Red Light-inhibited Mesocotyl Elongation in Maize Seedlings: I. The Auxin Hypothesis.

Red light-inhibited mesocotyl elongation, which occurs in intact Zea mays L. seedlings, was studied in excised segments which included the coleoptile (or parts therefrom) and apical centimeter of the mesocotyl. Experiments took into account, first, the ability of the segments to regenerate auxin supply sites, and, second, that auxin uptake can be greatly reduced if there is no cut surface, apical to the elongating cells, to act as a port of entry. In all cases, auxin completely reversed the inhibition of elongation by light. The results support the hypothesis that light regulates mesocotyl elongation by controlling auxin supply from the coleoptile. Sucrose concentration had no effect on auxin reversal of light-inhibited elongation, but relatively high concentrations of gibberellic acid (10 mum) could substitute for auxin in this system.

Mechanical inhibition of hypocotyl elongation induces radial enlargement: implications for cytokinin action.

Excised elongating segments from 3-day-old soybean (Glycine max var. Wayne) seedlings radially enlarged when auxin-promoted elongation was mechanically inhibited. Growth was similar to segments treated with auxin plus cytokinin. This observation suggests that cytokinin does not necessarily directly "reorient" cell enlargement. Cytokinin-induced radial cell growth may be a secondary effect of cytokinin's inhibition of auxin-promoted elongation.

Auxin-regulated Wall Loosening and Sustained Growth in Elongation.

It is proposed that auxin regulates and coordinates both wall loosening and the supply of wall materials in elongation. The tenets of the proposal allowed testable predictions. It was determined that, if the cell walls of Glycine max L. var. Wayne hypocotyl segments are maintained in a loosened state (by excising the segments directly into pH 4 medium), exogenous auxin induced only the second response. It was also predicted and confirmed that elongating systems, e.g. pea epicotyl, with certain early auxin-induced growth kinetics (an initial high non-steady-state rate followed immediately by a drop to a lower steady-state rate) would show a transient second response (in addition to the usual transient first response) when stimulated by pH 4 medium. Finally, it is pointed out that recent results which establish the existence of auxin-induced elongation-associated proteins support the proposition that auxin coordinates wall loosening and the supply of wall materials in elongation.

Separation of two responses to auxin by means of cytokinin inhibition.

A continuous growth apparatus was used to measure the effect of cytokinin on auxin-induced elongation. The soybean hypocotyl segments elicited a bi-phasic response to auxin that appeared to be two overlapping responses. The first response, which began 12 min after auxin addition, was not inhibited by cytokinin, even after long preincubation in cytokinin, but the second response to auxin, which began about 30 min after auxin addition, was completely inhibited by cytokinin. Such overlapping reactions are shown, depending on the amount of overlap, to yield a variety of summation reactions, many of which resemble rate-time curves that have been previously reported. We have shown that the transient first phase of auxin-induced elongation is very similar to acid-activated growth, while the second phase is long lasting and very likely identical to the long-term response to auxin, as extensively studied in Avena, soybean, and other elongating cells.

Characterization of 5.8S ribosomal ribonucleic acid in Neurospora crassa.

Neurospora crassa ribosomes contain a species of ribonucleic acid (RNA) of molecular weight 54,000, similar to 5.8S ribosomal RNA previously described for other eukaryotic organisms. The 5.8S RNA from N. crassa was found to be released by heat treatment at 60 C from 25S ribosomal RNA but not from 18S ribosomal RNA. The base composition of N. crassa 5.8S RNA was similar to that of 5.8S RNA from Saccharomyces cerevisiae, but differed from animal 5.8S RNA. During the course of this study, it was discovered that N. crassa 25S ribosomal RNA had a number of internal cleavages that may exist in vivo.

Mitochondrial tyrosyl transfer ribonucleic Acid in soybean seedlings.

