Cell wall pectins and xyloglucans are internalized into dividing root cells and accumulate within cell plates during cytokinesis.

Recently, we have reported that cell wall pectins are internalized into apical meristem root cells. In cells exposed to the fungal metabolite brefeldin A, all secretory pathways were inhibited, while endocytic pathways remained intact, resulting in accumulation of internalized cell wall pectins within brefeldin A-induced compartments. Here we report that, in addition to the already published cell wall epitopes, rhamnogalacturonan I and xyloglucans also undergo large-scale internalization into dividing root cells. Interestingly, multilamellar endosomes were identified as compartments internalizing arabinan cell wall pectins reactive to the 6D7 antibody, while large vacuole-like endosomes internalized homogalacturonans reactive to the 2F4 antibody. As all endosomes belong topographically to the exocellular space, cell wall pectins deposited in these "cell wall islands", enclosed by the plasma-membrane-derived membrane, are ideally suited to act as temporary stores for rapid formation of cell wall and generation of new plasma membrane. In accordance with this notion, we report that all cell wall pectins and xyloglucans that internalize into endosomes are highly enriched within cytokinetic cell plates and accumulate within brefeldin A compartments. On the other hand, only small amounts of the pectins reactive to the JIM7 antibody, which are produced in the Golgi apparatus, localize to cell plates and they do not accumulate within brefeldin A compartments. In conclusion, meristematic root cells have developed pathways for internalization and recycling of cell wall molecules which are relevant for plant-specific cytokinesis.

Immunocytochemistry of pectins in shoot apical meristems: consequences for intercellular adhesion.

The nature of pectins (acidic, methyl-, or acetyl-esterified) in the shoot meristem of Sinapis alba was assessed by immunocytochemistry with the 2F4 monoclonal antibody in light and electron microscopy. This antibody is specific for "egg-boxes"--the polygalacturonic acid conformation induced by calcium as described in Liners et al. (Plant Physiol. 99: 1099-1104, 1992). Hardly any acidic pectin was detected in meristem walls; the pectins were largely methyl-esterified and esterified by acetyl groups and/or other esters. After in situ chemical or enzymatic de-esterification, labeling was distributed over the primary wall and the middle lamella of meristematic cells. Acidic pectin and Ca2+-cross-linked homogalacturonans were absent from the pit fields, where plasmodesmata traverse the middle lamella. The type and distribution of pectins are discussed in relation to cellular adhesion between active meristem cells.

Distinction between cultivated and wild chicory gene pools using AFLP markers.

The cultivation area of industrial chicory, Cichorium intybus L. cv Sativum, coincides with the natural distribution area of its wild relative, C. intybus L., which could lead to gene flow between wild and cultivated types. The genetic diversity within and between the two types has therefore been studied using AFLP genotyping of samples from 12 wild populations collected in Belgium and ten commercial varieties. The genotyping of 233 individuals allowed the identification of 254 AFLP markers. Similar levels of genetic diversity were observed within wild populations and cultivated varieties, suggesting the absence of any strong bottleneck in the history of the cultivated types. The phylogenetic analysis pointed to a monophyletic origin of cultivated varieties as compared to the local wild populations studied, hence the two types of chicory form two separate gene pools. The genotyping of some individuals sampled in ruderal sites clearly showed that they belong to the cultivated gene pool, which suggests the existence of feral or weedy types. The low differentiation observed among wild populations indicates that gene flow might be important in this species.

Polyamines and pectins. II. Modulation of pectic-signal transduction.

A previous study had shown that polyamines adsorb selectively on plant cell walls according to the valence of the polyamine (Messiaen et al. 1997, Plant Physiol. 113: 387-395). In this study, the adsorption of polyamines onto isolated carrot cell walls and onto pure polygalacturonic acid was investigated in the presence of competing mono- and divalent cations (Na+ and Ca2+). Putrescine (Put2+) was unable to remove all the calcium (Ca2+) from cell walls or from polygalacturonic acid. Spermidine (Spd3+) and spermine (Spm4+) adsorbed on all galacturonates and were able to remove Ca2+ completely from both the walls and the pure polygalacturonates. Therefore, Spd3+ and Spm4+, unlike Put2+, prevented polygalacturonic acid from adopting the Ca(2+)-induced supramolecular conformation recognized by the 2F4 anti-pectin monoclonal antibody. We show that the signal transduction cascade otherwise initiated in plant cells by Ca(2+)-bound alpha-1,4-oligogalacturonides was indeed blocked by both Spd3+ and Spm4+, but not by Put2+. The mobilization of cytosolic free Ca2+ and the cytosolic acidification usually observed after treatment with pectic fragments did not occur and the subsequent activation of phenylalanine ammonia-lyase was suppressed. It is hypothesized that the disruption by Spd3+ and Spm4+ of the Ca(2+)-induced supramolecular conformation of pectic fragments was the cause of the inhibition of the pectic signal. We conclude that polyamines can act on plant cell physiology by modulating the transduction of the pectic signal.

