Pectin esterification is spatially regulated both within cell walls and between developing tissues of root apices

Monoclonal antibodies recognizing un-esterified (JIM5) and methyl-esterified (JIM7) epitopes of pectin have been used to locate these epitopes by indirect immunofluorescence and immunogold electron microscopy in the root apex of carrot (Daucus carota L.). Both antibodies labelled the walls of cells in all tissues of the developing root apex. Immunogold labelling observed at the level of the electron microscope indicated differential location of the pectin epitopes within the cell walls. The un-esterified epitope was located to the inner surface of the primary cell walls adjacent to the plasma membrane, in the middle lamella and abundantly to the outer surface at intercellular spaces. In contrast, the epitope containing methyl-esterified pectin was located evenly throughout the cell wall. In root apices of certain other species the JIM5 and JIM7 epitopes were found to be restricted to distinct tissues of the developing roots. In the root apex of oat (Avena sativa L.), JIM5 was most abundantly reactive with cell walls at the region of intercellular spaces of the cortical cells. JIM7 was reactive with cells of the cortex and the stele. Neither epitope occurred in walls of the epidermal or root-cap cells. These pattern of expression were observed to derive from the very earliest stages of the development of these tissues in the oat root meristem and were maintained in the mature root. In the coleoptile and leaf tissues of oat seedlings, JIM5 labelled all cells abundantly whereas JIM7 was unreactive. Other members of the Gramineae and also the Chenopodiaceae are shown to express similar restricted spatial patterns of distribution of these pectin epitopes in root apices.Abbreviations CDTA 1,2-diaminocyclohexane tetraacetic acid - RG rhamnogalacturonan J.P.K. was supported by the Agricultural and Food Research Council Cell Signalling and Recognition Programme. We thank J. Cooke and N. Stacey for technical assistance, H.A. Schols, Drs. P. Albersheim and A. Darvill for pectic polysaccharides, and Dr. R.R. Selvendran and M. McCann for useful discussions.     

Occurrence of cell surface arabinogalactan-protein and extensin epitopes in relation to pericycle and vascular tissue development in the root apex of four species

Monoclonal antibodies recognizing two classes of developmentally regulated plant cell surface components – arabinogalactan-proteins (AGPs) and extensins – have been used to immunolabel cells at the root apices of four species with different characteristics of pericycle and vascular tissue development. Root apices of pea (Pisum sativum L.), radish (Raphanus sativus L.), carrot (Daucus carota L.) and onion (Allium cepa L.) were immunolabelled with the anti-AGP monoclonal antibodies JIM4 and JIM13 and anti-extensin monoclonal antibodies JIM11, JIM12, JIM19 and JIM20. All of these antibodies recognized subsets of pericycle cells in at least one, but never all, of these species. The restricted patterns of epitope occurrence also reflected vascular cell development. The differences in patterns of antibody recognition in the four species are discussed in relation to the possible roles of these cell surface molecules in cell differentiation and root patterning events. Received: 11 March 1997 / Accepted: 20 May 1997     

Developmentally regulated epitopes of cell surface arabinogalactan proteins and their relation to root tissue pattern formation

Two polymorphic forms of an extracellular arabinogalactan protein (AGP1 and AGP2), obtained from the conditioned media of two carrot suspension-cultured cell lines, have been identified in terms of binding of the anti-plasma membrane antibodies JIM4 and MAC207. AGP1 and AGP2 have been used as immunogens to generate further anti-AGP monoclonal antibodies. JIM14 identified an epitope carried by AGP2 and also by glycoproteins of low molecular weight localized to the plant cell wall. In addition, further antibodies (JIM13 and JIM15) identified carbohydrate epitopes of the AGPs that also occur on plasma membrane glycoproteins and are expressed by patterns of cells that reflect cell position at the carrot root apex. Indirect immunofluorescence microscopy indicated that JIM13 recognized the surface of cells forming the epidermis and cells marking the region and axis of the future xylem. JIM15 recognized a pattern of cells directly complementary to the JIM13 pattern. The panel of anti-AGP monoclonal antibodies now available indicates groups of cells within the root meristem that may reflect an early pre-pattern of the tissues of the mature root structure and suggests extensive modulation of cell surface AGPs during cell development and the positioning of cells within the apex.     

