[show abstract][hide abstract] ABSTRACT: Phyllotaxis, the regular arrangement of leaves and flowers around the stem, is a key feature of plant architecture. Current models propose that the spatiotemporal regulation of organ initiation is controlled by a positive feedback loop between the plant hormone auxin and its efflux carrier PIN-FORMED1 (PIN1). Consequently, pin1 mutants give rise to naked inflorescence stalks with few or no flowers, indicating that PIN1 plays a crucial role in organ initiation. However, pin1 mutants do produce leaves. In order to understand the regulatory mechanisms controlling leaf initiation in Arabidopsis (Arabidopsis thaliana) rosettes, we have characterized the vegetative pin1 phenotype in detail. We show that although the timing of leaf initiation in vegetative pin1 mutants is variable and divergence angles clearly deviate from the canonical 137° value, leaves are not positioned at random during early developmental stages. Our data further indicate that other PIN proteins are unlikely to explain the persistence of leaf initiation and positioning during pin1 vegetative development. Thus, phyllotaxis appears to be more complex than suggested by current mechanistic models.
[show abstract][hide abstract] ABSTRACT: Although genetic control of morphogenesis is well established, elaboration of complex shapes requires changes in the mechanical properties of cells. In plants, the first visible sign of leaf formation is a bulge on the flank of the shoot apical meristem. Bulging results from local relaxation of cell walls, which causes them to yield to internal hydrostatic pressure. By manipulation of tissue tension in combination with quantitative live imaging and finite-element modeling, we found that the slow-growing area at the shoot tip is substantially strain-stiffened compared with surrounding fast-growing tissue. We propose that strain stiffening limits growth, restricts organ bulging, and contributes to the meristem's functional zonation. Thus, mechanical signals are not just passive readouts of gene action but feed back on morphogenesis.
[show abstract][hide abstract] ABSTRACT: The dynamic segregation of membrane components within microdomains, such as the sterol-enriched and sphingolipid-enriched membrane rafts, emerges as a central regulatory mechanism governing physiological responses in various organisms. Over the past five years, plasma membrane located raft-like domains have been described in several plant species. The protein and lipid compositions of detergent-insoluble membranes, supposed to contain these domains, have been extensively characterised. Imaging methods have shown that lateral segregation of lipids and proteins exists at the nanoscale level at the plant plasma membrane, correlating detergent insolubility and membrane-domain localisation of presumptive raft proteins. Finally, the dynamic association of specific proteins with detergent-insoluble membranes upon environmental stress has been reported, confirming a possible role for plant rafts as signal transduction platforms, particularly during biotic interactions.
Current opinion in plant biology 09/2011; 14(6):642-9. · 10.33 Impact Factor
[show abstract][hide abstract] ABSTRACT: The multicellular nature of plants requires that cells should communicate in order to coordinate essential functions. This is achieved in part by molecular flux through pores in the cell wall, called plasmodesmata. We describe the proteomic analysis of plasmodesmata purified from the walls of Arabidopsis suspension cells. Isolated plasmodesmata were seen as membrane-rich structures largely devoid of immunoreactive markers for the plasma membrane, endoplasmic reticulum and cytoplasmic components. Using nano-liquid chromatography and an Orbitrap ion-trap tandem mass spectrometer, 1341 proteins were identified. We refer to this list as the plasmodesmata- or PD-proteome. Relative to other cell wall proteomes, the PD-proteome is depleted in wall proteins and enriched for membrane proteins, but still has a significant number (35%) of putative cytoplasmic contaminants, probably reflecting the sensitivity of the proteomic detection system. To validate the PD-proteome we searched for known plasmodesmal proteins and used molecular and cell biological techniques to identify novel putative plasmodesmal proteins from a small subset of candidates. The PD-proteome contained known plasmodesmal proteins and some inferred plasmodesmal proteins, based upon sequence or functional homology with examples identified in different plant systems. Many of these had a membrane association reflecting the membranous nature of isolated structures. Exploiting this connection we analysed a sample of the abundant receptor-like class of membrane proteins and a small random selection of other membrane proteins for their ability to target plasmodesmata as fluorescently-tagged fusion proteins. From 15 candidates we identified three receptor-like kinases, a tetraspanin and a protein of unknown function as novel potential plasmodesmal proteins. Together with published work, these data suggest that the membranous elements in plasmodesmata may be rich in receptor-like functions, and they validate the content of the PD-proteome as a valuable resource for the further uncovering of the structure and function of plasmodesmata as key components in cell-to-cell communication in plants.
