a–e). Transverse sections show auxin transport from internal (L2) cell layers to L1 and from adjacent cells of L1 converging at the site of leaf initiation (f–i). P1, P2 designate plastochron number. D2 indicates the disc of insertion for phytomer 2. L1 and L2 indicate cell layer. The dashed line in (g) indicates the L1 layer. Individual cells are outlined in bright-field image in (h) (white, L1; blue, L2; black, P2). Asterisks indicate PIN1-depleted areas. Images in (a, b, d, f, g) have bright-field overlay. Bars: (a, b, f) 100 lm; (g–i) 20 lm. Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust
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lower end of the PO, such that auxin transport is inferred to converge at the tip of the incipient leaf (Lee et al., 2009; Fig. S1e–f). Immunolocalization of PIN1 in transverse sections through the shoot apex at the level of the midrib of the incipient leaf (Fig. 2f–i) revealed that auxin is also transported from internal (L2) cell layers of the SAM to the L1 layer, and from adjacent cells of the L1, converging at the site of leaf initiation (Fig. 2g). Subsequently, PIN1 is diverted internally to the L2 layers where it marks the provascular strand of the midrib. PIN1 accumulation is high in the adaxial sides of leaf buttresses and leaf primordia (Fig. 2a). Basal to this is a darker, PIN1-depleted region (denoted by asterisks in Fig. 2a–c); leaf outgrowth occurs along this axis of high versus low PIN1 accumulation. We investigated PIN1 localization during recruitment of lateral and marginal leaf domains, to determine if founder cell recruitment in lateral domains of the SAM is correlated with PIN1-mediated auxin transport. Lateral sections of seedling apices (i.e. longitudinal sections cut perpendicular to the midribmargin axis) show ZmPIN1a-YFP accumulation in the basal membranes of at least three tiers of L1 cells stacked directly above the emerging leaf buttress (Figs 2b,d,e, S1j–l). Thus, recruitment of lateral leaf founder cells in maize correlates with similar patterns of PIN1 accumulation observed during leaf initiation at the midrib domain (modeled in Fig. 5a). However, PIN1 localization in the P0 L2 directing auxin into the L1 (as described in Fig. 2f–i) was not observed in lateral domains. The extended and reiterative localization of PIN1-mediated, basipetal auxin transport in lateral domains about the circumference of the shoot apex is correlated with the formation of multiple, parallel, provascular strands (labeled as ‘pv’ in Fig. 2) in the young primordia. More uniform PIN1 accumulation is noted in younger primordial domains (Scarpella et al., 2006). Beginning at the P0 midrib (Fig. 2a), the canalization of PIN1 into provascular strands proceeds toward the margins as the leaf develops (Fig. 2f). By P3, provascular strands are apparent in lateral domains, while the margins have a more uniform localization of PIN1 (Fig. 3h). PIN1 accumulation becomes restricted to discreet provascular strands, which extend internally at the node (Fig. 2a–c). Provascular strands precede the eventual formation of lateral veins during later plastochrons; this described pattern of PIN1 localization suggests a model for the development of the monocot pattern of parallel leaf venation (modeled in Fig. 5b). A different pattern of PIN1 localization is associated with the recruitment of founder cells that give rise to the sheath margins (Fig. 3). The sheath margins comprise the final lateral domain to be recruited, and are derived from the SAM flank opposite to the midrib domain (Scanlon & Freeling, 1997). Clonal sector analyses show that the extended edges of the maize sheath margins are completely L1-derived (Poethig & Szymkowiak, 1995), and this dermal tissue is likewise devoid of vasculature (Scanlon et al., 1996). At the SAM flanks corresponding to the initiating margins of P1, P2 and P3 leaf primordia, PIN1 accumulates in the outer membranes of L2 cells that are adjacent to L1 cells (Fig. 3c–k). This polarized pattern of PIN1 localization indicates that auxin is transported from L2 cells to the L1 cells that will give rise to the sheath margins. These observations may explain the fused leaf New Phytologist (2015) 205: 306–315 www.newphytologist.com
