The gene MACCHI-BOU 4/ENHANCER OF PINOID encodes a NPH3-like protein and reveals similarities between organogenesis and phototropism at the molecular level

Intercellular transport of the phytohormone auxin is a significant factor for plant organogenesis. To investigate molecular mechanisms by which auxin controls organogenesis, we analyzed the macchi-bou 4 (mab4) mutant identified as an enhancer of pinoid (pid). Although mab4 and pid single mutants displayed relatively mild cotyledon phenotypes, pid mab4 double mutants completely lacked cotyledons. We found that MAB4 was identical to ENHANCER OF PINOID (ENP), which has been suggested to control PIN1 polarity in cotyledon primordia. MAB4/ENP encodes a novel protein, which belongs to the NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) family thought to function as a signal transducer in phototropism and control lateral translocation of auxin. MAB4/ENP mRNA was detected in the protodermal cell layer of the embryo and the meristem L1 layer at the site of organ initiation. In the mab4 embryo, the abundance of PIN1:GFP was severely decreased at the plasma membrane in the protodermal cell layer. In addition, subcellular localization analyses indicated that MAB4/ENP resides on a subpopulation of endosomes as well as on unidentified intracellular compartments. These results indicate that MAB4/ENP is involved in polar auxin transport in organogenesis.     

PIN-FORMED1 and PINOID regulate boundary formation and cotyledon development in Arabidopsis embryogenesis

In dicotyledonous plants, two cotyledons are formed at bilaterally symmetric positions in the apical region of the embryo. Single mutations in the PIN-FORMED1 (PIN1) and PINOID (PID) genes, which mediate auxin-dependent organ formation, moderately disrupt the symmetric patterning of cotyledons. We report that the pin1 pid double mutant displays a striking phenotype that completely lacks cotyledons and bilateral symmetry. In the double mutant embryo, the expression domains of CUP-SHAPED COTYLEDON1 (CUC1), CUC2 and SHOOT MERISTEMLESS (STM), the functions of which are normally required to repress growth at cotyledon boundaries, expand to the periphery and overlap with a cotyledon-specific marker, FILAMENTOUS FLOWER. Elimination of CUC1, CUC2 or STM activity leads to recovery of cotyledon growth in the double mutant, suggesting that the negative regulation of these boundary genes by PIN1 and PID is sufficient for primordium growth. We also show that PID mRNA is localized mainly to the boundaries of cotyledon primordia and early expression of PID mRNA is dependent on PIN1. Our results demonstrate the redundant roles of PIN1 and PID in the establishment of bilateral symmetry, as well as in the promotion of cotyledon outgrowth, the latter of which involves the negative regulation of CUC1, CUC2 and STM genes, which are boundary-specific downstream effectors.     

Shoot apical meristem and cotyledon formation during Arabidopsis embryogenesis: interaction among the CUP-SHAPED COTYLEDON and SHOOT MERISTEMLESS genes

The shoot apical meristem and cotyledons of higher plants are established during embryogenesis in the apex. Redundant CUP-SHAPED COTYLEDON 1 (CUC1) and CUC2 as well as SHOOT MERISTEMLESS (STM) of Arabidopsis are required for shoot apical meristem formation and cotyledon separation. To elucidate how the apical region of the embryo is established, we investigated genetic interactions among CUC1, CUC2 and STM, as well as the expression patterns of CUC2 and STM mRNA. Expression of these genes marked the incipient shoot apical meristem as well as the boundaries of cotyledon primordia, consistent with their roles for shoot apical meristem formation and cotyledon separation. Genetic and expression analyses indicate that CUC1 and CUC2 are redundantly required for expression of STM to form the shoot apical meristem, and that STM is required for proper spatial expression of CUC2 to separate cotyledons. A model for pattern formation in the apical region of the Arabidopsis embryo is presented.     

