Accumulation of extracellular proteins bearing unique proline-rich motifs in intercellular spaces of the legume nodule parenchyma

Summary. Nodulins encoding repetitive proline-rich cell wall proteins (PRPs) are induced during early interactions with rhizobia, suggesting a massive restructuring of the plant extracellular matrix during infection and nodulation. However, the proteins corresponding to these gene products have not been isolated or characterized, nor have cell wall localizations been confirmed. Posttranslational modifications, conformation, and interactions with other wall polymers are difficult to predict on the basis of only the deduced amino acid sequence of PRPs. PsENOD2 is expressed in nodule parenchyma tissue during nodule organogenesis and encodes a protein with distinctive PRP motifs that are rich in glutamate and basic amino acids. A database search for the ENOD2 signature motifs indicates that similar proteins may have a limited phylogenetic distribution, as they are presently only known from legumes. To determine the ultrastructural location of the proteins, antibodies were raised against unique motifs from the predicted ENOD2 sequence. The antibodies recognized nodule-specific proteins in pea (Pisum sativum), with a major band detected at 110 kDa, representing a subset of PRPs from nodules. The protein was detected specifically in organelles of the secretory pathway and intercellular spaces in the nodule parenchyma, but it was not abundant in primary walls. Similar proteins with an analogous distribution were detected in soybean (Glycine max). The use of polyclonal antibodies raised against signature motifs of extracellular matrix proteins thus appears to be an effective strategy to identify and isolate specific structural proteins for functional analysis. Correspondence and reprints: Delaware Biotechnology Institute, Newark, DE 19711, U.S.A.     

Peptides isolated from cell walls of Medicago truncatula nodules and uninfected root

The hydroxyproline-rich root nodules of legumes provide a microaerobic niche for symbiotic nitrogen-fixing Rhizobacteria. The contributions of the cell wall and associated structural proteins, particularly the hydroxyproline-rich glycoproteins (HRGPs), are therefore of interest. Our approach involved identification of the protein components by direct chemical analysis of the insoluble wall. Chymotryptic peptide mapping showed a "P3-type" extensin containing the highly arabinosylated Ser-Hyp4-Ser-Hyp-Ser-Hyp4-Tyr3-Lys motif as a major component. Cell wall amino acid analyses and quantitative hydroxyproline arabinoside profiles, predominantly of tri- and tetraarabinosides, confirmed this extensin as the major structural protein in the cell walls of both root nodules and uninfected roots. On the other hand, judging from the Pro, Glu and non-glycosylated Hyp content, the nodule-specific proline-rich glycoproteins, such as the early nodulins (ENOD-PRPs), are present in much lesser amounts. Although we isolated no PRP peptides from nodule cell walls, a single PRP peptide from root cell walls confirmed the presence of a PRP in roots and represented the first direct evidence for a crosslinked PRP in muro. Compared with root cell walls (approximately 7% protein dry weight) nodule cell walls contained significantly more protein (approximately 13% dry weight) with an overall amino acid and peptide composition indicating the presence of structural protein unrelated to the HRGPs.     

Chemical composition and ultrastructure of broad bean (<Emphasis Type="Italic">Vicia faba</Emphasis> L.) nodule endodermis in comparison to the root endodermis

