Metabolism of α-Ketoglutarate by Roots of Woody Plants

Abstract

The uptake and metabolism of a-ketoglutarate.5-'4C by peach, apple, and privet root tissues were studied over various time intervals. As much as 80 % of the absorbed l 4C appeared as 14CO, in 320 minutes in peach roots. Apple and privet roots were less effective in this oonversion with the bulk of the 14C found in the organic acid fraction. This indicates differences in organic acid metabolism among species of woody plants. The 14C accumulated in malate earlier and in larger quantities than in citrate. Both glutamate and aspartate were labeled in 10 minutes and glutamate was labeled as early as 3 minutes. The labeling pattern does not clearly distinguish between the synthesis of glutamate by glutamic dehydrogenase or by transamination with oxaloacetate. The rapid metabolism of ey.ketoglutarate to glutamate by the 3 sp,ecies studied indicates the presence of enzyme systems important in amino acid synthesis in the roots of woody plants. Zimmerman (9) has emphasized that photosyn-thates translocated downward in phloem tissue provide carbon skeletons for the synthesis of amino a,cids in the root system. at-Ketoglutarate as an iintermediate in the citric acid cycle is rapidly metabolizefl in intact tissues and mitochondria to CO2. Glutamate and its amide may also arise directly from a-ketogflutarate metabolism. Some amino aciidls are uised in root growth and some aire found in the translocating stream. Nitrogen for growth of aerial tissues comes from the root system andl in matulre woo(dy plants is translocated over considerable (listances. mansy species of woody perenniall plants ,glutamate, aspartate, an(d their amides may be important translocatory forms of nitrogen (2, 8). Bollard (1) has found that as much as 90 % of the niitrogeni extracted fro}m the xylem of the apple can be accouinted for in these amino aci,ds. If tupwardl nitrogen translocation occuirs primarily in the xylem, then it canl be assiumed that these aminio acids are physiologically important in upward niitrogen transport. The synthesis of glutamate and aspartate in root systems is of interest in this connection. This report presen.ts the resuilts of an investigation of the distribution and oxidation of carbon-5 of a-ketoglutarate-5-14,C after incubationm with excised roots of 3 species of woody plants. Materials and Methods Plant Materials. Peach seeds (Prunus persic(a L. var. Eliberta) with the seed coat removed were sterilized by immersion in 0.25 % sodium hypo-chlori,te for 30 seconds. The seeds were rinsed and.then cultured on sterile 0.5 % agar with ntitrient solution. The seedlings were grown for 32 days in a growth chamber tinder 3000 ft-c of lighit. Apple roots (Pyrus malus L. var. M. Merton 106) were obtained from 1-year-old rooted cuttings grown for 30 to 37 days in nonsterile medium. Miist-propagated privet (Ligustrzun vulgarac L.) cuttings were used. Incubation Pr-ocedirc. Replicate samples (0.5-1 g) of the distal 1 to 2 cm of the roots were placed in No. 15 me-ditim fritted glass filter funnels containing 2.5 Muc of a-ketogilutarate-5-_4C in 5 ml of 0.05 Mr potassium phosphate buffer, p)H 6.1. Ai,r was pulled through the hase of the filter flunnels to Aerate the roots suspended in solutiotl. Respired CO., was carried in the aiir streaml and hubbled throuigh 90 mnl of I H' Hyamine hyxdroxide. The absorbed CO. wa,s counted for radioactivity uising ,a Paclkar(d liquid scintilllationi spectrometer. Analytical l Methods. At predeitermiined times, an aeration vessel was disconniected from the system , substrate was drained o.ff, and 'the roots were rinsed with dce-ionized water to remove the Inoni-absorbed acid. The roots were transferred to 50 ml of boilling 100 % ethanlol for 10 mintutes. The tissnes were then homogenized wtith a Virtis blender and centrifutged at 10,000 X g. The residues were successively washed 4 times wiith 80 % (v/v) ethano-l. I'he extracts were combined and evaporated to dryness at 350 itl a rotary evaporator. The solutble extract was then dissolved in water and f.actionated sequentially on 1 X 8 cm coltmns of Dowex 50 (H+) and Dowex 1 (Formate) resins (4). The amino acid fraction eluted from the Dowex 50 (H-4+) column was separated into acidic amino acids (glutama,te and aspartate) and the remain,ing amino acids, by passage over a Dowex 1 (Acetate) column. This chromatography separates the initial extract into acidic amino acids, other amino acids, organic acids, and sugars. Ali-quots of these fractions were assayed for radioactivity. The insoluble residue remaining after centrifugation was filtered, heated to dryness, and assayed for radioactivity. 619