BACKGROUND
plant roots have two important functions – to anchor the plant in the soil and to absorb water and mineral nutrients.
during normal development, a plant produces a primary root during embryogenesis  <cit> , followed by lateral and adventitious roots, which are formed later during seedling or adult development  <cit> .
ar often develop spontaneously from the above-ground plant parts  <cit>  as a plant’s bauplan unfolds.
however, in some cases ar development is an adaptive reaction of plants to a stress, such as wounding or flooding.
ar also form in in vitro cultures of plant parts  and represent a partial regeneration of the plant from which the cells or tissue were taken.
generally, ar develop from a variety of stem tissues such as the pericycle, the vascular parenchyma, the phloem and cambium  <cit> .
grafting is widely used in various aspects of plant biological research  <cit> .
it is routinely used in relation to asexual reproduction and is also used for the enhancement of resistance, improvement of quality and to increase the production of agronomically important plants.
in recent years, this method has demonstrated an exchange of genetic information between a scion and stock and is thus a useful tool to study horizontal gene transfer  <cit> .
in the specific case of tomato, which is the focus of the present study, grafting is used to improve the quality of tomato cultivars and their tolerance to different environmental factors such as low temperatures and salinity .
among the various cellular events that are triggered by grafting, we focused on ar development with particular emphasis on the distribution of the pectic and agp epitopes during this process in both the ar and the scion tissues surrounding the developing ar.
homogalacturonan , a major pectin of the dicotyledonous primary cell walls  <cit> , contributes to cell extension, wall porosity and plant defense responses  <cit> .
the structure of hg is built from linear chains of galacturonic acid residues, to which methyl or acetyl groups   <cit>  or other monosaccharides such as xylose or apiose  may be added  <cit> .
the degree of esterification  influences hg properties and can be modified by pectin methylesterases   <cit> .
moreover, the de status varies during the life of cells and tissues  <cit> .
studies of the distribution of hg with different de have been used to explore the development and senescence of plant structures, the ripening and softening of fruit and vegetable tissues, the effects of fungal infection, as well as in the analysis of various species- or taxa-specific cell wall composition .
these studies are necessary to understand the cell wall structure and changes in its composition in relation to developmental processes  <cit> .
agp belong to the hydroxyproline-rich glycoprotein superfamily with a high level of type ii arabinogalactan glycosylation  <cit> .
agp are widespread in plants and their presence has been detected in the cell walls, plasma membranes and in extracellular secretions.
such a ubiquitous presence suggests that agp are important for plant cell structure and function, as they are.
involved in processes such as cell expansion, division and death, seed germination, pollen-tube growth and resistance to infection  <cit> .
information concerning the chemical composition of root cell walls has come only from studies on primary roots  <cit> .
studies on sugar beet roots showed a variable distribution of wall epitopes such as jim <dig>  jim <dig>  lm <dig> and lm <dig>  whose expression depended on the stage of root development and the tissue studied  <cit> .
it was shown for several species that the lm <dig> anti-xylogalacturonan antibody was associated with the detachment of root cap cells  <cit> .
in the case of the primary roots of daucus carota, zea mays, pisum sativum, brassica napus, benicasa hispida, alnus spp., oryza sativa and arabidopsis thaliana, the presence of different pectin and agp epitopes has been analysed .
the differential distribution of the agp epitopes that are recognised by jim <dig>  jim <dig>  jim <dig> and jim <dig> antibodies were described for roots of carrot, pea, radish and onion  <cit> .
many of the analysed agp epitopes were specific for root cap cells  <cit> , while others were specific for rhizodermal  <cit>  and elongating cells  <cit> , the root pericycle, the endodermis or young xylem cells  <cit> .
from the data summarised above, it appears that the chemical composition with particular emphasis on the pectic and agp epitopes of primary roots is well described.
in contrast, the different pectic and agp epitopes in the ar tissues and in the tissues that are penetrated by ar during their development and emergence from parental tissues has not been described to date.
thus, the aims of the study were: 1/to identify any morpho-histological changes that accompany ar development, 2/to analyse the chemical composition of the cell walls of ar and surrounding tissues, 3/to check whether adventitious-derived lr have a distribution pattern that is similar to the pectic and agp epitopes in ar.
methods
plant material and sample preparation
seeds of solanum lycopersicon l. ‘moneymaker’ were germinated in petri dishes with wet blotting paper for 5– <dig> d at  <dig> ±  <dig> °c in darkness.
germinated seedlings were transferred to pots with soil and grown at a temperature of  <dig> ±  <dig> °c, relative humidity 35% and  <dig> h photoperiod.
30–40-d-old plants with undamaged cotyledons and an epicotyl length of about 1– <dig>   <dig> cm were autografted, as described in  <cit> .
the middle of the epicotyl was cut transversely with a razor blade and the apical part  was carefully placed and aligned on the basal part .
toothpicks were used to support the grafted stems and the grafted area was protected by a parafilm tube.
in order to prevent excessive wilting, plants were watered and enclosed within a plastic cover to increase the relative humidity for  <dig> d.
fragments of grafted stems were collected for analysis during two time periods – from  <dig> to  <dig> d and between  <dig> to  <dig> d after grafting.
concurrently, control plants of similar ages were cut and put in beakers with tap water to check whether there were any differences between the developing and emerging ar from plants that had been wounded by cutting and the ar that were developing on grafted stems.
the latter were designated for further investigations.
analysis of plants morphology was carried out using an olympus  szh <dig> stereomicroscope.
surface staining with aqueous solutions of  <dig>  05%  toluidine blue  <dig>  and  <dig>  02%  ruthenium red  were applied to the hand-cut sections .
fragments of the grafted epicotyls were fixed in a mixture of 3%  paraformaldehyde ,  <dig>  25%  glutaraldehyde  in phosphate-buffered saline , ph  <dig>   <dig>  samples were de-aerated in fixative for  <dig> h and incubated in fixative at  <dig> °c overnight.
after rinsing with pbs , the material was dehydrated in an ethanol series  and embedded in steedman’s wax  <cit> .
longitudinal sections  were cut using a zeiss  hyrax m <dig> rotary microtome and collected on microscopic slides covered with mayer’s albumin or coated with poly-l-lysine .
histochemistry
sections were de-waxed, rehydrated in a successive ethanol series  and designated for the following histochemical schedules:
periodic acid-schiff’s  method to detect starch, cellulose and carboxylated polysaccharides
sections were oxidised in a  <dig>  5%  aqueous periodic acid  solution for  <dig> h at room temperature, washed in running water for  <dig> min and rinsed once with distilled water.
next, the slides were placed in schiff’s reagent  for  <dig> min in darkness, rinsed with distilled water and transferred to a  <dig>  5%  sodium sulphite solution for 1– <dig> min.
after washing with running tap water for  <dig> min, sections were placed in a  <dig>  5%  toluidine blue  <dig> aqueous solution for  <dig> s to visualise the meristematic cells of the developing ar.
they were then dehydrated in an ethanol series  and 100% isopropanol for  <dig> min.
slides were shaken dry and mounted in euparal .
sudan iii staining to detect lipid substances
sections were stained with a  <dig>  5% %  sudan iii  solution  was then added and the solution was filtered again).
slides were stained for a minimum  <dig> h , rinsed with 50%  ethanol, rinsed with distilled water and mounted in 50%  glycerol.
aniline blue staining to determine callose localisation
sections were stained with  <dig>  1%  methyl blue  in a  <dig>   <dig> m k2hpo <dig> solution for  <dig> min, rinsed three times with distilled water and mounted in 50%  glycerol.
immunocytochemistry
for the immunolabelling procedure, sections were de-waxed and rehydrated in an ethanol series .
the area occupied by the sections on the microscope slides was marked using a hydrophobic pap pen .
the detailed steps of procedure were performed exactly as described by sala et al  <cit> .
the primary rat monoclonal antibodies  that were used are listed in table  <dig>  the secondary antibody that was used was alexafluor  <dig> goat anti-rat .
sections were stained with  <dig>  05%  toluidine blue o in pbs for  <dig> min to quench tissue autofluorescence.
negative controls were performed by omitting the primary antibody step, thereby obtaining no fluorescence signal in the control set of sections.table  <dig> list of primary rat monoclonal antibodies used in the current study for the detection of agp and pectins in adventitious roots
pectins
agps
all observations and photography were carried out using a nikon eclipse ni-u microscope equipped with a nikon digital ds-fi1-u <dig> camera with corresponding software , and a maximum excitation wavelength of  <dig> nm  or  <dig> nm .
photographs and diagrams were edited using the coreldrawx <dig> graphics program.
