Arabinogalactan protein-rich cell walls, paramural deposits and ergastic globules define the hyaline bodies of rhinanthoid Orobanchaceae haustoria.
ABSTRACT: BACKGROUND AND AIMS:Parasitic plants obtain nutrients from their hosts through organs called haustoria. The hyaline body is a specialized parenchymatous tissue occupying the central parts of haustoria in many Orobanchaceae species. The structure and functions of hyaline bodies are poorly understood despite their apparent necessity for the proper functioning of haustoria. Reported here is a cell wall-focused immunohistochemical study of the hyaline bodies of three species from the ecologically important clade of rhinanthoid Orobanchaceae. METHODS:Haustoria collected from laboratory-grown and field-collected plants of Rhinanthus minor, Odontites vernus and Melampyrum pratense attached to various hosts were immunolabelled for cell wall matrix glycans and glycoproteins using specific monoclonal antibodies (mAbs). KEY RESULTS:Hyaline body cell wall architecture differed from that of the surrounding parenchyma in all species investigated. Enrichment in arabinogalactan protein (AGP) epitopes labelled with mAbs LM2, JIM8, JIM13, JIM14 and CCRC-M7 was prominent and coincided with reduced labelling of de-esterified homogalacturonan with mAbs JIM5, LM18 and LM19. Furthermore, paramural bodies, intercellular deposits and globular ergastic bodies composed of pectins, xyloglucans, extensins and AGPs were common. In Rhinanthus they were particularly abundant in pairings with legume hosts. Hyaline body cells were not in direct contact with haustorial xylem, which was surrounded by a single layer of paratracheal parenchyma with thickened cell walls abutting the xylem. CONCLUSIONS:The distinctive anatomy and cell wall architecture indicate hyaline body specialization. Altered proportions of AGPs and pectins may affect the mechanical properties of hyaline body cell walls. This and the association with a transfer-like type of paratracheal parenchyma suggest a role in nutrient translocation. Organelle-rich protoplasts and the presence of exceptionally profuse intra- and intercellular wall materials when attached to a nitrogen-fixing host suggest subsequent processing and transient storage of nutrients. AGPs might therefore be implicated in nutrient transfer and metabolism in haustoria.
Project description:Rhinanthus minor is a root hemiparasitic plant that attacks a wide range of host species which are severely damaged by the parasite. Rhinanthus minor also attempts unsuccessfully to form connections to a range of non-hosts which in contrast are not damaged by the parasite; however, the underlying physiological basis of these differences is not fully understood.Biomass of host-parasite combinations was studied, and histology, electron microscopy and FT-IR microspectroscopy were used to determine the cellular-level interactions between Rhinanthus haustoria (the parasite's connective structure) and the roots of a range of potential host species.Two distinct defence responses were observed in the non-host forbs Plantago lanceolata and Leucanthemum vulgare. Firstly, L. vulgare was able to encapsulate the parasite's invading structures preventing it from gaining access to the stele. This was supported by FT-IR microspectroscopy, used to monitor lignification in response to Rhinanthus haustoria. Secondly, host cell fragmentation was observed at the interface between the parasite and P. lanceolata. Growth data confirmed the non-host status of the two forbs whilst, in contrast, grasses and a legume which were good hosts showed no evidence of defence at the host/parasite interface.Variable resistance to Rhinanthus is shown for the first time to be controlled by cellular-level resistance to haustoria by either cell fragmentation or lignification at the host/parasite interface.
