Immunogold labelling of arabinoxylans in the plant cell walls of normal and bm3 mutant maize

Summary— Polyclonal antibodies directed against α, l -1.2-arabinofuranosyl poly-β,d -1.4-xylopyranosyl (degree of polymerization 130) have been raised from rabbits. The immunogold labelling in transmission electron microscopy (TEM) evidenced the arabinoxylans of the plant cell walls. Comparison between the stems of normal and mutant bm3 maize demonstrated a greater accessibility of arabinoxylans in the walls of the mutant maize. The method, specific and swift, allows us to specify the repartition in the different parts of the stem: sclerenchyma, fibers, parenchyma.     

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.     

Relationship between chemical composition and in situ rumen degradation characteristics of maize silages in dairy cows

Several in situ studies have been conducted on maize silages to determine the effect of individual factors such as maturity stage, chop length and ensiling of maize crop on the rumen degradation but the information on the relationship between chemical composition and in situ rumen degradation characteristics remains scarce. The objectives of this study were to determine and describe relationships between the chemical composition and the rumen degradation characteristics of dry matter (DM), organic matter (OM), CP, starch and aNDFom (NDF assayed with a heat stable amylase and expressed exclusive of residual ash) of maize silages. In all, 75 maize silage samples were selected, with a broad range in chemical composition and quality parameters. The samples were incubated in the rumen for 2, 4, 8, 16, 32, 72 and 336 h, using the nylon bag technique. Large range was found in the rumen degradable fractions of DM, OM, CP, starch and aNDFom because of the broad range in chemical composition and quality parameters. The new database with in situ rumen degradation characteristics of DM, OM, CP, starch and aNDFom of the maize silages was obtained under uniform experimental conditions; same cows, same incubation protocol and same chemical analysis procedures. Regression equations were developed with significant predictors (P<0.05) describing moderate and weak relationships between the chemical composition and the washout fraction, rumen undegradable fraction, potentially rumen degradable fraction, fractional degradation rate and effective rumen degradable fraction of DM, OM, CP, starch and aNDFom.     

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 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     

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.     

Extracellular cross-linking of maize arabinoxylans by oxidation of feruloyl esters to form oligoferuloyl esters and ether-like bonds

Primary cell walls of grasses and cereals contain arabinoxylans with esterified ferulate side chains, which are proposed to cross‐link the polysaccharides during maturation by undergoing oxidative coupling. However, the mechanisms and control of arabinoxylan cross‐linking in vivo are unclear. Non‐lignifying maize (Zea mays L.) cell cultures were incubated with l‐ [1‐³H]arabinose or (E)‐[U‐¹⁴C]cinnamate (radiolabelling the pentosyl and feruloyl groups of endogenous arabinoxylans, respectively), or with exogenous feruloyl‐[³H]arabinoxylans. The cross‐linking rate of soluble extracellular arabinoxylans, monitored on Sepharose CL‐2B, peaked suddenly and transiently, typically at ～9 days after subculture. This peak was not associated with appreciable changes in peroxidase activity, and was probably governed by fluctuations in H₂O₂ and/or inhibitors. De‐esterified arabinoxylans failed to cross‐link, supporting a role for the feruloyl ester groups. The cross‐links were stable in vivo. Some of them also withstood mild alkaline conditions, indicating that they were not (only) based on ester bonds; however, most were cleaved by 6 m NaOH, which is a property of p‐hydroxybenzyl–sugar ether bonds. Cross‐linking of [¹⁴C]feruloyl‐arabinoxylans also occurred in vitro, in the presence of endogenous peroxidases plus exogenous H₂O₂. During cross‐linking, the feruloyl groups were oxidized, as shown by ultraviolet spectra and thin‐layer chromatography. Esterified diferulates were minor oxidation products; major products were: (i) esterified oligoferulates, released by treatment with mild alkali; and (ii) phenolic components attached to polysaccharides via relatively alkali‐stable (ether‐like) bonds. Thus, feruloyl esters participate in polysaccharide cross‐linking, but mainly by oligomerization rather than by dimerization. We propose that, after the oxidative coupling, strong p‐hydroxybenzyl–polysaccharide ether bonds are formed via quinone‐methide intermediates.     

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.     

Secondary cell wall patterning—connecting the dots,pits and helices

All plant cells are encased in primary cell walls that determine plant morphology, but also protect the cells against the environment. Certain cells also produce a secondary wall that supports mechanically demanding processes, such as maintaining plant body stature and water transport inside plants. Both these walls are primarily composed of polysaccharides that are arranged in certain patterns to support cell functions. A key requisite for patterned cell walls is the arrangement of cortical microtubules that may direct the delivery of wall polymers and/or cell wall producing enzymes to certain plasma membrane locations. Microtubules also steer the synthesis of cellulose—the load-bearing structure in cell walls—at the plasma membrane. The organization and behaviour of the microtubule array are thus of fundamental importance to cell wall patterns. These aspects are controlled by the coordinated effort of small GTPases that probably coordinate a Turing''s reaction–diffusion mechanism to drive microtubule patterns. Here, we give an overview on how wall patterns form in the water-transporting xylem vessels of plants. We discuss systems that have been used to dissect mechanisms that underpin the xylem wall patterns, emphasizing the VND6 and VND7 inducible systems, and outline challenges that lay ahead in this field.     