Ultrastructure and development of apoplastic barriers within indeterminate root nodules formed by Vicia faba L. were examined by light and electron microscopy. The nodule outer cortex is separated from the inner cortex by a heavily suberized nodule endodermis, which matures in submeristematic regions and possesses suberin lamellae. Unsuberized passage cells are present near vascular strands, which are surrounded by a vascular endodermis attached on the inner side of the nodule endodermal cell walls. The vascular endodermis appears immediately below the meristematic apex in developmental state I (Casparian bands), gradually develops suberin lamellae, and attains developmental state II at the base of the nodule. For chemical analysis apoplastic barrier tissues were dissected after enzymatic digestion of non-impregnated tissues. Root epidermal and endodermal cell walls as well as nodule outer cortex could be isolated as pure fractions; nodule endodermal cell walls could not be separated from vascular endodermal cell walls and enclosed xylem vessels. Gas chromatography-flame ionization detection and gas chromatography-mass spectrometry were applied for quantitative and qualitative analysis of suberin and lignin in isolated cell walls of these tissues. The suberin content of isolated endodermal cell walls of nodules was approximately twice that of the root endodermal cell walls. The suberin content of the nodule outer cortex and root epidermal cell walls was less than one-tenth of that of the nodule endodermal cell wall. Substantial amounts of lignin could only be found in the nodule endodermal cell wall fraction. Organic solvent extracts of the isolated tissues revealed long-chain aliphatic acids, steroids, and triterpenoid structures of the lupeol type. Surprisingly, extract from the outer cortex consisted of 89% triterpenoids whereas extracts from all other cell wall isolates contained not more than 16% total triterpenoids. The results of ultrastructural and chemical composition are in good correspondence and underline the important role of the examined tissues as apoplastic barriers.     

PRPs localized to the middle lamellae are required for cortical tissue integrity in Medicago truncatula roots

Key message

A family of repetitive proline-rich proteins interact with acidic pectins and play distinct roles in legume root cell walls affecting cortical and vascular structure.

Abstract

A proline-rich protein (PRP) family, composed of tandemly repeated Pro-Hyp-Val-X-Lys pentapeptide motifs, is found primarily in the Leguminosae. Four distinct size classes within this family are encoded by seven tightly linked genes: MtPRP1, MtPRP2 and MtPRP3, and four nearly identical MtPRP4 genes. Promoter fusions to β-glucuronidase showed strong expression in the stele of hairy roots for all 4 PRP genes tested, with additional expression in the cortex for PRP1, PRP2 and PRP4. All except MtPRP4 are strongly expressed in non-tumorous roots, and secreted and ionically bound to root cell walls. These PRPs are absent from root epidermal cell walls, and PRP accumulation is highly localized within the walls of root cortical and vascular tissues. Within xylem tissue, PRPs are deposited in secondary thickenings where it is spatially exclusive to lignin. In newly differentiating xylem, PRPs are deposited in the regularly spaced paired-pits and pit membranes that hydraulically connect neighboring xylem elements. Hairpin-RNA knock-down constructs reducing PRP expression in Medicago truncatula hairy root tumors disrupted cortical and vascular patterning. Immunoblots showed that the knockdown tumors had potentially compensating increases in the non-targeted PRPs, all of which cross-react with the anti-PRP antibodies. However, PRP3 knockdown differed from knockdown of PRP1 and PRP2 in that it greatly reduced viability of hairy root tumors. We hypothesize that repetitive PRPs interact with acidic pectins to form block-copolymer gels that can play distinct roles in legume root cell walls.

     

PRGL: A cell wall proline-rich protein containning GASA domain in Gerbera hybrida

PRPs (proline-rich proteins) are a group of cell wall proteins characterized by their proline and hy- droproline-rich repetitive peptides. The expression of PRPs in plants is stimulated by wounding and environmental stress. GASA (gibberellic acid stimulated in Arabidopsis) proteins are small peptides sharing a 60 amino acid conserved C-terminal domain containing twelve invariant cysteine residues. Most of GASAs reported are localized to apoplasm or cell wall and their expression was regulated by gibberellins (GAs). It has been reported that, in French bean, these two proteins encoding by two distinct genes formed a two-component chitin-receptor involved in plant-pathogen interactions when plant was infected. We cloned a full-length cDNA of PRGL (proline-rich GASA-like) gene which encodes a protein containing both PRP and GASA-like domains. It is demonstrated that PRGL is a new protein with characteristics of PRP and GASA by analyzing its protein structure and gene expression.     

