The soluble leucine pool in maize root tips.

Abstract. The incorporation of 14C-leucine and 14C-amino acid mixture into protein in unfertilized eggs and developing embryos of the brown alga Fucus vesiculosus L. was studied. Bacterial contamination was initially a problem, but it was found that the addition of 40 jug/ml chloramphenicol to the incubation medium would inhibit bacterial protein synthesis without affecting early development of the Fucus embryos. The kinetics of uptake and incorporation of 14C-leucine into the trichloroacetic acid-soluble and -insoluble fractions indicated that the exogenous precursor did not equilibrate with the main soluble leucine pool before incorporation into protein. Uptake and incorporation of leucine by embryos 90 to 175 minutes old were proportional to exogenous leucine concentration over the range 5 X 10-6 M to 5 X 10-3 M.Unfertilized eggs will incorporate 14C-leucine into protein. Gamete release was obtained as described by McCully (11). Male receptacles were placed in petri dishes at 150 in the light, and antheridia were usually extruded from the conceptacles within 12 to 24 hours. The female receptacles were placed in sea water in petri dishes at 150 in the light, and oogonia were usually released wvithin 4 to 6 hours.The eggs were released from the oogonia shortly thereafter.Just prior to fertilization, antheridia were rinsed from the male receptacles with sea water, releasing the sperm. A small volume of sperm suspension was added to a suspension of eggs, and the mixture was swirled. The gametes were kept at 150 in the light for at least 0.5 hour to allow ample time for fertilization to occur. Then the zygotes and sperm were rinsed through a Nitex nylon filter (Tobler, Ernst and

Section: Methodssupporting

confidence: 77%

Amino Acid Incorporation in Developing Fucus Embryos

Peterson

Torrey

1968

“…1). Corn roots are known to absorb solutes more rapidly following washing (14) or aging (19). We have observed this …”

Section: Resultssupporting

confidence: 56%

“…Toxin increased the rates of leucine uptake and incorporation and increased the size of the soluble leucine pool (as defined by Oaks) (19) (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

Effects of Helminthosporium carbonum Toxin on Absorption of Solutes by Corn Roots

Yoder

Scheffer²

1973

Susceptible corn roots exposed to the host-selective toxin of Helminthosporium carbonum took up and retained more NO3-, Na+, Cl-, 3-o-methylglucose, and leucine than did control roots. Stimulatory effects on uptake were more pronounced with freshly cut roots than with roots that were washed and aged.Solutes were accumulated against a concentration gradient, and toxin-treated tissues developed a steeper gradient than did control tissues. Toxin affected both the low and high affinity uptake systems for Na+ and C1. Toxin did not affect uptake of Na2_, K+, Ca'+, phosphate ion (H2PO4-and HP04-), S04-, and glutamic acid. No toxin-induced leakage of any solute tested was detected within 5 to 6 hr after initial exposure to toxin. The data suggest that toxin from H. carbonum does not cause the general plasma membrane derangement caused by other host-selective toxins. Instead, H. carbonum toxin may cause specific changes in characteristics of the plasmalemma, which result in increased uptake of certain solutes.Helminthosporium carbonum Ullstrup race 1 produces a toxic compound which selectively affects the corn genotypes that are hosts of the fungus. Susceptible genotypes are those with a homozygous recessive factor at the locus conditioning disease reaction. Several lines of evidence show that the toxin is required for pathogenicity. All visible and known biochemical symptoms of infection are reproduced by the toxin (26). Thus, an understanding of disease and disease resistance should be possible from studies of toxic action. Metabolites with selective toxicity are now known from nine plant-infecting fungi, including Helminthosporium victoriae, Helminthosporium maydis race T, Periconia circinata, Alternaria kikuchiana, and Phyllosticta maydis (26,29 (28). Likewise, the apparent effect on nitrate reductase is secondary, because the increase in activity was traced to toxin-stimulated uptake of substrate, not to increased synthesis of the enzyme (31). Some of the other stimulatory effects of HC toxin may be secondary to initial toxic lesions. Metabolic activity induced by toxin at damaging concentrations is followed some hours later by decreased activity, cell leakiness, and death.The purpose of this work was to determine effects of toxin on uptake and retention of representative solutes in addition to nitrate. Nitrite, C1-, SO,-, Na+, K+, Ca2+, phosphate ion (H2PO-and HP042-), 3-o-methylglucose, leucine, and glutamic acid were selected for further study. HC toxin is selective for susceptible corn in its effects on NO3-uptake, and the increase in uptake is the earliest effect of toxin reported to date (31). An abstract describing some of the work has been published (30).Plant cells are thought to have at least two uptake systems for each of several ions (2, 12). Mechanism 1 of ion transport has a high affinity for ions and functions when ion concentrations are below 0.5 mM; this system is generally thought to be located in the plasmalemma, and it accumulates ions against a concentration gradient. Ion transport mech...