Phosphorylation of Conserved PIN Motifs Directs Arabidopsis PIN1 Polarity and Auxin Transport

Polar cell-to-cell transport of auxin by plasma membrane–localized PIN-FORMED (PIN) auxin efflux carriers generates auxin gradients that provide positional information for various plant developmental processes. The apical-basal polar localization of the PIN proteins that determines the direction of auxin flow is controlled by reversible phosphorylation of the PIN hydrophilic loop (PINHL). Here, we identified three evolutionarily conserved TPRXS(N/S) motifs within the PIN1HL and proved that the central Ser residues were phosphorylated by the PINOID (PID) kinase. Loss-of-phosphorylation PIN1:green fluorescent protein (GFP) (Ser to Ala) induced inflorescence defects, correlating with their basal localization in the shoot apex, and induced internalization of PIN1:GFP during embryogenesis, leading to strong embryo defects. Conversely, phosphomimic PIN1:GFP (Ser to Glu) showed apical localization in the shoot apex but did not rescue pin1 inflorescence defects. Both loss-of-phosphorylation and phosphomimic PIN1:GFP proteins were insensitive to PID overexpression. The basal localization of loss-of-phosphorylation PIN1:GFP increased auxin accumulation in the root tips, partially rescuing PID overexpression-induced root collapse. Collectively, our data indicate that reversible phosphorylation of the conserved Ser residues in the PIN1HL by PID (and possibly by other AGC kinases) is required and sufficient for proper PIN1 localization and is thus essential for generating the differential auxin distribution that directs plant development.     

Regulation of auxin response by the protein kinase PINOID

Arabidopsis plants carrying mutations in the PINOID (PID) gene have a pleiotropic shoot phenotype that mimics that of plants grown on auxin transport inhibitors or of plants mutant for the auxin efflux carrier PINFORMED (PIN), with defects in the formation of cotyledons, flowers, and floral organs. We have cloned PID and find that it is transiently expressed in the embryo and in initiating floral anlagen, demonstrating a specific role for PID in promoting primordium development. Constitutive expression of PID causes a phenotype in both shoots and roots that is similar to that of auxin-insensitive plants, implying that PID, which encodes a serine-threonine protein kinase, negatively regulates auxin signaling.     

The GURKE gene encoding an acetyl-CoA carboxylase is required for partitioning the embryo apex into three subregions in Arabidopsis

In Arabidopsis, three major regions, which ultimately develop into the two cotyledons, the cotyledon boundaries and the shoot apical meristem (SAM), are formed at the apex of the globular stage embryo. To reveal the molecular mechanism underlying this pattern formation, we isolated a cotyledon-defective mutant from EMS mutagenized lines. This mutant completely lacks cotyledons in the most severe cases, and is allelic to gurke (gk), which was previously reported as a mutant defective in apical patterning of the embryo. To evaluate the morphological effects of the mutation in the GK gene, we investigated the expression patterns in gk embryos of SHOOT MERISTEMLESS (STM), AINTEGUMENTA (ANT) and CUP-SHAPED COTYLEDON1 (CUC1), which are markers of the SAM, cotyledons and cotyledon boundaries, respectively. Expression of all these genes largely overlapped in gk, suggesting a failure to partition the apex of the embryo into the three subregions. Enlargement of the CUC1 expression domain was also observed and may explain the inhibition of cotyledon development in gk. Moreover, we cloned the GK gene, and confirmed that it encodes ACC1, an acetyl-CoA carboxylase which catalyzes malonyl-CoA synthesis. Our results suggest that metabolites derived from malonyl-CoA are required for partitioning of the apical part of the embryo.     

Ribosomal protein L27a is required for growth and patterning in Arabidopsis thaliana

Ribosomal proteins are integral to ribosome biogenesis, and function in protein synthesis. In higher eukaryotes, loss of cytoplasmic ribosomal proteins results in a reduced growth rate as well as developmental defects. To what extent and how ribosomal proteins affect development is currently not known. Here we describe a semi-dominant mutation in the cytoplasmic ribosomal protein gene RPL27aC that affects multiple aspects of plant shoot development, including leaf patterning, inflorescence and floral meristem function, and seed set. In the embryo, RPL27aC is required to maintain the growth rate and for the transition from radial to bilateral symmetry associated with initiation of cotyledons. rpl27ac-1d embryos undergo stereotypical patterning to establish a globular embryo. However, a temporal delay in initiation and outgrowth of cotyledon primordia leads to development of an enlarged globular embryo prior to apical domain patterning. Defects in embryo development are coincident with tissue-specific ectopic expression of the shoot meristem genes SHOOT MERISTEMLESS (STM) and CUP-SHAPED COTYLEDON2 (CUC2), in addition to delayed expression of the abaxial gene FILAMENTOUS FLOWER (FIL) and mis-regulation of the auxin efflux effector PIN-FORMED1 (PIN1). Genetic interactions with other ribosomal protein mutants indicate that RPL27aC is a component of the ribosome. We propose that RPL27aC regulates discrete developmental events by controlling spatial and temporal expression of developmental patterning genes via an as yet undefined process involving the ribosome.     