New Phytologist margin phenotype conditioned by inhibition of auxin transport; shoots cultured in the presence of the auxin transport inhibitor NPA form tubular bases in which the margins fail to separate and exhibit ectopic KNOX accumulation (Scanlon, 2003). In this view, auxin is transported from the L2-derived cells to the L1-derived sheath margins, and is required for margin separation (modeled in Fig. 5a–c). Accumulation of leaf patterning transcripts in the maize leaf base We reasoned that genes functioning in auxin biosynthesis, dorsiventral patterning, and lateral organ boundary specification are good candidates to be involved in patterning the outgrowth of the DOI into two overlapping edges of the sheathing maize leaf base. The auxin biosynthetic YUCCA genes are required for leaf margin development in Arabidopsis (Wang et al., 2011). In situ hybridization of the maize YUCCA homolog SPARSE INFLORESCENCE1 (SPI1) (Gallavotti et al., 2008) reveals SPI1 expression encircling the SAM during the early stages of leaf initiation, and persisting in the margin domain later in primordium development (Fig. 4a). At the P1 stage, SPI1 is detected in the margin flanks of the developing primordium. At P2 a discernible bump demarcates the DOI (D2 in Fig. 4a). SPI1 transcripts accumulate in the DOI, from which the margins will initiate in later plastochrons. By late P3, sheath margins have begun to emerge from the leaf base. Although no SPI1 transcript is detected in the already elaborated P3 leaf margin, SPI1 transcript accumulates in the P3 DOI, from which as yet unelaborated sheath margin will continue to form. SPI1 transcript accumulates most strongly in the L1 layer of incipient maize leaf margins, although weaker signal is detected in L2 cells (Fig. 4a). When considered with regard to the leaf margin ZmPIN1 localization data described previously (Fig. 3), these data suggest that polarized PIN1 expression in the L2 cells combined with localized L1-specific auxin biosynthesis in the incipient leaf margins may act to concentrate auxin in the L1 cells that give rise to the sheath margins. Intriguingly, whereas the synthetic auxin response reporter DR5-RFP generally co-localizes with ZmPIN1a-YFP maxima in incipient leaves, in the tips of leaf primordia, and in vascular traces, no DR5-RFP signal is correlated with ZmPIN1a-YFP or SPI1 transcript localization near maize sheath margins (Fig. 3a, b). Taken together, these data suggest that auxin maxima generated by auxin transport and/or localized biosynthesis are correlated with recruitment of the midrib, lateral, and marginal leaf founder cell domains (modeled in Fig. 5a,b), although domainspecific differences in the tissue layer localization of PIN1a and of the auxin response reporter DR5 are observed and SPI1 transcript accumulation is primarily detected in the lateral and marginal domains. The accumulation of AUXIN RESPONSE FACTOR3a (ARF3A) transcripts in abaxial domains of young maize leaf primordia and in developing vasculature has been described previously (Douglas et al., 2010). ARF3a expression in the margin domain is first detected as a patch of transcript accumulation in Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust
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Fig. 3 Auxin dynamics during leaf margin initiation. (a) Co-localization of ZmPIN1a YFP and DR5-RFP (an auxin response reporter containing a synthetic auxin promoter) in the maize seedling shoot apex. (b) DR5-RFP expression in same sample as (a). (c) Enlargement of the boxed region in (a), which includes the base of P1 and the disc of insertion (DOI) of P2. Note, DR5-RFP is not detected in the P2 margin domain. (d–k) PIN1 immunolocalization in maize shoot apices. (a–g) Median longitudinal sections through the midrib-to-margin axis. (h–k) Transverse sections through the shoot apex at the level of P2. (e–g, i–k) Enlargements of boxed regions in (d) and (h), respectively. P1, P2 and P3 designate plastochron number; white arrowheads indicate P2 margin domain. Arrows and black arrowheads indicate the direction of auxin transport inferred from PIN1 localization. In the margin domain, the direction of auxin transport is from the inner cell layers (L2) to the outer cell layer (L1). Dashed lines in (e) and (i) demarcate the L1 layer. Individual cells are outlined in brightfield images (f, j: white, L1; blue, L2; black, P3). Images in (a–e, h, i) have bright-field overlay. Bars: (a, b) 100 lm; (c, e–g, i–k) 300 lm; (d, h) 50 lm.