Plant cell wall glycoproteins and their genes

At least two main groups of glycoproteins can be distinguished in plant cell walls: extensins which are insoluble cell wall proteins; and soluble arabinogalactan proteins (AGPs) which have a high carbohydrate content such that protein content constitutes in some cases only 5 % of the glycoprotein weight. These two groups of proteins together with other cell wall proteins more or less glycosylated, such as proline-rich proteins (PRPs), hybrid PRP (HyPRPs) and expansins, are reviewed and compared with similar proteins present in other cell compartments. Different patterns of N- or O-glycosylation are analysed. In some cases, these cell wall proteins or proteins related to them present patterns of glycosylation that act as epitopes recognizable by IgE in allergic responses.     

Comparative localization of three classes of cell wall proteins.

The localization of the cell wall proline-rich proteins (PRPs), and the gene expression of the cell wall glycine-rich proteins (GRPs) and the hydroxyproline-rich glycoproteins (HRGPs) were examined in several dicot species. The PRPs are accumulated in the corner walls of the cortex where several cells are joined together and in the protoxylem cell walls of 3-day-old soybean root. In 1-month-old soybean plants, the PRPs are specifically deposited in xylem vessel elements of the young stem, and they are accumulated in both phloem fibers and xylem vessel elements and fibers of the older stem. Likewise, the PRPs are localized in xylem vessel elements and fibers in tomato, petunia, potato and tobacco stems. They are also found in outer and inner phloem fiber cell walls of tomato stem and in outer phloem fiber cell walls of petunia stem. The gene expression of the HRGPs and the GRPs is developmentally regulated in tomato, petunia and tobacco stems. HRGP mRNAs are abundant in outer and inner phloem regions, while GRP mRNAs are present mostly in primary xylem and in the cambium region. Immunocytochemical localization showed that the GRPs have a localization pattern similar to that of the PRPs in tomato, petunia and tobacco stems.     

Silica deposition by a strongly cationic proline-rich protein from systemically resistant cucumber plants

Infection of one leaf of cucumber (Cucumis sativa) plants can render other leaves resistant to various pathogens. This so-called systemic acquired resistance (SAR) can be functionally mimicked by certain chemicals. All these treatments enhanced expression of a gene encoding a novel proline-rich protein (PRP1) which has C-terminal repetitive sequences containing an unusually high amount of lysine and arginine residues. Antibodies raised against a synthetic peptide derived from four of the repetitive sequences cross-reacted mainly with a cell wall polypeptide of an apparent MW of 8 kDa. The protein accumulated upon SAR induction, though it does not appear to take part in oxidative protein cross-linking, at least in the hypocotyl tissue. The synthetic peptide derived from the repetitive sequences was able to polymerize orthosilicic acid to insoluble silica, a property not resulting directly from the primary protein sequence, but rather from the high positive charge density. Our results suggest that during induction of SAR, the synthesis of a strongly cationic PRP prepares the cell walls for enhanced silica deposition which is known to participate in cell wall reinforcement, localized at the site of attempted penetration of fungi into epidermal cells. Constitutive accumulation of related PRPs may function in silica deposition during certain developmental stages, a process important for various physiological functions of green plants.     

Ultrastructure and functioning of the transport system of the leguminous root nodule

Summary The structure of the vascular tissues of nitrogen-fixing nodules of 27 genera of legumes and some non-legumes has been investigated by light microscopy. Pisum and Trifolium nodules have been examined by electron microscopy.Attention is directed to the presence of a pericycle in the vascular bundles of the nodules. In 7 of the legumes the pericycle cells possess a wall labyrinth consisting of branched filiform protuberances. The ultrastructure of the pericycle cell cytoplasm is described: its most striking feature is its abundant rough endoplasmic reticulum. These cells surround the xylem and phloem of the bundles, and are in turn surrounded by a layer of endodermal cells with Casparian strips. The pericycle cells develop their wall labyrinth in the levels of the nodule at which the bacterial tissue becomes pigmented; in nodule senescence their cytoplasm is disrupted level with the breakdown of the bacterial tissue.A pathway for symplastic lateral transfer of assimilates exists, from the sieve elements through the pericycle, endodermis and cortex to the bacterial tissue. The apoplast within the endodermis consists largely of the pericycle wall labyrinth and the xylem. The ultrastructure of the Casparian strip resembles that of roots.Intact, detached nodules can be induced to bleed a fluid from their severed vascular tissue. This fluid is exceptionally rich in organic nitrogen, particularly amides, but does not appear to contain sugars. Comparison between its amino acid composition and that of other parts of the nodule suggests that an active uptake or secretion of nitrogenous compounds precedes export from the nodule. Special functions are suggested for the nodule endodermis and the pericycle cells in this export process.     