POPCORN functions in the auxin pathway to regulate embryonic body plan and meristem organization in Arabidopsis

The shoot and root apical meristems (SAM and RAM) formed during embryogenesis are crucial for postembryonic plant development. We report the identification of POPCORN (PCN), a gene required for embryo development and meristem organization in Arabidopsis thaliana. Map-based cloning revealed that PCN encodes a WD-40 protein expressed both during embryo development and postembryonically in the SAM and RAM. The two pcn alleles identified in this study are temperature sensitive, showing defective embryo development when grown at 22°C that is rescued when grown at 29°C. In pcn mutants, meristem-specific expression of WUSCHEL (WUS), CLAVATA3, and WUSCHEL-RELATED HOMEOBOX5 is not maintained; SHOOTMERISTEMLESS, BODENLOS (BDL) and MONOPTEROS (MP) are misexpressed. Several findings link PCN to auxin signaling and meristem function: ectopic expression of DR5(rev):green fluorescent protein (GFP), pBDL:BDL-GFP, and pMP:MP-β-glucuronidase in the meristem; altered polarity and expression of pPIN1:PIN1-GFP in the apical domain of the developing embryo; and resistance to auxin in the pcn mutants. The bdl mutation rescued embryo lethality of pcn, suggesting that improper auxin response is involved in pcn defects. Furthermore, WUS, PINFORMED1, PINOID, and TOPLESS are dosage sensitive in pcn, suggesting functional interaction. Together, our results suggest that PCN functions in the auxin pathway, integrating auxin signaling in the organization and maintenance of the SAM and RAM.     

Coordination of apical and basal embryo development revealed by tissue-specific GNOM functions

Flowering-plant embryogenesis generates the basic body organization, including the apical and basal stem cell niches, i.e. shoot and root meristems, the major tissue layers and the cotyledon(s). gnom mutant embryos fail to initiate the root meristem at the early-globular stage and the cotyledon primordia at the late globular/transition stage. Tissue-specific GNOM expression in the gnom mutant embryo revealed that both apical and basal embryo organization depend on GNOM provascular expression and a functioning apical-basal auxin flux: GNOM provascular expression in gnom mutant background resulted in non-cell-autonomous reconstitution of apical and basal tissues which could be linked to changes in auxin responses in those tissues, stressing the importance of apical-basal auxin flow for overall embryo organization. Although reconstitution of apical-basal auxin flux in gnom results in the formation of single cotyledons (monocots), only additional GNOM epidermal expression is able to induce wild-type apical patterning. We conclude that provascular expression of GNOM is vital for both apical and basal tissue organization, and that epidermal GNOM expression is required for radial-to-bilateral symmetry transition of the embryo. We propose GNOM-dependent auxin sinks as a means to generate auxin gradients across tissues.     

single cotyledon (sic) mutants of pea and their significance in understanding plant embryo development

Angiosperms are divided into two distinct classes—the dicotyledons (dicots) and monocotyledons (monocots)—based in part on the number of cotyledons in mature embryos. In this paper, we describe single‐cotyledon pea mutants, termed sic (single cotyledon), all of which show a degree of fusion between the cotyledons. The fusion in sic1 is along the margin of one cotyledon and is less complete than in sic2 embryos, but the effects of the mutations are additive in the double mutant. Occasionally sic2 mutants will show fusion of the two cotyledons into one cylindrical embryo in which the shoot apex becomes surrounded by the cotyledons. Both sic1 and sic2 mutants produce fertile plants. In the sic3 embryo, a single cotyledon is generated under the shoot apex that breaks the vascular connection between root and shoot, causing embryo lethality. The pattern of cotyledon development in all these mutants is identified by in situ mRNA hybridization and antibody labeling, using the storage protein vicilin as a cotyledon‐specific marker. These patterns indicate that the joining of the cotyledons was due to zonal growth. The results indicate that there are genes in pea that influence the positioning and the morphology of the cotyledon. A model for cotyledon development in pea is proposed that is based on the regulation of the positioning of cell clusters by the sic genes. Dev. Genet. 25:11–22, 1999. © 1999 Wiley‐Liss, Inc.     