“…Although it has been shown that key enzymes isolated from plants are inhibited by amino acids (2,18) evidence for the operation of such control by end product inhibition in living cells of higher plants is based primarily on isotope competition experiments (5,6,(15)(16)(17). These experiments have required several hours for completion, and therefore, have not distinguished b.tween control arising from end product inhibition, a rapid response in microorganisms, and that due to enzyme repression, a slow response (4,20).…”

mentioning

confidence: 99%

Influence of Cycloheximide on the Synthesis and Utilization of Amino Acids in Suspension Cultures

Fletcher

Beevers

1971

Cells from 4-days old suspension cultures of Paul's Scarlet rose were incubated with acetate-U-'4C for 10 minutes. After washing, cells were incubated for 2 hours in growth medium in the presence and absence of cycloheximide. The 14C content of individual amino acids in the soluble form and in protein were determined at the end of the 10 minute pulse and at intervals thereafter in control cells and those treated with cycloheximide. During the period following the pulse there was a 3-fold increase in the "4C content of protein in control cells; no such increase occurred in the presence of cycloheximide.In the control cells there was a net increase in the 14C content of eight individual amino acids during this period. For six of these, the corresponding increases in the presence of cycloheximide were curtailed by the following amounts: arginine 98%, lvsine 94%, isoleucine 93%, threonine 82%, valine 49%, and proline 36%. This is interpreted to mean that the synthesis of each of these amino acids was slowed down when their incorporation into protein was prevented.is emphasized by the work of Holleman and Key (14) in which they estimated that exogenously supplied leucine (5 mm solution) caused a 64-fold expansion of the protein precursor pool of leucine in soybean hypocotyls during a 5-hr incubation period. It is apparent that if this occurred during an isotope competition experiment, the nonradioactive amino acids entering the tissue from an exogenous source would seriously dilute amino acids synthesized endogenously from a radioactive precursor. Such dilution would reduce the specific radioactivity of the amino acids in the protein precursor pools and lead to a decreased incorporation of 14C into the protein of the treated tissue. It follows that in those isotope competition studies, where no consideration was given to changes in the soluble amino acids (5, 6, 15), the reduced incorporation of "C into the protein may not be due directly to the regulation of amino acid synthesis.The purpose of the present investigation was to determine if the synthesis of amino acids is subject to rapid control when protein synthesis is stopped. To achieve this, suspension cultures of Paul's Scarlet rose were exposed to a pulse of acetate-U-`C and its incorporation into soluble-and protein-bound amino acids was followed in control cells and in samples treated with cycloheximide, an inhibitor of protein synthesis (7,8,11).Experiments with microorganisms have shown that the rate of synthesis of individual amino acids is closely regulated by the amount present in the cell (19,20). Commonly, an exogenous supply of an amino acid will prevent endogenous synthesis by inhibition of specific enzymes in the synthetic pathway (end product inhibition) or by repression of synthesis of the enzymes required (20).Although it has been shown that key enzymes isolated from plants are inhibited by amino acids (2, 18) evidence for the operation of such control by end product inhibition in living cells of higher plants is based primarily on isoto...