Cell-specific expression of two arabinogalactan protein epitopes recognized by monoclonal antobodies JIM8 and JIM13 in maize roots

Summary The cell-specific expression of two arabinogalactan protein (AGP) epitopes recognized by monoclonal antibodies JIM8 and JIM13 is reported in maize roots. Employing immunofluorescence and immunogold electron microscopy, the JIM8 antibody was shown to label exclusively protophloem sieve elements, while the JIM13 antibody labelled sieve elements very strongly and adjacent pericycle and companion cells, as well as sloughing root cap cells less strongly. Since the labelling of sieve elements with JIM8 antibody was specific and did not spread to other cell types during root development, it is concluded that this AGP epitope can serve as a specific marker of these specialized cells within the maize root. In the case of the AGP epitope recognized by JIM13 antibody, part of the immunofluorescence label was also found to be associated with cytoplasmic strands in the pericycle and sloughing root cap cells. Immunogold-labelling of sieve elements revealed the association of both AGP epitopes (JIM8 and JIM13) with cortical sieve element reticulum and plasma membranes. Labelling of sieve element reticulum was prominent at its domains of adhesion to the plasma membrane, P-type plastids, and mitochondria. Based on our subcellular studies, we propose a new function of AGP epitopes in endomembrane recognition and adhesion within the sieve elements of maize roots.Abbreviations AGP arabinogalactan protein - SER sieve element reticulum     

DISTRIBUTION AND RELATIONSHIP OF CELL DIVISION AND MATURATION EVENTS IN PISUM SATIVUM (FABACEAE) SEEDLING ROOTS

Pea roots have open apical organization, where discrete initial cells do not exist. Differentiation of all tissues occurs in cylinders and vascular sectors that blend gradually with each other. This study reports the distribution of dividing cells and their relationship to maturation events in the 2 mm root tip, and in the 8–10 and 18–20 mm segments. Up to 200 μm from the root body/cap junction, cell division is uniformly distributed throughout all meristem regions. By 350 to 500 μ, xylem tracheary elements and cells of the pith parenchyma and middle cortex have stopped dividing. At this level cell division is almost entirely restricted to two cylinders, one composed of the inner root cap, the epidermis, and the outer cortex (outer cortex cylinder) and another composed of cells of the inner cortex, the pericycle and vascular tissue (inner cortex cylinder). When the protophloem matures, all cells in the phloem sector of the inner cortex cylinder, including the 1 layered pericycle, the endodermis and the phloem parenchyma, stop dividing. The 3–4 layered pericycle in the xylem sectors continues dividing until about 10 mm from the body/cap junction following the maturation of the protoxylem tracheary elements.     

The extent and differences in mitotic activity of the root tip ofVicia faba L.

Mitotic activity does not stop for different meristematic cells of the root apex at the same distance from the initials. The differences are connected with the functional heterogeneity of the apical meristem of the root. The arrangement of vascular bundles,i.e. the alternation of independent xylem and phloem groups, is of major importance. In broad bean roots, the protophloem sieve elements stop dividing first. The centre of the stelei. e. late metaxylem elements stop dividing next. Division in the stele gradually ceases centrifugally, while it ceases centripetally in the peripheral part of the root. The cylindrical region with prolonged cell division includes internal layers of the cortex including endodermis, pericycle and adjoining cells of the stele. Proximally apical meristem is reduced to isolated strands of cells adjacent to the protoxylem poles. Pericycle cells stop dividing last at a distance of approx. 9–10 mm from the initials. The number of the division cycles is limited and is specific for individual cell types. Epidermal and cortical cells divide in broad bean roots transversely approximately seven times, cells of late metaxylem approximately five times. Root apical meristem is an asynchronous cell population with a different duration of the mitotic cycle. We determined local variations in the duration of the mitotic cycle in the apical meristem of broad bean root by means of colchicine-induced polyploidy. The cells of the quiescent centre had the longest mitotic cycle after colchicine treatment. The region of the proper root adjacent to the quiescent centre was mixoploid (2n and 4n). Isolated cells with a long cycle occurred also in the cortex and in the central cylinder. Cells with a division cycle of 18h were found in the root cap, in the epidermis, in the cortex and in the central cylinder. Relatively numerous cells with the shortest division cycle, approx. 12 h, occurred farther of the quiescent centre in the epidermis, in the cortex, in the pericycle, and in adjacent layers of the stele through-out the entire meristematic region. The results derived from the analysis of the apical meristem are discussed in connection with the ontogenesis of different types of cells taking part in the primary structure of the root.     

