Cytochemical localization of pectinases and cellulases in developing laticifers of Maclura tinctoria and Ficus montana (Moraceae)

Abstract

Laticifers are latex‐producing structures which occur in some plant families. In Moraceae (the mulberry family), laticifers are of the non‐articulated and branched type and are distributed throughout the whole plant, i.e., they consist of a very long single cell that branches, forming a continuous network throughout the plant tissues. Given that the laticifer wall of Moraceae species is under continuing expansion and consists mainly of pectin and cellulose, the action of pectinases and cellulases must be of great importance in the laticifer development. Thus, this work aimed to verify the subcellular producing sites of pectinases and cellulases and their role in the laticifer development in two Moraceae species, using as a tool cytochemical analysis associated with transmission electron microscopy. Reproductive meristems of Maclura tinctoria (L.) D.Don ex Steud. and Ficus montana Burm.f. were collected, fixed in Karnovsky's solution and stored in 0.1 M phosphate buffer (pH 7.2) at 4°C. For pectinase detection the samples were incubated in 0.1 M sodium acetate buffer (pH 5.0) containing 0.5% of pectin for 20 min. at room temperature. To detect cellulase the samples were incubated in 0.05 M citrate buffer (pH 4.8) containing 0.02% of carboxymethylcellulose for 10 min. at room temperature. The materials from both treatments were transferred to Benedict's reagent heated to 80°C for 10 min., and then washed in 0.1 M phosphate buffer and postfixed in 1% osmium tetroxide for 2 h at 4°C. The samples were washed in distilled water, dehydrated, embedded in Araldite resin and sectioned at ca. 70 nm thick. The obtained sections were stained with 2% uranyl acetate for 25 min and Reynolds lead citrate for 5 min. and observed in a Jeol 100CX II transmission electron microscope. The cytolocalization controls of pectinases and cellulases followed the same methods but without the incubation of the samples in pectin and carboxymethylcellulose, respectively. Pectinases and cellulases were found in regions of the laticifer wall close to the middle lamella in both species (Figures 1 and 2). The pectinases should act on the dissolution of the middle lamella, enabling intrusive apical growth of laticifers through the plant tissues. The occurrence of pectinases is expected for the development of non‐articulated laticifers but it is the first record of cellulase presence in this type of laticifer. Cellulases have been reported in laticifers of the articulated type, in which they operate in the degradation of the end cell walls. In the case of the non‐articulated laticifers like those of Moraceae, it is likely that cellulases act on the partial disassembly of cellulosic components of the wall near to the middle lamella facilitating the intrusive growth. Both pectinases and cellulases are synthesized in the endoplasmic reticulum (Figure 3) and transported to the cell wall by exocytosis or stored in the vacuole (Figure 4) of Maclura tinctoria and Ficus montana . The production of these enzymes in the reticulum has been recorded for the secretory cavities of Citrus ; however, this is the first study in which were identified the organelles involved in the synthesis of pectinases and cellulases in laticifers.