Hormonal responses during early embryogenesis in ...

Regulation of Fertilization and Early Seed Development

Hormonal responses during early embryogenesis in maize Junyi Chen*, Andreas Lausser* and Thomas Dresselhaus*1 *Cell Biology and Plant Biochemistry, Biochemie-Zentrum Regensburg, University of Regensburg, Universitatsstrasse ¨ 31, 93053 Regensburg, Germany

Biochemical Society Transactions

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Abstract Plant hormones have been shown to regulate key processes during embryogenesis in the model plant Arabidopsis thaliana, but the mechanisms that determine the peculiar embryo pattern formation of monocots are largely unknown. Using the auxin and cytokinin response markers DR5 and TCSv2 (twocomponent system, cytokinin-responsive promoter version #2), as well as the auxin efflux carrier protein PIN1a (PINFORMED1a), we have studied the hormonal response during early embryogenesis (zygote towards transition stage) in the model and crop plant maize. Compared with the hormonal response in Arabidopsis, we found that detectable hormone activities inside the developing maize embryo appeared much later. Our observations indicate further an important role of auxin, PIN1a and cytokinin in endosperm formation shortly after fertilization. Apparent auxin signals within adaxial endosperm cells and cytokinin responses in the basal endosperm transfer layer as well as chalazal endosperm are characteristic for early seed development in maize. Moreover, auxin signalling in endosperm cells is likely to be involved in exogenous embryo patterning as auxin responses in the endosperm located around the embryo proper correlate with adaxial embryo differentiation and outgrowth. Overall, the comparison between Arabidopsis and maize hormone response and flux suggests intriguing mechanisms in monocots that are used to direct their embryo patterning, which is significantly different from that of eudicots.

Introduction The life cycle of the two major groups of flowering plants (angiosperms), named monocots and eudicots respectively, starts from a single cell, the fertilized egg cell or zygote. During the process of embryogenesis, this single cell develops into a functional multicellular organism, which contains the basic body plan to form a complex adult plant. Various genetic studies on embryogenesis in the eudicot model plant Arabidopsis thaliana revealed that the phytohormone auxin represents a key factor for embryo pattern formation as mutations of genes involved in auxin biosynthesis and response result in severe defects at all stages of embryo development (for a review, see [1]). Moreover, long-term interference of endogenous embryonic auxin gradients by application of synthetic auxins or auxin transport inhibitors also caused serious embryo defects including ball-shaped embryos without any discernable apical–basal axis and cup-shaped embryos with abnormal apical structure and a non-functional root pool [2]. Cytokinins represent another major group of plant hormones which play regulatory roles in various processes of plant growth and development and interact with auxin-response pathways [3–5]. During Arabidopsis embryogenesis, for example, the antagonistic Key words: auxin, cytokinin, DR5, embryo patterning, endosperm development, PINFORMED1 (PIN1), two-component system (TCS), Zea mays. Abbreviations: ARR, ARABIDOPSIS RESPONSE REGULATOR; AtPIN1, Arabidopsis thaliana PINFORMED1; BETL, basal endosperm transfer layer; DAP, days after pollination; ESR, embryo surrounding region; RAM, root apical meristem; SAM, shoot apical meristem; TCSv2, two-component system, cytokinin-responsive promoter version #2; ZmPIN1a, Zea mays PINFORMED1a. To whom correspondence should be addressed (email [email protected]).

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Biochem. Soc. Trans. (2014) 42, 325–331; doi:10.1042/BST20130260

interaction between auxin and cytokinin was shown to be crucial for specifying the embryonic root-stem cell niche [5]. As indicated above, almost all of our current knowledge on the role of plant hormones in embryogenesis is based on the study of the model plant Arabidopsis. Studies on auxin during vegetative and reproductive development in maize (Zea mays) suggest that basic molecular mechanisms that control auxin biosynthesis, transport and signal transduction are conserved between monocots and dicots [6–11]. However, considering that the cell division pattern during embryogenesis and the final shape of the embryo are dramatically different between monocots and eudicots [12], there is an exigency to study the distribution pattern of auxin and cytokinin and their role also during embryogenesis in monocots (especially in crop species such as maize and rice). In the present short article, we summarize our investigation of the dynamic distribution of the auxin efflux carrier ZmPIN1a (Zea mays PINFORMED1a), as well as auxin and cytokinin responses during early embryogenesis and endosperm development in maize. On the basis of these findings, we propose a model and compare the similarities and differences of hormonal response behaviour during early embryogenesis between monocots and eudicots.