RESULTS
adventitious roots – morphology
the ar that originate from tomato stems emerge in three different ways , depending on the stage of stem development and the type of procedure that is used to induce their growth .
in the first way, emergence was through epidermis disruption  and took place in cut stems or ‘young’ scions approx.
5– <dig> d after the graft was prepared and when the secondary growth of stem had not yet commenced.
the cortex and collenchyma had also become disrupted and loosened .
the surface of emergent roots was covered by pectic substances .
in the second way, the scion tissues formed an envelope-like structure around the new root , which eventually detached .
the ‘envelope’ consisted of parenchymatous cells which originated from the proliferation of the collenchyma .
the root and these parenchymatous cells remained in close contact during the early stage of root growth .
the formation of the “envelope” occurred in older grafts, i.e.
at approx.
<dig> d or more after grafting, when the stems were exhibiting secondary growth.
the ar that were surrounded by this parenchymatous ‘envelope’ had a bulbous outline .
in the third way, which was specific to cut stems only, numerous ar emerged through eye-like openings that had formed on the stem and were filled with friable cells .
the epidermis became disrupted and large isodiametric cells protruded from the stem .
even when the primordium was still at a relatively early stage of growth, the opening was already established .
the cells filling the opening originated from dedifferentiated collenchyma cells, but sub-epidermis and cambium, through their cell division, contributed as well .fig.
<dig> adventitious roots that formed on tomato stems – morphology and three pathways of their emergence.
a1-a <dig>  emergence by means of epidermis disruption observed in ‘young’ grafts or cut stems .
a <dig>  multiple ar on a scion that had already emerged  or were about to .
a <dig>  an empty space  is formed between the ar  and the scion as a result of tissue loosening or lysis.
a <dig>  ar are covered with pectic substances at the root tip.
b1-b <dig>  occurrence of an envelope enclosing ar observed on ‘older’ grafts only .
b <dig>  envelope surrounds the developing ar primordia  but may be detached , leaving intact roots .
b <dig>  transverse section through an adventitious root  enclosed within an envelope consisting of large parenchymatous cells ; inset 1: the envelope originates from scion parenchyma cell division  and further cell proliferation; inset 2: root and envelope cells remain in close contact.
b <dig>  ars formed on stems with secondary growth have a specific shape  at the primordium stage and have a shape that is different from that of the ars that formed on young stems.
c1-c <dig>  occurrence of an oculus-like opening observed on cut stems only .
c <dig>  oculus-like openings are visible before root emergence ; their size is variable and they comprise of one or more emerging ars .
c <dig>  openings filled with large, loosely attached callus cells  are established at an early stage of ars development .
c <dig>  callus cells are of a parenchymatous origin .
asterisk – callus cells, dag – days after grafting.
scale:  <dig>  mm ,  <dig> mm ,  <dig>  mm
adventitious roots – histological type i and ii
at the earliest stage of ar development, initial cells, which were rich in starch, showed cell divisions, thereby forming multicellular complexes .
the cell walls became thickened at the boundaries between the developing ar and the scion tissues .
the root primordium consisted of actively dividing meristematic cells, each having a dense cytoplasm, a large nucleus and reduced starch content .
as development proceeded, the boundaries between the root and scion cells were composed mainly of insoluble polysaccharides, as was revealed by a pas reaction, and became thicker .
within a given scion, multiple ar at different stages of development were found .
moreover, lr formed from the pericycle of the ar , independently of the point of emergence of the latter roots.
there was no significant occurrence of callose during ar or lr emergence.
callose was present, however, in some areas within the thick layer between the ar and the scion tissues , or at the border between the ar and lr .
in the ar, callose was detected in the primary pit fields and cell plates of dividing cells .
when the ar or lr were still enclosed within the maternal tissues, no lipid substances were detected in their cell walls .
after root emergence, deposition of polyphenolics in the rhizodermal walls commenced and cells beneath the rhizodermis divided periclinally  giving rise to the periderm .
identification of this tissue was based on its phenotypic features such as the presence of lipid substances within the cell wall, the presence of polyphenols and cell shape.fig.
<dig> histology of adventitious roots from tomato-grafted stems.
a division of the initial cells  and thickened walls at the ar/scion tissue border ; asterisk – multicellular complexes.
b meristematic cells of a young primordium  and scion cells .
c ar bearing lr initials  and cell divisions in the parenchyma  next to a developing ar ; inset 1: lr pericycle origin ; inset 2: thick layer between the ar and scion .
d callose in the phloem  and the layer between the ar  and scion ; inset 1: callose in the primary pit fields  and cell plate ; inset 2: callose  between the ar/lr border.
e polyphenols in the rhizodermis  and periclinal cell divisions beneath ; inset: older ar, lipids in the phellem .
f transverse section, ar  enclosed within the envelope covered by lipid substances , no lipids in the rhizodermis ; inset: envelope cuticle .
g, h ar emerging by means of epidermis disruption; the root cap file starts at the edge of the primordium  and comprises  <dig> to  <dig> cell files , adjacent to layer of dead cells .
i ar primordium  emerging within an envelope; root cap cells  adjacent to a layer of dead cells .
j root cap cells of various sizes  next to dead cells .
k root cap cells  after emergence.
ar – adventitious root, c – cambium, col – collenchyma, cor – cortex, e – endodermis, lr – lateral root, p – pericycle, rc – root cap.
scale:  <dig> μm ,  <dig> μm ,  <dig> μm ;  <dig> μm
when the developing ar were surrounded by an envelope of parenchyma, lipid substances were detected only in the cuticle, and were deposited on the outer periclinal cell walls of the so-called “envelope” .
cells of a root cap could already be distinguished within the dome-shaped primordium , which was formed of two or more cell layers .
in roots that were emerging within their “envelope”, the outermost root cells were of various sizes and shapes .
although the boundary between the emerging root and scion tissues was marked by a thickened polysaccharide layer, the outermost root cap cells resembled the cortical cells of the scion .
after emergence, the root cap remained attached to the root tip .
adventitious roots – distribution of pectic epitopes
at an early stage of ar development, a pectic epitope that was recognised by the lm <dig> antibody  was detected in the walls of all of the initial cells .
however, the fluorescence signal was most intense at the border between the cambium/ar and the ar/cortex of the scion, thus suggesting a higher amount of the epitope in these regions.
in contrast, in the newly formed cell walls, the occurrence of the lm <dig> epitope was hardly detectable .
as ar growth proceeded, the lm <dig> epitope was still detected in the initial cells but its distribution was not uniform – in some walls, the signal was more or less punctate, in other walls, its distribution was continuous within the wall and had an intense fluorescence signal .
the lm <dig> epitope occurred abundantly in the layer between the ar and the cortex cells of the scion; it was also detected in the periclinal walls of the root epidermis as well as in the periclinal walls and intercellular spaces of the cortical cells .
in the walls of the initial cells of older ar primordial, the lm <dig> epitope was abundant and distributed within the wall in a continuous manner.
this was in contrast to the other cells of the primordium where the fluorescence signal relating to this epitope was weak and was distributed in a more or less punctate manner .
the lm <dig> epitope also occurred in a thick extracellular layer that was formed between the cells of the developing ar and the cortex of the scion .fig.
