ZmPIN1-mediated auxin transport is related to cellular differentiation during maize embryogenesis and endosperm development

Abstract

To study the influence of PINFORMED1 (PIN1)-mediated auxin transport during embryogenesis and endosperm development in monocots, the expression pattern of the three identified ZmPIN1 genes was determined at the transcript level. Localization of the corresponding proteins was also analyzed during maize (Zea mays) kernel development. An anti-indole-3-acetic acid (IAA) monoclonal antibody was used to visualize IAA distribution and correlate the direction of auxin active transport, mediated by ZmPIN1 proteins, with the actual amount of auxin present in maize kernels at different developmental stages. ZmPIN1 genes are expressed in the endosperm soon after double fertilization occurs; however, unlike other tissues, the ZmPIN1 proteins were never polarly localized in the plasma membrane of endosperm cells. ZmPIN1 transcripts and proteins also colocalize in developing embryos, and the ZmPIN1 proteins are polarly localized in the embryo cell plasma membrane from the first developmental stages, indicating the existence of ZmPIN1-mediated auxin fluxes. Auxin distribution visualization indicates that the aleurone, the basal endosperm transfer layer, and the embryo-surrounding region accumulate free auxin, which also has a maximum in the kernel maternal chalaza. During embryogenesis, polar auxin transport always correlates with the differentiation of embryo tissues and the definition of the embryo organs. On the basis of these reports and of the observations on tissue differentiation and IAA distribution in defective endosperm-B18 mutant and in N-1-naphthylphthalamic acid-treated kernels, a model for ZmPIN1-mediated transport of auxin and the related auxin fluxes during maize kernel development is proposed. Common features between this model and the model previously proposed for Arabidopsis (Arabidopsis thaliana) are discussed.

Figure 1.

ZmPIN1 quantitative expression analysis. Quantitative real-time PCR analysis reveals that ZmPIN1 genes show differential expression patterns during kernel development. All three genes are up-regulated after double fertilization of the maize embryo sac: ZmPIN1c shows an increase in expression levels (7-fold increase) in 3-DAP kernels, while ZmPIN1a expression increases during later stages. The average value from three replicates and two biological samples was estimated for the three ZmPIN1 mRNAs. This value was subsequently normalized by applying the Pfaffl (2001) method to the transcript level of the housekeeping genes 18S and GAPC2, in order to avoid differences in the RNA extraction and cDNA synthesis. Bar diagrams show standardized average levels of ZmPIN1 transcripts, expressed as fold change with respect to the mRNA levels measured in ovaries from unfertilized ears. sd bars are indicated.

Figure 2.

In situ hybridization of ZmPIN1 mRNAs during maize kernel development. All the images represent longitudinal sections of B73 inbred kernels labeled with an antisense mRNA probe that hybridizes to all three ZmPIN1 transcripts. As soon as the double fertilization occurs, ZmPIN1 genes start to be expressed around the free dividing nuclei that form the syncytium (A), while at the end of cellularization phase, the endosperm shows a gradient of ZmPIN1 expression. The transcripts are most abundant in the basal part of the endosperm (B), a region that will differentiate in the BETL domain and in the cells of the ESR. At the end of differentiation, both these domains show a high expression of ZmPIN1 (C). In BETL cells, ZmPIN1 transcripts are very abundant and compartmentalized on the inner side of the cells (D and E); they also mark the above-located conductive cells (C). In the ESR domain, the ZmPIN1 hybridization signal is particularly strong during early stages of embryogenesis (C, F, and G). During embryogenesis, ZmPIN1 mRNAs are mainly expressed in the embryo proper at the proembryo stage (F and G). Later on, at the early transition stage, ZmPIN1 transcripts mark both the central group of cells in the embryo proper and the differentiating protoderm (H), while no ZmPIN1 expression is detectable in the suspensor (H). The arrow in H indicates the protoderm layer. During the late transition stage, ZmPIN1 mRNAs are mainly localized on the adaxial surface where SAM bulges out (I) and in the scutellum tip (J), whereas very weak hybridization signal is detectable on the scutellum abaxial side (J). A high expression of ZmPIN1 is detectable in the scutellum provascular tissues and in the RAM (K). During the morphogenetic phase, SAM produces five to six leaf primordia and ZmPIN1 genes are expressed in the corpus of the SAM, in the developing primordia, and in the vasculature of the differentiated leaves (L and M). The signal is still present in the RAM and coleorhiza (L). CC, Conductive cells; col, coleoptile; ep, embryo proper; scu, scutellum; se, starchy endosperm; su, suspensor. Bars = 200 μ m in L; 100 μ m in C, G, H to K, and M; 50 μ m in A, B, D, and F; and 25 μ m in E.