In contrast with endosperm development, an auxin response is not detectable during early embryogenesis in maize Studies on wheat embryos indicate that heterogeneous auxin distribution plays a major role in the shift from radial symmetry to bilateral polarity during monocot embryogenesis [13,14]. In situ hybridization studies of C The

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the auxin efflux carrier protein genes ZmPIN1 and corresponding immunolocalization assays using an antiAtPIN1 (Arabidopsis thaliana PINFORMED1) antibody revealed that polar auxin transport correlates with the differentiation of embryonic tissues and defining embryo organs in maize [15]. These few studies on the role of auxin in monocot embryogenesis are mainly focused on later stages of embryogenesis. So far, little is known about auxin biosynthesis, polar auxin transport and response during early embryogenesis and endosperm formation in monocots. We used the marker lines DR5:mRFP-ER (which contains the synthetic auxin-responsive promoter DR5 [16]) to visualize the temporal and spatial distribution of auxin responses as well as ZmPIN1a:ZmPIN1a–YFP to investigate polar auxin transport mediated by ZmPIN1a [6,17]. These marker lines should provide a first clue to the role of auxin-dependent processes during early embryogenesis and endosperm development in maize. Our results show that DR5 activity was evident in the endosperm, but not in the embryo shortly after fertilization [zygote to 4 DAP (days after pollination)] (Figures 1A and 1B). At the early pro-embryo stage (approximately 5–6 DAP), strong DR5 activity was pronounced in a defined endosperm region at the adaxial side above the embryo proper. This region is likely to represent the major auxin biosynthesis site in the developing endosperm (Figures 1C and 1D). Intriguingly, the auxin response in the endosperm extended from a central region (with a maximal auxin response) towards the surface of the adaxial side of the embryo proper (Figures 1C–1F). At the middle transition stage (7– 8 DAP), the auxin response in the endosperm expanded and fully covered the surface of the embryo proper at the late transition stage (8 DAP) (Figures 1G–1L). Whereas the auxin response was visible in the embryo proper surrounding region, signals in endosperm cells around the suspensor were not detected (Figures 1I and 1J). The earliest auxin response inside the embryo was detectable at the late transition stage (approximately 8 DAP) in the most apical cells of the scutellum (Figures 1K and 1L). The auxin efflux carrier ZmPIN1a exhibited a partly correlated distribution pattern during early embryo development. At 4–5 DAP, the strongest ZmPIN1a signals were detected in the maternal ovary tissues from the micropylar region towards the endosperm-outgrowth site (Figures 1M and 1N). We also observed a maximum of DR5 activity at this maternal region, but relatively weaker signals at the endosperm outgrowth site and inside the endosperm (results not shown). These observations indicate an auxin flux from the maternal chalazal region to the very early stages of endosperm development. A strong auxin response at the endosperm outgrowth site coincides with active endosperm growth and spreads at this region during very early developmental stages. When the auxin response in the endosperm reached the surface of the proembryo, ZmPIN1a signals were restricted to a few cells at the adaxial side of the embryo proper and most strongly at the apical epidermal L1 cell layer. These cells are in contact C The


with endosperm cells displaying a strong auxin response (Figures 1O and 1P). At the early transition stage, ZmPIN1a signals were basally localized in the apical cells of the embryo proper, except in the cells at the very abaxial side of the embryo proper (Figures 1Q and 1R). This indicates an auxin flux directed downwards at the adaxial part of the apical embryo proper. During the late transition stage (approximately 8 DAP), ZmPIN1a proteins were polar-localized mainly at the basal cell membranes of the internal L3 layer. The signals were detected from the abaxial subtip region of the scutellum towards the most basal part of the embryo proper. This indicates an auxin flux directed downwards towards the RAM (root apical meristem) and even towards basal cell layers that may represent the founder cells of the coleorhiza. ZmPIN1a signals were absent from the SAM (shoot apical meristem), weak in the RAM, but strong in the regions above and below the RAM initiation site (Figures 1S–1V). At the late transition stage (8 DAP), strong ZmPIN1a signals also appeared in the adaxial side endosperm above the auxin-response maximum region (Figures 1W and 1X). This observed pattern of DR5:mRFP-ER activity and ZmPIN1a:ZmPIN1a–YFP localization deviates in some aspects from published data using in situ hybridization and immunolocalization assays [15]. First, DR5 expression is not found in the ESR (embryo surrounding region) [18], but is at a high level outside the ESR in the adaxial endosperm, which is different from the finding that high levels of auxin exist in the ESR according to auxin immunolocalization data [15]. Secondly, evident localization of ZmPIN1a in BETL (basal endosperm transfer layer) [18], ESR or SAM before the late transition stage was not observed. This indicates that ZmPIN1 localization in these regions may be due to PIN1b/PIN1c localization. Thirdly, at the late transition stage, ZmPIN1a proteins are detected inside the embryo from the subtip region to the most basal part of the embryo proper, whereas ZmPIN1 proteins detected by immunolocalization were reported to be localized at the SAM and along the scutellum, but not at the basal part of the embryo proper [15]. Taken together, these results show that an auxin response was not detectable during early embryogenesis in maize until the late transition stage when the embryo already consists of hundreds of cells. Here, a significant auxin response was detected at the adaxial surface of the embryo proper. ZmPIN1a was polar-localized at the basal site of L1 and L2 layer cells, pointing towards the inside of the apical embryo proper region. Auxin signalling from the adaxial endosperm continues from the early pro-embryo stage towards the transition stage, which is correlated with adaxial apical embryo cellular differentiation and outgrowth.