the DOI of the P2 primordium (marked as D2 in Fig. 4b). By P3, the DOI forms a prominent bulge just above where it inserts into the node (D3). Two stripes of ARF3a expression in the DOI appear to mark the abaxial domains of the as yet uninitiated inner and outer sheath margins, and predict the emergence of these margin domains. In P4 leaves, ARF3a transcripts are abaxialized in the elaborated margins of the primordial blade and sheath, although a stripe of ARF3a expression persists in the P4 DOI. Our previous analyses of NPA-treated maize shoots demonstrated that the P4-staged DOI still contains pre-marginal domains that have not yet emerged from the node (Scanlon, 2003). We suggest that this stripe of ARF3a transcript accumulation in the P4 DOI Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust
demarcates the abaxial domain of the as yet unelaborated outer leaf margin. HOMEODOMAIN-LEUCINE ZIPPERIII (HD-ZIPIII) genes have well-described functions during adaxial patterning and vascular development (McConnell & Barton, 1998; McConnell et al., 2001; Emery et al., 2003; Juarez et al., 2004; Itoh et al., 2008). Transcript localization of the maize genes ROLLED1 (RLD1) (Juarez et al., 2004) and ZmPHABULOSA1 (ZmPHAB) (Juarez et al., 2004; Zhang et al., 2012) were re-examined by in situ hybridization. Both RLD1 and ZmPHAB1 are expressed in adaxial domains of leaf primordia and in developing vascular bundles, as described previously (Fig. 4c,d); however, transcripts New Phytologist (2015) 205: 306–315 www.newphytologist.com
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also accumulate in the DOI of P2 and P3 leaf primordia, a pattern that has not been described previously. Bands of RLD1 and ZmPHAB transcript accumulation are detected on the distal side of the P2 DOI. An equivalent patch of ZmPHAB1 expression is also observed in the P3 DOI of the seedling sample shown in Fig. 4(d), which has still not elaborated P3 marginal domains from this flank of the SAM. By contrast, the slightly older P3 primordium in Fig. 4(c) has begun elaboration of the inner sheath margin, and shows corresponding adaxial RLD1 expression (green arrowhead). The unelaborated outer margin is marked by a stripe of RLD1 expression in the DOI (green arrow in Fig. 4c). These results suggest that dorsiventrality of sheath margin founder cells is specified within the DOI, before their outgrowth. The CUP-SHAPED COTYLEDON (CUC) gene family performs evolutionarily conserved functions during SAM development, organ separation, and leaf margin dissection/serration (Olsen et al., 2005; Aida & Tasaka, 2006). First identified in fused cotyledon and SAM maintenance mutants of Arabidopsis and Petunia hybrida (Souer et al., 1996; Aida et al., 1997), CUC genes are expressed at lateral organ boundaries and several CUC genes are targeted for post-transcriptional regulation by microRNA164 (miR164) (Rhoades et al., 2002; Mallory et al., 2004). Fig. 4(e) presents an in situ hybridization of a maize CUC gene, a putative homolog of the Arabidopsis gene CUC2 (Aida & Tasaka, 2006). In the maize SAM, CUC2 accumulates above the incipient (P0) leaf primordium (Fig. 4e). In elaborated primordia, CUC2 accumulates in notches marking the boundaries between leaf primordia and the SAM (in P1), or between leaf primordia and the stem (for older leaf primordia). CUC2 transcripts are likewise detected above the incipient DOI of the P1, and in the distal boundaries between the P2 and P3 discs of insertion and the stem. Intriguingly, a patch of CUC2 accumulation is New Phytologist (2015) 205: 306–315 www.newphytologist.com
Fig. 4 Accumulation of leaf patterning transcripts in maize shoot apices. In situ hybridizations of the accumulation of maize gene transcripts (a) SPARSE INFLORESCENCE1 (SPI), (b) AUXIN RESPONSE FACTOR3a (ARF3a), (c) ROLLED LEAF1 (RLD1), (d) PHABULOSA1 (PHB1), and (e) CUP-SHAPED COTYLEDON2 (CUC2) are shown in 14-d-old seedlings. Color-coded stages of leaf development denote plastochron stages 0 (yellow), 1 (blue), 2 (red), 3 (green) and 4 (dark blue). D, disc of insertion; arrowheads, elaborated margins; arrows, specific examples of expression within the DOI. Bar, 100 lm.