Soybean seedlings were examined for the presence of mitochondrial tRNA. Tyrosyl transfer tRNAs from whole cells, from a well characterized mitochondrial preparation, and from a snake venom phosphodiesterase-treated mitochondrial preparation, were compared by reverse phase chromatography. It was concluded that none of the three previously reported tRNA(Tyr) species were mitochondrial. Rather, a fourth tRNA(Tyr) species, eluting somewhat later, was of mitochondrial origin. Mitochondrial tRNA(Tyr) was chromatographically similar to Escherichia coli tRNA(Tyr).

The fractionation of transfer ribonucleic Acid from roots of pea seedlings.

Isoaccepting transfer RNA species for several amino acids were fractionated by reverse phase column chromatography. Transfer RNA from dividing cells of pea (Pisum sativum) root was compared to that from nondividing cells, and no relative quantitative or qualitative differences were noted for the transfer RNA species for leucine, lysine, proline, threonine, methionine, serine, and phenylalanine. However, certain artifactual differences for serine and phenylalanine were noted. Quantitative differences were observed in tyrosyl-transfer RNA's. Ribonuclease action on tRNA did not contribute to the tRNA species observed.

Comparison of Auxin-induced and Acid-induced Elongation in Soybean Hypocotyl.

Acid-induced growth was compared to auxin-induced growth. After a transient pH 4-induced increase in the elongation rate was completed, auxin could still induce an enhanced rate of elongation in soybean (Glycine max) hypocotyl segments. This auxin response occurred both when the medium was changed to pH 6 before auxin addition, and when the auxin was added directly to the pH 4 medium. This postacid response to auxin was persistent, and quite unlike a postacid response to acid, which was again shortlived. One mm N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7) inhibited the first response to auxin (the first response to auxin being similar to the acid response), but not the second response. This did not appear to be simply a hydrogen ion neutralizing effect, however, since a 50-fold increase in buffer concentration at pH 6 did not inhibit the first response. Decrease in the pH of the external medium, previously shown to occur with excised soybean hypocotyl segments, was not affected by auxin. Furthermore, this pH drop, during which the cells appear to be adjusting their external pH to about 5.4, did not result in an increased rate of elongation. Addition of auxin after the equilibrium pH had been attained did not alter the pH, but it did increase the rate of elongation, eliciting a normal auxin response. It was concluded that hydrogen ions do not mediate in long term auxin-induced elongation in soybean hypocotyl.

Two elongation responses to auxin respond differently to protein synthesis inhibition.

The first and second responses to auxin react differently to the inhibition of protein synthesis by cycloheximide. It was determined that the protein with the shortest half-life, among the several necessary for the first response, is different from its counterpart among the several necessary for the second response. Specifically, the protein half-lives are 28 minutes and 11 minutes for the first and second responses, respectively.

The Isolation and Characterization of Adenosine Monophosphate-rich Polynucleotides Synthesized by Soybean Hypocotyl Cells: Their Relation to Messenger Ribonucleic Acid.

Plant ribonucleic acids which have high adenosine monophosphate concentrations were studied. Purified deoxyribonucleic acid-like ribonucleic acid and tenaciously bound ribonucleic acid fractions both contained poly-adenosine monophosphate sequences (those from the latter being longer than those from the former); without these poly-adenosine monophosphate sequences their base compositions were the same. The average poly-adenosine monophosphate sequence from purified tenaciously bound ribonucleic acid was 160 residues long, as measured by gel electrophoresis. However, base hydrolysis and chromatography indicated one 3'-nucleoside (adenosine) per 71 nucleotides, giving a chain length of 72 residues. The dominant species in the cytoplasm, as measured by radioactive precursor incorporation, was tenaciously bound ribonucleic acid, whereas deoxyribonucleic acid-like ribonucleic acid was present in greater amounts in the nucleus. This work provides evidence that deoxyribonucleic acid-like ribonucleic acid and tenaciously bound ribonucleic acid represent forms of messenger ribonucleic acid in soybean, with deoxyribonucleic acid-like ribonucleic acid residing in the nucleus, perhaps as the messenger ribonucleic acid precursor, and tenaciously bound ribonucleic acid residing, as the active messenger ribonucleic acid, in the cytoplasm.