Cytokinin activity is detectable during early endosperm development, but not during early embryogenesis in maize To explore the role of another important plant hormone, cytokinin, during early embryogenesis in maize, we analysed


Figure 1 Auxin response and flux during early embryogenesis and endosperm development in maize (A–L) DR5:mRFP-ER auxin response during early seed development in maize. (A and B) DR5 activity is evident at 4 DAP in the endosperm. (C and D) At the early pro-embryo stage (5–6 DAP), strong DR5 activity is pronounced at a defined endosperm region at the adaxial side above the embryo proper. The white broken line indicates the outline of the embryo, and the yellow broken line indicates the outline of the ESR. (E and F) Endosperm-derived auxin response signals spread to the adaxial surface of the embryo proper (arrowhead) at 5–6 DAP, but are not detectable inside the embryo. (G and H) Endosperm DR5 activity expands at the late pro-embryo/early transition stage (7 DAP) and covers the surface of the embryo proper at its adaxial side. At the late transition stage (8 DAP), the whole embryo proper is surrounded by DR5 activity signals (I and J). The first evident DR5 activity in the embryo is detected at the very tip of the scutellum (arrowhead) (K and L). Note that (I and J) and (K and L) represent similar stages at different magnification. (M–X) ZmPIN1a–YFP localization during early embryogenesis in maize. (M and N) At 4–5 DAP, ZmPIN1a signals are strongest at the maternal chalazal region to the endosperm outgrowth site. (O and P) At the pro-embryo stage (6 DAP), ZmPIN1a is present in a few cells at the adaxial side of the embryo proper with the strongest signals at the apical epidermal L1 cell layer. (Q and R) At the early transition stage (7 DAP), ZmPIN1a proteins are mainly localized at the basal pole of the upper cells of the embryo proper, except for the cells at its very abaxial side. (S–V) During the late transition stage (8 DAP), ZmPIN1a proteins are polar-localized in the basal cell membranes along the inside area of the embryo. The signals are detected from the abaxial subtip region of the scutellum to the most basal part of the embryo proper. (U) and (V) are two focal planes of one embryo. (W) Additional ZmPIN1a signals appear at the adaxial side of the endosperm above the auxin-response maximum region (see X). (X) Magnified images of encircled regions of (W) show ZmPIN1a in the endosperm. (A, C, E, G, I, K, M, O, Q and S) Fluorescence micrographs. (B, D, F, H, J, L, N, P, R and T–X) Fluorescence images merged with their respective brightfield images. Abbreviations: ds, degenerated synergid; en, endosperm; ep, embryo proper; esr, embryo surrounding region; nu, nucellus; ram, root apical meristem; sam, shoot apical meristem; scu, scutellum; su, suspensor. Scale bars, 100 μm.

transgenic plants carrying an RFP (tdTomato) reporter containing an NLS (nuclear localization signal) controlled by the TCSv2 promoter (two-component system, cytokininresponsive promoter version #2). Signals of the cytokininresponse reporter TCSv2:NLS-tdTomato were detected in the developing endosperm shortly after fertilization. At 3

DAP (before initiation of endosperm cellularization), strong signals were detected in nuclei of the chalazal endosperm and weaker signals in nuclei at the micropylar pole of the endosperm (Figures 2A and 2B). Strong signals were also detected in maternal tissues (nucellus and inner integument) at the micropylar region of the ovule surrounding the C The


