Immunogold labelling of xylans and arabinoxylans in the plant cell walls of maize stem

Summary— Polyclonal antibodies against 4-O-methyl-glucuronoxylan and α L-1-3 arabinofuranosyl poly-β-d-1-4-xylopyranosyl were raised from rabbits. An immunocytochemical technique was used to localize xylans and arabinoxylans in the plant cell walls of the apical internode of two maize lines of different digestibility. The sclerenchyma, fibres and xylem (lignified tissues) and the parenchyma (non-lignified tissue) were studied. The arabinoxylans were more heavily labelled than the xylans in the lignified tissues of the less digestible maize whereas in the more digestible line the labelling of the two polysaccharides was similar. The xylans and arabinoxylans were localized in the secondary cell wall. In both maize lines, labelling increased from the base upwards of the apical internode, reflecting the changes in growth stage.     

淹水玉米幼苗根尖分生细胞内Ca2+超微细胞化学定位

采用焦锑酸钾沉淀法,对遭受淹水胁迫的玉米幼苗初生根根尖分生细胞内钙离子分布变化情况进行了电镜细胞化学观察。在正常状态下,根尖分生细胞内Ca^2＋沉淀颗粒的分布较少．主要位于细胞核和细胞质中。在淹水1h后,根尖分生细胞内呈现有大量Ca^2＋沉淀颗粒分布,细胞核和细胞质中分布的Ca^2＋沉淀颗粒密度,远大于正常细胞。随着淹水时间的延长,根尖分生细胞的细胞核和细胞质中分布的Ca^2＋沉淀颗粒呈现不断增多的趋势,而液泡中分布的Ca^2＋沉淀颗粒则逐步明显减少。根据实验结果本文对受淹根尖分生细胞的死亡与Ca^2＋分布变化的关系进行了研究。     

Immunocytochemical detection of lignin-related epitopes in cell walls in bryophytes and the charalean alga Nitella

Lignins are complex phenolic heteropolymers present in xylem and sclerenchyma cell walls in tracheophytes. The occurrence of lignin-like polymers in bryophytes is controversial. In this study two polyclonal antibodies against homoguaiacyl (G) and guaiacyl/syringyl (GS) synthetic lignin-like polymers that selectively labelled lignified cell walls in tracheophytes also bound to cell walls in bryophytes, the GS antibody usually giving a stronger labelling than the G antibody. In contrast to tracheophytes, the antibody binding in liverworts and mosses was not tissue-specific. In the hornworts Megaceros flagellaris and M. fuegiensis the pseudoelaters and spores were labelled more intensely than the other cell types with the GS antibody. The cell walls in Nitella were labelled with both antibodies but no binding was observed in Coleochaete. The results suggest that the ability to incorporate G or GS moieties in cell walls is a plesiomorphy (primitive character) of the land plant clade.     

Immunocytochemical localization of cell wall polysaccharides in the root tip ofAvena sativa

Summary Two polyclonal antisera, anti-xyloglucan (anti-XG) and anti-polygalacturonic acid/rhamnogalacturonan I (anti-PGA/RG-I), which recognize, respectively, noncellulosic -(14)-D-glucan containing polysaccharides and the unesterified forms of the acidic pectic polysaccharide polygalacturonic acid/rhamnogalacturonan I, were used to localize epitopes recognized by the two antisera in the root tip of oat (Avena sativa). Immunoblot analysis shows that epitopes recognized by the anti-XG antibodies are present in both the mixed linkage -(13)-(14)-D-glucans (MG) and in xyloglucan (XG). Immunogold electron microscopy shows that the cell walls of meristematic, cortical, epidermal, columella, and peripheral cells contain significant amounts of such epitopes. In contrast, the molecules that carry these MG/XG epitopes appear to be sparse in the expanded middle lamella of meristematic cells, but dense in the expanded middle lamella of peripheral root cap cells. This finding suggests that the porosity of the middle lamella is altered in peripheral root cap cells to facilitate mucilage secretion. In contrast, few PGA/RG-I epitopes were detected in any cell walls of any of the cell types examined. Double immunogold labeling experiments revealed an intriguing localization pattern of MG/XG and of PGA/RG-I epitopes in the peripheral mucilage-secreting cells of the root cap. Whereas MG/XG epitopes were abundant in the cell wall, they were sparse in both the secreted mucilage and in intracellular secretory vesicles. In marked contrast, PGA/RG-I epitopes were detected at high density in intracellular secretory vesicles, but unexpectedly, were quite sparse in both the cell wall and in the mucilage. These immunolabeling patterns are consistent with the hypotheses that the synthesis and secretion of particular -D-glucans is subject to both activation and down-regulation during cell development and differentiation and that post-secretory alterations of pectic polysaccharides, such as enzymatic release of RG-I-type mucilage molecules from PGA/RG-I precursors, may occur in the peripheral cell walls of the oat root cap.Abbreviations MG mixed linkage -(13)-(14)-D-glucan - PGA/RG-I polygalacturonic acid/rhamnogalacturonan I - SEPS sycamore extracellular polysaccharides - TGN trans Golgi network - XG xyloglucan     