Acetylene reduction (nitrogen fixation) and metabolic activities of soybean having various leaf and nodule water potentials.

An apparatus was designed that permitted acetylene reduction (N(2) fixation) by root nodules to be measured in situ simultaneously with net photosynthesis, dark respiration, and transpiration of the shoot in soybean plants (Glycine max [L.] Merr. var. Beeson). Tests showed that acetylene reduction was linear with time for at least 5 hours, except for the first 30 to 60 minutes. Endogenous ethylene production did not affect the measurements. Successive determinations of acetylene reduction could be made without apparent aftereffects on the plant.This apparatus was used to investigate the effects of soil flooding and desiccation on acetylene reduction under conditions where soil, nodule, and leaf water potentials could be measured. No acetylene reduction was detectable in flooded soil or in soil desiccated to a water potential of -19.5 bars. Between these extremes, acetylene reduction displayed a sharp optimum. Removing the soil eliminated the inhibitory effects of flooding, suggesting that rates of gas exchange were restricted between the nodules and the atnosphere at soil water potentials above -2 bars.As the soil desiccated further, acetylene reduction decreased, and the decrease was correlated with decreases in photosynthesis and transpiration. Although dark respiration was inhibited, it was not affected to the extent that acetylene reduction, photosynthesis, or transpiration were. Consequently, it was concluded that photosynthesis, transpiration, or some direct effect on the nodules other than that caused by respiration were most likely to account for the inhibition of acetylene reduction at soil water potentials below -2 bars.

Limitation of acetylene reduction (nitrogen fixation) by photosynthesis in soybean having low water potentials.

The role of photosynthesis and transpiration in the desiccation-induced inhibition of acetylene reduction (nitrogen fixation) was investigated in soybean (Glycine max [L.] Merr. var. Beeson) using an apparatus that permitted simultaneous measurements of acetylene reduction, net photosynthesis, and transpiration. The inhibition of acetylene reduction caused by low water potentials and their aftereffects could be reproduced by depriving shoots of atmospheric CO(2) even though the soil remained at water potentials that should have favored rapid acetylene reduction. The inhibition of acetylene reduction at low water potentials could be partially reversed by exposing the shoots to high CO(2) concentrations. When transpiration was varied independently of photosynthesis and dark respiration in plants having high water potentials, no effects on acetylene reduction could be observed. There was no correlation between transpiration and acetylene reduction in the CO(2) experiments. Therefore, the correlation that was observed between transpiration and acetylene reduction during desiccation was fortuitous. We conclude that the inhibition of shoot photosynthesis accounted for the inhibition of nodule acetylene reduction at low water potentials.

ATP-Dependent Calcium Transport in Plasmalemma Preparations from Soybean Hypocotyls : EFFECT OF HORMONE TREATMENTS.

A plasmalemma-enriched membrane preparation from etiolated soybean (Glycine max L., cv. Wayne) hypocotyls possesses an active ATP-dependent calcium pump which leads to calcium accumulation when assayed by the methods of Gross and Marmé (1978, Proc Natl Acad Sci USA 75: 1232-1236). Two-hour treatment of segments from the elongating zone of the hypocotyl with growth-promoting concentrations of indoleacetic acid gives up to 100 percent increase in the calcium transport activity. Conversely, similar pretreatment with zeatin or other cytokinins is inhibitory. In the meristematic and maturing zones of the hypocotyl, zeatin has the opposite effect of promoting calcium transport activity. One facet of cell-growth regulation may lie with hormonally mediated changes in efflux pumping of calcium.

Red Light-inhibited Mesocotyl Elongation in Maize Seedlings: II. Kinetic and Spectral Studies.