Constitutive expression of cell wall invertase genes increases grain yield and starch content in maize

Grain size, number and starch content are important determinants of grain yield and quality. One of the most important biological processes that determine these components is the carbon partitioning during the early grain filling, which requires the function of cell wall invertase. Here, we showed the constitutive expression of cell wall invertase–encoding gene from Arabidopsis, rice (Oryza sativa) or maize (Zea mays), driven by the cauliflower mosaic virus (CaMV) 35S promoter, all increased cell wall invertase activities in different tissues and organs, including leaves and developing seeds, and substantially improved grain yield up to 145.3% in transgenic maize plants as compared to the wild‐type plants, an effect that was reproduced in our 2‐year field trials at different locations. The dramatically increased grain yield is due to the enlarged ears with both enhanced grain size and grain number. Constitutive expression of the invertase‐encoding gene also increased total starch content up to 20% in the transgenic kernels. Our results suggest that cell wall invertase gene can be genetically engineered to improve both grain yield and grain quality in crop plants.     

The connection of cytoskeletal network with plasma membrane and the cell wall

The cell wall provides external support of the plant cells, while the cytoskeletons including the microtubules and the actin filaments constitute an internal framework. The cytoskeletons contribute to the cell wall biosynthesis by spatially and temporarily regulating the transportation and deposition of cell wall components. This tight control is achieved by the dynamic behavior of the cytoskeletons, but also through the tethering of these structures to the plasma membrane. This tethering may also extend beyond the plasma membrane and impact on the cell wall, possibly in the form of a feedback loop. In this review, we discuss the linking components between the cytoskeletons and the plasma membrane, and/or the cell wall. We also discuss the prospective roles of these components in cell wall biosynthesis and modifications, and aim to provide a platform for further studies in this field.     

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.     

Effect of auxin and abscisic acid on cell wall extensibility in maize coleoptiles

Plastic and elastic in-vitro extensibilities (E _pland E _el ) of cell walls from growing maize (Zea mays L.) coleoptile segments were measured by stretching frozen-thawed tissue, pre-extended to its in-vivo length, at constant force (creep test) in a custom-buildt extensiometer, equipped with a linear-displacement transducer. The indole-3-acetic acid (IAA)-induced change of E _pl (E _pl ) is strictly correlated with the growth rate for a period of 3–4 h. Subsequently, E _plremains constant while the growth rate is slowing down. Since this discrepancy can be accounted for by a growth-dependent reduction of osmotic pressure, it is concluded that E _plrepresents quantitatively the relative increase of in-vivo extensibility (cell wall loosening) involved in IAA-mediated cell growth over a much longer time. On the other side it is argued that the growth rate may not be strictly correlated with wall extensibility during long-term growth. Abscisic acid (ABA) inhibits segment growth induced by auxin, fusicoccin, or exogenous acid, and this effect can be quantitatively attributed to an ABA-mediated reduction of cell wall extensibility as determined by the E _plmeasurement. Both, IAA and ABA have no effect on total protein synthesis, RNA synthesis, and amount of osmotic solutes. Fusicoccin-induced proton excretion is only slightly inhibited by ABA. In contrast to ABA, growth inhibition by cycloheximide (CHI) is always much larger than the concomitant reduction of E _pl , indicating that a further growth parameter is also involved in the inhibition of cell growth by CHI. E _el is not affected by either IAA, ABA, or CHI. It is concluded that E _pl as determined by the applied method, represents a relative measure of the actual in-vivo extensibility of the growing cell wall at the very moment when the tissue is killed, rather than an average extensibility accumulated over some immediate-past period of time as suggested by Cleland (1984, Planta 160, 514–520). Hence, we further draw the conclusion that IAA and ABA control of cell growth can entirely be attributed to a modulation of cell wall extensibility by these hormones in maize coleoptiles.Abbreviations ABA ±abscisic acid - CHI cycloheximide - E _el , E_pl elastic and plastic in vitro extensibilities, respectively (E _el+E_pl=E_tot>) - FC fusicoccin - IAA indole-3-acetic acid     

酸性培养基对喜树叶片细胞壁降解的影响

在组织培养过程中对喜树叶片外植体进行解剖学观察。发现在组织培养条件下,喜树叶片在培养基的酸性环境中细胞壁呈现模糊微弱降解、明显降解和完全降解直至消失的解剖学特征。在同样的组培条件和相同的时间内,同一喜树叶片不同部位出现细胞壁程度不同的降解和消失现象,可能是喜树叶片因上表皮凸凹不平进而导致其不同部位与酸性培养基接触的程度不同,因而使培养基中的酸性物质对喜树叶片上表皮的不同部位影响出现异质化。本文对培养基中的酸性成分对喜树叶片细胞壁降解的影响有了进一步的认识和理解。