白鲜根的发育解剖学研究

应用半薄切片、常规石蜡切片并结合离析法,对药用植物白鲜(Dictamnus dasycarpus Turcz.)根的发生发育过程进行了研究。结果表明:白鲜根的发生发育过程包括4个阶段,即原分生组织阶段、初生分生组织阶段、初生结构阶段以及次生结构阶段。原分生组织位于根冠内侧及初生分生组织之间,衍生细胞分化为初生分生组织。初生分生组织由原表皮、基本分生组织以及中柱原组成。原表皮分化为表皮,基本分生组织分化为皮层,中柱原分化为维管柱,共同组成根的初生结构;在初生结构中,部分表皮细胞外壁向外延伸形成根毛,皮层中分布有油细胞,内皮层有凯氏带,初生木质部为二原型或偶见三原型,外始式;根初生结构有髓或无。次生结构来源于原形成层起源的维管形成层的活动以及中柱鞘起源的木栓形成层的活动;白鲜次生韧皮部宽广,其中多年生根中可占根横切面积的85%,另外除基本组成分子外,还分布有油细胞;周皮发达,木栓层厚;初生皮层、次生木质部和次生韧皮部薄壁细胞中常充满丰富的淀粉粒。     

Distribution of pectic epitopes in cell walls of the sugar beet root

Immunolabelling techniques with antibodies specific to partially methyl-esterified homogalacturonan (JIM5: unesterified residues flanked by methylesterified residues. JIM7: methyl-esterified residues flanked by unesterified residues), a blockwise de-esterified homogalacturonan (2F4), 1,4-galactan (LM5) and 1,5-arabinan (LM6) were used to map the distribution of pectin motifs in cell walls of sugar beet root (Beta vulgaris). PME and alkali treatments of sections were used in conjunction with JIM5-7 and 2F4. The JIM7 epitope was abundant and equally distributed in all cells. In storage parenchyma, the JIM5 epitope was restricted to some cell junctions and the lining of intercellular spaces while in vascular tissues it occurred at cell junctions in some phloem walls and in xylem derivatives. After secondary wall formation, the JIM5 epitope was restricted to inner cell wall regions between secondary thickenings. The 2F4 epitope was not detected without de-esterification treatment. PME treatments prior to the use of 2F4 indicated that HG at cell corners was not acetylated. The LM5 epitope was mainly present in the cambial zone and when present in storage parenchyma, it was restricted to the wall region closest to the plasma membrane. The LM6 epitope was widely distributed throughout primary walls but was more abundant in bundles than in medullar ray tissue and storage parenchyma. These data show that the occurrence of oligosaccharide motifs of pectic polysaccharides are spatially regulated in sugar beet root cell walls and that the spatial patterns vary between cell types suggesting that structural variants of pectic polymers are involved in the modulation of cell wall properties.     

An AGP epitope distinguishes a central metaxylem initial from other vascular initials in theArabidopsis root

Summary The developmental expression of an arabinogalactan protein (AGP) recognised by a monoclonal antibody, JIM 13, is described inArabidopsis roots. It is expressed in the single initial of the central metaxylem vessel that lies immediately above the four central cells of the quiescent centre. AGP expression spreads in a non-clona] fashion to neighbouring cell files as they mature. The AGP first appears in other pre-metaxylem elements and subsequently in the layer of parenchyma cells on either side of the xylem axis. Later, it spreads to the entire ring of eight endodermal cell files and to those pericycle cell files next to the xylem pole. It is postulated that this epitope reflects an underlying set of cell signalling events that may be involved in defining local positional values of cells important in establishing cellular patterns in the root.     

Localization of cell wall polysaccharides in nonarticulated laticifers ofAsclepias speciosa Torr.