Rheological analysis of viscoelastic cell wall changes in maize coleoptiles as affected by auxin and osmotic stress

The effects of auxin and osmotic stress on elongation growth of maize (Zea mays L.) coleoptile segments are accompanied by characteristic changes in the extensibility of the growth-limiting cell walls. At full turgor auxin causes growth by an increase in wall extensibility (wall looseining). Growth can be stopped by an osmotically produced step-down in turgor of 0.45 MPa. Under these conditions auxin causes the accumulation of a potential for future wall extension which is released after restoration of full turgor. Turgor reduction causes a reversible decrease in wall extensibility (wall stiffening) both in the presence and absence of auxin. These changes in vivo are correlated with corresponding changes in the rheological properties of the cell walls in vitro which can be traced back to specific modifications in the shape of the hysteretic stress-strain relationship. The longitudinally load-bearing walls of the coleoptile demonstrate almost perfect viscoelasticity as documented by a nearly closed hysteresis loop. Auxin-mediated wall loosening causes an increase of loop width and thus affects primarily the amount of hysteresis in the isolated wall. In contrast, turgor reduction by osmotic stress reduces loop length and thus affects primarily the amount of viscoelastic wall extensibility. Pretreatment of segments with anoxia and H₂O₂ modify the hysteresis loop in agreement with the conclusion that the wall-stiffening reaction visualized under osmotic stress in vivo is an O₂-dependent process in which O₂ can be substituted by H₂O₂. Cycloheximide specifically inhibits auxin-mediated wall loosening without affecting wall stiffening, and this is mirrored in specific changes of the hysteresis loop. Corroborating a previous in vivo study (Hohl et al. 1995, Physiol. Plant. 94: 491–498) these results show that cell wall stiffening in vivo can also be demonstrated by Theological measurements with the isolated cell wall and that this process can be separated from cell wall loosening by specific changes in the shape of the hysteresis loop.     

Effect of anti-wall protein antibodies on auxin-induced elongation, cell wall loosening, and β-D-glucan degradation in maize coleoptile segments

Antiserum raised against the LiCl extract of maize shoot cell walls suppresses auxin-induced elongation of maize coleoptile segments. A series of polyclonal antibodies were raised against protein fractions separated from the LiCl extract of maize ( Zea mays L. cv. B73 x Mo17) coleoptiles by SP-Sephadex and Bio-Gel P-150 chromatography. To understand the role of cell wall proteins in growth regulation, the effect of these antibodies on auxin-induced elongation and changes in the cell walls of maize coleoptiles was examined. Four of the fractions prepared reacted with the antiserum raised against the total LiCl extract and effectively suppressed its growth-inhibiting activity. Only these fractions contained the proteins responsible for eliciting growthinhibiting antibodies. The antibodies capable of growth inhibition of auxin-induced elongation of segments also inhibited auxin-induced cell wall loosening (decrease in the minimum stress-relaxation time of the cell walls) of segments. The antibodies raised against one of the protein fractions separated by SP-Sephadex inhibited the autolytic reactions of isolated cell walls and the auxin-induced decrease in (1→3), (1→4)-β-D-glucans in the cell walls. Thus, the degradation of β-D-glucans by cell wall enzymes may be associated with the cell wall loosening that is responsible for cell elongation. Because the other antibodies did not influence the auxin-induced degradation of (1→3), (1→4)-β-D-glucanses, β-D-glucanases and other cell wall enzymes may cooperate in regulation of cell elongation in maize coleoptiles.     