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Figure 2 Cytokinin response during early embryogenesis and endosperm development in maize The cytokinin response is visualized by the activity of the TCSv2 promoter driving the nuclear-localized RFP tdTomato reporter. (A and B) TCSv2 activity in the endosperm before cellularization at 3 DAP. (C and D) At 3 DAP, strong signals are detected in the nucellus and integumental cells at the micropylar area, but not inside the embryo. (E and F) At 4 DAP, TCSv2 activity is still absent from the embryo, but strong signals are still detectable at the inner integument. (G–L) TCSv2 activities in the endosperm chalazal area and the BETL at the pro-embryo stage (6 DAP; G and H), the early transition stage (7 DAP; I and J) and towards the late transition stage (8 DAP; K and L). The broken line indicates the BETL region. (M and N) At the late transition stage, the first TCSv2 activities in the embryo are detected at the tip and adaxial scutellum epidermis as well as the initiation site of the SAM. (O–R) At the coleoptilar stage (9–10 DAP), signals become stronger at the epidermis of the adaxial scutellum and the SAM. (O and P) and (Q and R) are two focal planes of one embryo. (A, C, E, G, I, K, M, O and Q) Fluorescence micrographs. (B, D, F, H, J, L, N, P and R) Flurorescence images merged with their respective brightfield images. Abbreviations: betl, basal endosperm transfer layer; col, coleoptile; ds, degenerated synergid; en, endosperm; ep, embryo proper; ii, inner integument; nu, nucellus; sam, shoot apical meristem; scu, scutellum; su, suspensor. Scale bars, 100 μm.

developing embryo (Figures 2A–2D). However, a significant signal inside the very early embryo could not be detected. At 4 DAP, reporter signals remained in the inner integument and were not detectable in the pro-embryo (Figures 2E and 2F). The degenerated synergid cell shows strong autofluorescence. TCSv2 reporter activity was very strong in the endosperm at 6 DAP, especially at the chalazal part of the endosperm. Strong signals were also detected at the differentiated BETL (Figures 2G and 2H). During progression of endosperm development, signals were gradually reduced (7–8 DAP), but remained detectable at the adaxial side of the chalazal region of the endosperm. At this stage, the embryo had developed already into the late transition phase. Signals at the opposite pole of the endosperm, at the BETL region, remained strong as well (Figures 2G–2L). High cytokinin levels (referred to by TCSv2 reporter activity) at the chalazal endosperm and BETL region may be correlated with fast cell division and growth at the apical and basal part of the endosperm. Similarly to DR5 activity, the earliest TCSv2 activity in the embryo was detected at the late transition stage (8 DAP). Signals were first visible at the tip (L1 layer) of the adaxial C The


epidermis of the scutellum, as well as the initiation site of the SAM (Figures 2M and 2N), and became stronger in these cells at 9–10 DAP (coleoptilar stage) (Figures 2O– 2R). Interestingly, auxin and cytokinin appeared to act antagonistically at the scutellum tip. Whereas auxin reporter activity accumulated at the very most tip of the scutellum and was especially strong at the abaxial half of the tip region (Figures 1K and 1L, and results not shown), weak cytokinin signals were detected at the very tip of the scutellum. In particular, signals appeared weak at the adaxial half of the tip and were nearly absent from the abaxial half of this region (here the auxin response was strongest). Strong cytokinin signals were also detected at the subtip region of the adaxial epidermis of the scutellum (Figures 2O–2R). In Arabidopsis, auxin and cytokinin activity levels exhibit an inverse profile in the root-stem cell system. Auxin antagonizes cytokinin output in the basal cell lineage by regulating transcriptional activity of ARR (ARABIDOPSIS RESPONSE REGULATOR) genes ARR7 and ARR15, which are feedback repressors of cytokinin signalling. Moreover, transient and antagonistic interaction between


auxin and cytokinin is critical for specifying the first root-stem cell niche [5]. Thus, in the tip region of the scutellum of maize, the abaxial tip region showing a significant auxin response may repress cytokinin activity in a similar way. The resulting difference in the auxin/cytokinin ratio between adaxial–abaxial tip and apical compared with subapical regions may thus also be correlated with adaxial–abaxial axis formation and cell differentiation in monocots.