Summary Asclepias speciosa Torr, has latex-containing cells known as nonarticulated laticifers. In stem sections of this species, we have analyzed the cell walls of nonarticulated laticifers and surrounding cells with various stains, lectins, and monoclonal antibodies. These analyses revealed that laticifer walls are rich in (1→4) β-D-glucans and pectin polymers. Immunolocalization of pectic epitopes with the antihomogalacturonan antibodies JIM5 and JIM7 produced distinct labeling patterns. JIM7 labeled all cells including laticifers, while JIM5 only labeled mature epidermal cells and xylem elements. Two antibodies, LM5 and LM6, which recognize rhamnogalacturonan I epitopes distinctly labeled laticifer walls. LM6, which binds to a (l→5) α-arabinan epitope, labeled laticifer walls more intensely than walls of other cells. LM5, which recognizes a (1→4) β-D-galac-tan epitope, did not label laticifer segments at the shoot apex but labeled more mature portions of laticifers. Also the LM5 antibody did not label cells at the shoot apical meristem, but as cells grew and matured the LM5 epitope was expressed in all cells. LM2, a monoclonal antibody that binds to β-D-glucuronic acid residues in arabinogalactan proteins, did not label laticifers but specifically labeled sieve tubes. Sieve tubes were also specifically labeled byRicinus communis agglutinin, a lectin that binds to terminal β-D-galactosyl residues. Taken together, the analyses conducted showed that laticifer walls have distinctive cytochemical properties and that these properties change along the length of laticifers. In addition, this study revealed differences in the expression of pectin and arabinogalactan protein epitopes during shoot development or among different cell types.     

Tissue-specific subcellular immunolocalization of a myosin-like protein in maize root apices

Summary Using a heterologous myosin antibody raised against the whole molecule of bovine muscle myosin, we have identified a myosin-like protein in maize. Immunoblots of subcellular fractions isolated from roots identified one distinct band at about 210 kDa in the microsomal protein fraction and one band at about 180 kDa in the soluble protein fraction. Indirect immunofluorescence was performed using maize root apex sections to reveal endocellular distributions of the myosin-like protein. Both diffuse and particulate labelling patterns were observed throughout the cytoplasm of all root cells. In mitotic cells, myosin-like protein was excluded from spindle regions. Amyloplast surfaces were labelled prominently in cells of the root cap statenchyma and in all root cortex cells. On the other hand, myosin-like protein was prominently enriched at cellular peripheries in cells of the pericycle and outer stele in the form of continuous peripheral labelling. From all root apex tissues, phloem elements showed the most abundant presence of myosinlike protein.Abbreviations AFs actin filaments - MTs microtubules Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

Common components of the infection thread matrix and the intercellular space identified by immunocytochemical analysis of pea nodules and uninfected roots

Three rat hybridoma cell lines have been isolated which produce monoclonal antibodies identifying a noduleenhanced, soluble component of Pisum sativum root nodules. These antibodies each recognized a protease-sensitive band (M_r 95K) on SDS-polyacrylamide gels. The 95K antigen was resolved by isoelectric focusing into acidic and neutral components which were separately detected by AFRC MAC 236 and MAC 265 respectively. The third antibody (MAC 204) reacted with both acidic and neutral components through an epitope that was sensitive to periodate oxidation. These monoclonal antibodies were used for immunogold localizations at light and electron microscopic levels. In each case, the antigen was shown to be present in the matrix that surrounds the invading rhizobia in infection threads and infection droplets, as well as in the intercellular spaces between plant cell walls of nodules and also of uninfected roots. By contrast, a fourth monoclonal antibody, AFRC JIM 5, labelled a pectic component in the walls of infection threads, and JIM 5 was also found to label the middle lamella of plant cell walls, especially at three-way junctions between cells. The composition and structure of the infection thread lumen is thus comparable to that of an intercellular space.     