Figure 3.

ZmPIN1 protein localization during maize endosperm development. All images portray maize B73 kernel sections labeled with the anti-AtPIN1 antibody plus a secondary antibody carrying the Alexa568 fluorochrome. A, H, and I present laser confocal images, while B to G present epifluorescence images acquired with a Leica DC300F camera. Nuclei in B and E are blue due to DAPI staining. During the first events of endosperm development, ZmPIN1 proteins are detectable in the nuclear cytoplasmic domains of syncytial endosperm, where proteins are localized in endomembrane systems (A). ZmPIN1 proteins mark the formation of the first anticlinal cell plasma membrane at the beginning of the cellularization phase, while endomembranes of the syncytium are still labeled (B). The arrow in B indicates the newly formed anticlinal plasma membrane. At the end of the cellularization phase, ZmPIN1 proteins are detectable both in endomembranes and cell plasma membranes without any detectable polarity (C). A gradient of protein expression is evident: the proteins are more abundant in the basal-chalazal region of the endosperm (C), where BETL and ESR will differentiate. Both transfer layer and ESR cells show high ZmPIN1 levels (D and E), but these domains are characterized by two different ZmPIN1 localization patterns. In BETL and inner conductive cells, the proteins are localized in all the cell membranes, without any detectable polarization, suggesting a homeostasis of auxin levels between the cells of this endosperm domain (D, F, and H). In ESR, ZmPIN1s are detectable almost exclusively in the cell endomembranes (D, G, and I), suggesting a lack of auxin efflux mediated by PIN1 proteins from these cells. CC, Conductive cells; ep, embryo proper. Bars = 200 μ m in E; 100 μ m in A, C, D, F, and G; 75 μ m in I; 50 μ m in B; and 40 μ m in H.

Figure 4.

ZmPIN1 protein localization during maize embryogenesis. All images portray maize B73 embryo sections labeled with the anti-AtPIN1 antibody plus a secondary antibody carrying the Alexa568 fluorochrome. G to I and K present laser confocal images, while A to F and J present epifluorescence images acquired with a Leica DC300F camera. Green and blue color in B and J represents tissue autofluorescence collected with GFP and DAPI filters, respectively. E and F represent bigger magnification of D, while H reports a detail of K. During the proembryo stage, ZmPIN1 proteins mark a group of cells placed in the middle of the proembryo, without any evident polarity, while no signal is detectable in the suspensor (A and B). In the early transition phase, ZmPIN1 proteins are expressed in the upper cells of the proembryo and detectable and polarized only in the anticlinal membranes of differentiating embryo protoderm, indicating an auxin flux directed upward and converging toward the embryo tip (C). Later on, at the transition stage, ZmPIN1 proteins mark the initials of the SAM and inner tissues of the developing scutellum (D). The SAM initials are characterized by a central group of cells presenting apolar ZmPIN1 protein localization (E), while along the scutellum, the proteins mark the apical cell membranes, indicating the presence of an acropetal auxin flux directed toward the tip of the scutellum (F). Arrowheads in F indicate the polarization of ZmPIN1 proteins in apical cell membranes. At the late coleoptilar stage, an inversion of ZmPIN1 polarization is evident in the scutellum: after scutellum vasculature differentiation, the ZmPIN1 proteins are localized in the basal cell membranes, suggesting an auxin flux directed downward (G). Arrowheads in G indicate the polarization of ZmPIN1 proteins in basal cell plasma membranes. ZmPIN1 protein relocation at the level of the scutellum corresponds to the establishment of a continuous basally polarized localization of the ZmPIN1 proteins at the level of embryo axis (G). Therefore, two basipetal auxin fluxes can be detected at this stage: one from the scutellum tip to the SAM and RAM, and another from the SAM toward the root pole (G and I). Maize SAM initiates up to six true leaf primordia prior to seed dormancy, and during these processes, ZmPIN1s localize in all the cell membranes of the central group of cells in the SAM, while in the L1 outer layer ZmPIN1s localize only at the level of the incipient primordia (H, I, and K). ZmPIN1 proteins are basally localized in primordia provasculature, suggesting auxin fluxes directed from the primordia to the inner tissues of the SAM and then to the root pole (I and K). ZmPIN1 protein polarization also suggests basipetal auxin transport from the SAM to the RAM (I and J), and the proteins are also detectable in the coleorhiza (I and J) and at the sites of seminal root initiation (J). Moreover, when coleoptile is fully developed, a longitudinal section shows that ZmPIN1 proteins mark the apical cell membranes, suggesting auxin fluxes directed toward the coleoptile tip (K). clh, Coleorhiza; col, coleoptile; ep, embryo proper; I1, incipient primordium; P1 and P2, leaf primordia; scu, scutellum; sr, seminal root; su, suspensor. Bars = 100 μ m in D, G, I, and J; 75 μ m in K; 50 μ m in A to C, E, and F; and 20 μ m in H.