Project description:The origin of novel traits is recognized as an important process underlying many major evolutionary radiations. We studied the genetic basis for the evolution of haustoria, the novel feeding organs of parasitic flowering plants, using comparative transcriptome sequencing in three species of Orobanchaceae. Around 180 genes are upregulated during haustorial development following host attachment in at least two species, and these are enriched in proteases, cell wall modifying enzymes, and extracellular secretion proteins. Additionally, about 100 shared genes are upregulated in response to haustorium inducing factors prior to host attachment. Collectively, we refer to these newly identified genes as putative "parasitism genes." Most of these parasitism genes are derived from gene duplications in a common ancestor of Orobanchaceae and Mimulus guttatus, a related nonparasitic plant. Additionally, the signature of relaxed purifying selection and/or adaptive evolution at specific sites was detected in many haustorial genes, and may play an important role in parasite evolution. Comparative analysis of gene expression patterns in parasitic and nonparasitic angiosperms suggests that parasitism genes are derived primarily from root and floral tissues, but with some genes co-opted from other tissues. Gene duplication, often taking place in a nonparasitic ancestor of Orobanchaceae, followed by regulatory neofunctionalization, was an important process in the origin of parasitic haustoria.
Project description:Molecular phylogenetic analyses have greatly advanced our understanding of phylogenetic relationships in Orobanchaceae, a model system to study parasitism in angiosperms. As members of this group may lack some genes widely used for phylogenetic analysis and exhibit varying degrees of accelerated base substitution in other genes, relationships among major clades identified previously remain contentious. To improve inferences of phylogenetic relationships in Orobanchaceae, we used two pentatricopeptide repeat (PPR) and three low-copy nuclear (LCN) genes, two of which have been developed for this study. Resolving power and level of support strongly differed among markers. Despite considerable incongruence among newly and previously sequenced markers, monophyly of major clades identified in previous studies was confirmed and, especially in analyses of concatenated data, strongly supported after the exclusion of a small group of East Asian genera (Pterygiella and Phtheirospermum) from the Euphrasia-Rhinanthus clade. The position of the Orobanche clade sister to all other parasitic Orobanchaceae may indicate that the shift to holoparasitism occurred early in the evolution of the family. Although well supported in analyses of concatenated data comprising ten loci (five newly and five previously sequenced), relationships among major clades, most prominently the Striga-Alectra clade, the Euphrasia-Rhinanthus clade, and the Castilleja-Pedicularis clade, were uncertain because of strongly supported incongruence also among well-resolving loci. Despite the limitations of using a few selected loci, congruence among markers with respect to circumscription of major clades of Orobanchaceae renders those frameworks for detailed, species-level, phylogenetic studies.
Project description:Root hemiparasites from the rhinanthoid clade of Orobanchaceae possess metabolically active glandular trichomes that have been suggested to function as hydathode trichomes actively secreting water, a process that may facilitate resource acquisition from the host plant's root xylem. However, no direct evidence relating the trichomes to water secretion exists, and carbon budgets associated with this energy-demanding process have not been determined.Macro- and microscopic observations of the leaves of hemiparasitic Rhinanthus alectorolophus were conducted and night-time gas exchange was measured. Correlations were examined among the intensity of guttation, respiration and transpiration, and analysis of these correlations allowed the carbon budget of the trichome activity to be quantified. We examined the intensity of guttation, respiration and transpiration, correlations among which indicate active water secretion.Guttation was observed on the leaves of 50 % of the young, non-flowering plants that were examined, and microscopic observations revealed water secretion from the glandular trichomes present on the abaxial leaf side. Night-time rates of respiration and transpiration and the presence of guttation drops were positively correlated, which is a clear indicator of hydathode trichome activity. Subsequent physiological measurements on older, flowering plants indicated neither intense guttation nor the presence of correlations, which suggests that the peak activity of hydathodes is in the juvenile stage.This study provides the first unequivocal evidence for the physiological role of the hydathode trichomes in active water secretion in the rhinanthoid Orobanchaceae. Depending on the concentration of organic elements calculated to be in the host xylem sap, the direct effect of water secretion on carbon balance ranges from close to neutral to positive. However, it is likely to be positive in the xylem-only feeding holoparasites of the genus Lathraea, which is closely related to Rhinanthus. Thus, water secretion by the hydathodes might be viewed as a physiological pre-adaptation in the evolution of holoparasitism in the rhinanthoid lineage of Orobanchaceae.