A comparison of auxin and cytokinin activity during early embryogenesis in maize and Arabidopsis In contrast with maize, a large number of studies were conducted to uncover the role of plant hormones for embryo pattern formation in Arabidopsis. Similarly, the hormoneresponsive promoters DR5 and TCS were used to monitor the dynamic distribution patterns of auxin and cytokinin respectively. During embryogenesis in Arabidopsis, first DR5 reporter activity was detected in the smaller apical cell immediately after asymmetric first division of the zygote [2]. Later on, the signal intensity in the developing embryo proper rapidly increased, whereas only very weak signals were detected in the suspensor cells. The auxin-response maximum at the apical embryo was correlated with PIN7 polar localization at the apical side of the suspensor cells from the first zygotic division towards the 32-cell stage. The resulting apical–basal auxin gradient triggers the specification of the apical embryo structure. From the one-cell towards the 32-cell stage, PIN1 proteins were localized at all apical embryo cell boundaries without any detectable polarity. At the 32-cell stage, PIN1 became polar-localized at the basal side of provascular cells. This coincided with the shift of auxin maximum to the basal embryo pole named as the hypophysis (Figure 3, upper panels). Analysis of pin quadruple mutants identified PIN-dependent transport as an essential part of the mechanism for apical–basal axis establishment during embryogenesis. In contrast with Arabidopsis, activity of both hormone reporters were not detected during early embryogenesis in maize. Shortly after fertilization (from the zygote stage to 3– 4 DAP), evident DR5 and TCSv2 activities were detected in the endosperm, but not in the embryo. From the early proembryo stage (5–6 DAP) to the transition stage (7–8 DAP), strong DR5 activity from the endosperm was pronounced and spread at the adaxial surface of the embryo proper. Meanwhile, ZmPIN1a proteins were basally accumulated at a corresponding adaxial apical embryo proper region, indicating a possible auxin flux from the endosperm directed downwards from the (adaxial) apical L1 region of the embryo towards inner cell layers (Figure 3, lower panels). The results discussed in the present article indicate that auxin signalling from the adaxial endosperm probably represents a mechanism to pattern asymmetric embryo structures during early embryogenesis in maize. This is

supported by a number of observations. First, a continuous auxin signal at the surface of the adaxial embryo side from the early pro-embryo stage towards the transition stage is correlated in this region with specification of the embryo proper, differentiation of the SAM and outgrowth of the scutellum. Secondly, ZmPIN1a activity at the embryo stage is related to endosperm-derived auxin response at the adaxial surface of the embryo. Strongest ZmPIN1a signals appeared at the apical epidermal L1 cell layer, and ZmPIN1a proteins were polar-localized at the basal cell membrane at this stage. This suggests an auxin influx from the apical surface via the adaxial L1 epidermal cells towards inner cells of the L2 and L3 layers respectively. The resulting auxin gradient may trigger embryo specification and differentiation in this region. Thirdly, grasses have evolved a specific endospermderived structure, i.e. the ESR, which appears to lack auxin signals. This region might function to maintain the auxin gradient and to isolate the embryonic suspensor region free from exogenous auxin-derived signals. So far, little is known about the function of the ESR, which is established at the early pro-embryo stage (4–6 DAP) and which is characterized by cells with dense cytoplasm and extensive rough endoplasmic reticulum [18]. We observed that the ESR seems to prevent auxin expanding to the basal embryo proper and embryo suspensor surface to the middle transition stage (Figures 1C–1H), thus keeping auxin signals localized at the apical embryo surface until early patterning decisions have been accomplished (Figures 1G and 1H). Moreover, the embryo seems to exhibit a tendency to grow towards the area of auxin maxima as the late transition stage as the embryo appears bent and the embryo proper grows towards the most adaxial endosperm area showing the strongest auxin response (Figures 1G–1J, 2K and 2L). The ESR may thus function to establish a buffer zone between the embryo and the most adaxial endosperm. It may retard development of the embryo proper to reach the most adaxial region, thus keeping the adaxial–abaxial auxin gradient provided by the endosperm until proper adaxial embryo differentiation and outgrowth takes place. Similarly to auxin, cytokinin responses appear much later during embryogenesis in maize. The earliest TCS activity in Arabidopsis embryos appeared in the hypophysis at the 32-cell stage. During the triangular/transition stage, the hypophysis divides asymmetrically into a small apical lensshape cell and a large basal cell. The large basal daughter cell with higher DR5 activity repressed TCS activity, whereas the apical lens-shape cell with lower DR5 activity retained TCS activity. At this stage, additional auxin maxima were formed at the two flanks of the apical domain, where the cotyledons initiate [19] (Figure 3, upper panel). In maize, the earliest DR5 and TCSv2 activities in the embryo appeared at the late transition stage. TCSv2 activity was first visible at the tip (L1 layer) of the adaxial scutellum epidermis as well as the SAM initiation site. At the developing scutellum tip, DR5 activity exhibited an inversed profile with TCSv2 activity (Figure 3, lower panel). Thus we also observed some similarities between maize and Arabidopsis embryogenesis. First, in C The