Electron microscopic immunocytochemical localization of proline-rich proteins in normal mouse parotid salivary glands

Summary Rabbit polyclonal antibodies against isoproterenol-induced mouse proline-rich proteins (PRPs) were used to localize PRPs in the parotid salivary glands of normal adult BALB/c mice. The antibodies recognized both acidic-type and basic-type PRPs. Immunoblotting experiments revealed that the glands contained an acidic-type and a basic-type PRP. Parotid gland tissue was fixed with Karnosky's fixative and embedded in Lowicryl resin at low temperature. PRPs were localized at the electron microscope level using an indirect post-embedding staining technique with protein A-gold. The secretion granules of the acinar cells were strongly labelled. Pre-absorption of the antibody with purified acidic-type and basic-type PRPs indicated that the basic-type PRP is mainly located at the periphery of the granules but that the acidic-type PRP is more evenly distributed within the granules. Pre-absorption of the antibody with -amylase did not affect the staining pattern, suggesting minimal cross-reactivity. PRPs were also detected within the rough endoplasmic reticulum and the Golgi apparatus of acinar cells, within the granules of the proacinar cells and in the lumena of the ducts, but not within the intercalated or striated duct cell granules.     

The distribution of aquaporin subtypes (PIP1, PIP2 and gamma-TIP) is tissue dependent in soybean (Glycine max) root nodules

BACKGROUND AND AIMS The inner cortical cells (IC-cells) of legume root nodules have been previously shown to regulate the resistance to nodule O2 diffusion by a rapid contraction/expansion mechanism, which controls the volume of intercellular spaces and their occlusion by a liquid phase. The expression of aquaporins in IC-cells was also found to be involved in this nodule O2 diffusion mechanism. The aim of this study was to compare the expression of plasma membrane intrinsic proteins (PIP) aquaporin isoforms with tonoplast intrinsic protein (gamma-TIP) in both IC-cells and adjacent cell types. METHODS: Using immunogold labelling in ultra-thin sections of Glycine max nodules, the expression of two PIP isoforms was observed and compared with the gamma-TIP pattern. KEY RESULTS: The plasma membrane aquaporins PIP1 and PIP2 were expressed more in IC-cells and endodermis than in pericycle and infected cells. The tonoplast aquaporin gamma-TIP has shown a distribution pattern similar to that of the PIPs. CONCLUSIONS: PIPs and gamma-TIP aquaporins are highly expressed in both plasmalemma and tonoplast of nodule IC-cells. This distribution is consistent with the putative role of water fluxes associated with the regulation of nodule conductance to O2 diffusion and the subsequent ATP-dependent nitrogenase activity. In the endodermis, these aquaporins might also be involved in nutrient transport between the infected zone and vascular traces.     

Structure, organization, and regulation of a hamster proline-rich protein gene. A multigene family

Genomic DNA fragments bearing proline-rich protein (PRP) genes expressed specifically in hamster parotid glands have been isolated and characterized. Complete exonic sequences as well as intronic and a considerable portion of the flanking sequences are reported for a PRP gene, H29. H29 is interrupted by three intervening sequences, with consensus splice junctions, and it likely encodes the acidic hamster PRP Hp43a. Exceedingly high homology of the 5'-untranslated region and the sequence encoding the signal peptide is observed with other PRPs of all species studied. Significant homology was also detected among the repetitive sequences of the mature acidic PRPs from human, mouse, hamster, and rat. This conservation of the internal repeats of the PRPs suggested that proline-rich protein gene evolution involved intragenic duplication of internal repeats and gene duplication and conversion. Both hamster and mouse PRP genes (H29 and mouse proline-rich protein gene, respectively) share considerable sequence similarity in the 5'-flanking regions for about 100 base pairs upstream. The remainder of the upstream sequences were heterologous except for three oligonucleotide regions with 60-70% sequence conservation. These three regions are thought to be involved in the regulation of the tissue-specific PRP gene induction.     