Developmental regulation of pectic polysaccharides in the root meristem of Arabidopsis

JIM 5, an antibody that recognizes a relatively unesterifiedpectic epitope, distinguishes between dividing (meristematic)and non-dividing (central cells of the quiescent centre) cellsin the Arabidopsis root tip, indicating that non-dividing cellwalls contain higher levels of relatively unesterified pectinthan dividing cells. JIM 7, an antibody that recognizes a relativelymethyl esterified epitope, labels all cell walls uniformly throughoutthe root, suggesting that there is little variation in the relativelymethyl esterified pectic component in the two cell types. Theseobservations suggest that the characteristics of cell wallsin the root tip result in part from modulations in the amountof unesterified and non-methyl esterified pectin. Key words: Pectin, quiescent centre, roots, Arabidopsis     

Carbohydrate antigens and lectin receptors of the plasma membrane of carrot cells

Summary A monoclonal antibody (JIM 1) has been derived, subsequent to immunization of rats with carrot protoplasts and a hybridoma screen of protoplast immunoagglutination, that recognizes a determinant at the outer face of the plasma membrane of carrot cells. The binding of JIM 1 is readily inhibitable by -D-galactosyl residues. Although weakly cross-reacting with an extracellular arabinogalactan protein, isolated from the conditioned medium of suspension-cultured carrot cells, JIM 1 does not recognize arabinogalactan proteins associated with the plasma membrane. The plasma membrane antigen recognized by JIM 1 was of low molecular weight and was sensitive to both periodate treatment and a protease. JIM 1 therefore defines a new class of galactosyl-residue containing plant cell surface antigen, distinct from the arabinogalactan proteins. However, the extracellular arabinogalactan protein and related plasma membrane-associated glycoproteins are demonstrated to bind the anti-galactose plant lectin peanut agglutinin.Abbrevations AGP arabinogalactan protein - McAb monoclonal antibody - PNA peanut agglutinin     

Life Cycle,Pathogenicity, Histopathology,and Host Range of Race 5 of the Barley Root-Knot Nematode

The optimum temperature for development of race 5 of Meloidogyne naasi was 26 C. A life cycle was completed in 34 days. Growth of sorghum was suppressed when inoculated with M. naasi. Observations of M. naasi-infected sorghum roots demonstrated that roots were penetrated just behind the root cap; giant cells were generally initiated either in the procambial region or in very young phloem. When giant cells developed in the cortex, corresponding areas of the vascular system did not have an endodermis, pericycle, or phloem fibers. Nineteen plant species were tested for suitability as hosts for race 5 of M. naasi. Reproduction occurred on 11 of 12 monocotolydenous hosts and none of 7 dicotolydenous hosts. Reproduction often occurred without gall development.     

Symplastic communication between primary and developing lateral roots of Arabidopsis thaliana

The functional symplastic connections between primary and developinglateral roots of Arabidopsis were studied non-invasively usingconfocal laser scanning microscopy (CLSM), following ester-loadingof the phloem with carboxyfluorescein (CF). Prior to the formationof lateral primordia in the pericycle, the phloem of the primaryroot behaved as an isolated conducting domain. However, thedifferentiation of phloem connector elements within the dividingpericycle allowed the rapid establishment of intercellular communicationbetween the phloem and the cells of the lateral primordium.This communication was often established prior to the completeemergence of the lateral root from the parent root. Shortlyafter its emergence, functional conducting phloem became differentiatedwithin the developing lateral root. A progressive isolationbetween the phloem and surrounding cells at the base of thelateral root was observed as the lateral continued to grow;the new phloem conducting CF to the elongation zone where itwas unloaded symplastically from the protophloem into surroundingcells of the cortex and stele, a feature mirroring the patternfound near the apex of growing primary roots. Anomalous patternsof intercellular communication were found which indicated thatpreviously functional symplastic pathways may have become sealedoff following the emergence of some of the lateral roots. Key words: Arabidopsis, carboxyfluorescein, confocal laser scanning microscopy (CLSM), intercellular transport, lateral roots, phloem (unloading), symplast     

Soluble Signals from Cells Identified at the Cell Wall Establish a Developmental Pathway in Carrot