noted in the upper region of the P4 DOI, extending to the boundary between the inner and outer P4 sheath margins. Finally, a stripe of CUC2 expression near the basal region of the P4 DOI presumably marks the boundary between the unelaborated P4 outer sheath margin and the incipient axillary meristem. Intriguingly, CUC2 expression in the SAM peripheral zone and in the DOI of initiating leaf primordia appears to be complementary to that of ARF3a, in keeping with the proposed model wherein auxin negatively regulates CUC2 (Bilsborough et al., 2011). A model for patterning of the sheathing leaf base in maize Our CT imaging data, PIN1a localizations, and in situ hybridizations of leaf patterning transcripts suggest a model for patterning the distinctive parallel venation and sheathing leaf bases that characterize the monocot maize leaf (Fig. 5). In keeping with previous fate map analyses demonstrating the basipetal pattern of maize leaf development (Poethig, 1984; Poethig & Szymkowiak, 1995; Scanlon & Freeling, 1997), our CT analyses illustrate that the distal blade domain of the maize leaf emerges first from the SAM and its margins do not overlap the shoot apex (Fig. 1; modeled in Fig. 5a). Beginning in later stages of plastochron 3, the sheath margins begin to emerge from the DOI as two separate, overlapping fronts of tissue growth to give rise to the wrapped, left and right edges of the sheathing leaf base (Fig. 1; modeled in Fig. 5a). This front of growth continues in plastochron 4 as the remaining portions of the sheath margins emerge. Analyses of polar auxin transportinhibited maize seedlings reveal that complete emergence of the basal-most sheath margins is not completed until plastochron 4 (Scanlon, 2003). Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust
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Fig. 5 Models for ontogeny of the sheathing maize leaf base. (a) Models portray two angled vantage points of three successive stages in the development of a single maize leaf. Leaves are green; hatched edges delineate imaginary lines of dissection of apical regions of leaf primordia; purple delineates expression of CUP-SHAPED COTYLEDON2 (CUC2), an organ-boundary marker; disc of insertion (DOI) is light blue. At the P2 stage the emerged primordium is entirely composed of leaf blade, whose margins do not overlap. In late P3, the left and right sheath margins emerge from the DOI as two fronts of tissue growth, which are already overlapping upon emergence. Pre-wrapped sheath tissue continues to develop from the DOI as late as P4. (b, c) Schematic models of longitudinal (b) and transverse (c) sections of the maize shoot apical meristem (SAM) showing PIN1-mediated basipetal auxin transport within the L1 layer of the SAM toward the site of leaf initiation during recruitment of leaf founder cells in the maize leaves. At the site of leaf initiation at the midrib, PIN1 then directs transport of auxin internally, to generate a provascular strand that will form the midvein (large blue bar in (b); large blue dot in (c)). Subsequent and reiterative PIN1-mediated auxin transport in successive lateral domains generates a series of additional provascular strands (smaller blue bars in b; smaller dots in c) that will eventually give rise to a pattern of parallel lateral veins in the young primordium. Note that the full complement of provascular strands is not presented in this schematic. Finally, and in contrast to the recruitment of midrib and lateral leaf domains, the recruitment of margin founder cells (stippled in b and c) correlates with PIN1-mediated auxin transport from the L2 layer toward L1.
Different patterns of founder cell recruitment in eudicots and monocots result in differences in leaf morphology and venation (Kaplan, 1973, 2001; Sack & Scoffoni, 2013). In eudicots, each auxin maximum generated by polarized PIN1 in the L1 is restricted to a small part of the SAM and redirection of auxin to internal cell layers specifies the midvein (Reinhardt et al., 2000, 2003b; Benkova et al., 2003; Scarpella et al., 2006). In maize, the generation of each auxin maximum extends around the circumference of the SAM (Figs 2, 3) and the primordium emerges as an encircling ridge, rather than a localized bump. PIN1 Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust
relocalization within the ground tissue of the encircling primordium canalizes auxin flow to generate distinct provascular strands. This PIN1 relocalization is observed in transverse shoot apical sections (Figs 2f, 3h), wherein PIN1 localization is uniformly distributed within the ground tissue of more recently recruited margin domains of P2–P3 primordia, but is reorganized into punctate provascular strands nearer to the midrib domains of the same primordia. In this way, a series of parallel veins is patterned within the primordium (modeled in Fig. 5b,c). Thus, the parallel leaf venation that characterizes monocot leaves arises as a consequence of the extended initialization of lateral leaf domains during recruitment of maize leaf founder cells, as opposed to the post-primordial formation of eudicot lateral leaf domains. A different pattern of PIN1a localization characterizes the recruitment of the DOI that gives rise to the sheathing margins of the monocot leaf base. Unlike the L1-localized PIN1a accumulation during recruitment of midrib and lateral leaf domains, recruitment of the maize sheath margins correlates with PIN1amediated auxin transport from the L2 internal layers toward the L1 layer of the marginal meristem flank (Fig. 3; modeled in Fig. 5b,c). In addition, the basipetal relocalization of PIN1 to generate provascular strands is not observed in this sheath marginal region (Fig. 3). Consequently, the extended, overlapping margin domains of the maize sheath are devoid of lateral veins (Scanlon et al., 1996). Our PIN1 localization data indicate that PIN1 acts to create auxin maxima in the L1 layer during all stages of maize leaf initiation, although the direction of transport differs in different leaf domains. In addition, Spi1 transcript accumulation is associated with leaf initiation around the periphery of the SAM, and persists in the DOI until initiation of the leaf margins at P4. The observation that Spi1 is strongest in L1, combined with PIN1 localization data, suggests that Spi1 and PIN1 act synergistically to concentrate auxin within specific regions of L1, and are consistent with previous studies showing that auxin transport and localized auxin biosynthesis act synergistically to promote lateral organ initiation in maize (Gallavotti et al., 2008). These findings also support the idea that L1 plays a crucial role in controlling the initiation of lateral organs in maize, as has been shown in eudicots (Reinhardt et al., 2003a). Analyses of adaxial- and abaxial-specific transcript accumulation further indicate that maize dorsiventral leaf identities are specified during primordium initialization, and that dorsiventral leaf identities of the basal sheath margins are assigned within the pre-primordial DOI, beginning as early as P2 (Fig. 4b–d). The abaxializing transcript ARF3a marks the abaxial sides of the incipient sheath margins, whereas Rld1 and PHB transcripts accumulate on the adaxial sides. All three transcripts maintain these polarized expression patterns as the margins emerge from the DOI. CUC2 functions to repress growth of boundary cells in eudicots Arabidopsis and petunia (Aida et al., 1997; Douglas et al., 2010). Accumulated transcripts of a putative maize ortholog of CUC2 delineate boundaries between leaf primordia and the SAM (Fig. 4e; modeled in Fig. 5). CUC2 also accumulates in the DOI New Phytologist (2015) 205: 306–315 www.newphytologist.com
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Ontogeny of the sheathing leaf base in maize (Zea mays) Robyn Johnston, Samuel Leiboff and Michael J. Scanlon Department of Plant Biology, Cornell University, Ithaca, NY 14853, USA
Summary Author for correspondence: Michael J. Scanlon Tel: +1 607 254 1156 Email: [email protected] Received: 10 May 2014 Accepted: 23 July 2014
New Phytologist (2015) 205: 306–315 doi: 10.1111/nph.13010
Key words: auxin, disc of insertion (DOI), founder cells, leaf margin, maize (Zea mays), monocot.
Leaves develop from the shoot apical meristem (SAM) via recruitment of leaf founder cells. Unlike eudicots, most monocot leaves display parallel venation and sheathing bases wherein the margins overlap the stem. Here we utilized computed tomography (CT) imaging, localization of PIN-FORMED1 (PIN1) auxin transport proteins, and in situ hybridization of leaf developmental transcripts to analyze the ontogeny of monocot leaf morphology in maize (Zea mays). CT imaging of whole-mounted shoot apices illustrates the plastochron-specific stages during initiation of the basal sheath margins from the tubular disc of insertion (DOI). PIN1 localizations identify basipetal auxin transport in the SAM L1 layer at the site of leaf initiation, a process that continues reiteratively during later recruitment of lateral leaf domains. Refinement of these auxin transport domains results in multiple, parallel provascular strands within the initiating primordium. By contrast, auxin is transported from the L2 toward the L1 at the developing margins of the leaf sheath. Transcripts involved in organ boundary formation and dorsiventral patterning accumulate within the DOI, preceding the outgrowth of the overlapping margins of the sheathing leaf base. We suggest a model wherein sheathing bases and parallel veins are both patterned via the extended recruitment of lateral maize leaf domains from the SAM.
