The O-Hyp glycosylation code in tobacco and Arabidopsis and a proposed role of Hyp-glycans in secretion

Most aspects of plant growth involve cell surface hydroxyproline (Hyp)-rich glycoproteins (HRGPs) whose properties depend on arabinogalactan polysaccharides and arabinosides that define the molecular surface. Potential glycosylation sites are defined by an O-Hyp glycosylation code: contiguous Hyp directs arabinosylation. Clustered non-contiguous Hyp directs arabinogalactosylation. Elucidation of this code involved a single species, tobacco (Nicotiana tabacum) BY-2 cells. However, recent work suggests species variation, perhaps tissue specific Hyp glycosylation. Thus, the extent to which the Hyp glycosylation code is 'global' needs testing. We compared the ability of distantly related Arabidopsis cell cultures to process putative HRGP glycosylation motifs encoded by synthetic genes. The genes included: repetitive Ser-Pro, Ser-Pro2, Ser-Pro4 and an analog of the tomato arabinogalactan-protein, LeAGP-1DeltaGPI. All were expressed as enhanced green fluorescent protein (EGFP) fusion glycoproteins, designated: AtSO-EGFP (O=Hyp), AtSO2-EGFP, AtSO4-EGFP and AtEGFP-LeAGP-1DeltaGPI, respectively. The Arabidopsis glycosylation patterns were essentially similar to those observed in Nicotiana: non-contiguous Hyp residues in AtSO-EGFP were glycosylated exclusively with arabinogalactan polysaccharides while contiguous Hyp in AtSO2-EGFP and AtSO4-EGFP was exclusively arabinosylated. Mixed contiguous and non-contiguous Hyp residues in AtEGFP-LeAGP-1DeltaGPI were also arabinosylated and arabinogalactosylated consistent with the code. However, slightly more arabinogalactosylated Hyp and less non-glycosylated Hyp in AtEGFP-LeAGP-1DeltaGPI than tobacco NtEGFP-LeAGP-1DeltaGPI suggested Arabidopsis prolyl hydroxylases have a slightly broader specificity. Arabidopsis Hyp-arabinogalactans differed from tobacco in decreased glucuronic acid content and lack of rhamnose. Yields of the EGFP fusion glycoproteins were dramatically higher than targeted EGFP lacking Hyp-glycomodules. This validates earlier suggestions that the glycosylation of proteins facilitates their secretion.     

Engineering ‘designer’ glycomodules for boosting recombinant protein secretion in tobacco hairy root culture and studying hydroxyproline‐O‐glycosylation process in plants

The key technical bottleneck for exploiting plant hairy root cultures as a robust bioproduction platform for therapeutic proteins has been low protein productivity, particularly low secreted protein yields. To address this, we engineered novel hydroxyproline (Hyp)‐O‐glycosylated peptides (HypGPs) into tobacco hairy roots to boost the extracellular secretion of fused proteins and to elucidate Hyp‐O‐glycosylation process of plant cell wall Hyp‐rich glycoproteins. HypGPs representing two major types of cell wall glycoproteins were examined: an extensin module consisting of 18 tandem repeats of ‘Ser‐Hyp‐Hyp‐Hyp‐Hyp’ motif or (SP4)₁₈ and an arabinogalactan protein module consisting of 32 tandem repeats of ‘Ser‐Hyp’ motif or (SP)₃₂. Each module was expressed in tobacco hairy roots as a fusion to the enhanced green fluorescence protein (EGFP). Hairy root cultures engineered with a HypGP module secreted up to 56‐fold greater levels of EGFP, compared with an EGFP control lacking any HypGP module, supporting the function of HypGP modules as a molecular carrier in promoting efficient transport of fused proteins into the culture media. The engineered (SP4)₁₈ and (SP)₃₂ modules underwent Hyp‐O‐glycosylation with arabino‐oligosaccharides and arabinogalactan polysaccharides, respectively, which were essential in facilitating secretion of the fused EGFP protein. Distinct non‐Hyp‐O‐glycosylated (SP4)₁₈‐EGFP and (SP)₃₂‐EGFP intermediates were consistently accumulated within the root tissues, indicating a rate‐limiting trafficking and/or glycosylation of the engineered HypGP modules. An updated model depicting the intracellular trafficking, Hyp‐O‐glycosylation and extracellular secretion of extensin‐styled (SP4)₁₈ module and AGP‐styled (SP)₃₂ module is proposed.     

