Exocellular Enzymes of Corn Roots

Abstract

Little attention has been given to the occurrence of exocellular hydrolytic enzymes associated with the roots of higher plants. Such enzymes, if present, could solubilize macromolecular constituents of organic soils, permitting uptake and utilization by the plant of the resultant low molecular weight organic compounds. The present work supports this concept by demonstrating that several exocellular hydrolytic enzymes are associated with the roots of intact corn seedlings. Distinction has been made between exo-cellular enzymes released into the culture media and enzymes apparently bound to the root surface. A previous paper from this laboratory (17) re-viewedl earlier studies and presented evidence for the occurrence of exocellular enzymnes associated with the primary cell w all of corn coleoptiles. Of particular relevance to the present work is a report (36) of the secretion of peroxi(lase, aci(l phosphatase, indolyl-acetic aci(l oxidase, and amylase into the culture media bathing plant tissue cultures. In addition, reports have appeared (2, 6, 14) of the occurrence of an exo-cellular invertase, possibly involved in sucrose metabolism by roots. In the present work it is reporte(I that invertase, cellobiase, adenosine tri phosphatase, pyrophosphatase, ancl nuclease activity is observed when appropriate substrates are added to nutrient solution bathing the roots of intact seedling corn plants. In addition, in-vertase and nuclease are (letectecl as soluble enzymes in the nutrient solution. Materials and Methods Corn seeds (Zea ways L. var. Michigan hybrid 350) were soaked in running water overnight, then surface sterilized by soaking in 1% sodium p-toluene-sulfonchloramide (chlorazenie) for 5 minutes and finally , washed thoroughly wxith sterile glass distilled water. The soaked seeds were germinate(l between sterilized paper towels in a moist chamber at room temperature an(l used at an age of 4 to 5 days. Two incubation methods were used, A) the sub-strate was addle(l to the nutrient in which the seedling roots were growing, an(l B) the seedlings were removed from the nutrient and the substrate then added. MWethod A measured total enzymatic activity of the roots while method BP measure(l activity of enzymes present in solution. 60 For procedure A, 8 intact seedlings, each 80 mml in length, were washed with glass distilled water, then incubated at room temperature in 15 X 100 mm tubes. The tubes contained seedlings, substrate, and necessary cofactors, in 13 nml of inorganic nutrient solution (37). Aseptic air was supplied at the bottom of the tube by means of a fine glass tube. At intervals, 1 nl of reaction mixture was removed an(l the amount of substrate hydrolyzed(l determined by standard methods with minor modifications. For procedure BP 8 seedlings were suspended in tubes containing 13 nml of inorganic nutrient solution. Samples of 1 nml of the nutrient were removed from the tubes, at varying time intervals, and incubated with the appropriate substrate and cofactors. Rates of reaction were determined as for B above. No(direct comparison may be made between the activities observed using method A and B because incubation conditions in the A series were selected to be optimal for root growth, and not for the activity being tested, while incubation conditions for B were optimal for the enzymic assay. Assav .ilctliods for Proced(lrc 4. For all assays a total volume of 13 nml of mineral nutrient was used. To this was added, in mmnnoles: sucrose 0.95, for inver-tase assay; cellobiose 0.95, for cellobiase assay; ATP 0.026 and M\gSO4 0.07 for adenosine triphosphatase assay; sodlium pyrophosphate 0.02 and _MgSO4 0.02 for pyrophosphatase assay; yeast RNA 13 nmg plus NaCl 37 mg for ribonuclease assay; salmon sperm DNA 13 nmg plus-MgSO4 13 nmg for deoxyribonuclease assay; ancl denature(l bovine serum albumin 13 mig for protease assay. After incubation at roomi temperature , for the indicated time, the amount of substrate hydrolyzed was measured as descril)e(l below.