Figure 5.

IAA localization during maize kernel development. An anti-IAA monoclonal antibody was employed to determine the auxin distribution and accumulation in developing maize endosperm and embryos. IAA accumulation is detectable in the BETL domain at 8 DAP (B) and 12 DAP (C and D), in conductive cells at 12 DAP (C), and in the chalazal maternal tissues from 6 to 16 DAP (A–C), while only a weak signal is visible in the starchy endosperm (B, C, and E). Free IAA accumulation is detectable in ESR cells (F and G) and in the aleurone layer (E). Embryos at the globular stage are characterized by high auxin accumulation at the top of the embryo proper, while a very low signal is detectable in the suspensor (F). At the early and late transition stages, the auxin accumulation is evident in the protodermal layer and inner cells of the embryo proper, whereas suspensor shows the IAA minimum (G). The coleoptilar stage embryos are characterized by gradients of IAA distribution: the signal is high in the tip of the scutellum and in all the protoderm cells at the adaxial side, while it decreases toward the suspensor (H). Auxin maxima are detectable at the tip of the developing coleoptile, in the SAM, and in embryo root pole (H). During the SAM morphogenetic phase, auxin accumulation is evident in the tip of leaf primordia (I and J), in the corpus of the SAM (I and J), and in the RAM (J). al, Aleurone; CC, conductive cells; cha, chalaza; col, coleoptile; ep, embryo proper; I1, incipient primordium; P1, leaf primordium; pro, protoderm; scu, scutellum; se, starchy endosperm; su, suspensor. Bars = 100 μ m in A to C, F to H, and J; and 50 μ m in D, E, and I.

Figure 6.

IAA accumulation and ZmPIN1 and BETL1 transcript localization in de*-B18 endosperm transfer layer cells. Longitudinal sections of kernels at 12 DAP of B73 (A–C) and de*-B18 (D–F) were labeled with an antisense mRNA probe that hybridizes to all three ZmPIN1 transcripts (A and D) and with an antisense BETL1 probe (B and E; Hueros et al., 1995). Anti-IAA monoclonal antibody was used to determine the auxin distribution and accumulation in developing BETL cells (C and F). Unlike in B73 wild-type kernels (A), de*-B18 maize mutant kernels lack ZmPIN1 expression at the level of BETL cells (D) and show an altered differentiation pattern of these cells, which are round shaped and not polarized (D). Compared with the wild type (B), also the BETL1 gene is not expressed in mutant transfer and conductive cells (E). These alterations are accompanied by a reduction of auxin accumulation in BETL cells (C and F), while IAA accumulates as in the wild type in the chalazal maternal region. cha, Chalaza; se, starchy endosperm. Bars = 100 μ m in B, C, E, and F; and 50 μ m in A and D.

Figure 7.