Cells in a plant differentiate according to their positions and use cell-cell communication to assess these positions. Similarly, single cells in suspension cultures can develop into somatic embryos, and cell-cell communication is thought to control this process. The monoclonal antibody JIM8 labels an epitope on cells in specific positions in plants. JIM8 also labels certain cells in carrot embryogenic suspension cultures. We have used JIM8 and secondary antibodies coupled to paramagnetic beads to label and immunomagnetically sort single cells in a carrot embryogenic suspension culture into pure populations. Cells in the JIM8(+) population develop into somatic embryos, whereas cells in the JIM8(-) population do not form somatic embryos. However, certain cells in JIM8(+) cultures (state B cells) undergo asymmetric divisions, resulting in daughter cells (state C cells) that do not label with JIM8 and that sort to JIM8(-) cultures. State C cells are competent to form somatic embryos, and we show here that a conditioned growth medium from a culture of JIM8(+) cells allows state C cells in a JIM8(-) culture to go on and develop into somatic embryos. JIM8 labels cells in suspension cultures at the cell wall. Therefore, a cell with a role in cell-cell communication and early cell fate selection can be identified by an epitope in its cell wall.     

THE ONTOGENY OF THE PRIMARY THICKENING MERISTEM OF ATRIPLEX HORTENSIS L. (CHENOPODIACEAE)

Seedlings of Atriplex hortensis were studied to ascertain; 1) in which organ the primary thickening meristem (PTM) first differentiates; 2) the direction of differentiation of the PTM, and 3) the pattern of differentiation of conjunctive tissue. The PTM initially differentiates in pericycle of the primary root base 11 days after emergence of the primary root. It then differentiates in the transition region of the hypocotyl, mostly in cells of pericycle between pairs of vascular bundles. In the upper hypocotyl, PTM differentiates by day 20 in the inner layer of cortical parenchyma. In the epicotyl, PTM apparently differentiates in the inner layer of cortex, by day 24. Desmogic xylem differentiates from radial files of internal conjunctive tissue cells and desmogic phloem differentiates opposite desmogic xylem strands from newly formed cells of external conjunctive tissue. No interfascicular cambium differentiates in the root, hypocotyl, or epicotyl.     

Immunolocalization of Arabinogalactan Proteins and Pectins in Floral Buds of Cucumber (Cucumis sativus L.) During Sex Determination

Arabinogalactan proteins (AGPs) and pectins were detected in the floral buds of cucumber (Cucumis sativus L.) during its sex determination using the following monoclonal antibodies: MAC 207 (recognizes AGP epitopes); JIM 8 (recognizes a subset ofAGP epitopes); and JIM 5 and JIM 7 (epitopes of pectins esterified to various degrees). In the stem apex meristem (SAM) of the cucumber, epitopes of MAC 207, JIM 7, and JIM 5 were localized in the cells from second to third peripheral layers when the sex organ primodium began to differentiate; epitopes of MAC 207 and JIM 5 were also detected in the ragged edge cells. A very dense labeling signal with MAC 207 was observed in the carpel and pistil primodium. The AGP epitopes recognized by JIM 8 were localized in the anther of the male flower and the anther-like portion of the stagnant stamen of the female flower. This suggests that the AGPs and pectins in the SAM of the cucumber are closely associated with the differentiation of the SAM, from meristematic cells to floral primodium. The subset of AGPs recognized by JIM 8 may play an important role in stamen formation.     

Expression of the JIM8 cell wall epitope in carrot somatic embryogenesis

Certain single cells in carrot (Daucus carota L.) suspension cultures react with the monoclonal antibody JIM8, and it has been proposed that these cells represent a transitional stage in somatic embryo formation. Shortly after isolation of the single cells by sieving, up to 80% of the cells react with JIM8. Within 4 d, JIM8 labelling becomes restricted to 1% of the single cells. To obtain evidence for the proposed correlation between expression of the JIM8 cell wall epitope and somatic embryo formation the developmental fate of carrot single cells labelled with JIM8 was determined by cell tracking. The results, obtained by recording 43 000 cells, show that only few JIM8-labelled cells give rise to embryos, and most somatic embryos develop from cells devoid of the JIM8 cell wall epitope. We therefore conclude that the presence of the JIM8 cell wall epitope does not coincide with the ability of single suspension cells to form embryos.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - AGP arabino galactan protein - B5-0 Gamborg's B5 medium - B5-0.2 Gamborg's B5 medium supplemented with 0.2 M 2,4-D - FITC fluoresceïn isothiocyanate - PBS phosphate-buffered saline