<dig> immunolabelling of adventitious roots during development – pectin epitopes.
a initial stage, lm <dig> epitope in walls between the cambium/initial cells/cortex , weak signal in division walls .
b punctate  or continuous  distribution as divisions proceed, abundant occurrence at ar/cortex boundary, in the walls and intercellular spaces of cortex .
c lm <dig> present in the extracellular layer ; inset: magnification of c , weak signal in primordium.
d initial stage, lm <dig> epitope present in initials  and multicellular complex ; inset: magnification of d , weak signal in the division walls  in contrast to the walls of the cell complex .
e cortex adjacent to a developing root, lm <dig> localised in the walls, middle lamellae  and intercellular spaces .
f lm <dig> epitope abundant in the ground tissue  in contrast to the central region  and epidermis , strong labelling in the cortex walls, intercellular spaces and the extracellular layer ; inset: magnification of f , epitope distribution .
g, h the extracellular layer.
i. parenchyma next to an lr primordium  and thickening of the extracellular matrix .
i’.
i fluorescence, lm <dig> epitope present in the cortical cells adjacent to a primordium .
j lm <dig> between the root and scion , no fluorescence signal in sections without contact .
k lm <dig> in the rhizodermis .
l magnification of k , lm <dig> detected in the outer periclinal walls  and a weak signal in the anticlinal walls .
m root cap, lm <dig> present in the outer periclinal walls , weak signal in the inner cells .
ar – adventitious root, c – cambium, col – collenchyma, cor – cortex, f – fibre, rc – root cap, sc – scion.
scale:  <dig> μm,  <dig> μm ,  <dig> μm ,  <dig> μm
early in ar development, the pectic epitope that was recognised by the lm <dig> antibody  was less abundant than the lm <dig> epitope and was distributed in a punctate manner in the walls of the initial cells .
in the walls of the multicellular complexes, which often accompanied the initiation of the primordium, the lm <dig> epitope was distributed in the primary walls, but was absent in the newly formed cell walls within a complex .
in the walls of the cortical cells, which were adjacent to a developing root, the lm <dig> epitope occurred abundantly and was distributed in a punctate manner throughout the primary walls and the middle lamellae; it was also detected in the intercellular spaces .
in older primordia, the lm <dig> epitope distribution was more widespread than the lm <dig> epitope .
the lm <dig> epitope was detected in the walls of the differentiating cells in the ground tissue of the primordium, in contrast to its central region and epidermis, from which this epitope was absent .
in between the ar and the cortex, there was a thick extracellular layer where the lm <dig> epitope was especially abundant ; however, it was not the only component .
in the ar that developed within the scion tissues, the pectic epitope that was recognised by the lm <dig> antibody  occurred only in the walls of root cap cells, which were adjacent to the primordium .
in the part of the mature ar that was enclosed within the scion, the lm <dig> epitope was detected in the layer between the rhizodermis and the adjacent scion cells .
its distribution was not uniform, and the signal was most intense where the contact between the ar and the scion was closest, but was absent from sections where the contact was not so tight .
in the ar that had emerged, the lm <dig> epitope was detected in the cell walls of the epidermis.
here, however, its distribution was discontinuous  and its occurrence was largely restricted to the outer periclinal cell walls; a weak fluorescence signal was also detected in the anticlinal walls .
within the root cap, only small amounts of the lm <dig> epitope were detected in the cell walls, in contrast to the larger amounts that were observed in the outer periclinal cell walls of the root as a whole .
adventitious roots – distribution of agp epitopes
at the earliest stages of ar development, the agp epitope that was recognised by the jim <dig> antibody was not detected in the initial cells or during their subsequent divisions .
the jim <dig> epitope did appear, however, at the root primordium stage, though here it occurred less abundantly than it did in the scion collenchyma or cortical cells .
jim <dig> was not observed in meristematic cells near the border of the root nor in the root cap cells within the primordium ; however, it was present in cells from the other files, and here the level of the jim <dig> epitope increased parallel with the level of cell vacuolisation .
the epitope was present in the cell wall and/or plasma membrane  as well as in the cytoplasm .
there was a difference in epitope distribution – from the cell wall/plasma membrane to the cytoplasm – between cells of different cell files .
the jim <dig> antibody signal was also detected in the layer between the emerging root and the scion cells .fig.
<dig> immunolabelling of adventitious roots during development – agp epitopes.
a primordium stage of ar, jim <dig> epitope occurrence in ground and vascular tissue and in the layer between the ar and the scion ; inset: absence of the jim <dig> epitope during the initial stages of ar development .
b magnification of a ., abundant occurrence of the jim <dig> epitope in the scion cells ; increase of the jim <dig> presence in particular ar cells , its absence from root cap and meristematic cells .
b’.
bright field of b. c magnification of a , the jim <dig> epitope is either localised in the wall , cytoplasm  or is absent , depending on the cell file.
c’.
bright field of c. d primordium stage of ar with the jim <dig> epitope occurrence in the ground tissue and some root cap cells; inset: the jim <dig> epitope present in the layer between the ar and the scion .
e magnification of d .
tonoplast localisation of the jim <dig> epitope .
f punctate distribution of the jim <dig> epitope in the cells of an ar root primordium , no signal in scion tissues ; inset: the jim <dig> epitope is present at the division stage of the initial cells with localisation in walls/plasmalemma  of the complex .
g the jim <dig> epitope presence in the tonoplast  and in the cytoplasm  next to the vacuole ;vacuole inset 1: bright field of g; inset 2: abundant jim <dig> epitope occurrence in the root cap cells  and in the meristematic cell files beneath , the right side of the dotted line.
f – fibre, col – collenchyma, cor – cortex, xy – xylem vessels.
scale:  <dig> μm ,  <dig> μm
the agp epitope that was recognised by jim <dig> antibody showed a temporal and spatial distribution similar to that of the jim <dig> epitope, with one exception; it did occur in some root cap cells .
there was also a correlation between the increase in the jim <dig> epitope and the level of cell vacuolisation but, in contrast to jim <dig>  the jim <dig> epitope was mainly present in the tonoplast, as well as in the large and small vacuoles .
at the earlier stage of ar development, the agp epitope that was recognised by the jim <dig> antibody was detected in the walls/plasma membrane of the dividing initial cells  as a punctate signal and this type of distribution was displayed throughout the entire root development.
at the primordium stage, the jim <dig> epitope occurred in all of the cells of the developing root with the exception of the future vasculature.
this was in contrast to the scion tissues, collenchyma and cortex, where it was hardly present .
in contrast to the jim <dig> and jim <dig> epitopes, the jim <dig> epitope occurred in the root cap cells and in the highly meristematic cell file next to the root cap .
the occurrence of the jim <dig> epitope was associated with the tonoplast, cytoplasm and plasma membrane .
lateral roots – distribution of pectic and agp epitopes
upon the emergence of the lr, the lm <dig> epitope was found in the walls of the cells enclosing the future vascular tissue and in the outer periclinal walls of the rhizodermis  .
in the other cells of the primordium, the lm <dig> epitope was weakly expressed and was distributed in either a punctuate or a continuous manner .
the signal of the lm <dig> antibody was, however, sporadically punctuate .
the lm <dig> epitope was hardly present in the primordium with the exception of some ground tissue cells .
the lm <dig> epitope was absent in the lateral root primordium .fig.
<dig> immunolabelling of lateral roots upon emergence – pectic and agp epitopes.
a localisation of lm <dig> in the cells adjacent to the future vascular tissue and in the outer periclinal walls of rhizodermis , weak signal in other tissues .
b lm <dig> at the border of root cap and other cells .
c punctate signal of lm <dig> .
d low  or no occurrence  of the lm <dig> epitope in lr  and presence in the ground tissue .
e occurrence of jim <dig> in the ar cells  and ground tissue of an lr .
f magnification of e , tonoplast localisation of jim <dig> in the ground tissue  and some epidermal cells .
in ar, occurrence in the cortex walls and/or plasmalemma  and in the extracellular layer .
f’.
bright field of f, vacuolisation degrees of epidermal , meristematic and ground tissue cells .
g the jim <dig> epitope in the ar cells  and ground tissue of the lr; wall distribution in differentiated cells  and cytoplasmic localisation in meristematic cells .
h magnification of g , jim <dig> in cytoplasm , tonoplast , plasmalemma and/or cell wall , abundant occurrence in remnants of collapsed cells .
h’.
bright field of h. i the jim <dig> epitope in ar  and lr cells with an abundant cytoplasmic distribution on the periphery of a primordium  in contrast to wall localisation in the ground tissue .
j magnification of i , continuous  or punctate  tonoplast localisation of jim <dig>  in the lr epidermis, epitope detected in the plasmalemma, cytoplasm  and in the extracellular layer ; in ar punctate distribution in plasmalemma and/or wall .
j’.
bright field of j. scale:  <dig> μm ,  <dig> μm
the agp epitope that was recognised by the jim <dig> antibody occurred abundantly in the ar tissue .
within the lateral root, however, a complete absence of jim <dig> epitope was observed in the cells that were localised on the periphery and in the central region of the primordium zones that corresponded to the future epidermis, stele and root apical meristem.
this contrasted with the ground tissue where jim <dig> occurred abundantly .
in the differentiated ground tissue cells at the base of the lr primordium, the jim <dig> epitope was present in the cell walls .
the distribution of the jim <dig> epitope within the other cells of ground tissue that had meristematic features  was restricted to the tonoplast .
within the layer of the rhizodermis, the jim <dig> epitope was detected only in the tonoplast of some cells .