Differential organ-specific response to salt stress and water deficit in nodulated bean (Phaseolus vulgaris)

Nodulated bean plants were exposed to mild salt stress or water deficit in such a way that the nodule's nitrogen‐fixing activity was reduced to about 25–30% that of controls. Water‐deprived plants showed a slight decrease in the weight of the aerial part, whereas the photosynthetic parameters were not significantly affected. In contrast, salt‐stressed plants displayed a reversible decrease in the quantum yield of photosystem II photochemistry. Five water‐deficit responsive cDNA clones encoding one lipid transfer protein, two late‐embryogenesis abundant (LEA) proteins and two proline‐rich proteins (PRPs) showed different organ‐specific expression patterns depending on the kind of stress applied. PRPs and one LEA protein, PvLEA‐18, exhibited the highest expression in nodules. Anti‐PvLEA‐18 antibodies were used to immunolocalize the protein in the nodule. PvLEA‐18 was localized in the cytoplasm and nucleus of nodule cortex cells, and preferentially in cells of the vascular bundles, showing enhanced accumulation under water deficit. To our knowledge, this is the first time that a LEA protein has been identified in legume nodules.     

Movement of fluorescent dyes Lucifer Yellow (LYCH) and carboxyfluorescein (CF) in Medicago truncatula Gaertn. roots and root nodules

Lucifer Yellow (LYCH) and carboxyfluorescein (CF) served in Medicago truncatula roots and root nodules as the markers of apoplastic and symplastic transport, respectively. The aim of this study was to understand better the water and photoassimilate translocation pathways to and within nodules. The present study shows that in damaged roots LYCH moves apoplastically through the vascular elements but it was not detected within the nodule vascular bundles. In intact roots, the outer cortex was strongly labeled but the dye was not present in the interior of intact root nodules. The inwards movement of LYCH was halted in the endodermis. When the dye was introduced into a damaged nodule by infiltration, it spread only in the cell walls and the intercellular spaces up to the inner cortex. Our research showed that in addition to the outer cortex, the inner tissue containing bacteroid-infected cells is also an apoplastic domain. Our results are consistent with the hypothesis that nodules do not receive water from the xylem but get it and photoassimilates from phloem. A comparison between using LYCH and LYCH followed by glutaraldehyde fixation indicates that glutaraldehyde is responsible for fluorescence of some organelles within root nodule cells. The influence of the fixation on nodule fluorescence has not been reported before but must be taken into consideration to avoid errors. An attempt was made to follow carboxyfluorescein (6(5) CF) translocation from leaflets into roots and root nodules. In root nodules, CF was present in all or a couple of vascular bundles (VB), vascular endodermis and some adjacent cells. The leakage of CF from the VBs was observed, which suggests symplastic continuity between the VBs and the nodule parenchyma. The lack of CF in inner tissue was observed. Therefore, photoassimilate entry to the infected region of nodule must involve an apoplastic pathway.     

Proline-rich proteins and glycoproteins: expression of salivary gland multigene families

Our recent research interests have focused on a group of unusual proteins and glycoproteins high in proline content, or the so-called proline-rich proteins (PRPs). The PRPs are tissue-specific expressions of salivary gland multigene families. Normally PRPs are not detected or are present in very low amounts in rat, mouse and hamster salivary glands, but these unusual proteins are dramatically induced by treatment with the catecholamine isoproterenol. The structures and organizations of several PRP mRNAs and PRP genes have been determined. The amino acid sequences of all PRPs show 4 distinct regions, namely, a signal peptide, a transition region, a repeat region and a carboxyl-terminal region. Glycoproteins induced by isoproterenol treatment may be N-glycosylated or O-glycosylated. The N-glycosylated glycoprotein GP-158 from rat submandibular glands has a 12 amino acid glycopeptide which repeats possibly 49 times. Proline-rich proteins of the parotid glands of rats and mice are also greatly induced by dietary tannins. The apparent unique occurrence of PRPs in saliva suggests that one biological role is to neutralize the detrimental effects of dietary tannins and other polyphenols. The upstream regions of the mouse and hamster PRP genes contain cyclic AMP-regulated sequences as demonstrated by deletions and transient transfections. The PRP multigene family members of mouse are all located on chromosome 8.     

