ИСТИНА

The plant cell wall behaves as an ion exchanger where fixed charges (ionogenic groups) interact with exchangeable ions in the surrounding solution. Ionogenic groups of wall structural polymers are known to affect many properties of primary cell wall, including pH and ionic status of matrix, mechanical and porosity properties. These properties in turn are likely to influence cell wall links, hydration capacity, the movement and access of cell wall-modifying proteins through the matrix. In spite of numerous studies the chemical composition and the precise organization of unique pollen wall are still a matter of conjecture. Using isolated pollen wall we have analyzed here for the first time, to our knowledge, the qualitative and quantitative composition of ionogenic groups in structural polymers of exine and intine in Lilium longiflorum Thunb. 2-layer pollen wall (including intine and exine) was isolated by sequential extraction of protoplast components and soluble components of pollen walls. Pollen grains were washed with diethyl ether, fixed by a mixture of absolute ethanol and acetone, and then treated step by step with 2% SDS, 1% NaOH, 1% HCl (the each for 30 min). After purification and washing one part of the sample was dried and used for potentiomeric titration. Another part of the sample was used for exine isolation by enzymatic digestion (cellulase and pectinase, 1%, 24 h at 25°C). The preparation was washed, incubated in 2% SDS, washed again and dried. Determination of qualitative and quantitative composition of ionogenic groups in the 2-layer pollen wall and exine was done according to the potentiometric titration method described earlier [1]. The total amount of the amine groups in the samples was determined by the method of nonaqueous titration. The composition of ionogenic groups in the intine was estimated based on mass ratio measurements for 2-layer pollen wall and exine. Cytological study of the preparations showed, that 2-layer wall and exine maintained the original morphology of the untreated pollen grain. The exine was free from intine fragments. Staining for DNA (DAPI) also showed the complete removal of nuclei and their fragments from isolated wall. It testifies about enough high degree of purification of walls from protoplast components. The results of potentiometric titration show that there are four ionogenic groups in the polymeric structure of 2-layer pollen wall and three groups in the exine. In according to known procedure [1] the amount of the functional group of each type was estimated, and the corresponding values of pKa were calculated. As discussed previously [1] the groups with the pKa ~ 2 are amine groups, the ones with pKa ~ 4 are carboxyl groups of galacturonic acid (GA), the ones with pKa ~ 8 are the carboxyl groups of hydroxycinnamic acid, and the ones with pKa ~10 are the phenolic groups. There are three cation exchangeable groups in the intine - two different carboxyl groups, and phenolic groups, - and one group exchanges anions (amine groups). Exine includes carboxyl groups of hydroxycinnamic acid, phenolic groups, and also amine groups. In all cases the total amount of cation (Scat) exchange groups is much greater (5 times and more) than anion one (San). These results show that the intine and exine are the natural ion exchangers that possess predominantly cation exchange properties. In the exine the amounts of the carboxyl groups of hydroxycinnamic acid and the phenolic groups was about 1400 and 500 mkmol per g dry wt of the exine respectively. These results are in consistent with known data on sporopollenin content. In the intine the content of the phenolic groups is approximately 2 times smaller than in the exine, and the amount of the carboxyl groups of hydroxycinnamic acid and amine groups is app. equally. It should be noted that the question on phenolic compounds in the intine structural polymers was not examined earlier. At the same time in primary walls of somatic cells the important structural function of hydroxycinnamic acids is revealed. The occurrence of the hydroxycinnamic acids in ester/ether; linkage to arabinoxylans, to pectic polysaccharides and to xyloglucans is well established. It is likely that there are some covalent cross-links between hydroxycinnamic acids and glycans in dehydrated pollen grain. These links may be hydrolyzed at the initial stage of pollen rehydration, and as consequence a loosening of the structural polymers of intine will take place.