The AGC kinase,PINOID, blocks interactive ABCB/PIN auxin transport

Plant growth and development is determined by intracellular and intercellular auxin gradients that are controlled at first hand by auxin efflux catalysts of the ABCB/PGP and PIN families. ABCB transport activity was shown to be counter-actively regulated by protein phosphorylation by the AGC protein kinase, PINOID (PID), that is coordinated by interaction with the immunophilin-like FKBP42, TWISTED DWARF1 (TWD1). In contrast, PID was shown to determine PIN polarity, however, the direct impact of PID on PIN activity has yet not been tested. Co-expression in yeast indicates that PID had no effect on PIN1,2 alone but specifically inhibits interactive ABCB1-PIN1/PIN2 auxin efflux in an action that is dependent on its kinase activity. PIN1-PID co-transfection in N. benthamiana revealed that PID blocks PIN1-mediated auxin efflux without changing PIN1 location. In summary, these data provide evidence that PID phosphorylation does not only determine PIN polarity but also has a direct impact on transport activity of the activity of the binary PIN-ABCB1 complex.     

An expanded role for the TWN1 gene in embryogenesis: defects in cotyledon pattern and morphology in the twn1 mutant of Arabidopsis (Brassicaceae)

The suspensor is a specialized basal structure that differentiates early in plant embryogenesis to support development of the embryo proper. Suspensor differentiation in Arabidopsis is maintained in part by the TWIN1 (TWN1) gene, which suppresses embryogenic development in suspensor cells: twn1 mutants produce supernumerary embryos via suspensor transformation. To better understand mechanisms of suspensor development and further investigate the function of TWN1, we have characterized late-embryo and post-embryonic development in the twn1 mutant, using seedling culture, microscopy, and genetics. We report here that the twn1 mutation disrupts cotyledon number, arrangement, and morphology and occasionally causes partial conversion of cotyledons into leaves. These defects are not a consequence of suspensor transformation. Thus, in addition to its basal role in suspensor differentiation, TWN1 influences apical pattern and morphology in the embryo proper. To determine whether other genes can similarly affect both suspensor and cotyledon development, we looked for twinning in Arabidopsis mutants previously identified by their abnormal cotyledon phenotypes. One such mutant, amp1, produced a low frequency of twin embryos by suspensor transformation. Our results suggest that mechanisms that maintain suspensor identity also function later in development to influence organ formation at the embryonic shoot apex. We propose that TWN1 functions in cell communication pathways that convey local positional information in both the apical and basal regions of the Arabidopsis embryo.     

Microsurgery reveals regional capabilities for pattern reestablishment in somatic carrot embryos

The extent to which regions of a somatic embryo were committed to a particular developmental fate was explored by surgically removing portions of somatic embryos and observing patterns of regeneration. Through a variety of excisions that resulted in tissue slices ranging from less than 10% to nearly 90% of the original embryo mass, we observed only a few cases where such isolates completely abandoned preexisting patterns of organized growth. Instead, most subcultured portions of the embryonic axis restored all, or part of, a missing complement of the organism. At the shoot apex, a single lost cotyledon was replaced by new cotyledonary structures, although these usually occurred as multiple pairs of cotyledons. If both cotyledons were removed, secondary axes, each with its own cotyledons, typically formed at the embryo midlength. When embryos were divided into shoot and root pieces, the shoot pole usually regenerated a new root, while the original root and rapidly elongated and matured days earlier than uncut controls. Surprisingly, cotyledon regeneration from excised root sections occurred at much greater frequency when the root piece comprised only 10-25% of the embryo mass; larger portions of the root pole rarely produced recognizable shoot structures. These studies indicate that several discrete regions of the embryo are committed to specific types of patterned growth, and that continuity between certain of these regions is required for the maintenance of axial polarity.     