Introduction Leaves evolved multiple times within the land plants (Townsley & Sinha, 2012). Despite the abundance of leaf morphological diversity found in nature, all leaves develop from founder cells recruited from the periphery of a stem cell niche called the shoot apical meristem (SAM) (Kaplan, 2001). One of the earliest described molecular markers of leaf initiation is the downregulation of KNOTTED1-like homeobox (KNOX) protein accumulation in the founder cells of the incipient leaf (P0), a process that requires transport of the plant hormone auxin (Smith et al., 1992; Scanlon, 2003; Hay et al., 2006). Among the angiosperms, eudicot leaves typically have nonsheathing bases and branched venation. By contrast, most monocot leaves have parallel veins and a sheathing base that surrounds the stem at the node, with a prominent distal lamina or leaf blade (reviewed in Kaplan, 1973). Distinct patterns of KNOX down-regulation and leaf founder cell recruitment in monocot and eudicot SAMs correlate with morphological differences in mature leaves within these clades. In eudicots, local polarized PIN1 expression in the L1 layer of the SAM peripheral zone creates an auxin maximum at the incipient (P0) leaf primordium (Reinhardt et al., 2003b). Subsequent alterations in PIN1 expression and localization at these convergence points redirect the flow of auxin from the L1 to internal cell layers, creating a canalized pattern of auxin flow that will pattern the 306 New Phytologist (2015) 205: 306–315 www.newphytologist.com
central midvein of the new primordium (Reinhardt et al., 2000; Benkova et al., 2003). Auxin-mediated KNOX down-regulation occurs at the leaf initiation site (Hay et al., 2006). In eudicots such as Arabidopsis, the domain of KNOX down-regulation and founder cell recruitment is localized to only a small portion of the SAM (Long et al., 1996), such that a nonsheathing, peg-like leaf primordium eventually emerges from the SAM. These findings have led to models in which the lateral domains comprising the Arabidopsis leaf lamina are initiated after founder cell recruitment from a peg-like primordium, while the nonsheathing leaf base comprises a limited lateral region near the point of insertion at the node (Kaplan, 1973, 2001; Sack & Scoffoni, 2013). In Arabidopsis, PIN-FORMED1 (PIN1)-mediated auxin transport initiates from the margins of the leaf primordium toward the midvein, thereby generating a series of vascular traces connecting the expanding lateral domains of the growing primordium to the midvein (Scarpella et al., 2006). The resulting eudicot leaf has branched venation, and does not form a sheathing leaf base at the insertion point on the node. By contrast, initiation of the sheathing monocot leaf coincides with KNOX down-regulation around the entire circumference of the maize SAM (Smith et al., 1992), such that the lateral domains of the monocot blade and sheathing leaf base are initiated during founder cell recruitment as opposed to during primordial stages. The margin regions of the basal sheath are completely L1-derived in maize. Fate mapping demonstrates that the margins are Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust
New Phytologist Scanlon MJ, Schneeberger RG, Freeling M. 1996. The maize mutant narrow sheath fails to establish leaf margin identity in a meristematic domain. Development 122: 1683–1691. Scarpella E, Marcos D, Friml J, Berleth T. 2006. Control of leaf vascular patterning by polar auxin transport. Genes & Development 20: 1015–1027. Sharman BC. 1942. Developmental anatomy of the shoot of Zea mays L. Annals of Botany 6: 245–282. Shimizu R, Ji J, Kelsey E, Ohtsu K, Schnable PS, Scanlon MJ. 2009. Tissue specificity and evolution of meristematic WOX3 function. Plant Physiology 149: 841–850. Smith LG, Greene B, Veit B, Hake S. 1992. A dominant mutation in the maize homeobox gene, Knotted-1, causes its ectopic expression in leaf-cells with altered fates. Development 116: 21–30. Souer E, van Houwelingen A, Kloos D, Mol J, Koes R. 1996. The no apical meristem gene of Petunia is required for pattern formation in embryos and flowers and is expressed at meristem and primordia boundaries. Cell 85: 159–170. Townsley BT, Sinha NR. 2012. A new development: evolving concepts in leaf ontogeny. Annual Review of Plant Biology 63: 535–562. Wang W, Xu B, Wang H, Li J, Huang H, Xu L. 2011. YUCCA genes are expressed in response to leaf adaxial-abaxial juxtaposition and are required for leaf margin development. Plant Physiology 157: 1805–1819. Zhang X, Douglas RN, Strable J, Lee M, Buckner B, Janick-Buckner D, Schnable PS, Timmermans MC, Scanlon MJ. 2012. Punctate vascular expression1 is a novel maize gene required for leaf pattern formation that functions downstream of the trans-acting small interfering RNA pathway. Plant Physiology 159: 1453–1462.
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Supporting Information Additional supporting information may be found in the online version of this article. Fig. S1 Additional images showing PIN1 localization associated with leaf initiation. Movie S1 Computated tomography video of a paramarginal view of the maize seedling shoot. Movie S2 Computated tomography video of a serial transverse optical section of the maize shoot. Movie S3 Computated tomography three-dimensional reconstructed video of a maize seedling apex. Please note: Wiley Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.
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