Project description:Using a strategy consisting of (i) the isolation of cell walls from synchronously differentiating cells of Zinnia, (ii) the generation of mAbs with an antibody phage display method, and (iii) screening with a subtraction method, we isolated mAbs recognizing vascular development-specific cell wall components without prior antigen identification. One of the isolated mAbs, designated CN 8, recognized a cell wall component contained in the hemicellulosic fraction. Immunohistochemical analyses showed that the CN 8 epitope was localized to the cell wall of immature tracheary elements and xylem parenchyma cells. In immature tracheary elements, the CN 8 epitope had a polarized localization pattern regardless of whether the cells are formed as parts of vessels in situ or as single tracheary elements in vitro, suggesting that cell polarity autonomously formed on the cell wall may function in tracheary element differentiation.
Project description:Arabinogalactan-proteins (AGPs) are highly glycosylated hydroxyproline-rich glycoproteins present in plant cell walls. AGPs are characterized by arabinose-/galactose-rich side chains, which define their interactive molecular surface. Fucose residues are found in some dicotyledon AGPs, and AGP fucosylation is developmentally regulated. We previously identified Arabidopsis thaliana FUT4 and FUT6 genes as AGP-specific fucosyltransferases (FUTs) based on their enzymatic activities when heterologously expressed in tobacco (Nicotiana tabacum) BY2 suspension-cultured cells. Here, the functions of FUT4 and FUT6 and the physiological roles of fucosylated AGPs were further investigated using Arabidopsis fut4, fut6, and fut4/fut6 mutant plants. All mutant plants showed no phenotypic differences compared to wild-type plants under physiological conditions, but showed reduced root growth in the presence of elevated NaCl. However, roots of wild-type and fut4 mutant plants contained terminal fucose epitopes, which were absent in fut6 and fut4/fut6 mutant plants as indicated by eel lectin staining. Monosaccharide analysis showed fucose was present in wild-type leaf and root AGPs, but absent in fut4 leaf AGPs and in fut4/fut6 double mutant leaf and root AGPs, indicating that FUT4 was required for fucosylation of leaf AGPs while both FUT4 and FUT6 contributed to fucosylation of root AGPs. Glycome profiling of cell wall fractions from mutant roots and leaves showed distinct glycome profiles compared to wild-type plants, indicating that fucosyl residues on AGPs may regulate intermolecular interactions between AGPs and other wall components. The current work exemplifies the possibilities of refinement of cell wall structures by manipulation of a single or a few cell wall biosynthetic genes.
Project description:BACKGROUND: Arabinogalactan proteins (AGPs) are cell wall proteoglycans that have been shown to be important for pollen development. An Arabidopsis double null mutant for two pollen-specific AGPs (agp6 agp11) showed reduced pollen tube growth and compromised response to germination cues in vivo. A microarray experiment was performed on agp6 agp11 pollen tubes to search for genetic interactions in the context of pollen tube growth. A yeast two-hybrid experiment for AGP6 and AGP11 was also designed. RESULTS: The lack of two specific AGPs induced a meaningful shift in the gene expression profile. In fact, a high number of genes showed altered expression levels, strengthening the case that AGP6 and AGP11 are involved in complex phenomena. The expression levels of calcium- and signaling-related genes were found to be altered, supporting the known roles of the respective proteins in pollen tube growth. Although the precise nature of the proposed interactions needs further investigation, the putative involvement of AGPs in signaling cascades through calmodulin and protein degradation via ubiquitin was indicated. The expression of stress-, as well as signaling- related, genes was also changed; a correlation that may result from the recognized similarities between signaling pathways in both defense and pollen tube growth.The results of yeast two-hybrid experiments lent further support to these signaling pathways and revealed putative AGP6 and AGP11 interactors implicated in recycling of cell membrane components via endocytosis, through clathrin-mediated endosomes and multivesicular bodies. CONCLUSIONS: The data presented suggest the involvement of AGP6 and AGP11 in multiple signaling pathways, in particular those involved in developmental processes such as endocytosis-mediated plasma membrane remodeling during Arabidopsis pollen development. This highlights the importance of endosomal trafficking pathways which are rapidly emerging as fundamental regulators of the wall physiology.