The latest hype on Hyp-O-glycosylation codes

Contemporary glycobiology reflects the intense interest in glycoproteins and their biological roles. Addition of saccharides by N- or O-glycosylation is precise rather than random and forms a uniquely interactive molecular surface. We designate these well conserved glycomotifs as glycomodules to emphasize their functional significance. Thus, elucidation of the glycosylation codes that determine saccharide addition is a significant goal. The focus here is on the Hyp O-glycosylation of cell wall proteins. This involves two consecutive posttranslational modifications, proline hydroxylation and glycosylation. Peptide sequence rather than conformation seems to determine these modifications. Hyp glycosylation occurs in two distinct modes: Hyp arabinosylation and Hyp galactosylation. The Hyp contiguity hypothesis predicts arabinosylation of contiguous Hyp residues and galactosylation of clustered non-contiguous Hyp. Elucidation of Hyp glycosylation codes involves the design and expression of putative glycomotifs as simple repetitive peptides. Thus, repetitive (Ser-Hyp), directed Hyp galactosylation resulting in the exclusive addition of arabinogalactan polysaccharide to all the non-contiguous Hyp residues. and a new AGP. Another repetitive peptide from gum arabic glycoprotein, containing both contiguous and non-contiguous Hyp, directed both modes of Hyp glycosylation. Furthermore, expression of the (Ser-Hypx)n series confirmed the arabinosylation of contiguous Hyp. Thus, the Hyp contiguity hypothesis is a useful predictive tool in the functional genomics toolbox.     

Characterization of the arabinogalactan protein 31 (AGP31) of Arabidopsis thaliana: new advances on the Hyp-O-glycosylation of the Pro-rich domain

Proteins are important actors in plant cell walls because they contribute to their architecture and their dynamics. Among them, hydroxyproline (Hyp)-rich glycoproteins constitute a complex family of O-glycoproteins with various structures and functions. In this study, we characterized an atypical Hyp-rich glycoprotein, AGP31 (arabinogalactan protein 31), which displays a multidomain organization unique in Arabidopsis thaliana, consisting of a short arabinogalactan protein (AGP) motif, a His stretch, a Pro-rich domain, and a C-terminal PAC (PRP-AGP containing Cys) domain. The use of various mass spectrometry strategies was innovative and powerful: it permitted us to locate Hyp residues, to demonstrate the presence of carbohydrates, and to refine their distribution over the Pro-rich domain. Most Hyp were isolated within repeated motifs such as KAOV, KSOV, K(PO/OP)T, K(PO/OP)V, T(PO/OP)V, and Y(PO/OP)T. A few extensin-like motifs with contiguous Hyp (SOOA and SOOT) were also found. The Pro-rich domain was shown to carry Gal residues on isolated Hyp but also Ara residues. The existence of new type Hyp-O-Gal/Ara-rich motifs not recognized by the β-glucosyl Yariv reagent but interacting with the peanut agglutinin lectin was proposed. In addition, the N-terminal short AGP motif was assumed to be substituted by arabinogalactans. Altogether, AGP31 was found to be highly heterogeneous in cell walls because arabinogalactans could be absent, Hyp-O-Gal/Ara-rich motifs of different sizes were observed, and truncated forms missing the C-terminal PAC domain were found, suggesting degradation in muro and/or partial glycosylation prior to secretion.     

