The Influence of 0.03% Carbon Dioxide on Protein Metabolism of Etiolated Avena sativa Coleoptiles

Abstract

The influence of indoleacetic acid, 0.03 % C02, and malate on protein metabolism of etiolated Avena sativa coleoptile sections has been investigated. All three were found to elevate both the rate of incorporation of labeled leucine into protein, and the level of soluble protein. The synergistic relationship between IAA and 0.03% CO2 in stimulating the growth of etiolated Avena coleoptile sections has been previously described (1), and the involvement of protein synthesis in auxin-stimulated growth is well documented (4,10,13). Experiments reported here were designed to study the separate and combined influence of IAA and CO2 on coleoptile protein metabolism, and to test the proposal that CO2-stimulated growth is mediated by a process involving both dark CO2 fixation and CO2-stimulated protein synthesis (16). The results suggest that both IAA and CO2 stimulate protein synthesis, that CO2-stimulated protein synthesis involves dark CO2 fixation, and that elevated rates of protein synthesis result in increased incorporation of labeled bicarbonate into protein. It is proposed that CO2-stimulated growth is dependent on C02-stimulated protein synthesis. MATERIALS AND METHODS"4C-sodium bicarbonate (59 mc/mmole) and L-"C(U)leucine (331 mc/mmole) were purchased from Amersham Searle; cycloheximide and IAA were purchased from the Sigma Chemical Company. Seeds of Avena sativa var. 'Victory' were grown and harvested as described previously (1). Except where mentioned, the 20-mm coleoptile section below the 3-mm tip was used. Incubation Conditions. Weighed batches of tissue in 50-ml light-tight incubation tubes were submerged in 10 ml of phosphate buffer (25 mm, pH 7.5) and incubated at 25 C in the presence or absence of various test substances. In experiments involving 14C-leucine, the buffer was aerated with air or CO2-free air at a rate of 10 I/hr batch of tissue; incubations involving "4C-bicarbonate were in closed tubes without aeration. The CO2 concentration used had no measurable influence on the pH of the incubation medium, and was measured with an infrared gas analyzer.Protein Determination. After incubation, tissue was washed thoroughly with water, frozen for 3 to 4 hr, thawed, and then ground vigorously in a Potter-Elvehjem tissue homogenizer for 5 min with 5 ml of phosphate buffer (1 mM, pH 7.5). The homogenate was centrifuged at 13,000g for 15 min, the supernatant fluid was collected, and the extraction procedure was repeated two more times with pH 7.5 phosphate buffer at 25 mm and 0.10 M. The three supernatant fluids were then combined. An equal volume of saturated ammonium sulfate was added to the combined extracts and, after 12 hr at 4 C, the precipitate was collected by centrifugation at 25,000g for 30 min. The pellet was washed with 5 ml of saturated ammonium sulfate containing nonradioactive leucine (0.1% w/v), centrifuged at 25,000g for 30 min, and then dissolved in phosphate buffer (25 mm, pH 7.5). Aliquots of this solution were used for soluble protein determination by the standard method of Lowry...

Metabolic Changes in Gladiolus Cormels During the Break of Dormancy: The Role of Dark CO2 Fixation

“…Hart and Berrie (7) found that 3% CO2 in the atmosphere abolishes the inhibition of A. fatua seed germination by light. The growth of various types of excised plant tissues-roots (18,19), carrot disks (18), and Avena coleoptiles (1), is promoted by supplementing the medium with bicarbonate or a suitable acid. Johnson and Rayle (11) found that fusicoccin and IAA promote dark fixation in Avena coleoptiles.…”

“…Numerous observations were made on the promotion of growth of excised plant tissues by the supply of bicarbonate or, alternatively, a suitable tricarboxylic acid cycle acid in the growth medium (1,18,19). Bacterial mutants lacking PEP2 carboxylase also require malate for growth (13).…”

“…In the present paper an attempt is made to assess the endogenous function of dark CO2 fixation in preparation for cormel germination. 1 Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel 50200, 1981 Series No. 1 16E.…”

Gaseous factors involved in the enhanced elongation of rice coleoptiles under water

“…Carbon dioxide has been known to stimulate growth of various kinds of plant tissues and organs; for example, eoleoptiles (Evans, 1974), hypocotyls (Goto & Esashi, 1974) and seed tissues (Esashi & Katoh, 1975). Evans, Ray & Reinhold (1971) suggested that the stimulation of elongation by CO2 is an acid effect, and some others (Bown & Aung, 1974;Haschke & Luttge, 1977) reported that CO2 fixation may be involved in C02-stimulated elongation. In Table 2, the elongation-stimulating effects of CO2 were accompanied by increases in dry weight of the eoleoptiles and these effects required O2 at concentrations above 0.08 m-' m"^ (Fig.…”

A Synergistic Stimulation of Avena sativa Coleoptile Elongation by Indoleacetic Acid and Carbon Dioxide

“…Splittstoesser (14) proposed that growth stimulation of etiolated plant tissues by atmospheric CO2 involved dark CO2 fixation and the consequent synthesis of 4 This reaction is essentially irreversible, and in vivo oxaloacetate is readily converted to malate and aspartate. In addition, the enzyme has a high affinity for CO2 which allows it to fix CO2 at concentrations less than 0.03% (12).…”