An update on receptor-like kinase involvement in the maintenance of plant cell wall integrity

Background

Plant cell walls form the interface between the cells and their environment. They perform different functions, such as protecting cells from biotic and abiotic stress and providing structural support during development. Maintenance of the functional integrity of cell walls during these different processes is a prerequisite that enables the walls to perform their particular functions. The available evidence suggests that an integrity maintenance mechanism exists in plants that is capable of both detecting wall integrity impairment caused by cell wall damage and initiating compensatory responses to maintain functional integrity. The responses involve 1-aminocyclopropane-1-carboxylic acid (ACC), jasmonic acid, reactive oxygen species and calcium-based signal transduction cascades as well as the production of lignin and other cell wall components. Experimental evidence implicates clearly different signalling molecules, but knowledge regarding contributions of receptor-like kinases to this process is less clear. Different receptor-like kinase families have been considered as possible sensors for perception of cell wall damage; however, strong experimental evidence that provides insights into functioning exists for very few kinases.

Scope and Conclusions

This review examines the involvement of cell wall integrity maintenance in different biological processes, defines what constitutes plant cell wall damage that impairs functional integrity, clarifies which stimulus perception and signal transduction mechanisms are required for integrity maintenance and assesses the available evidence regarding the functions of receptor-like kinases during cell wall integrity maintenance. The review concludes by discussing how the plant cell wall integrity maintenance mechanism could form an essential component of biotic stress responses and of plant development, functions that have not been fully recognized to date.     

The novel herbicide oxaziclomefone inhibits cell expansion in maize cell cultures without affecting turgor pressure or wall acidification

Oxaziclomefone [OAC; IUPAC name 3-(1-(3,5-dichlorophenyl)-1-methylethyl)-3,4-dihydro-6-methyl-5-phenyl-2H-1,3-oxazin-4-one] is a new herbicide that inhibits cell expansion in grass roots. Its effects on cell cultures and mode of action were unknown. In principle, cell expansion could be inhibited by a decrease in either turgor pressure or wall extensibility. Cell expansion was estimated as settled cell volume; cell division was estimated by cell counting. Membrane permeability to water was measured by a novel method involving simultaneous assay of the efflux of (3)H(2)O and [(14)C]mannitol from a 'bed' of cultured cells. Osmotic potential was measured by depression of freezing point. OAC inhibited cell expansion in cultures of maize (Zea mays), spinach (Spinacia oleracea) and rose (Rosa sp.), with an ID(50) of 5, 30 and 250 nm, respectively. In maize cultures, OAC did not affect cell division for the first 40 h. It did not affect the osmotic potential of cell sap or culture medium, nor did it impede water transport across cell membranes. It did not affect cells' ability to acidify the apoplast (medium), which may be necessary for 'acid growth'. As OAC did not diminish turgor pressure, its ability to inhibit cell expansion must depend on changes in wall extensibility. It could be a valuable tool for studies on cell expansion.     

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.     

Immunocytochemical localization of protamine in the spermatids of the ram

Pure ram protamine isolated from epididymal spermatozoa was used to raise antisera in castrated rabbits. The antibodies were visualized in the electron microscope using the method of Moriarty and Halmi [1972] with either peroxidase or coupling with colloidal gold. The gold gave better contrast but lower afinity than the peroxidase method. With the use of fixation according to Thiery and Rambourg [1976] and thick sections treated with hydrogen peroxide, it was possible to detect the protamine in the cytoplasm near the flagellum and the chromatoid body of step 12 spermatids. It was concluded that protamine enters the nucleus and concentrates at that step in the ram.     

The state of cell wall pectin monitored by wall associated kinases: A model

The Wall Associated Kinases (WAKs) bind to both cross-linked polymers of pectin in the plant cell wall, but have a higher affinity for smaller fragmented pectins that are generated upon pathogen attack or wounding. WAKs are required for cell expansion during normal seedling development and this involves pectin binding and a signal transduction pathway involving MPK3 and invertase induction. Alternatively WAKs bind pathogen generated pectin fragments to activate a distinct MPK6 dependent stress response. Evidence is provided for a model for how newly generated pectin fragments compete for longer pectins to alter the WAK dependent responses.     