Chlamydomonas reinhardtii has multiple prolyl 4-hydroxylases, one of which is essential for proper cell wall assembly

Prolyl 4-hydroxylases (P4Hs) catalyze formation of 4-hydroxyproline (4Hyp), which is found in many plant glycoproteins. We cloned and characterized Cr-P4H-1, one of 10 P4H-like Chlamydomonas reinhardtii polypeptides. Recombinant Cr-P4H-1 is a soluble 29-kD monomer that effectively hydroxylated in vitro both poly(l-Pro) and synthetic peptides representing Pro-rich motifs found in the Chlamydomonas cell wall Hyp-rich glycoprotein (HRGP) GP1. Similar Pro-rich repeats that are likely to be Cr-P4H-1 substrates are also present in the cell wall HRGP GP2 and probably GP3. Suppression of the gene encoding Cr-P4H-1 by RNA interference led to a defective cell wall consisting of a loose network of fibrils resembling the inner and outer W1 and W7 layers of the wild-type wall, while the layers forming the dense central triplet were absent. The lack of Cr-P4H-1 most probably affected 4Hyp content of the major HRPGs of the central triplet, GP1, GP2, and GP3. The reduced 4Hyp levels in these HRGPs can also be expected to affect their glycosylation and, thus, the interactive properties and stabilities of their fibrous shafts. Interestingly, our RNA interference data indicate that the nine other Chlamydomonas P4H-like polypeptides could not fully compensate for the lack of Cr-P4H-1 activity and are therefore likely to have different substrate specificities and functions.     

Contiguous hydroxyproline residues direct hydroxyproline arabinosylation in Nicotiana tabacum

Hydroxyproline (Hyp) O-glycosylation characterizes the hydroxyproline-rich glycoprotein (HRGP) superfamily of the plant extracellular matrix. Hyp glycosylation occurs in two modes: Arabinosylation adds short oligoarabinosides (Hyp-arabinosides) while galactosylation leads to the addition of larger arabinogalactan polysaccharides (Hyp-polysaccharides). We hypothesize that sequence-dependent glycosylation of small peptide motifs results in glycomodules. These small functional units in combination with other repetitive peptide modules define the properties of HRGPs. The Hyp contiguity hypothesis predicts arabinosylation of contiguous Hyp residues and galactosylation of clustered noncontiguous Hyp residues. To determine the minimum level of Hyp contiguity that directs arabinosylation, we designed a series of synthetic genes encoding repetitive (Ser-Pro(2))(n), (Ser-Pro(3))(n), and (Ser-Pro(4))(n). A signal sequence targeted these endogenous substrates to the endoplasmic reticulum/Golgi for post-translational proline hydroxylation and glycosylation in transformed Nicotiana tabacum cells. The fusion glycoproteins also contained green fluorescence protein, facilitating their detection and isolation. The (Ser-Pro(2))(n) and (Ser-Hyp(4))(n) fusion glycoproteins yielded Hyp-arabinosides but no Hyp-polysaccharide. The motif (Ser-Pro(3))(n) was incompletely hydroxylated, yielding mixed contiguous/noncontiguous Hyp and a corresponding mixture of Hyp-arabinosides and Hyp-polysaccharides. These results plus circular dichroic spectra of the glycosylated and deglycosylated (Ser-Pro(2))(n), (Ser-Pro(3))(n), and (Ser-Pro(4))(n) modules corroborate the Hyp contiguity hypothesis and indicate that Hyp O-glycosylation is indeed sequence-driven.     

Identification and characterization of peptides that interact with hepatitis B virus via the putative receptor binding site

A direct involvement of the PreS domain of the hepatitis B virus (HBV) large envelope protein, and in particular amino acid residues 21 to 47, in virus attachment to hepatocytes has been suggested by many previous studies. Several PreS-interacting proteins have been identified. However, they share few common sequence motifs, and a bona fide cellular receptor for HBV remains elusive. In this study, we aimed to identify PreS-interacting motifs and to search for novel HBV-interacting proteins and the long-sought receptor. PreS fusion proteins were used as baits to screen a phage display library of random peptides. A group of PreS-binding peptides were obtained. These peptides could bind to amino acids 21 to 47 of PreS1 and shared a linear motif (W1T2X3W4W5) sufficient for binding specifically to PreS and viral particles. Several human proteins with such a motif were identified through BLAST search. Analysis of their biochemical and structural properties suggested that lipoprotein lipase (LPL), a key enzyme in lipoprotein metabolism, might interact with PreS and HBV particles. The interaction of HBV with LPL was demonstrated by in vitro binding, virus capture, and cell attachment assays. These findings suggest that LPL may play a role in the initiation of HBV infection. Identification of peptides and protein ligands corresponding to LPL that bind to the HBV envelope will offer new therapeutic strategies against HBV infection.     

