Multiple isozymes of endo-β-d-mannanase in dry and imbibed seeds

A gel overlay method for detecting isozymes of endo-β-d-mannanase (EC 3.2.1.78) separated by isoelectric focusing has been developed for use with plant extracts. The seeds of 19 selected species of monocots, dicots, and gymnosperms (and all cultivars thereof tested) expressed endo-β-d-mannanase activity when imbibed and most also contained enzyme activity in the dry state. Generally, the number of isozymes increased when the seed imbibed water. This was most evident when the seed was left intact instead of being separated into the embryo and nutritive tissue (if present) prior to imbibition. Usually, more isozymes were present in the endosperm/megagametophyte than in the embryo regardless of whether the seed was intact or separated. In some cases, isozymes were recovered from the incubation water in which the seeds or seed parts had been imbibed. Such isozymes were considered to be mature forms of the enzyme that were present extracellularly and had leaked from the cell walls into the surrounding medium. The roots, shoots, and leaves of flowering alfalfa also contained endo-β-d-mannanase. In the roots there was one very active isozyme, identical in pI to that present in the imbibed alfalfa seed.