Planit Physiol. (1969) 44, 1371-1377 PLANT Tol. 44 No. 10 PHYSIOLOGSY October 1969 Comparative Studies of Effect of Auxin and Ethylene on Permeability and Synthesis of RNA and Protein' Joseph A. Sacher and Seppo 0. Salminen2 Department of Botany, California State College, Los Angeles, California 90032 Received January 7, 1969. Abstract. The effects of ethylene on permeability and RNA and protein synthesis were assayed over a 6 to 26 hr period in tissue sections from avocado (Persea gratissima Gaertn. F., var. Fuerte), both pulp and peel of banana (Musa sapientum L., var. Gros Michel), bean endocarp (Phaseolus vulgaris L., var. Kentucky Wonder Pole beans) and leaves of Rhoeo discolor. Ethylene had no effect on permeability in 4 of the 5 tissues, but sometimes enhanced solute uptake in banana peel; it had either no effect or an inhibitory effect on synthesis of RNA and protein in sections from fruits of avocado and banana. Auxin (a-naphthalene acetic acid) stimulated synthesis of RNA and protein in bean endocarp and Rhoeo leaf sections, whereas ethylene inhibited both basal and auxin-induced synthesis. It is conoluded that in these tissues the auxin effect is not an ethylene effect. The results of many investigations have shown production above the threshold level precedes onset that auxin may stimulate synthesis of ethylene of the respiratory climacteric by 4 hr in banana (9). (12, 16). It was concluded that auxin-induced syn- For banana tissue, however, marked changes in thesis of ethylene is the basis for the effect of high membrane permeability are initiated about 2 days concentrations (10-5 M or greater) of auxin on before the onset of the climacteric, manifested first inhibition of growth of etiolated sections of pea and by a gradual and then a rapid increase in the per- sunflower stems (10). The stimulation of RNA centage of the tissue volume that is free space synthesis in senescent bean abscission zone explants (22,25). This evidence was considered supportive by ethylene has been reported (1, 2), and led to the of the Blackman and Parija (6) hypothesis that suggestion that some effects of auxin on RNA syn- permeability changes are causative of the climacteric. thesis may be owed to auxin enhancement of ethvlene The onset of the increase in free space in avocado, synthesis. however, coincides with the onset of the respiratory In line with the facts that einhanced ethylene increase (27, and Wong and Sacher, unpublished synthesis is associated with the respiratory climac- data). Other workers have also reported a large teric of some fleshy fruits, and that application of increase in leakage or free space during the climac- ethylene initiates the climacteric, ethylene has been teric in avocado (4,20,29) and banana (3,21). regarded as a product of ripening by some (5) and as a ripening hormone by others (7,18). Some investigators have examined the causal Ethylene relations between ethylene and membrane perme- abilitv. Isolated mitochondria are induced to swell 1 Supported by National Science Foundation Grant by a high concentration of ethylene (15). Also, GB-6985. ethylene affects the rate of volume change induced 2 Present address: Department of Agronomy, Uni- in mitochondria by ADP and ATP and increases versity of California, Riverside, ATPase activity of isolated mitochondria from a 1371 1372 PLANT PHYSIOLOGY variety of tissues. For this effect the integrity of with humidified, ethylene-free air or an air-ethylene the mitochondria is essential, as ethylene does not mixture which provided a turnover of the gaseouis affect activity of the purified enzyme (19). Ethylene environment about every 1.5 min. Periodic moni- had no effect on leakage of solutes from pea stem toring with a Beckman GC-4 gas chromatograph sections (10), while treatment of canteloupe tissue showed no detectable ethylene (<0.01 ppm) in the discs with ethylene caused an increased permeability continuous flow-control flasks. The ethylene mix- to water (26). ture provided was 30 ppm. The present investigations were conducted to After incubation at 250 the tissue was washed for ascertain the effects of ethylene on RNA and protein 1 hr in running tap water, blotted and placed into 3 synthesis and membrane permeability in tissue sec- volumes of absolute ethanol. Extraction and assay tions from 5 kinds of fruit and leaf tissues. Ethylene of RNA and protein and radioassay of dual labeled had no adverse effect on membrane properties of samples were conducted as described previouslv (23). these tissues to uridine or L-leucine, an,d either had no effect on RNA and protein synthesis or cauised Results inhibition of synthesis in some experiments. The results also indicate that auxin-induced synthesis of Effect of Ethylene on Memibrane