in the cells adjacent to a protruding lr primordium, the jim <dig> epitope signal was observed in the plasma membrane and/or primary cell walls, as well as in the thick layer between the borders of the lateral roots and ar .
an arabinogalactan protein epitope that was recognised by the jim <dig> antibody was observed in similar regions of the lateral root primordia , and also occurred in the tonoplast and cell cytoplasm .
in contrast to jim <dig>  the jim <dig> epitope was also present in some of the meristematic cells at the developing root tip .
the remnants of cortical cells that had collapsed as a result of the emergence of the lateral root were also rich in the jim <dig> epitope .
the arabinogalactan protein epitope that was recognised the by jim <dig> antibody was detected in all of the tissues of the developing lateral root, either as a continuous or punctuate signal, although in the central region of the primordium, the fluorescence signal was punctate and mostly weak .
in contrast to the jim <dig> and jim <dig> epitopes, the jim <dig> epitope occurred abundantly in the meristematic cells at the root tip, where it was detected in the tonoplast .
in.the rhizodermis, the epitope was detected in the cytoplasmic compartments and in the plasma membrane .
in the ar cells, the occurrence of the jim <dig> epitope was observed less easily than the jim <dig> and jim <dig> epitopes .
discussion
the distribution of the lm <dig> and lm <dig> epitopes during ar primordium development indicates that these epitopes have distinctive localisations at the corresponding developmental stages in both the developing ar and the scion.
at the earliest phase of ar development, the lm <dig> epitope occurred abundantly, while at a more advanced stage, its presence decreased and its distribution became less uniform.
concurrently, the lm <dig> epitope increased and, at the primordium stage, it was more widespread than the lm <dig> epitope.
similarities in the distribution also concerned the ar/cortex border where both epitopes were abundantly detected in a thick layer, whereas neither occurred, or were only faintly detected, in the new cell walls of the divided cells.
this is consistent with observations that meristematic cells have methyl-esterified hg domains whereas differentiating cells exhibit more non-methyl-esterified domains  <cit> .
a distinct distribution of methyl- and/or non-methyl-esterified hg was also reported during the somatic embryogenesis of various plants  <cit>  and the embryogenesis of the fern ceratopteris richardii  <cit>  who used jim <dig> and jim <dig> antibodies, showed that methyl- and un-esterified pectins were located at the interface between the mother and emerging lr of onion.
it was shown that these same antibodies strongly labelled the space that was created between the primordium and the main root cells, thus indicating that a release and/or accumulation of pectic fragments occurred at this interface.
the results presented here are in accordance with the findings described above because the jim <dig> and jim <dig> antibodies display some similarities to lm <dig> and lm <dig> antibodies  <cit> , which were used in our work.
lm <dig> and lm <dig> antibodies, as well as jim <dig> and jim <dig>  share epitopes that have a partial degree of esterification  <cit> .
differences and similarities in the lm <dig> and lm <dig> epitope distribution at the primordium stage of development are schematically depicted in fig.
6a.fig.
<dig> schematic representation of a pectic and b agp epitope distribution in tissues of the primordium
xylogalacturonan, which is represented by the lm <dig> epitope, was present in the root cap cells of ar during the primordium stage but not at a more advanced stage of development.
this epitope is thought to be specifically associated with the root cap cells in a range of angiosperm species  <cit> , thus indicating that roots that have a different origin have the same root cap markers.
it must be noted that, in our system, during more advanced stages of ar development, the root cap cells were marked only in the outer pericinal walls, in contrast to the cytoplasmic and wall distribution in the roots of pisum sativum, daucus carota, zea mays and arabidopsis  <cit> .
the lm <dig> epitope is not only a marker of root cap cells, but is also specifically associated with the separation process that results in complete cap cell detachment  <cit> .
the occurrence of xylogalacturonan was observed during organogenesis in an in vitro culture of wheat, where it was present in detached or loosely connected callus cells  <cit>  and also at the surface of loosely attached cells in embryogenic carrot suspension cell cultures  <cit> .
moreover, the lm <dig> epitope was detected in the inner parenchyma cells that are loosened and subsequently crushed by the growing cotyledons during pea testa development  <cit> .
it is obvious that during the development and emergence of ar, the cell walls of the cortical cells are destroyed and pile up as the root primordium advances through the cortical tissue  <cit> , thus forming a thick layer between the root and maternal tissues.
in our model, the cortex, collenchyma and epidermis are the tissues through which ar has to protrude – yet, we found no lm <dig> epitopes in the scion cells surrounding the developing ar.
that observation indicates that root emergence is not accompanied by any significant cell detachment.
in the case of the agp epitopes, it appeared that their presence and distribution depended on the stage of ar development and the cell type.
jim <dig> and jim <dig> antibodies were detected in the more advanced primordium stage of ar development, whereas the jim <dig> epitope was present from the earliest division events.
results from various studies concerning the presence of agp epitopes in the roots of a range of species are listed in  <cit> .
jim <dig> and jim <dig> epitopes were found in the border and border-like cells of the root caps of arabidopsis thaliana, pisum sativum and brassica napus  <cit> , whereas jim <dig> and jim <dig> occurred in the elongating cells of arabidopsis thaliana  <cit>  and in the root apical meristem of daucus carota  <cit> .
in our work, the distribution patterns of the jim <dig> and jim <dig> epitopes were similar – both appeared at the advanced developmental stage but were absent from the meristematic cell files of the primordium; their amounts increased simultaneously with the degree of cell vacuolisation and finally, jim <dig> and jim <dig> epitopes were less abundant in ar cells compared to the parenchymatous cells of the scion.
the jim <dig> epitope, however, was detected at the earliest developmental stages, when the divisions of the initial cells commenced.
by contrast, it was hardly present in the scion tissues.
later, the jim <dig> epitope occurred in all of the cells of primordia, especially in the root cap and meristematic cell files.
although these results may indicate differences between various species or between the type of roots , they still point to the important role of agp in root tissue development.
in brasica napus flowers, a temporal and spatial regulation of the plasma membrane agp epitope that is recognised by the jim <dig> antibody was postulated  <cit> .
during embryogenesis, the jim <dig> epitope was at first expressed in the cells of the proembryo, disappeared from the embryo proper but remained in the suspensor.
during the differentiation of the stamens and carpels, the jim <dig> occurrence exhibited a temporal sequence of distribution as cell differentiation progressed  <cit> .
this indicates that the jim <dig> epitope specifies positionally defined tissues or cell types, just like the other agp epitopes jim <dig>  <cit>  or jim <dig>  <cit>  in other species or organs.
thus, it was suggested that agp may act as cell-position marker and convey information that is essential for cell patterning or the establishment of symmetry  <cit> .
although all three antibodies that were used, jim <dig>  jim <dig> and jim <dig>  labelled some cytoplasmic compartments with the most evident localisation in the tonoplast, only the jim <dig> epitope was present in the meristematic cell files next to the root cap.
jim <dig> and jim <dig> epitopes appeared in those vacuolated cells that were differentiating.
this observation may suggest that these agp can mark cells before any phenotypic changes of cell differentiation are visible  <cit> .
therefore, our results may point to characteristic agp expression pattern that may be involved in adventitious or lateral root development.
the distribution of all of the agp epitopes that were applied within primordium in the presented study is schematically presented in fig.
6b.
comparison of ar to lr
the comparison of lr and ar at the corresponding stages of development showed some differences as well as similarities in the occurrence of the pectic and agp epitopes.
no xylogalacturonan was detected in the lr; moreover, the lm <dig> epitope  was hardly present in lr in comparison to ar.
the lm <dig> epitope  occurred in the outer periclinal cell walls of the primordium and was especially abundantly in the regions where lr had already emerged.
agp epitopes had a similar distribution pattern in both types of roots.
however, they were more expressed in the lr than in the ar.
ar share the same function as lr but develop from aerial tissues  <cit> .
the partial similarities in cell components that were observed can be explained in two ways – it is either the origin that influences the cell composition or it is the environment surrounding the developing primordial that is the influence.
more studies should be conducted to verify these two assumptions.