Isoproterenol increases sorting of parotid gland cargo proteins to the basolateral pathway

Exocrine cells have an essential function of sorting secreted proteins into the correct secretory pathway. A clear understanding of sorting in salivary glands would contribute to the correct targeting of therapeutic transgenes. The present work investigated whether there is a change in the relative proportions of basic proline-rich protein (PRP) and acidic PRPs in secretory granules in response to chronic isoproterenol treatment, and whether this alters the sorting of endogenous cargo proteins. Immunoblot analysis of secretory granules from rat parotids found a large increase of basic PRP over acidic PRPs in response to chronic isoproterenol treatment. Pulse chase experiments demonstrated that isoproterenol also decreased regulated secretion of newly synthesized secretory proteins, including PRPs, amylase and parotid secretory protein. This decreased efficiency of the apical regulated pathway may be mediated by alkalization of the secretory granules since it was reversed by treatment with mild acid. We also investigated changes in secretion through the basolateral (endocrine) pathways. A significant increase in parotid secretory protein and salivary amylase was detected in sera of isoproterenol-treated animals, suggesting increased routing of the regulated secretory proteins to the basolateral pathway. These studies demonstrate that shifts of endogenous proteins can modulate regulated secretion and sorting of cargo proteins. amylase; parotid secretory protein; polarized secretion     

Immunological comparison of proline-rich proteins from human and primate parotid secretion.

Antisera raised in response to proline-rich proteins purified from parotid secretions of man and the primate Macaca fascicularis were employed to investigate the interrelationships of these proteins by immunodiffusion, immunoelectrophoresis and the combined use of disc gel acrylamide electrophoresis with radial immunodiffusion. The major human proline-rich proteins, PRP I, PRP II, PRP III and PRP IV as well as several minor proline-rich proteins cross-react with antiserum to PRP I or PRP III. Similarly primate parotid saliva contains several components cross-reacting with antiserum directed against a purified primate proline-rich protein, MPRP. Antiserum to PRP I or PRP III cross-reacted with MPRP and primate parotid saliva protein, whereas antiserum to MPRP cross-reacted only with human parotid saliva protein and not with the isolated human proline-rich proteins. The immunological relationships of these salivary proline-rich proteins within and between species suggest their origin from a common precursor molecule.     

Involvement of a maize proline-rich protein in secondary cell wall formation as deduced from its specific mRNA localization

A clone encoding a proline-rich protein (ZmPRP) has been obtained from maize root by differential screening of a maturing elongation root cDNA library. The amino acid sequence deduced from the full-length cDNA contains a putative signal peptide and a highly repetitive sequence containing the PEPK motif, indicating that the ZmPRP mRNA may code for a cell wall protein. The PEPK repeat is also found in a previously reported wheat sequence but differs from the repeated sequences found in hydroxyproline-rich glycoproteins (HRGP) and in dicot proline-rich proteins (PRP). In the maize genome, the ZmPRP protein is encoded by a single gene that is expressed in maturing regions of the root, in the hypocotyl and in the pericarp. In these organs, the ZmPRP mRNA accumulates in the xylem and surrounding cells, and in the epidermis. No ZmPRP mRNA was found in the phloem. The pattern of mRNA accumulation is very similar to the one observed for genes coding for proteins involved in lignin biosynthesis and, like most cell wall proteins, ZmPRP synthesis is also induced by wounding. These data support the hypothesis that ZmPRP is a member of a new class of fibrous proteins involved in the secondary cell wall formation in monocot species.