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Figure 3 Comparative models of AtPIN1/ZmPIN1a protein localization as well as auxin and cytokinin responses during early embryogenesis in Arabidopsis and maize Successive stages of Arabidopsis embryogenesis are according to [23] and the maize embryogenesis stages are according to [24,25]. The Arabidopsis model is generated on the basis of many previous studies, and the maize model is proposed on the basis of the data discussed in the present article. Red lines indicate the localizations of AtPIN1/ZmPIN1a proteins; blue arrows indicate auxin flux mediated by polar localization of AtPIN1/ZmPIN1a proteins; orange dots represent DR5 reporter activity; green dots represent TCSv2 reporter activity. Embryonic stages are indicated. Abbreviations: ac, apical cell; bc, basal cell; ep, embryo proper; hy, hypophysis; pd, protoderm; ram, root apical meristem; sam, shoot apical meristem; scu, scutellum; su, suspensor; zy, zygote.

both systems, the auxin-response maximum appeared at the tip of the emerging cotyledons. Secondly, auxin-response maxima coincide with either regional auxin biosynthesis or polar auxin transport mediated by the auxin efflux carrier PIN proteins. Thirdly, antagonistic interaction exists between auxin and cytokinin, and this is important for different cell specification in a relatively small region.

Concluding remarks Monocotyledonous plant species generate embryos containing a single cotyledon, a number of embryonic leaves and lateral root anlagen, thus they differ significantly from eudicot species such as Arabidopsis that produce more simply C The


structured embryos. Monocots must therefore have evolved different patterning mechanisms to regulate embryogenesis. The plant hormone auxin has been identified as the key regulator that controls most of the embryo patterning steps in Arabidopsis. In the present article, we report that the adaxial endosperm continuously generates a strong auxin response during early embryogenesis in maize. In addition, the endosperm-derived structure ESR appears to play an important role in maintaining the auxin gradient provided by the endosperm, and this is correlated with early embryo pattern formation in maize. The grass endosperm is usually considered a nutrient tissue, which may provide nutrition to the developing embryo. Our findings suggest novel aspects of endosperm and ESR function in providing and distributing


auxin signals that regulate early embryo patterning in maize. Moreover, extensive communication was reported during reproduction between and among maternal and gametophytic cells during gamete formation, pollen germination, tube growth and guidance, and double fertilization processes (for a review, see [20]). This comparative report indicates that communication also takes place after fertilization during early seed development between endosperm and embryo respectively, and brings a new perspective to the role of the endosperm for embryo patterning. It will be interesting now to find out whether other monocot species, especially the economically important grasses such as wheat, rice and barley, involve similar hormonal responses during early embryogenesis. An exciting challenge will be to explore the molecular mechanisms of auxin perception and signal transduction at the adaxial apical embryo proper. Is there an ABP1 (auxin-binding protein 1)-like auxin receptor [21] at the apical epidermal L1 cell layer? Are Rho GTPase-based cell-surface auxin signalling pathways [22] or a different molecular framework involved to modulate ZmPIN1a polar localization at the adaxial apical embryo proper? With the completion of the maize genome sequence and the development of novel genetic and cell biology tools, such as the marker lines used in this study, more research opportunities are now available to also study ‘difficult’ plant species including the important crop plant maize. We expect many exciting findings and the elucidation of the basic principles of hormone regulation during patterning of early embryogenesis in the grasses in the near future.

Acknowledgements We are grateful to David Jackson (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, U.S.A.) for providing seeds of the DR5:mRFPER, ZmPIN1a:ZmPIN1a–YFP and TCSv2:NLS-tdTomato marker lines. Ursula Wittmann and Gunther ¨ Peissig are acknowledged for plant care.

Funding This work is supported by grants from the German Research Foundation (DFG) [grant numbers SFB924 TP A03 and SPP 1365/2 DR 334/10-1] to T.D.

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