Immunocytochemical localization of 3-methylcrotonyl-CoA carboxylase in cultured ependymal, microglial and oligodendroglial cells

To evaluate the ability of ependymal, microglial and oligodendroglial cells to degrade leucine, the presence of 3-methylcrotonyl-CoA carboxylase (MCC) was investigated in cultures of these cells. MCC is a biotin-containing heterodimeric enzyme that is specific for the irreversible part of the leucine catabolic pathway. It has been reported previously that in cell culture MCC is expressed in astrocytes and a subpopulation of neurones. In the present study ependymal, microglial and oligodendroglial cell cultures, derived from the brains of newborn rats, were examined for the expression of MCC by RT-PCR, western blotting and immunocytochemistry. The results of RT-PCR and western blotting showed the presence of mRNA as well as protein of both subunits of MCC in ependymal, microglial and oligodendroglial cell cultures. Immunocytochemical investigation of the cellular and subcellular distribution of MCC demonstrated a mitochondrial location of MCC in all neuroglial cell types investigated. The ubiquitous expression of MCC in glial cells demonstrates the ability of the cells to engage in the catabolism of leucine transported into the brain, mainly for the generation of energy.     

A receptor-like kinase controls the amplitude of secondary cell wall synthesis in rice

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ZmXTH1, a new xyloglucan endotransglucosylase/hydrolase in maize, affects cell wall structure and composition in Arabidopsis thaliana

Xyloglucan endotransglucosylase/hydrolases (XTHs; EC 2.4.1.207and/or EC 3.2.1.15 [EC] 1) are enzymes involved in the modificationof cell wall structure by cleaving and, often, also re-joiningxyloglucan molecules in primary plant cell walls. Using a poolof antibodies raised against an enriched cell wall protein fraction,a new XTH cDNA in maize, ZmXTH1, has been isolated from a cDNAexpression library obtained from the elongation zone of themaize root. The predicted protein has a putative N-terminalsignal peptide and possesses the typical domains of this enzymefamily, such as a catalytic domain that is homologous to thatof Bacillus macerans β-glucanase, a putative N-glycosylationmotif, and four cysteine residues in the central and C terminalregions of the ZmXTH1 protein. Phylogenetic analysis of ZmXTH1reveals that it belongs to subgroup 4, so far only reportedfrom Poaceae monocot species. ZmXTH1 has been expressed in Pichiapastoris (a methylotrophic yeast) and the recombinant enzymeshowed xyloglucan endotransglucosylase but not xyloglucan endohydrolaseactivity, representing the first enzyme belonging to subgroup4 characterized in maize so far. Expression data indicate thatZmXTH1 is expressed in elongating tissues, modulated by cultureconditions, and induced by gibberellins. Transient expressionassays in onion cells reveal that ZmXTH1 is directed to thecell wall, although weakly bound. Finally, Arabidopsis thalianaplants expressing ZmXTH1 show slightly increased xyloglucanendohydrolase activity and alterations in the cell wall structureand composition. Key words: Cell elongation, cell wall, plant transformation, XEH, XET, XTH, Zea mays     

Fracture mechanics of the cell wall of Chara corallina

Previous mechanical studies using algae have concentrated on cell extension and growth using creep-type experiments, but there appears to be no published study of their failure properties. The mechanical strength of single large internode cell walls (up to 2 mm diameter and 100 mm in length) of the charophyte (giant alga) Chara corallina was determined by dissecting cells to give sheets of cell wall, which were then notched and fractured under tension. Tensile tests, using a range of notch sizes, were conducted on cell walls of varying age and maturity to establish their notch sensitivity and to investigate the propagation of cracks in plant cell walls. The thickness and stiffness of the walls increased with age whereas their strength was little affected. The strength of unnotched walls was estimated as 47 ± 13 MPa, comparable to that of some grasses but an order of magnitude higher than that published for model bacterial cellulose composite walls. The strength was notch-sensitive and the critical stress intensity factor K _1c was estimated to be 0.63 ± 0.19 MNm^−3/2, comparable to published values for grasses. Received: 4 April 2000 / Accepted: 21 July 2000     

Degradation of lignified secondary cell walls of lucerne (Medicago sativa L.) by rumen fungi growing in methanogenic co-culture