Effects of NPA treatment on embryo and endosperm differentiation, ZmPIN1 expression, and auxin accumulation. The figure represents Calcofluor staining (A–J), ZmPIN1 in situ hybridization (K–N), ZmPIN1 immunolocalization (O–T), and IAA localization (U–X) on longitudinal sections of kernels at different developmental stages collected from plants watered for 2 weeks with 80 or 120 μm NPA. Q and R present laser confocal images, while A to J, O, P, S, and T present epifluorescence images acquired with a Leica DC300F camera. Blue color in T represents tissue autofluorescence collected with a DAPI filter. P reports a detail of O. The serial sections through a 17-DAP embryo collected from a plant watered for 19 d with 80 μm NPA (A–G) show that NPA induces the formation of a newly semicircular ridge of cells above the meristem, while the two leaf primordia only partially enclose the SAM. At the same stage, NPA treatments result in abnormal scutellum growth with complete loss of symmetry (H) and altered differentiation of vascular bundles in both scutellum (H and I) and embryonic root (J). Growth of misspecified apical structures is observed also in a 9-DAP embryo treated for 15 d with 120 μm NPA (Q), while the most severe phenotype, with two embryos in the same kernel, was observed in 11-DAP seeds treated for 15 d with 80 μm NPA (W). NPA treatments induce ZmPIN1 gene and protein ectopic expression. In 15-DAP kernels treated for 18 d with 80 μm NPA, ZmPIN1 transcripts (L) and proteins (O and P) mark the pluristratified aleurone, while no hybridization signal is detectable in the mock control treated with DMSO only for 18 d (K). ZmPIN1 protein plasma membrane insertion is observable in some aleurone cells (O and P). ZmPIN1 ectopic expression is detectable in the maternal tissues corresponding to the chalaza (M). At the proembryo stage (9-DAP embryo treated for 15 d with 120 μm NPA), ZmPIN1 protein localization is restricted to the apical part of the embryo proper (Q), while at the early transition stage (11-DAP embryo treated for 15 d with 120 μm NPA), the proteins were not inserted in the plasma membranes of the outer cell layer at the protoderm differentiation stage (R). See Figure 4, A to C, for ZmPIN1 protein localization in untreated embryos at the proembryo and early transition stages. During the embryo morphogenetic phase (15-DAP embryo treated for 19 d with 120 μm NPA), ZmPIN1 transcripts are detectable in the whole embryo (N) and in particular in embryo mesocotyl and hypocotyl (N), regions in which no hybridization signal is detectable in untreated kernels (Fig. 2M). In 15-DAP embryo treated for 18 d with 80 μm NPA, ZmPIN1 proteins mark all the cell plasma membranes without any evident polarity (S), and an altered localization of ZmPIN1 proteins is also detectable in the scutellum provascular tissues and in the abnormal embryonic root (T). See Figure 2, I and J, for ZmPIN1 protein localization in untreated embryos. Inhibition of polar auxin transport causes alteration of auxin gradients inside the kernel and embryo. In 13-DAP kernels treated for 15 d with 120 μm NPA, a very high IAA accumulation is visible in the altered aleurone (U and V), while a uniform IAA distribution is evident in the transfer cells toward the central endosperm (U). NPA treatments result in loss of the auxin gradient inside the embryo and in a uniform IAA distribution in both the embryo and the scutellum, with a higher IAA level in the coleorhiza of 15-DAP embryos treated for 19 d with 120 μm NPA (X). See Figure 5 for auxin accumulation patterns in untreated endosperms and embryos. White arrowheads indicate the semicircular structure that surrounds the NPA-treated SAM, while red arrowheads point to the abnormal, enlarged, and irregular vascular tissues. Blue asterisks mark two abnormal structures flanking the embryo proper. al, Aleurone; cha, chalaza; clh, coleorhiza; col, coleoptile; hyp, hypocotyl; mes, mesocotyl; P1 and P2, first and second leaf primordia; scu, scutellum; se, starchy endosperm; su, suspensor. Bars = 200 μ m in A to G and U; 100 μ m in H to J, M to O, and W; 75 μ m in Q and R; 50 μ m in K, L, S, T, V, and X; and 20 μ m in P.

Figure 8.

Overview of successive stages of maize embryogenesis and model for the ZmPIN1-mediated auxin transport during maize embryogenesis. Medial longitudinal sections of proembryo, transition, coleoptilar, and L1 and L2 stage maize embryos are shown, adapted from Bommert and Werr (2001). The localization of ZmPIN1 proteins in embryo plasma membrane is reported in red, and arrows indicate the polar auxin fluxes deduced by our results. After the first division of the zygote, a series of cell divisions in unpredictable planes gives rise, at the proembryo stage, to the small embryo proper and the larger suspensor. During the transition stage, adaxial/abaxial polarity is manifested by outgrowth of the scutellum at the abaxial side of the embryo. By the late transition stage, the SAM is evident as a group of cytoplasm-rich cells on the adaxial side. At the coleoptilar stage, the coleoptile becomes evident histologically above the SAM. Activity of the SAM is detected at leaf stage 1 (L1) by emergence of the first leaf primordium opposite the coleoptile. Note that the shoot-root axis is oriented in an oblique angle relative to the apical-basal polarity of the proembryo. The second leaf is initiated opposite the first leaf. The RAM is protected by the coleorhiza. clh, Coleorhiza; col, coleoptile; ep, embryo proper; I1, incipient primordium; P1 and P2, leaf primordia; scu, scutellum; su, suspensor.