PLoS ONE 01/2011; 6(4):e18880. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Remorins are plant-specific proteins found associated with plasma membrane microdomains, called lipid rafts. Recently, we have shown that this lipid raft marker also accumulates at plasmodesmata, likely within the plasma membrane lining these structures. Here, we have investigated the gene expression and protein accumulation patterns of remorin at the organ and cell type levels. We show that remorin level is significantly increased in dehiscent, mature and ageing tissues, as well as in source parts of the leaves, where mature branched plasmodesmata are in majority. These results suggest that remorin predominantly associates with mature branched plasmodesmata.
[show abstract][hide abstract] ABSTRACT: One of the most striking aspects of plant diversity is variation in leaf shape. Much of this diversity is achieved by the modulation of leaf blade dissection to form lobes or leaflets. Here, we show that the phytohormone auxin is a crucial signal regulating the partitioned outgrowth necessary to develop a dissected leaf. In developing leaves, the asymmetric distribution of auxin, driven by active transport, delineates the initiation of lobes and leaflets and specifies differential laminar outgrowth. Furthermore, homologous members of the AUX/indole-3-acetic acid (IAA) gene family mediate the action of auxin in determining leaf shape by repressing outgrowth in areas of low auxin concentration during both simple and compound leaf development. These results provide molecular evidence that leaflets initiate in a process reminiscent of organogenesis at the shoot apical meristem, but that compound and simple leaves regulate marginal growth through an evolutionarily conserved mechanism, thus shedding light on the homology of compound and simple leaves.
Development 10/2009; 136(17):2997-3006. · 6.60 Impact Factor
[show abstract][hide abstract] ABSTRACT: Remorins (REMs) are proteins of unknown function specific to vascular plants. We have used imaging and biochemical approaches and in situ labeling to demonstrate that REM clusters at plasmodesmata and in approximately 70-nm membrane domains, similar to lipid rafts, in the cytosolic leaflet of the plasma membrane. From a manipulation of REM levels in transgenic tomato (Solanum lycopersicum) plants, we show that Potato virus X (PVX) movement is inversely related to REM accumulation. We show that REM can interact physically with the movement protein TRIPLE GENE BLOCK PROTEIN1 from PVX. Based on the localization of REM and its impact on virus macromolecular trafficking, we discuss the potential for lipid rafts to act as functional components in plasmodesmata and the plasma membrane.
The Plant Cell 06/2009; 21(5):1541-55. · 9.25 Impact Factor
[show abstract][hide abstract] ABSTRACT: Many patterning events in plants are regulated by the phytohormone auxin. In fact, so many things are under the influence of auxin that it seems difficult to understand how a single hormone can do so much. Auxin moves throughout the plant via a network of specialized membrane-bound import and export proteins, which are often differentially expressed and polarized depending on tissue type. Here, we review simulation models of pattern formation that are based on the control of these transporters by auxin itself. In these transport-feedback models, diversity in patterning comes not from the addition of more morphogens, but rather by varying the mechanism that regulates the transporters.
Plant Cell and Environment 06/2009; 32(9):1258-71. · 5.14 Impact Factor
[show abstract][hide abstract] ABSTRACT: In Arabidopsis thaliana, auxin is a key regulator of tissue patterning in the developing embryo. We have identified a group of proteins that act downstream of auxin accumulation in auxin-mediated root and vascular development in the embryo. Combined mutations in OBERON1 (OBE1) and OBERON2 (OBE2) give rise to obe1 obe2 double mutant seedlings that closely phenocopy the monopteros (mp) mutant phenotype, with an absence of roots and defective development of the vasculature. We show that, in contrast to the situation in mp mutants, obe1 obe2 double mutant embryos show auxin maxima at the root pole and in the provascular region, and that the SCF(TIR1) pathway, which translates auxin accumulation into transcriptional activation of auxin-responsive genes, remains intact. Although we focus on the impact of obe mutations on aspects of embryo development, the effect of such mutations on a broad range of auxin-related gene expression and the tissue expression patterns of OBE genes in seedlings suggest that OBE proteins have a wider role to play in growth and development. We suggest that OBE1 and OBE2 most likely control the transcription of genes required for auxin responses through the action of their PHD finger domains.
