Enzymology of Glutamine Metabolism Related to Senescence and Seed Development in the Pea ( Pisum sativum L.)

Abstract

The metabolism of glutamme in the leaf and subtended fruit of the aging pea (Pisum sativum L. cv. Burpeeana) has been studied in relation to changes in the protein, chlorophyll, and free amino acid content of each organ during ontogenesis. Glutamine synthetase IEC 6.3.1.21 acthvity was measured during development and senescence in each organ. Glutamate synthetase IEC 2.6.1.531 activity was followed in the pod and cotyledon during develpment and maturation. Maximal glutamine synthetase activity and free amino acid accmulation occurred together in the young leaf. Glutamine synthetase (in vitro) in leaf extracts greatly exceeded the requirement (in vivo) for reduced N in the organ. Glutamine synthetase activity, although declinig in the senescing leaf, was sufficient (in vitro) to produce glutamine from all of the N released during protein hydrolysis (in vivo). Maximal glutamine synthetase activity in the-d was recorded 6 days after the peak accumulation of the free amino acids in this organ. In the young pod, free amino acids accumulated as glutamate synthetase activity increased. Maximal pod glutamate synthetase actvity occurred shnultaneously with maxhnal leaf glutamine synthetase activity, but 6 days prior to the corresponding maximu of glutamine synthetase in the pod. Cotyledonary glutamate synthetase activity increased during the asshnila-tory phase of embryo growth which coincided with the loss of protein and free amino acids from the leaf and pod; maximal activity was recorded simultaneously with maximal pod glutamine synthetase. We suggest that the actity of glutamine synthetase in the supply organs (leaf, pod) funishes the translocated amide necessary for the N nutrition of the cotyledon. The subsequent activity of glutamate synthetase could provide a mechanism for the transfer of hiported amide N to aIpha amino N subsequenty used in protein syntbesis. In vito measurements of enzyme actii Indicate there was sufficient catalytic potential in vivo to accomplsh these proposed roles. During the course of fructification in pisum sativum, substantial amounts of reserve proteins are deposited over a brief period of time in the developing cotyledons (3, 5, 25). The synthesis of protein reserves creates a demand for the necessary amino acid precursors. This demand is met mainly by amino acids synthesized de novo utilizing reduced C and N imported by the seed (22, 37). Most of the N translocated to the ripening fruit is in the amides, glutamine and asparagine (2, 34, 37) and they are the main N donors for in situ synthesis of protein amino acids in the seed (19, 22). The importance of amides in the N nutrition of the ripening ovule is substantiated by the findings that asparagine and (espe-cially) glutamine stimulate growth and protein synthesis in cul-' tures (in vitro) of legume cotyledons and various plant embryos (26, 48). The subtending leaf (leaflets plus stipule) in the reproductive node and pod (carpel wall) surrounding the ripening ovules are the most important supply organs contributing the bulk of the reduced N imported by the developing cotyledons (19, 21, 35). Pate et aL (38) have stated that the role of the leaf in cycling solutes to its developing fruit is second only in importance to its role in photosynthetic C fixation. The pod is almost totally committed to the N nutrition of its developing ovules (16, 27, 36, 40). In a previous communication, we demonstrated the presence of proteolytic activity capable of releasing amino acids from the protein of agini leaves and pods (46). In this paper we report the presence of GS (EC 6.3.1.2) and GOGAT (EC 2.6.1.53) activity in aging supply organs and developing recipient organs of the pea. The respective catalytic potentials of these enzymes to incorporate N into translocated glutamine and subsequently utilize this N for assimilatory growth in the cotyledons are crucial to the N economy of the plant. MATERIALS AND METHODS Plants (P. sativum L. cv. Burpeeana) were grown under controlled environmental conditions and the age (days postanthesis) of individual organs was followed as before (46). All studies were conducted with the leaf (leaflets plus stipule) and subtended fruit (pod and cotyledons) of the lowest reproductive node of each plant. Organs were harvested, dissected and combined, and deter-minations of fresh weight, protein, Chl, and soluble a-amino N were made as described previously (46). Values given for these determinations were confirmed by triplicate analysis of six separately prepared samples.