CONCLUSIONS
we performed histo- and immunohistochemical analyses of the cell wall to study the development of tomato ar.
the ar that formed on the grafted stems differed from the ar that were induced by stem cutting in terms of the shape of the primordium and the way they emerged.
two hg epitopes, lm <dig> and lm <dig>  had different localisations at the corresponding ar developmental stages with an increase in lm <dig> occurrence along with the progress of ar growth.
of the three agp epitopes that were applied, jim <dig> and jim <dig> showed an association with the tonoplast in particular cells of the primordium, whereas the jim <dig> epitope occurred from the earliest stage, and therefore it can be considered to be a marker of initial cells.
finally, the differences observed between the ar and lr cell wall composition might be origin- or environment-dependent.
abbreviations
agpsarabinogalactan proteins
ar/aradventitious root
ccambium
colcollenchyma
corcortex
dagdays after grafting
dedegree of esterification
eendodermis
ffibre
hghomogalacturonan
lr/lrlateral root
ppericycle
pasperiodic acid-schiff
pbsphosphate-buffered saline
pcdprogrammed cell death
pmespectin methylesterases
rcroot cap
scscion
xyxylem vessels

 plant roots have two important functions – to anchor the plant in the soil and to absorb water and mineral nutrients. during normal development, a plant produces a primary root during embryogenesis, followed by lateral and adventitious roots, which are formed later during seedling or adult development. ar often develop spontaneously from the above-ground plant parts as a plant’s bauplan unfolds. however, in some cases ar development is an adaptive reaction of plants to a stress, such as wounding or flooding. ar also form in in vitro cultures of plant parts and represent a partial regeneration of the plant from which the cells or tissue were taken. generally, ar develop from a variety of stem tissues such as the pericycle, the vascular parenchyma, the phloem and cambium. grafting is widely used in various aspects of plant biological research. it is routinely used in relation to asexual reproduction and is also used for the enhancement of resistance, improvement of quality and to increase the production of agronomically important plants. in recent years, this method has demonstrated an exchange of genetic information between a scion and stock and is thus a useful tool to study horizontal gene transfer. in the specific case of tomato, which is the focus of the present study, grafting is used to improve the quality of tomato cultivars and their tolerance to different environmental factors such as low temperatures and salinity. among the various cellular events that are triggered by grafting, we focused on ar development with particular emphasis on the distribution of the pectic and agp epitopes during this process in both the ar and the scion tissues surrounding the developing ar. homogalacturonan, a major pectin of the dicotyledonous primary cell walls, contributes to cell extension, wall porosity and plant defense responses. the structure of hg is built from linear chains of galacturonic acid residues, to which methyl or acetyl groups or other monosaccharides such as xylose or apiose may be added. the degree of esterification influences hg properties and can be modified by pectin methylesterases. moreover, the de status varies during the life of cells and tissues. studies of the distribution of hg with different de have been used to explore the development and senescence of plant structures, the ripening and softening of fruit and vegetable tissues, the effects of fungal infection, as well as in the analysis of various species- or taxa-specific cell wall composition. these studies are necessary to understand the cell wall structure and changes in its composition in relation to developmental processes. agp belong to the hydroxyproline-rich glycoprotein superfamily with a high level of type ii arabinogalactan glycosylation. agp are widespread in plants and their presence has been detected in the cell walls, plasma membranes and in extracellular secretions. such a ubiquitous presence suggests that agp are important for plant cell structure and function, as they are. involved in processes such as cell expansion, division and death, seed germination, pollen-tube growth and resistance to infection. information concerning the chemical composition of root cell walls has come only from studies on primary roots. studies on sugar beet roots showed a variable distribution of wall epitopes such as jim jim lm and lm whose expression depended on the stage of root development and the tissue studied. it was shown for several species that the lm anti-xylogalacturonan antibody was associated with the detachment of root cap cells. in the case of the primary roots of daucus carota, zea mays, pisum sativum, brassica napus, benicasa hispida, alnus spp., oryza sativa and arabidopsis thaliana, the presence of different pectin and agp epitopes has been analysed. the differential distribution of the agp epitopes that are recognised by jim jim jim and jim antibodies were described for roots of carrot, pea, radish and onion. many of the analysed agp epitopes were specific for root cap cells, while others were specific for rhizodermal and elongating cells, the root pericycle, the endodermis or young xylem cells. from the data summarised above, it appears that the chemical composition with particular emphasis on the pectic and agp epitopes of primary roots is well described. in contrast, the different pectic and agp epitopes in the ar tissues and in the tissues that are penetrated by ar during their development and emergence from parental tissues has not been described to date. thus, the aims of the study were: 1/to identify any morpho-histological changes that accompany ar development, 2/to analyse the chemical composition of the cell walls of ar and surrounding tissues, 3/to check whether adventitious-derived lr have a distribution pattern that is similar to the pectic and agp epitopes in ar. methods plant material and sample preparation seeds of solanum lycopersicon l. ‘moneymaker’ were germinated in petri dishes with wet blotting paper for 5– d at ± °c in darkness. germinated seedlings were transferred to pots with soil and grown at a temperature of ± °c, relative humidity 35% and h photoperiod. 30–40-d-old plants with undamaged cotyledons and an epicotyl length of about 1– cm were autografted, as described in. the middle of the epicotyl was cut transversely with a razor blade and the apical part was carefully placed and aligned on the basal part. toothpicks were used to support the grafted stems and the grafted area was protected by a parafilm tube. in order to prevent excessive wilting, plants were watered and enclosed within a plastic cover to increase the relative humidity for d. fragments of grafted stems were collected for analysis during two time periods – from to d and between to d after grafting. concurrently, control plants of similar ages were cut and put in beakers with tap water to check whether there were any differences between the developing and emerging ar from plants that had been wounded by cutting and the ar that were developing on grafted stems. the latter were designated for further investigations. analysis of plants morphology was carried out using an olympus szh stereomicroscope. surface staining with aqueous solutions of 05% toluidine blue and 02% ruthenium red were applied to the hand-cut sections. fragments of the grafted epicotyls were fixed in a mixture of 3% paraformaldehyde, 25% glutaraldehyde in phosphate-buffered saline, ph samples were de-aerated in fixative for h and incubated in fixative at °c overnight. after rinsing with pbs, the material was dehydrated in an ethanol series and embedded in steedman’s wax. longitudinal sections were cut using a zeiss hyrax m rotary microtome and collected on microscopic slides covered with mayer’s albumin or coated with poly-l-lysine. histochemistry sections were de-waxed, rehydrated in a successive ethanol series and designated for the following histochemical schedules: periodic acid-schiff’s method to detect starch, cellulose and carboxylated polysaccharides sections were oxidised in a 5% aqueous periodic acid solution for h at room temperature, washed in running water for min and rinsed once with distilled water. next, the slides were placed in schiff’s reagent for min in darkness, rinsed with distilled water and transferred to a 5% sodium sulphite solution for 1– min. after washing with running tap water for min, sections were placed in a 5% toluidine blue aqueous solution for s to visualise the meristematic cells of the developing ar. they were then dehydrated in an ethanol series and 100% isopropanol for min. slides were shaken dry and mounted in euparal. sudan iii staining to detect lipid substances sections were stained with a 5% % sudan iii solution was then added and the solution was filtered again). slides were stained for a minimum h, rinsed with 50% ethanol, rinsed with distilled water and mounted in 50% glycerol. aniline blue staining to determine callose localisation sections were stained with 1% methyl blue in a m k2hpo solution for min, rinsed three times with distilled water and mounted in 50% glycerol. immunocytochemistry for the immunolabelling procedure, sections were de-waxed and rehydrated in an ethanol series. the area occupied by the sections on the microscope slides was marked using a hydrophobic pap pen. the detailed steps of procedure were performed exactly as described by sala et al. the primary rat monoclonal antibodies that were used are listed in table  the secondary antibody that was used was alexafluor goat anti-rat. sections were stained with 05% toluidine blue o in pbs for min to quench tissue autofluorescence. negative controls were performed by omitting the primary antibody step, thereby obtaining no fluorescence signal in the control set of sections.table list of primary rat monoclonal antibodies used in the current study for the detection of agp and pectins in adventitious roots pectins agps all observations and photography were carried out using a nikon eclipse ni-u microscope equipped with a nikon digital ds-fi1-u camera with corresponding software, and a maximum excitation wavelength of nm or nm. photographs and diagrams were edited using the coreldrawx graphics program.  adventitious roots – morphology the ar that originate from tomato stems emerge in three different ways, depending on the stage of stem development and the type of procedure that is used to induce their growth. in the first way, emergence was through epidermis disruption and took place in cut stems or ‘young’ scions approx. 