Red light induces two distinct inhibition responses in mesocotyls of etiolated corn seedlings. A light dose of 10 nanoeinsteins per square centimeter is saturating for the more sensitive response, whereas doses above 1,000 nanoeinsteins per square centimeter are required to exceed the threshold sensitivity of the less sensitive one. The sensitive response can be detected within 20 minutes of the onset of illumination whereas the other response does not become apparent until more than 4 hours after the beginning of irradiation. The reciprocity law is valid for the first response, but probably not for the second. An action spectrum for the first response shows two maxima, one at 640 nanometers and the other between 660 and 670 nanometers, with a pronounced minimum near 650 nanometers. The effects both of 640 and 665 nanometers of light were reversible by far red light, but doses of far red required for full reversibility were almost three orders of magnitude greater than the doses of red required either to saturate the initial inhibition or to reverse the effect of far red light. The results suggest that corn may contain a red-absorbing pigment other than phytochrome which in some way interacts with phytochrome in the inhibition of mesocotyl elongation by red light.

The inhibition of soybean metabolism by cadmium and lead.

Lead (300 mum) and cadmium (18 mum) inhibit pod fresh weight in soybeans (Glycine max L.) by 35%. Eighteen micromolar cadmium caused a 30% decline in nitrogenase activity by day 52 (the day on which maximum activity was measured) and a 71% inhibition by day 59. The heavy metals depressed photosynthetic rates; when photosynthesis was depressed by 60%, as measured on the day of peak photosynthesis activity, carbohydrate did not accumulate in the nodules. The reduction of pod fresh weight correlated with the effect of lead and cadmium on several other aspects of plant metabolism (shoot, root, leaf, and nodule dry weight; nodule ammonia, protein and carbohydrate content).

Fusarium tricinctum T-2 Toxin Inhibits Auxin-promoted Elongation in Soybean Hypocotyl.

T-2 toxin, a mycotoxin produced by Fusarium tricinctum, inhibited elongation of excised hypocotyl sections of Glycine max var. Hawkeye 63. Auxin-promoted elongation was inhibited more severely than was control elongation, and a 1 hour preincubation of 5 mum toxin prevented the induction of a faster rate of elongation by auxin. While the inhibition of elongation by cytokinin was similar to that of the toxin, the mode of action of the two compounds appeared to be different, i.e. their effects on elongation were additive, and only kinetin promoted radial enlargement. Toxin treatment did not diminish cytokinin-induced radial enlargement. The properties of the plasma membrane, as measured by electrolyte leakage, were not affected by the toxin.

Auxin Has No Effect on Modification of External pH by Soybean Hypocotyl Cells.

The cellular adjustment of the pH of the external environment of soybean (Glycine max) hypocotyl elongating cells, frequently assumed to be hydrogen ion secretion when the pH is lowered, is unaffected by auxin. These elongating cells actively adjust the external hydrogen ion concentration (from any pH in the range of 4-8) to pH 5.4 + 0.2. This pH adjustment occurs in a medium which does not contain potassium. Growth-optimum auxin concentrations have no effect on cellular pH adjustment of the external medium, whether added at the beginning of the experiment or after the equilibrium pH is attained. The pH adjustment by the cells occurs rapidly and in spite of the presence of a cuticle.

Additional evidence for separable responses to auxin in soybean hypocotyl.

Additional evidence for two separable responses to auxin is presented. The average of 24 control experiments indicated lag times of 12.4 and 35.4 min, and maximum rates of 0.57 and 0.54 mm hr(-1), for the first and second response, respectively. The auxin analog 4-azido-2-chlorophenoxyacetic acid increased the lag time of the second response (but not the first), resulting in the temporal separation of the two responses. Plots of elongation rates against time, taken from the literature, allowed the characterization of the two responses in monocotyls and dicotyls. Study of published rate-time elongation curves showed that the maximum rate of the first response is frequently greater than the maximum rate of the second response; however, the maximum rate of the second response has not yet been shown to exceed the maximum rate of the first response.