Evidence for a Protein Gel Structure Cross-linked by Metal Cations in the Intercellular Cement of Plant Tissue

Abstract

The factors which modify the action of chelating agents in dissolving the intercellular cement in plant tissue have been investigated. The action was assessed by determining the degree of separation of cells in treated pea root tips. Greatly increased separation was observed when heavy metal chelating agents were combined with EDTA (which, in addition to heavy metals, chelates Ca) indicating that two types of cations are involved in stabilizing the cement. After the cells had been separated by removal of the cations they could be recemented by treatment with divalent cations. In low concentrations only certain combinations of cations were effective, again pointing to the presence of more than one type of cation in the cement. Proteolytic enzymes or denaturing agents eliminated this capacity to recement. Pretreatment with divalent cations enhanced subsequent separation (in EDTA solution). This effect could be countered by treatment with monovalent cations. Pretreatment in buffers covering a wide range of pH resulted in increased separation both at pH 3 and at pH 11, suggesting an ampholytic component in the cement. Urea, when applied as a pretreatment, only enhanced separation when in high concentration. This effect was influenced by ionic strength. When applied in combination with EDTA, urea produced an optimum effect at low concentration (0.125 M.). Pretreatment in hot water also enhanced subsequent separation in EDTA solutions. High concentrations of KCl countered this effect. These findings point to the importance of H bonds in the intercellular cement. Thioglycollic acid, applied either as pretreatment or simultaneously with EDTA, lowered the EDTA concentration necessary to achieve separation, suggesting the presence of S—S bridges. The curve relating the effect of pretreatment at varying temperatures, either in H₂O or in apolar solvents, to temperature, showed a sharp break, suggesting that the melting-point of an organized gel might be involved. The conclusion is reached that the intercellular cement can be regarded as an oriented gel structure containing protein molecules cross-linked by two types of metallic ion, the metallic cross linkage being chelate in character.