PIN Auxin Efflux Carrier Polarity Is Regulated by PINOID Kinase-Mediated Recruitment into GNOM-Independent Trafficking in Arabidopsis

The phytohormone auxin plays a major role in embryonic and postembryonic plant development. The temporal and spatial distribution of auxin largely depends on the subcellular polar localization of members of the PIN-FORMED (PIN) auxin efflux carrier family. The Ser/Thr protein kinase PINOID (PID) catalyzes PIN phosphorylation and crucially contributes to the regulation of apical-basal PIN polarity. The GTP exchange factor on ADP-ribosylation factors (ARF-GEF), GNOM preferentially mediates PIN recycling at the basal side of the cell. Interference with GNOM activity leads to dynamic PIN transcytosis between different sides of the cell. Our genetic, pharmacological, and cell biological approaches illustrate that PID and GNOM influence PIN polarity and plant development in an antagonistic manner and that the PID-dependent PIN phosphorylation results in GNOM-independent polar PIN targeting. The data suggest that PID and the protein phosphatase 2A not only regulate the static PIN polarity, but also act antagonistically on the rate of GNOM-dependent polar PIN transcytosis. We propose a model that includes PID-dependent PIN phosphorylation at the plasma membrane and the subsequent sorting of PIN proteins to a GNOM-independent pathway for polarity alterations during developmental processes, such as lateral root formation and leaf vasculature development.     

The PINOID protein kinase regulates organ development in Arabidopsis by enhancing polar auxin transport

Arabidopsis pinoid mutants show a strong phenotypic resemblance to the pin-formed mutant that is disrupted in polar auxin transport. The PINOID gene was recently cloned and found to encode a protein-serine/threonine kinase. Here we show that the PINOID gene is inducible by auxin and that the protein kinase is present in the primordia of cotyledons, leaves and floral organs and in vascular tissue in developing organs or proximal to meristems. Overexpression of PINOID under the control of the constitutive CaMV 35S promoter (35S::PID) resulted in phenotypes also observed in mutants with altered sensitivity to or transport of auxin. A remarkable characteristic of high expressing 35S::PID seedlings was a frequent collapse of the primary root meristem. This event triggered lateral root formation, a process that was initially inhibited in these seedlings. Both meristem organisation and growth of the primary root were rescued when seedlings were grown in the presence of polar auxin transport inhibitors, such as naphthylphtalamic acid (NPA). Moreover, ectopic expression of PINOID cDNA under control of the epidermis-specific LTP1 promoter provided further evidence for the NPA-sensitive action of PINOID. The results presented here indicate that PINOID functions as a positive regulator of polar auxin transport. We propose that PINOID is involved in the fine-tuning of polar auxin transport during organ formation in response to local auxin concentrations.     

MACCHI-BOU 2 is required for early embryo patterning and cotyledon organogenesis in Arabidopsis

The phytohormone auxin is a key regulator of organogenesis in plants and is distributed asymmetrically via polar transport. However, the precise mechanisms underlying auxin-mediated organogenesis remain elusive. Here, we have analyzed the macchi-bou 2 (mab2) mutant identified in a pinoid (pid) enhancer mutant screen. Seedlings homozygous for either mab2 or pid showed only mild phenotypic effects on cotyledon positions and/or numbers. In contrast, mab2 pid double mutant seedlings completely lacked cotyledons, indicating a synergistic interaction. We found that mab2 homozygous embryos had defective patterns of cell division and showed aberrant cotyledon organogenesis. Further analysis revealed that the mab2 mutation affected auxin response but not auxin transport in the embryos, suggesting the involvement of MAB2 in auxin response during embryogenesis. MAB2 encodes an Arabidopsis ortholog of MED13, a putative regulatory module component of the Mediator complex. Mediator is a multicomponent complex that is evolutionarily conserved in eukaryotes and its regulatory module associates with Mediator to control the interaction of Mediator and RNA polymerase II. MAB2 interacts with a regulatory module component in yeast cells. Taken together, our data suggest that MAB2 plays a crucial role in embryo patterning and cotyledon organogenesis, possibly through modulating expression of specific genes such as auxin-responsive genes.