Regulation of plant peptide hormones and growth factors by post‐translational modification

The number, diversity and significance of peptides as regulators of cellular differentiation, growth, development and defence of plants has long been underestimated. Peptides have now emerged as an important class of signals for cell‐to‐cell communication over short distances, and also for long‐range signalling. We refer to these signalling molecules as peptide growth factors and peptide hormones, respectively. As compared to remarkable progress with respect to the mechanisms of peptide perception and signal transduction, the biogenesis of signalling peptides is still in its infancy. This review focuses on the biogenesis and activity of small post‐translationally modified peptides. These peptides are derived from inactive pre‐pro‐peptides of approximately 70–120 amino acids. Multiple post‐translational modifications (PTMs) may be required for peptide maturation and activation, including proteolytic processing, tyrosine sulfation, proline hydroxylation and hydroxyproline glycosylation. While many of the enzymes responsible for these modifications have been identified, their impact on peptide activity and signalling is not fully understood. These PTMs may or may not be required for bioactivity, they may inactivate the peptide or modify its signalling specificity, they may affect peptide stability or targeting, or its binding affinity with the receptor. In the present review, we will first introduce the peptides that undergo PTMs and for which these PTMs were shown to be functionally relevant. We will then discuss the different types of PTMs and the impact they have on peptide activity and plant growth and development. We conclude with an outlook on the open questions that need to be addressed in future research.     

Identification of three potent hydroxyproline O‐galactosyltransferases in Arabidopsis

Arabinogalactan proteins (AGPs) are plant‐specific extracellular glycoproteins implicated in a variety of processes during growth and development. AGP biosynthesis involves O‐galactosylation of hydroxyproline (Hyp) residues followed by a stepwise elongation of the complex sugar chains. However, functionally dominant Hyp O‐galactosyltransferases, such that their disruption produces phenocopies of AGP‐deficient mutants, remain to be identified. Here, we purified and identified three potent Hyp O‐galactosyltransferases, HPGT1, HPGT2 and HPGT3, from Arabidopsis microsomal fractions. Loss‐of‐function analysis indicated that approximately 90% of the endogenous Hyp O‐galactosylation activity is attributable to these three enzymes. AGP14 expressed in the triple mutant migrated much faster on SDS‐PAGE than when expressed in wild‐type, confirming a considerable decrease in levels of glycosylation of AGPs in the mutant. Loss‐of‐function mutant plants exhibited a pleiotropic phenotype of longer lateral roots, longer root hairs, radial expansion of the cells in the root tip, small leaves, shorter inflorescence stems, reduced fertility and shorter siliques. Our findings provide genetic evidence that Hyp‐linked arabinogalactan polysaccharide chains are critical for AGP function and clues to how arabinogalactan moieties of AGPs contribute to cell‐to‐cell communication during plant growth and development.     