Aims: To compare the abilities of the monocentric rumen fungi Neocallimastix frontalis, Piromyces communis and Caecomyces communis, growing in coculture with Methanobrevibacter smithii, to colonize and degrade lignified secondary cell walls of lucerne (alfalfa) hay. Methods and Results: The cell walls of xylem cylinders isolated from stems of lucerne contained mostly xylans, cellulose and lignin together with a small proportion of pectic polysaccharides. All of these major components were removed during incubation with the three fungi, and differing cell wall polysaccharides were degraded to different extents. The greatest dry weight loss was found with N. frontalis and least with C. communis, and scanning electron microscopy revealed that these extensively colonized different cell types. C. communis specifically colonized secondary xylem fibres and showed much less degradation than N. frontalis and P. communis. Conclusions: Neocallimastix frontalis and P. communis were efficient degraders of the cell walls of lucerne xylem cylinders. Degradation occurred of pectic polysaccharides, xylan and cellulose. Loss of lignin from the xylem cylinders probably resulted from the cleavage of xylan releasing xylan–lignin complexes. Significance and Impact of the Study: Unlike rumen bacteria, the rumen fungi N. frontalis, P. communis and C. communis are able to degrade lignified secondary walls in lucerne stems. These fungi could improve forage utilization by ruminants and may have potential in the degradation of lignocellulosic biomass in the production of biofuels.     

Differential cell wall degradation byErwinia chrysanthemi in petiole ofSaintpaulia ionantha

Summary Erwinia chrysanthemi is a soft-rot pathogenic enterobacterium that provokes maceration of host plant tissues by producing extracellular cell-wall-degrading enzymes, among which are pectate lyases, pectin methyl esterases, and cellulases. Cell wall degradation in leaves and petiole tissue of infectedSaintpaulia ionantha plants has been investigated in order to define the structural and temporal framework of wall deconstruction. The degradation of major cell wall components, pectins and cellulose, was studied by both classical histochemical techniques (Calcofluor and periodic acid-thiocarbohydrazide-silver proteinate staining) and immunocytochemistry (tissue printing for detection of pectate lyases; monoclonal antibodies JIM5 and JIM7 for detection of pectic substrates). The results show that the mode of progression of the bacteria within the host plant is via the intercellular spaces of the parenchyma leaf and the petiole cortex. Maceration symptoms and secretion of pectate lyases PelA, -D, and -E can be directly correlated to the spread of the bacteria. Wall degradation is very heterogeneous. Loss of reactivity with JIM5 and JIM7 was progressive and/or clearcut. The primary and middle lamella appear to be the most susceptible regions of the wall. The innermost layer of the cell wall frequently resists complete deconstruction. At the wall intersects and around intercellular spaces resistant domains and highly degraded domains occurred simultaneously. All results lead to the hypothesis that both spatial organisation of the wall and accessibility to enzymes are very highly variable according to regions. The use of mutants lacking pectate lyases PelA, -D, -E or -B, -C confirm the important role that PelA, PelD, and PelE play in the rapid degradation of pectins from the host cell walls. In contrast, PelB and PelC seem not essential for degradation of the wall, though they can be detected in leaves infected with wild-type bacteria. With Calcofluor staining, regularly localised cellulose-rich and cellulose-poor domains were observed in pectic-deprived walls.Abbreviations MAb monoclonal antibody - PATAg periodic acid-thiocarbohydrazide-silver proteinate     

Methane production and substrate degradation by rumen microbial communities containing single protozoal species in vitro

AIMS: To assess the effect of protozoal species on rumen fermentation characteristics in vitro. METHODS AND RESULTS: Entodinium caudatum, Isotricha intestinalis, Metadinium medium, and Eudiplodinium maggii from monofaunated wethers and mixed protozoa from conventional wethers were obtained by centrifugation, re-suspended at their normal densities in rumen fluid supernatants from defaunated or conventional wethers and incubated in vitro. The presence of protozoa increased the concentration of ammonia and altered the volatile fatty acids balance with more acetate and butyrate produced at the expense of propionate. Differences among species were observed, notably in the production of methane, which increased with E. caudatum as compared to other ciliates and to defaunated and mixed protozoa treatments (P < 0.05). The increased methanogenesis was not correlated to protozoal biomass indicating that the metabolism of this protozoan and/or its influence on the microbial ecosystem was responsible for this effect. CONCLUSIONS: Entodinium caudatum stimulated the production of methane, a negative effect that was reinforced by a concomitant increase in protein degradation. SIGNIFICANCE AND IMPACT OF THE STUDY: Comparison of individual species of protozoa highlighted the particular influence of E. caudatum on rumen fermentation. Its elimination (targeted defaunation) from the rumen could reduce methane production without affecting feed degradation.