5– d after the graft was prepared and when the secondary growth of stem had not yet commenced. the cortex and collenchyma had also become disrupted and loosened. the surface of emergent roots was covered by pectic substances. in the second way, the scion tissues formed an envelope-like structure around the new root, which eventually detached. the ‘envelope’ consisted of parenchymatous cells which originated from the proliferation of the collenchyma. the root and these parenchymatous cells remained in close contact during the early stage of root growth. the formation of the “envelope” occurred in older grafts, i.e. at approx. d or more after grafting, when the stems were exhibiting secondary growth. the ar that were surrounded by this parenchymatous ‘envelope’ had a bulbous outline. in the third way, which was specific to cut stems only, numerous ar emerged through eye-like openings that had formed on the stem and were filled with friable cells. the epidermis became disrupted and large isodiametric cells protruded from the stem. even when the primordium was still at a relatively early stage of growth, the opening was already established. the cells filling the opening originated from dedifferentiated collenchyma cells, but sub-epidermis and cambium, through their cell division, contributed as well.fig. adventitious roots that formed on tomato stems – morphology and three pathways of their emergence. a1-a emergence by means of epidermis disruption observed in ‘young’ grafts or cut stems. a multiple ar on a scion that had already emerged or were about to. a an empty space is formed between the ar and the scion as a result of tissue loosening or lysis. a ar are covered with pectic substances at the root tip. b1-b occurrence of an envelope enclosing ar observed on ‘older’ grafts only. b envelope surrounds the developing ar primordia but may be detached, leaving intact roots. b transverse section through an adventitious root enclosed within an envelope consisting of large parenchymatous cells; inset 1: the envelope originates from scion parenchyma cell division and further cell proliferation; inset 2: root and envelope cells remain in close contact. b ars formed on stems with secondary growth have a specific shape at the primordium stage and have a shape that is different from that of the ars that formed on young stems. c1-c occurrence of an oculus-like opening observed on cut stems only. c oculus-like openings are visible before root emergence; their size is variable and they comprise of one or more emerging ars. c openings filled with large, loosely attached callus cells are established at an early stage of ars development. c callus cells are of a parenchymatous origin. asterisk – callus cells, dag – days after grafting. scale: mm, mm, mm adventitious roots – histological type i and ii at the earliest stage of ar development, initial cells, which were rich in starch, showed cell divisions, thereby forming multicellular complexes. the cell walls became thickened at the boundaries between the developing ar and the scion tissues. the root primordium consisted of actively dividing meristematic cells, each having a dense cytoplasm, a large nucleus and reduced starch content. as development proceeded, the boundaries between the root and scion cells were composed mainly of insoluble polysaccharides, as was revealed by a pas reaction, and became thicker. within a given scion, multiple ar at different stages of development were found. moreover, lr formed from the pericycle of the ar, independently of the point of emergence of the latter roots. there was no significant occurrence of callose during ar or lr emergence. callose was present, however, in some areas within the thick layer between the ar and the scion tissues, or at the border between the ar and lr. in the ar, callose was detected in the primary pit fields and cell plates of dividing cells. when the ar or lr were still enclosed within the maternal tissues, no lipid substances were detected in their cell walls. after root emergence, deposition of polyphenolics in the rhizodermal walls commenced and cells beneath the rhizodermis divided periclinally giving rise to the periderm. identification of this tissue was based on its phenotypic features such as the presence of lipid substances within the cell wall, the presence of polyphenols and cell shape.fig. histology of adventitious roots from tomato-grafted stems. a division of the initial cells and thickened walls at the ar/scion tissue border; asterisk – multicellular complexes. b meristematic cells of a young primordium and scion cells. c ar bearing lr initials and cell divisions in the parenchyma next to a developing ar; inset 1: lr pericycle origin; inset 2: thick layer between the ar and scion. d callose in the phloem and the layer between the ar and scion; inset 1: callose in the primary pit fields and cell plate; inset 2: callose between the ar/lr border. e polyphenols in the rhizodermis and periclinal cell divisions beneath; inset: older ar, lipids in the phellem. f transverse section, ar enclosed within the envelope covered by lipid substances, no lipids in the rhizodermis; inset: envelope cuticle. g, h ar emerging by means of epidermis disruption; the root cap file starts at the edge of the primordium and comprises to cell files, adjacent to layer of dead cells. i ar primordium emerging within an envelope; root cap cells adjacent to a layer of dead cells. j root cap cells of various sizes next to dead cells. k root cap cells after emergence. ar – adventitious root, c – cambium, col – collenchyma, cor – cortex, e – endodermis, lr – lateral root, p – pericycle, rc – root cap. scale: μm, μm, μm; μm when the developing ar were surrounded by an envelope of parenchyma, lipid substances were detected only in the cuticle, and were deposited on the outer periclinal cell walls of the so-called “envelope”. cells of a root cap could already be distinguished within the dome-shaped primordium, which was formed of two or more cell layers. in roots that were emerging within their “envelope”, the outermost root cells were of various sizes and shapes. although the boundary between the emerging root and scion tissues was marked by a thickened polysaccharide layer, the outermost root cap cells resembled the cortical cells of the scion. after emergence, the root cap remained attached to the root tip. adventitious roots – distribution of pectic epitopes at an early stage of ar development, a pectic epitope that was recognised by the lm antibody was detected in the walls of all of the initial cells. however, the fluorescence signal was most intense at the border between the cambium/ar and the ar/cortex of the scion, thus suggesting a higher amount of the epitope in these regions. in contrast, in the newly formed cell walls, the occurrence of the lm epitope was hardly detectable. as ar growth proceeded, the lm epitope was still detected in the initial cells but its distribution was not uniform – in some walls, the signal was more or less punctate, in other walls, its distribution was continuous within the wall and had an intense fluorescence signal. the lm epitope occurred abundantly in the layer between the ar and the cortex cells of the scion; it was also detected in the periclinal walls of the root epidermis as well as in the periclinal walls and intercellular spaces of the cortical cells. in the walls of the initial cells of older ar primordial, the lm epitope was abundant and distributed within the wall in a continuous manner. this was in contrast to the other cells of the primordium where the fluorescence signal relating to this epitope was weak and was distributed in a more or less punctate manner. the lm epitope also occurred in a thick extracellular layer that was formed between the cells of the developing ar and the cortex of the scion.fig. immunolabelling of adventitious roots during development – pectin epitopes. a initial stage, lm epitope in walls between the cambium/initial cells/cortex, weak signal in division walls. b punctate or continuous distribution as divisions proceed, abundant occurrence at ar/cortex boundary, in the walls and intercellular spaces of cortex. c lm present in the extracellular layer; inset: magnification of c, weak signal in primordium. d initial stage, lm epitope present in initials and multicellular complex; inset: magnification of d, weak signal in the division walls in contrast to the walls of the cell complex. e cortex adjacent to a developing root, lm localised in the walls, middle lamellae and intercellular spaces. f lm epitope abundant in the ground tissue in contrast to the central region and epidermis, strong labelling in the cortex walls, intercellular spaces and the extracellular layer; inset: magnification of f, epitope distribution. g, h the extracellular layer. i. parenchyma next to an lr primordium and thickening of the extracellular matrix. i’. i fluorescence, lm epitope present in the cortical cells adjacent to a primordium. j lm between the root and scion, no fluorescence signal in sections without contact. k lm in the rhizodermis. l magnification of k, lm detected in the outer periclinal walls and a weak signal in the anticlinal walls. m root cap, lm present in the outer periclinal walls, weak signal in the inner cells. ar – adventitious root, c – cambium, col – collenchyma, cor – cortex, f – fibre, rc – root cap, sc – scion. scale: μm, μm, μm, μm early in ar development, the pectic epitope that was recognised by the lm antibody was less abundant than the lm epitope and was distributed in a punctate manner in the walls of the initial cells. in the walls of the multicellular complexes, which often accompanied the initiation of the primordium, the lm epitope was distributed in the primary walls, but was absent in the newly formed cell walls within a complex. in the walls of the cortical cells, which were adjacent to a developing root, the lm epitope occurred abundantly and was distributed in a punctate manner throughout the primary walls and the middle lamellae; it was also detected in the intercellular spaces. in older primordia, the lm epitope distribution was more widespread than the lm epitope. the lm epitope was detected in the walls of the differentiating cells in the ground tissue of the primordium, in contrast to its central region and epidermis, from which this epitope was absent. in between the ar and the cortex, there was a thick extracellular layer where the lm epitope was especially abundant; however, it was not the only component. in the ar that developed within the scion tissues, the pectic epitope that was recognised by the lm antibody occurred only in the walls of root cap cells, which were adjacent to the primordium. in the part of the mature ar that was enclosed