A gymnosperm extensin contains the serine-tetrahydroxyproline motif

The extensin family is a diverse group of hydroxyproline-rich glycoproteins located in the cell wall and characterized by repetitive peptide motifs glycosylated to various degrees. The origin of this diversity and its relationship to function led us earlier to compare extensins of the two major groups of angiosperms from which we concluded that the highly glycosylated Ser-Hyp₄ motif was characteristic of advanced herbaceous dicots, occurring rarely or not at all in a representative graminaceous monocot (Zea mays) and a chenopod (Beta vulgaris) representative of primitive dicots. Because these results could arise either from loss or acquisition of a characteristic feature, we chose a typical gymnosperm representing seed-bearing plants more primitive than the angiosperms. Thus, salt eluates of Douglas fir (Pseudotsuga menziesii) cell suspension cultures yielded two monomeric extensins differing in size and composition. The larger extensin reported earlier lacked the Ser-Hyp₄ motif, was rich in proline and hydroxyproline, and contained peptide motifs similar to the dicot repetitive proline-rich proteins. The smaller extensin monomer reported here (Superose-6 peak 2 [SP2]) was compositionally similar to typical dicot extensins such as tomato P1, mainly consisting of Hyp, Thr, Ser, Pro, Val, Tyr, Lys, His, abundant arabinose, and a small but significant galactose content. A chymotryptic peptide map (on Hamilton PRP-1) of anhydrous hydrogen fluoride-deglycosylated SP2 yielded eight peptides sequenced after further purification on a high-resolution fast-sizing column (polyhydroxyethyl aspartamide; Poly LC). Significantly, two of the eight peptides contained the Ser-Hyp₄ motif, consistent both with the SP2 amino acid composition as well as the presence of hydroxyproline tetraarabinoside as a small (4% of total Hyp) component of the hydroxyproline arabinoside profile; thus, hydroxyproline tetraarabinoside corroborates the presence of Ser-Hyp₄, in agreement with our earlier observation that Hyp contiguity and Hyp glycosylation are positively correlated. Interestingly, other peptide sequences indicate that SP2 contains motifs such as Ser-Hyp₃-Thr-Hyp-Tyr, Ser-Hyp₄-Lys, and (Ala-Hyp)_n repeats that are related to and typify dicot extensins P1, P3, and arabinogalactan proteins, respectively. Overall, these peptide sequences confirm our previous prediction that Ser-Hyp₄ is indeed an ancient motif and also strongly support our suggestion that the extensins comprise an extraordinarily diverse, but nevertheless phylogenetically related, family of cell wall hydroxyproline-rich glycoproteins.     

Glycosylation motifs that direct arabinogalactan addition to arabinogalactan-proteins

Hydroxyproline (Hyp)-rich glycoproteins (HRGPs) participate in all aspects of plant growth and development. HRGPs are generally highly O-glycosylated through the Hyp residues, which means carbohydrates help define the interactive molecular surface and, hence, HRGP function. The Hyp contiguity hypothesis predicts that contiguous Hyp residues are sites of HRGP arabinosylation, whereas clustered noncontiguous Hyp residues are sites of galactosylation, giving rise to the arabinogalactan heteropolysaccharides that characterize the arabinogalactan-proteins. Early tests of the hypothesis using synthetic genes encoding only clustered noncontiguous Hyp in the sequence (serine [Ser]-Hyp-Ser-Hyp)(n) or contiguous Hyp in the series (Ser-Hyp-Hyp)(n) and (Ser-Hyp-Hyp-Hyp-Hyp)(n) confirmed that arabinogalactan polysaccharide was added only to noncontiguous Hyp, whereas arabinosylation occurred on contiguous Hyp. Here, we extended our tests of the codes that direct arabinogalactan polysaccharide addition to Hyp by building genes encoding the repetitive sequences (alanine [Ala]-proline [Pro]-Ala-Pro)(n), (threonine [Thr]-Pro-Thr-Pro)(n), and (valine [Val]-Pro-Val-Pro)(n), and expressing them in tobacco (Nicotiana tabacum) Bright-Yellow 2 cells as fusion proteins with green fluorescent protein. All of the Pro residues in the (Ala-Pro-Ala-Pro)(n) fusion protein were hydroxylated and consistent with the hypothesis that every Hyp residue was glycosylated with arabinogalactan polysaccharide. In contrast, 20% to 30% of Pro residues remained non-hydroxylated in the (Thr-Pro-Thr-Pro)(n), and (Val-Pro-Val-Pro)(n) fusion proteins. Furthermore, although 50% to 60% of the Hyp residues were glycosylated with arabinogalactan polysaccharide, some remained non-glycosylated or were arabinosylated. These results suggest that the amino acid side chains of flanking residues influence the extent of Pro hydroxylation and Hyp glycosylation and may explain why isolated noncontiguous Hyp in extensins do not acquire an arabinogalactan polysaccharide but are arabinosylated or remain non-glycosylated.     