The Plant Journal 05/2009; 59(3):426-36. · 6.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: The plant hormone auxin mediates developmental patterning by a mechanism that is based on active transport. In the shoot apical meristem, auxin gradients are thought to be set up through a feedback loop between auxin and the activity and polar localization of its transporter, the PIN1 protein. Two distinct molecular mechanisms for the subcellular polarization of PIN1 have been proposed. For leaf positioning (phyllotaxis), an "up-the-gradient" PIN1 polarization mechanism has been proposed, whereas the formation of vascular strands is thought to proceed by "with-the-flux" PIN1 polarization. These patterning mechanisms intersect during the initiation of the midvein, which raises the question of how two different PIN1 polarization mechanisms may work together. Our detailed analysis of PIN1 polarization during midvein initiation suggests that both mechanisms for PIN1 polarization operate simultaneously. Computer simulations of the resulting dual polarization model are able to reproduce the dynamics of observed PIN1 localization. In addition, the appearance of high auxin concentration in our simulations throughout the initiation of the midvein is consistent with experimental observation and offers an explanation for a long-standing criticism of the canalization hypothesis; namely, how both high flux and high concentration can occur simultaneously in emerging veins.
Genes & development 03/2009; 23(3):373-84. · 12.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Plasmodesmata (Pds) traverse the cell wall to establish a symplastic continuum through most of the plant. Rapid and reversible deposition of callose in the cell wall surrounding the Pd apertures is proposed to provide a regulatory process through physical constriction of the symplastic channel. We identified members within a larger family of X8 domain-containing proteins that targeted to Pds. This subgroup of proteins contains signal sequences for a glycosylphosphatidylinositol linkage to the extracellular face of the plasma membrane. We focused our attention on three closely related members of this family, two of which specifically bind to 1,3-beta-glucans (callose) in vitro. We named this family of proteins Pd callose binding proteins (PDCBs). Yellow fluorescent protein-PDCB1 was found to localize to the neck region of Pds with potential to provide a structural anchor between the plasma membrane component of Pds and the cell wall. PDCB1, PDCB2, and PDCB3 had overlapping and widespread patterns of expression, but neither single nor combined insertional mutants for PDCB2 and PDCB3 showed any visible phenotype. However, increased expression of PDCB1 led to an increase in callose accumulation and a reduction of green fluorescent protein (GFP) movement in a GFP diffusion assay, identifying a potential association between PDCB-mediated callose deposition and plant cell-to-cell communication.
The Plant Cell 03/2009; 21(2):581-94. · 9.25 Impact Factor
[show abstract][hide abstract] ABSTRACT: Symplastic domains in plants are defined by spatial limitations on cell-to-cell communication through plasmodesmata (Pds) and establish tissue boundaries necessary for metabolic and developmental programming. With the exception of the physical closure of Pds by callose, the cues and the processes for creating symplastic domains remain poorly understood. Recently, we identified a novel family of eight proteins, called Pd-located protein 1 (PDLP1). These proteins span the plasma membrane within Pds and likely form part of a signal transduction system that perceives external signals to regulate molecular trafficking between cells. For two members of this family that have high expression in the shoot apex we show that they have defined and partially overlapping tissue-specific expression patterns that correlate in part with previously defined symplastic domains. The importance of non-cell-autonomous proteins in shoot development and of the spatial rules that govern leaf and floral development highlight the need to have a clearer understanding of symplastic domains.