Polyamines and Pectins (I. Ion Exchange and Selectivity).

The ion-binding and -exchange properties of putrescine, spermidine, and spermine on purified walls of carrot (Daucus carota L.) cell suspensions were investigated by producing ion-exchange isotherms and comparing them with the behavior of Na+, Mg2+, and Ca2+. The cation exchange capacity of the carrot cell walls was 0.8 equivalent kg-1 dry matter, and the ionic selectivity sequence of the walls for polyamines followed the sequence spermine4+ > spermidine3+ [almost equal to] Ca2+ > putrescine2+. The polyamines were subjected to only electroselectivity and probably did not induce any favorable supramolecular conformation of pectin like the one induced by Ca2+. Triangular ion exchanges were also performed with three diamines: ethanediamine, butanediamine, and octanediamine. The shorter the diamine, the higher the total adsorption and selectivity of the exchange. The lower selectivity of the cell wall for putrescine was partly attributed to its inability to access and displace Ca2+ from higher affinity sites within dimerized pectic sequences. The polyamine adsorption and exchange on pectic sequences could result in pectic signal modulation in pathogenesis and in differentiation.

Callose synthesis in spirostanol treated carrot cells is not triggered by cytosolic calcium, cytosolic pH or membrane potential changes.

Carrot (Daucus carota L.) cell suspensions were treated with a spirostanol saponin from Yucca. This saponin is an elicitor of callose synthesis. Irrespectively of the mode of action of spirostanol on the callose synthase activity itself, the spirostanol-induced callose synthesis in carrot is not preceded by changes in membrane potential, cytosolic free calcium or cytosolic pH. The inability of modulators of cytosolic free calcium content (verapamil, nifedipine and Br-A23187), EGTA and a proton pump inhibitor (vandate) to inhibit or induce callose formation is consistent with a calcium- and pH-independent mechanism for callose deposition.

Chicory seed lot variety identification by leucine-aminopeptidase and esterase zymogram analysis.

A method of identifying closely related chicory varieties has been developed using native polyacrylamide gel electrophoresis and subsequent leucine aminopeptidase and esterase staining of bulked seed sample extracts. The variety zymograms are analyzed according to a mobility-density scheme. This quick, easy and low-cost method can be a useful tool for chicory breeders.

Influence of the degree of polymerization of oligogalacturonates and of esterification pattern of pectin on their recognition by monoclonal antibodies.

The ability of galacturonic and oligogalacturonic acids with degrees of polymerization (DP) from 2 to 10 to inhibit the recognition of homopolygalacturonic acid by a monoclonal antibody specific for dimers of pectin (F Liners, J-J Letesson, C Didembourg, P Van Cutsem [1989] Plant Physiol 91: 1419-1424) has been tested by enzyme-linked immunosorbent assays. Oligomers of DP9 and above preincubated with the antibodies clearly inhibited the association between the antibodies and immobilized pectin. A minimum DP of nine consecutive galacturonic residues is thus necessary to be associated through calcium cations to form dimers. Randomly deesterified pectin was recognized by the antibody if its degree of methylesterification was <30%, whereas blockwise deesterified pectin was recognized up to 40% of methylesterification. The replacement of calcium ions by magnesium prevented the recognition of polygalacturonic acid by the antibody.

Monoclonal Antibodies against Pectin: Recognition of a Conformation Induced by Calcium.

Monoclonal antibodies have been produced that recognize a conformation of homopolygalacturonic acid (pectic acid) induced by an optimum concentration of calcium and sodium of about 1 and 150 millinormal, respectively. The epitope recognized is probably part of the dimers of pectin chains associated according to the ;egg box' model.

Proton-Metal Cation Exchange in the Cell Wall of Nitella flexilis.

When protons are exchanging for bivalent cations (Cu(2+), Zn(2+), or Ca(2+)) on the carboxylic groups of Nitella flexilis cell wall, the values of the respective global equilibrium constants do not change up to a protonation degree of 80%. These values drastically increase at higher proton concentrations and tend to approximately 3.4, which is the intrinsic pK value of the constitutive alpha-d-galacturonic acid monomer. These data suggest that the electric field in the matricial polymer and the cation bridges between pairs of negative sites have disappeared.