within the scion, the lm epitope was detected in the layer between the rhizodermis and the adjacent scion cells. its distribution was not uniform, and the signal was most intense where the contact between the ar and the scion was closest, but was absent from sections where the contact was not so tight. in the ar that had emerged, the lm epitope was detected in the cell walls of the epidermis. here, however, its distribution was discontinuous and its occurrence was largely restricted to the outer periclinal cell walls; a weak fluorescence signal was also detected in the anticlinal walls. within the root cap, only small amounts of the lm epitope were detected in the cell walls, in contrast to the larger amounts that were observed in the outer periclinal cell walls of the root as a whole. adventitious roots – distribution of agp epitopes at the earliest stages of ar development, the agp epitope that was recognised by the jim antibody was not detected in the initial cells or during their subsequent divisions. the jim epitope did appear, however, at the root primordium stage, though here it occurred less abundantly than it did in the scion collenchyma or cortical cells. jim was not observed in meristematic cells near the border of the root nor in the root cap cells within the primordium; however, it was present in cells from the other files, and here the level of the jim epitope increased parallel with the level of cell vacuolisation. the epitope was present in the cell wall and/or plasma membrane as well as in the cytoplasm. there was a difference in epitope distribution – from the cell wall/plasma membrane to the cytoplasm – between cells of different cell files. the jim antibody signal was also detected in the layer between the emerging root and the scion cells.fig. immunolabelling of adventitious roots during development – agp epitopes. a primordium stage of ar, jim epitope occurrence in ground and vascular tissue and in the layer between the ar and the scion; inset: absence of the jim epitope during the initial stages of ar development. b magnification of a., abundant occurrence of the jim epitope in the scion cells; increase of the jim presence in particular ar cells, its absence from root cap and meristematic cells. b’. bright field of b. c magnification of a, the jim epitope is either localised in the wall, cytoplasm or is absent, depending on the cell file. c’. bright field of c. d primordium stage of ar with the jim epitope occurrence in the ground tissue and some root cap cells; inset: the jim epitope present in the layer between the ar and the scion. e magnification of d. tonoplast localisation of the jim epitope. f punctate distribution of the jim epitope in the cells of an ar root primordium, no signal in scion tissues; inset: the jim epitope is present at the division stage of the initial cells with localisation in walls/plasmalemma of the complex. g the jim epitope presence in the tonoplast and in the cytoplasm next to the vacuole;vacuole inset 1: bright field of g; inset 2: abundant jim epitope occurrence in the root cap cells and in the meristematic cell files beneath, the right side of the dotted line. f – fibre, col – collenchyma, cor – cortex, xy – xylem vessels. scale: μm, μm the agp epitope that was recognised by jim antibody showed a temporal and spatial distribution similar to that of the jim epitope, with one exception; it did occur in some root cap cells. there was also a correlation between the increase in the jim epitope and the level of cell vacuolisation but, in contrast to jim the jim epitope was mainly present in the tonoplast, as well as in the large and small vacuoles. at the earlier stage of ar development, the agp epitope that was recognised by the jim antibody was detected in the walls/plasma membrane of the dividing initial cells as a punctate signal and this type of distribution was displayed throughout the entire root development. at the primordium stage, the jim epitope occurred in all of the cells of the developing root with the exception of the future vasculature. this was in contrast to the scion tissues, collenchyma and cortex, where it was hardly present. in contrast to the jim and jim epitopes, the jim epitope occurred in the root cap cells and in the highly meristematic cell file next to the root cap. the occurrence of the jim epitope was associated with the tonoplast, cytoplasm and plasma membrane. lateral roots – distribution of pectic and agp epitopes upon the emergence of the lr, the lm epitope was found in the walls of the cells enclosing the future vascular tissue and in the outer periclinal walls of the rhizodermis. in the other cells of the primordium, the lm epitope was weakly expressed and was distributed in either a punctuate or a continuous manner. the signal of the lm antibody was, however, sporadically punctuate. the lm epitope was hardly present in the primordium with the exception of some ground tissue cells. the lm epitope was absent in the lateral root primordium.fig. immunolabelling of lateral roots upon emergence – pectic and agp epitopes. a localisation of lm in the cells adjacent to the future vascular tissue and in the outer periclinal walls of rhizodermis, weak signal in other tissues. b lm at the border of root cap and other cells. c punctate signal of lm. d low or no occurrence of the lm epitope in lr and presence in the ground tissue. e occurrence of jim in the ar cells and ground tissue of an lr. f magnification of e, tonoplast localisation of jim in the ground tissue and some epidermal cells. in ar, occurrence in the cortex walls and/or plasmalemma and in the extracellular layer. f’. bright field of f, vacuolisation degrees of epidermal, meristematic and ground tissue cells. g the jim epitope in the ar cells and ground tissue of the lr; wall distribution in differentiated cells and cytoplasmic localisation in meristematic cells. h magnification of g, jim in cytoplasm, tonoplast, plasmalemma and/or cell wall, abundant occurrence in remnants of collapsed cells. h’. bright field of h. i the jim epitope in ar and lr cells with an abundant cytoplasmic distribution on the periphery of a primordium in contrast to wall localisation in the ground tissue. j magnification of i, continuous or punctate tonoplast localisation of jim in the lr epidermis, epitope detected in the plasmalemma, cytoplasm and in the extracellular layer; in ar punctate distribution in plasmalemma and/or wall. j’. bright field of j. scale: μm, μm the agp epitope that was recognised by the jim antibody occurred abundantly in the ar tissue. within the lateral root, however, a complete absence of jim epitope was observed in the cells that were localised on the periphery and in the central region of the primordium zones that corresponded to the future epidermis, stele and root apical meristem. this contrasted with the ground tissue where jim occurred abundantly. in the differentiated ground tissue cells at the base of the lr primordium, the jim epitope was present in the cell walls. the distribution of the jim epitope within the other cells of ground tissue that had meristematic features was restricted to the tonoplast. within the layer of the rhizodermis, the jim epitope was detected only in the tonoplast of some cells. in the cells adjacent to a protruding lr primordium, the jim epitope signal was observed in the plasma membrane and/or primary cell walls, as well as in the thick layer between the borders of the lateral roots and ar. an arabinogalactan protein epitope that was recognised by the jim antibody was observed in similar regions of the lateral root primordia, and also occurred in the tonoplast and cell cytoplasm. in contrast to jim the jim epitope was also present in some of the meristematic cells at the developing root tip. the remnants of cortical cells that had collapsed as a result of the emergence of the lateral root were also rich in the jim epitope. the arabinogalactan protein epitope that was recognised the by jim antibody was detected in all of the tissues of the developing lateral root, either as a continuous or punctuate signal, although in the central region of the primordium, the fluorescence signal was punctate and mostly weak. in contrast to the jim and jim epitopes, the jim epitope occurred abundantly in the meristematic cells at the root tip, where it was detected in the tonoplast. in.the rhizodermis, the epitope was detected in the cytoplasmic compartments and in the plasma membrane. in the ar cells, the occurrence of the jim epitope was observed less easily than the jim and jim epitopes. discussion the distribution of the lm and lm epitopes during ar primordium development indicates that these epitopes have distinctive localisations at the corresponding developmental stages in both the developing ar and the scion. at the earliest phase of ar development, the lm epitope occurred abundantly, while at a more advanced stage, its presence decreased and its distribution became less uniform. concurrently, the lm epitope increased and, at the primordium stage, it was more widespread than the lm epitope. similarities in the distribution also concerned the ar/cortex border where both epitopes were abundantly detected in a thick layer, whereas neither occurred, or were only faintly detected, in the new cell walls of the divided cells. this is consistent with observations that meristematic cells have methyl-esterified hg domains whereas differentiating cells exhibit more non-methyl-esterified domains. a distinct distribution of methyl- and/or non-methyl-esterified hg was also reported during the somatic embryogenesis of various plants and the embryogenesis of the fern ceratopteris richardii who used jim and jim antibodies, showed that methyl- and un-esterified pectins were located at the interface between the mother and emerging lr of onion. it was shown that these same antibodies strongly labelled the space that was created between the primordium and the main root cells, thus indicating that a release and/or accumulation of pectic fragments occurred at this interface. the results presented here are in accordance with the findings described above because the jim and jim antibodies display some similarities to lm and lm antibodies, which were used in our work. lm and lm antibodies, as well as jim and jim share epitopes that have a partial degree of esterification. differences and similarities in the lm and lm epitope distribution at the primordium stage of development are schematically depicted in fig. 6a.fig. schematic representation of a pectic and b agp epitope distribution in tissues of the primordium xylogalacturonan, which is represented by the lm epitope, was present in the root cap cells of ar during the primordium stage but not at a more advanced