MGOUN3, an Arabidopsis gene with TetratricoPeptide-Repeat-related motifs, regulates meristem cellular organization

In order to understand the functioning of apical meristems in Arabidopsis more clearly, a new mutant, mgoun3 (mgo3), affected in the structural organization and the functional regulation of both shoot and root meristems has been isolated. mgo3 plants display perturbations in leaf morphogenesis, in the spatial and the temporal formation of primordia, and frequent fasciation of the inflorescence stem. Cellular analysis showed that both cellular organization and cell identity patterning are impaired in the mutant meristems. The MGO3 gene has been isolated by positional cloning. The protein deduced from the cDNA sequence contains TetratricoPeptide Repeats (TPR) and Leucine-Rich Repeats (LRR), two motifs that are thought to act in protein-protein interactions. This gene appears to be unique in the Arabidopsis genome. Although the MGO3 protein presents TPR as in the Arabidopsis proteins HOBBIT and SPINDLY, the MGO3 motifs are more similar to those present in LGN-related proteins, which are regulators for some of the asymmetric cell divisions in animal development. These features suggest a key role for MGO3 in meristematic cell divisions and would be of interest for the comparison between plant and animal development.     

The mode of action of the plant antimicrobial peptide MiAMP1 differs from that of its structural homologue, the yeast killer toxin WmKT

The plant antimicrobial peptide MiAMP1 from Macadamia integrifolia and the yeast killer toxin peptide WmKT from Williopsis mrakii are structural homologues. Comparative studies of yeast mutants were performed to test their sensitivity to these two antimicrobial peptides. No differences in susceptibility to MiAMP1 were detected between wild-type and several WmKT-resistant mutant yeast strains. A yeast mutant MT1, resistant to MiAMP1 but unaffected in its susceptibility to plant defensins and hydrogen peroxide, also did not show enhanced tolerance towards WmKT. It is therefore probable that the Greek key beta-barrel structure shared by MiAMP1 and WmKT provides a robust structural framework ensuring stability for the two proteins but that the specific action of the peptides depends on other motifs.     

Cell adhesion proteins and α-fetoprotein. Similar structural motifs as prerequisites for common functions

This review summarizes and analyzes data on structural and functional relationships between cell adhesion proteins and alpha-fetoprotein (AFP), which play an important role in embryo- and carcinogenesis and act in synergism with growth factors. These two groups of proteins are mosaic, multimodular, and polyfunctional, and each of their modules can function independently through binding with its specific membrane receptor. Most cell adhesion proteins contain modules similar to epidermal growth factor (EGF) and also their repeats, which determine the involvement of these proteins in regulation of cell proliferation, differentiation, and apoptosis. These EGF-like modules are found to include short motifs similar to the fragment LDSYQCT of human AFP. Both direct and inverted AFP-like motifs are linked through a consensus octapeptide motif CXXGY/FXGX. Such AFP-like motifs of cell adhesion proteins and the tripeptide RGD found in AFP may be structural prerequisites for common functions of these groups of nonhomologous and unrelated proteins.     

MiAMP1, a novel protein from Macadamia integrifolia adopts a Greek key beta-barrel fold unique amongst plant antimicrobial proteins.