Project description:Changes in the composition of the cell walls are postulated to accompany changes in the cell's fate. We check whether there is a relationship between the presence of selected pectic, arabinogalactan proteins (AGPs), and extensins epitopes and changes in cell reprogramming in order to answer the question of whether they can be markers accompanying changes of cell fate. Selected antibodies were used for spatio-temporal immunolocalization of wall components during the induction of somatic embryogenesis. Based on the obtained results, it can be concluded that (1) the LM6 (pectic), LM2 (AGPs) epitopes are positive markers, but the LM5, LM19 (pectic), JIM8, JIM13 (AGPs) epitopes are negative markers of cells reprogramming to the meristematic/pluripotent state; (2) the LM8 (pectic), JIM8, JIM13, LM2 (AGPs) and JIM11 (extensin) epitopes are positive markers, but LM6 (pectic) epitope is negative marker of cells undergoing detachment; (3) JIM4 (AGPs) is a positive marker, but LM5 (pectic), JIM8, JIM13, LM2 (AGPs) are negative markers for pericycle cells on the xylem pole; (4) LM19, LM20 (pectic), JIM13, LM2 (AGPs) are constitutive wall components, but LM6, LM8 (pectic), JIM4, JIM8, JIM16 (AGPs), JIM11, JIM12 and JIM20 (extensins) are not constitutive wall components; (5) the extensins do not contribute to the cell reprogramming.
Project description:Alpha2u-globulin is an adult male rat-specific protein that accumulates spontaneously or inductively in the renal proximal tubular epithelium and forms microscopically observable deposits, which are generally referred to as "hyaline droplets," whereas a specific type of deposits is referred to as "eosinophilic bodies" by Japanese toxicologic pathologists. We compared hyaline droplets and eosinophilic bodies using special stains including immunostaining for ?2u-globulin and lysosome-associated membrane protein in spontaneously occurring and d-limonene-induced cases. Eosinophilic bodies appeared simultaneously and increased in parallel with the hyaline droplets in the induced case. In both of the spontaneous and induced cases, hyaline droplets and eosinophilic bodies were associated with ?2u-globulin and lysosomes, although there were differences in the forms and staining properties that probably reflected the purity or density of ?2u-globulin. According to the results, it is not necessary for eosinophilic bodies to be strictly distinguished from hyaline droplets, and it is reasonable to identify eosinophilic bodies as hyaline droplets in ?2u-globulin nephropathy in routine toxicity studies, as they have been recognized to be a sequence of changes associated with accumulation of ?2u-globulin.
Project description:Banana Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc) is soil-borne disease of banana (Musa spp.) causing significant economic losses. Extensins and arabinogalactan proteins (AGPs) are cell wall components important for pathogen defence. Their significance for Foc resistance in banana was not reported so far. In this study, two banana cultivars differing in Foc sensitivity were used to monitor the changes in transcript levels, abundance and distribution of extensins and AGPs after wounding and Foc inoculation. Extensins mainly appeared in the root cap and meristematic cells. AGPs recognized by JIM13, JIM8, PN16.4B4 and CCRC-M134 antibodies located in root hairs, xylem and root cap. Individual AGPs and extensins showed specific radial distribution in banana roots. At the transcript level, seven extensins and 23 AGPs were differentially expressed between two banana cultivars before and after treatments. Two extensins and five AGPs responded to the treatments at the protein level. Most extensins and AGPs were up-regulated by wounding and pathogen inoculation of intact plants but down-regulated by pathogen attack of wounded plants. Main components responsible for the resistance of banana were MaELP-2 and MaPELP-2. Our data revealed that AGPs and extensins represent dynamic cell wall components involved in wounding and Foc resistance.