[show abstract][hide abstract] ABSTRACT: One of the most striking features of plant architecture is the regular arrangement of leaves and flowers around the stem, known as phyllotaxis. Peaks in concentration of the plant hormone auxin, generated by the polar localization of the PIN1 auxin efflux carrier, provide the instructive signal for primordium initiation. This mechanism generates the spacing between neighboring primordia, which results in regular phyllotaxis. Studies of the role of auxin transport in phyllotactic patterning have focused on PIN1-mediated efflux. Recent computer simulations indicate an additional role for transporter-mediated auxin uptake. Mutations in the AUX1 auxin influx carrier have not, however, been reported to cause an aerial phenotype. Here, we study the role of AUX1 and its paralogs LAX1, LAX2, and LAX3. Analysis of the quadruple mutant reveals irregular divergence angles between successive primordia. A highly unusual aspect of the phenotype is the occurrence of clusters of primordia, in violation of classical theory. At the molecular level, the sharp peaks in auxin levels and coordinated PIN polarization are reduced or lost. In addition, the increased penetrance of the phenotype under short-day conditions suggests that the AUX LAX transporters act to buffer the PIN-mediated patterning mechanism against environmental or developmental influences.
Genes & Development 04/2008; 22(6):810-23. · 12.44 Impact Factor
[show abstract][hide abstract] ABSTRACT: Plasmodesmata provide the cytoplasmic conduits for cell-to-cell communication throughout plant tissues and participate in a diverse set of non-cell-autonomous functions. Despite their central role in growth and development and defence, resolving their modus operandi remains a major challenge in plant biology. Features of protein sequences and/or structure that determine protein targeting to plasmodesmata were previously unknown. We identify here a novel family of plasmodesmata-located proteins (called PDLP1) whose members have the features of type I membrane receptor-like proteins. We focus our studies on the first identified type member (namely At5g43980, or PDLP1a) and show that, following its altered expression, it is effective in modulating cell-to-cell trafficking. PDLP1a is targeted to plasmodesmata via the secretory pathway in a Brefeldin A-sensitive and COPII-dependent manner, and resides at plasmodesmata with its C-terminus in the cytoplasmic domain and its N-terminus in the apoplast. Using a deletion analysis, we show that the single transmembrane domain (TMD) of PDLP1a contains all the information necessary for intracellular targeting of this type I membrane protein to plasmodesmata, such that the TMD can be used to target heterologous proteins to this location. These studies identify a new family of plasmodesmal proteins that affect cell-to-cell communication. They exhibit a mode of intracellular trafficking and targeting novel for plant biology and provide technological opportunities for targeting different proteins to plasmodesmata to aid in plasmodesmal characterisation.
[show abstract][hide abstract] ABSTRACT: With the completion of the sequencing of the Arabidopsis genome and the recent advances in proteomic technology, the identification of proteins from highly complex mixtures is now possible. Rather than using gel electrophoresis and peptide mass fingerprinting, we have used multidimensional protein identification technology (MudPIT) to analyse the "tightly-bound" proteome for purified cell walls from Arabidopsis cell suspension cultures. Using bioinformatics for the prediction of signal peptides for targeting to the secretory pathway and for the absence of ER retention signal, 89 proteins were selected as potential extracellular proteins. Only 33% of these were identified in previous proteomic analyses of Arabidopsis cell walls. A functional classification revealed that a large proportion of the proteins were enzymes, notably carbohydrate active enzymes, peroxidases and proteases. Comparison of all the published proteomic analyses for the Arabidopsis cell wall identified 268 non-redundant genes encoding wall proteins. Sixty of these (22%) were derived from our analysis of tightly-bound wall proteins.
[show abstract][hide abstract] ABSTRACT: A current challenge in plant biology is to identify the structural and functional components of plasmodesmata (PDs). The use of plant tissue as a source material for plasmodesmal characterisation has had limited success, so we have explored the frequency and features of PDs occurring in suspension cell cultures of Arabidopsis thaliana. This material has the advantages of homogeneity, quantity, and ease of disruption. Using light and electron microscopy and immunostaining for callose and calreticulin, we showed that suspension cells laid down abundant PDs in division walls, and that vestiges of these structures were retained as half PDs even when the cell-to-cell contacts were disrupted during culture growth. Although callose was a reliable marker for PD distribution, which was deposited in an organised collar around the neck of PDs, it was not abundant in unstressed cells. Calreticulin and the chemical stain 3,3'-dihexyloxacarbocyanine iodide also provided useful markers when monitoring PDs in cell wall preparations by light microscopy. Purified cell walls were shown to be virtually free of contamination from cytoplasmic components, except for the presence of small amounts of cortical endoplasmic reticulum attached to PDs. Hence, clean cell walls from A. thaliana suspension cells provide a valuable resource for a proteomic approach to the analysis of plasmodesmal components.