Permeability. RNA and protein is not owed to an effect of auxin We have found that ethylene (7-45 ppm) has no on ethylene synthesis. effect on permeability of sections of Rhoeo leaves, bean endocarp or banana or avocado pulp tissue to Materials and Methods labeled uridine and leucine. An exception has been observed in the case of banana peel, which will be Tissue sections were prepared from fruits of taken up later. Data for uptake of labeled leucinie avocado (Persea gratissimla Gaertn. F., var. Fuerte), are shown in table 1. Uptake is measured as radio- banana (Musa sapientuin L., var. Gros Michel) and activity retained in both the ethanol soluble and pole beans (Phaseolus vzdlqaris L., var. Kentucky insoluble fractions of tissue homogenates after aln Wonder) and leaves of Rhoco discolor. exhaustive washing in running tap water to remove A whole fruit of avocado or banana was taken on radioactivity from the free space. Sections from stuccessive days prior to the onset of the climacteric. these 4 tissues tundergo a 4- to 50-fold increase in Tissue discs were sectioned (1.5 X 5 mm), washed solute uptake during a 15-hr period of aging (23), for 20 min in running tap water, blotted and duplicate or triplicate 1 g samples weiglhed out for each treat- ment. Sections 1.5 mm thick were similarly pre- Table I. Effect of Etlylene on Permeability of Sectionis pared from bean endocarp and the basal midrib Fron Lcaf and Frutit Tissuies to Leucine-U-l4C region of matuire Rhoeo leaves. For radioassay of Samples (1 g fresh wt) of tissue sections incubated RNA and protein synthesis, based onl the rate of in 125 ml Erlenmeyer flasks on filter paper wetted with incorporation of labeled precutrsors, the tissue sections 2.5 ml of dual labeled solutions containing 25 j,g/ml sulfate and to 6.1. Incubations were arranged on a dry filter paper in 125 ml flasks streptomycin buffered pH were 15 hr for bean, avccado, and banana sections, and and then 2.5 ml of the dual labeled were soluitions 6 hr for Rhoco leaf sections, which \ere pre-treated added. For trapping CO. during the incubations as described in table III. For bean and Rhoeo tissue 1.0 ml 10 % KOH was added to a center well and ethylene was added with a syringe through serum caps a fluted paper added. In all experiments the thick- to a final concentration of 7 or 45 ppm and 1 ml of ness of the sections allowed the upper cut surface to 10 % KOHI with fluted paper added to the flask be freely exposed to the gaseous environment, and center well. Banana and avocado sections were pro- the lower surface in contact with the labeled solution; vided with a 90 cc/Tiin flow of humidified air or a 30 ppm ethylene-air mixture. NAA was 10 ,u.g/ml. therefore it was not necessary to shake the flasks. The incubation solutions contained 25 ,ug/ml strepto- Total uptake of leucine-U-14C' mycin sulfate buffered to pH 6.1 with phosphate Expt. Tissue Control Ethylene NAA buffer and dual labeled with 5 uc uridine-5-T (28.8 C/mM) and 1 ,uc of iA-leucine-U-'4C (305 dp.X 10-3 mc/mai). For some experiments auxin (a-naphtha- 1 Bean 1730 ± 80 1722 ± 12 1820 ± 170 2 Rhoeo 1195 ± 17 1141 ± 11 1137 ± 10 lene acetic acid, NAA) was added to a final con- 3 Avocado 1248 ± 41 1296 ± 17 centration of 10 sg/ml. 4 Avocado 1440 -± 20 1470 ± 30 Incubation in Ethylene. Ethylene was provided 5 Banana 1280 ± 12 1350 ± 30 using either a closed flask or a continuous flow 6 Banana 1628 ± 3 1627 ± 20 ... system. For the former the flasks were sealed with 7 Banana peel 990 + 70 1027 ± 29 serum caps through which a volume of an air-,ethylene 1Measured as radioactivity in the ethanol soluble and mixture could be injected to give a final concentra- insoluble fractions of homogenates from tissue sec- tion of 7 or 45 ppm ethylene. An equal volume of tions which had been incubated in labeled solutions air was injected into the control flasks. In the and then washed in running water for 1 hr. Means continuous flow system the flasks were ventilated and standard errors for duplicate or triplicate samples. SACHER AND SALMINEN-ETHYLENE EFFECTS: PERMEABILITY AND RNA SYNTHESIS 1373 Table II. Coniparison of Effects of Ethylene and Auxin on Synthesis of RNA and Protein by Sections of Bean Endocarp Tissue sections were incubated for 15 hr as described in table I. NTAA was 10 ,g/ml and ethylene was 45 ppm. RNA' Proteinl dpm incorporated % o % of % of dpm total uptake zero zero Treatment Uridine Leucine tinme time ratio /Lg/g ,g1g Zero time ... ... ... ... 593 + 15 100 3816 ± 31 100 Water 0.224 0.005 0.705 + 0.005 486 ± 3 82 3523 ± 63 92 Ethylene 0.217 ± 0.012 0 0.678 ± 0.007 - 5 % 458 ± 0 77 3315 ± 0 87 NAA 0.570 + 0.030 + 139 % 0.899 ± 0.006 + 25 % 700 ±- 32 118 4336 ± 216 114 1 Means and standard errors for duplicate samples of tissue.