MiAMP1 is a recently discovered 76 amino acid residue, highly basic protein from the nut kernel of Macadamia integrifolia which possesses no sequence homology to any known protein and inhibits the growth of several microbial plant pathogens in vitro while having no effect on mammalian or plant cells. It is considered to be a potentially useful tool for the genetic engineering of disease resistance in transgenic crop plants and for the design of new fungicides.The three-dimensional structure of MiAMP1 was determined through homonuclear and heteronuclear ((15)N) 2D NMR spectroscopy and subsequent simulated annealing calculations with the ultimate aim of understanding the structure-activity relationships of the protein. MiAMP1 is made up of eight beta-strands which are arranged in two Greek key motifs. These Greek key motifs associate to form a Greek key beta-barrel.This structure is unique amongst plant antimicrobial proteins and forms a new class which we term the beta-barrelins. Interestingly, the structure of MiAMP1 bears remarkable similarity to a yeast killer toxin from Williopsis mrakii. This toxin acts by inhibiting beta-glucan synthesis and thereby cell wall construction in sensitive strains of yeast. The structural similarity of MiAMP1 and WmKT, which originate from plant and fungal phyla respectively, may reflect a similar mode of action.     

Programmed cell death in Ricinus and Arabidopsis: the function of KDEL cysteine peptidases in development

Programmed cell death (PCD) in plants is a prerequisite for development as well as seed and fruit production. It also plays a significant role in pathogen defense. A unique group of papain-type cysteine endopeptidases, characterized by a C-terminal endoplasmic reticulum (ER) retention signal (KDEL CysEP), is involved in plant PCD. Genes for these endopeptidases have been sequenced and analyzed from 25 angiosperms and gymnosperms. They have no structural relationship to caspases involved in mammalian PCD and homologs to this group of plant cysteine endopeptidases have not been found in mammals or yeast. In castor beans (Ricinus communis), the CysEP is synthesized as pre-pro-enzyme. The pro-enzyme is transported to the cytosol of cells undergoing PCD in ER-derived vesicles called ricinosomes. These vesicles release the mature CysEP in the final stages of organelle disintegration triggered by acidification of the cytoplasm resulting from the disruption of the vacuole. Mature CysEP digests the hydroxyproline (Hyp)-rich proteins (extensins) that form the basic scaffold of the plant cell wall. The KDEL CysEPs accept a wide variety of amino acids at the active site, including the glycosylated Hyp residues of the extensins. In Arabidopsis, three KDEL CysEPs (AtCEP1, AtCEP2 and AtCEP3) are expressed in tissues undergoing PCD. In transgenic Arabidopsis plants expressing β-glucuronidase under the control of the promoters for these three genes, cell- and tissue-specific activities were mapped during seedling, flower and seed development. KDEL CysEPs participate in the collapse of tissues in the final stage of PCD and in tissue re-modeling such as lateral root formation.     

Lectin receptor kinases participate in protein-protein interactions to mediate plasma membrane-cell wall adhesions in Arabidopsis

Interactions between plant cell walls and plasma membranes are essential for cells to function properly, but the molecules that mediate the structural continuity between wall and membrane are unknown. Some of these interactions, which are visualized upon tissue plasmolysis in Arabidopsis (Arabidopsis thaliana), are disrupted by the RGD (arginine-glycine-aspartic acid) tripeptide sequence, a characteristic cell adhesion motif in mammals. In planta induced-O (IPI-O) is an RGD-containing protein from the plant pathogen Phytophthora infestans that can disrupt cell wall-plasma membrane adhesions through its RGD motif. To identify peptide sequences that specifically bind the RGD motif of the IPI-O protein and potentially play a role in receptor recognition, we screened a heptamer peptide library displayed in a filamentous phage and selected two peptides acting as inhibitors of the plasma membrane RGD-binding activity of Arabidopsis. Moreover, the two peptides also disrupted cell wall-plasma membrane adhesions. Sequence comparison of the RGD-binding peptides with the Arabidopsis proteome revealed 12 proteins containing amino acid sequences in their extracellular domains common with the two RGD-binding peptides. Eight belong to the receptor-like kinase family, four of which have a lectin-like extracellular domain. The lectin domain of one of these, At5g60300, recognized the RGD motif both in peptides and proteins. These results imply that lectin receptor kinases are involved in protein-protein interactions with RGD-containing proteins as potential ligands, and play a structural and signaling role at the plant cell surfaces.