stage of development. this epitope is thought to be specifically associated with the root cap cells in a range of angiosperm species, thus indicating that roots that have a different origin have the same root cap markers. it must be noted that, in our system, during more advanced stages of ar development, the root cap cells were marked only in the outer pericinal walls, in contrast to the cytoplasmic and wall distribution in the roots of pisum sativum, daucus carota, zea mays and arabidopsis. the lm epitope is not only a marker of root cap cells, but is also specifically associated with the separation process that results in complete cap cell detachment. the occurrence of xylogalacturonan was observed during organogenesis in an in vitro culture of wheat, where it was present in detached or loosely connected callus cells and also at the surface of loosely attached cells in embryogenic carrot suspension cell cultures. moreover, the lm epitope was detected in the inner parenchyma cells that are loosened and subsequently crushed by the growing cotyledons during pea testa development. it is obvious that during the development and emergence of ar, the cell walls of the cortical cells are destroyed and pile up as the root primordium advances through the cortical tissue, thus forming a thick layer between the root and maternal tissues. in our model, the cortex, collenchyma and epidermis are the tissues through which ar has to protrude – yet, we found no lm epitopes in the scion cells surrounding the developing ar. that observation indicates that root emergence is not accompanied by any significant cell detachment. in the case of the agp epitopes, it appeared that their presence and distribution depended on the stage of ar development and the cell type. jim and jim antibodies were detected in the more advanced primordium stage of ar development, whereas the jim epitope was present from the earliest division events. results from various studies concerning the presence of agp epitopes in the roots of a range of species are listed in. jim and jim epitopes were found in the border and border-like cells of the root caps of arabidopsis thaliana, pisum sativum and brassica napus, whereas jim and jim occurred in the elongating cells of arabidopsis thaliana and in the root apical meristem of daucus carota. in our work, the distribution patterns of the jim and jim epitopes were similar – both appeared at the advanced developmental stage but were absent from the meristematic cell files of the primordium; their amounts increased simultaneously with the degree of cell vacuolisation and finally, jim and jim epitopes were less abundant in ar cells compared to the parenchymatous cells of the scion. the jim epitope, however, was detected at the earliest developmental stages, when the divisions of the initial cells commenced. by contrast, it was hardly present in the scion tissues. later, the jim epitope occurred in all of the cells of primordia, especially in the root cap and meristematic cell files. although these results may indicate differences between various species or between the type of roots, they still point to the important role of agp in root tissue development. in brasica napus flowers, a temporal and spatial regulation of the plasma membrane agp epitope that is recognised by the jim antibody was postulated. during embryogenesis, the jim epitope was at first expressed in the cells of the proembryo, disappeared from the embryo proper but remained in the suspensor. during the differentiation of the stamens and carpels, the jim occurrence exhibited a temporal sequence of distribution as cell differentiation progressed. this indicates that the jim epitope specifies positionally defined tissues or cell types, just like the other agp epitopes jim or jim in other species or organs. thus, it was suggested that agp may act as cell-position marker and convey information that is essential for cell patterning or the establishment of symmetry. although all three antibodies that were used, jim jim and jim labelled some cytoplasmic compartments with the most evident localisation in the tonoplast, only the jim epitope was present in the meristematic cell files next to the root cap. jim and jim epitopes appeared in those vacuolated cells that were differentiating. this observation may suggest that these agp can mark cells before any phenotypic changes of cell differentiation are visible. therefore, our results may point to characteristic agp expression pattern that may be involved in adventitious or lateral root development. the distribution of all of the agp epitopes that were applied within primordium in the presented study is schematically presented in fig. 6b. comparison of ar to lr the comparison of lr and ar at the corresponding stages of development showed some differences as well as similarities in the occurrence of the pectic and agp epitopes. no xylogalacturonan was detected in the lr; moreover, the lm epitope was hardly present in lr in comparison to ar. the lm epitope occurred in the outer periclinal cell walls of the primordium and was especially abundantly in the regions where lr had already emerged. agp epitopes had a similar distribution pattern in both types of roots. however, they were more expressed in the lr than in the ar. ar share the same function as lr but develop from aerial tissues. the partial similarities in cell components that were observed can be explained in two ways – it is either the origin that influences the cell composition or it is the environment surrounding the developing primordial that is the influence. more studies should be conducted to verify these two assumptions.  we performed histo- and immunohistochemical analyses of the cell wall to study the development of tomato ar. the ar that formed on the grafted stems differed from the ar that were induced by stem cutting in terms of the shape of the primordium and the way they emerged. two hg epitopes, lm and lm had different localisations at the corresponding ar developmental stages with an increase in lm occurrence along with the progress of ar growth. of the three agp epitopes that were applied, jim and jim showed an association with the tonoplast in particular cells of the primordium, whereas the jim epitope occurred from the earliest stage, and therefore it can be considered to be a marker of initial cells. finally, the differences observed between the ar and lr cell wall composition might be origin- or environment-dependent. abbreviations agpsarabinogalactan proteins ar/aradventitious root ccambium colcollenchyma corcortex dagdays after grafting dedegree of esterification eendodermis ffibre hghomogalacturonan lr/lrlateral root ppericycle pasperiodic acid-schiff pbsphosphate-buffered saline pcdprogrammed cell death pmespectin methylesterases rcroot cap scscion xyxylem vessels 
the adventitious roots of plants share the same function as primary and lateral roots, although their development is mainly an adaptive reaction to stress conditions. two pectic epitopes were developmentally regulated and the occurrence of the lm xylogalacturonan epitope in the root cap of the ar differed from other species described so far. jim and jim epitopes were detected in the more advanced stages of primordium development, whereas the jim epitope was present from the earliest division events of the initial ar cells. agp epitopes were abundantly present in the cytoplasmic compartments and were correlated with the degree of cell vacuolisation. arabinogalactan proteinsautograftingcell differentiationcell wallimmunohistochemistrylateral rootspectinstomatoesfaculty of biology and environmental protection, university of silesiagrant for young scientists 1m-0113-001- zfin00000040sala katarzyna issue-copyright-statement© the author 2017 pectins and arabinogalactan proteins are helpful markers of particular cellular events, such as programmed cell death, elongation, proliferation or other differentiation events that accompany ar development. however, little is known about the distribution of pectins and agps during ar ontogeny, either in the primordium or stem tissues from which ar arise or their correspondence with these events during lr formation. ar were developed from different stem tissues such as parenchyma, xylem rays and the cambium, depending on the stem age and treatment of the parental tissue.
 the adventitious roots of plants share the same function as primary and lateral roots, although their development is mainly an adaptive reaction to stress conditions. regeneration of grafted plants is often accompanied by ar formation thus making the grafting technique a good model for studying ar initiation and development and their means of emergence. pectins and arabinogalactan proteins are helpful markers of particular cellular events, such as programmed cell death, elongation, proliferation or other differentiation events that accompany ar development. however, little is known about the distribution of pectins and agps during ar ontogeny, either in the primordium or stem tissues from which ar arise or their correspondence with these events during lr formation.  ar were developed from different stem tissues such as parenchyma, xylem rays and the cambium, depending on the stem age and treatment of the parental tissue. immunochemical analysis of the presence of pectic and agp epitopes in ar and ar-associated tissues showed differential, tissue-specific distributions of these epitopes. two pectic epitopes were developmentally regulated and the occurrence of the lm xylogalacturonan epitope in the root cap of the ar differed from other species described so far. agp epitopes were abundantly present in the cytoplasmic compartments and were correlated with the degree of cell vacuolisation. jim and jim epitopes were detected in the more advanced stages of primordium development, whereas the jim epitope was present from the earliest division events of the initial ar cells. the comparison between ar and lr showed quantitative and qualitative differences.  the chemical compositions of adventitious and lateral root cells show differences that correlate with the different origins of these cells. in ar, developmental changes in the distribution of pectins and agp suggest the turnover of wall compounds. our data extend the knowledge about the distribution of pectin and agp during non-embryogenic root development in a species that is important from an agronomic point of view. keywords arabinogalactan proteinsautograftingcell differentiationcell wallimmunohistochemistrylateral rootspectinstomatoesfaculty of biology and environmental protection, university of silesiagrant for young scientists 1m-0113-001- zfin00000040sala katarzyna issue-copyright-statement© the author 2017 