Alfalfa meal and chloroform extracts of the meal have increased the growth and yield of several plant species. A crystalline substance isolated from the active fraction of alfalfa meal increased the dry weight and water uptake of rice seedlings when sprayed on the foliage or applied in nutrient culture. The substance was identified as triacontanol by mass spectrometry. Sprays containing this compound also increased the growth of corn, and barley grown in soil. Authentic triacontanol produced a similar response over a wide range of concentrations on rice grown in nutrient cultures and tomatoes grown in soil.

Triacontanol (TRIA) increases the dry weight and alters the metabolism of rice (Oryza sativa L.) seedlings within 10 min of application to either the shoots or roots. This activity is prevented if octacosanol (OCTA, C28 primary alcohol) is applied with the TRIA on the roots or shoots. Triacontanol activity is also stopped if the OCTA is applied at least 1 min before the TRIA on the opposite part of the seedling.Triacontanol rapidly elicits a second messenger that moves rapidly throughout the plant resulting in stimulation of growth (dry-weight increase) and water uptake. Octacosanol also produces a second messenger that inhibits TRIA activity. We have named the putative secondary messengers elicited by TRIA and OCTA, TRIM and OCTAM, respectively. The water-soluble TRIM extracted from plants treated with TRIA increases the growth of rice seedlings about 50% more than extracts from untreated plants, within 24 h of application. Both OCTAM and OCTA inhibit the activity of TRIA but not of TRIM.The TRIA messenger was isolated from rice roots within 1 min of a foliar application of TRIA. The TRIM elicited by TRIA will pass through a 4-mm column of water connecting cut rice shoots with their roots and can also be recovered from water in which cut stems of TRIA-treated plants have been immersed. Triacontanol applied to oat (Avena sativa L.) or tomato (Lycopersicon esculentum Mill.) shoots connected to rice roots by a 4-mm water column also results in the appearance of TRIM in rice roots.

Since the discovery of the plant growth-regulating properties of TRIA, a primary alcohol that is a natural constituent of plant waxes, and its second messenger L(+)-adenosine, the rapid response kinetics to these compounds have been enigmatic (Ries and Wert, 1988;Ries, 1991).TRIA increased the dry weight, free amino acids, reducing sugars, and soluble protein of rice (Oryza sativa L.) and maize (Zea mays L.) plants within 5 min (Ries, 1991). TRIA also elicited the appearance of L(+)-adenosine in the roots of plants whose shoots were sprayed with nanomolar concentrations within 1 min (Ries and Wert, 1988). This was the first evidence that L(+)-adenosine occurred in nature. Synthetic L(+)-adenosine increased the rate of growth of rice seedlings, as measured by total dry weight gain, by more than 50% within 24 h of foliar application of 0.01 to 100.0 kg L-' (3.7 X 10-" to 10-7 M), whereas D(-)-adenosine did not affect plant growth (Ries, 1991). 49site plant part, providing it was applied 1 min prior to TRIA application (Ries and Wert, 1988). TRIA applied to oat (Avena sativa L.) or tomato (Lycopersicon esculentum Mill.) shoots connected to rice roots by a 4-mm water column also resulted in the appearance of L(+)-adenosine (TRIM) in rice roots (Ries and Wert, 1988).In an attempt to determine other physiological responses to TRIA in addition to the elicitation of L(+)-adenosine, 20-to 25-d-old tomato seedlings were sprayed with water or TRIA, and after 1 min the plants were excised. Analysis of the diffusate from the excised shoots, as determined by HPLC and atomic absorption spectrophotometry, indicated large concentration differences in organic compounds and inorganic cations (unpublished data). The largest differences were in the cation concentration of the exudate from the stump of the excised tomato plant. Thus, the objective of this research was to use this observation to further elucidate the mode of action for the rapid responses of plants to TRIA and L(+)-adenosine.We present here evidence that foliar applications of both of these compounds at nanomolar concentrations cause rapid changes in soluble Ca2+, Mg2+, and K+ concentrations within xylem exudates from the stumps of excised stems and leaves. MATERIALS A N D METHODS Plant Crowth and TreatmentTomatoes (Lycopersicon esculentum Mill. cv Sunny), cucumbers (Cucumis sativa L. cv Flurry), and maize (Zea mays L.cv Pioneer 3780) were grown in a greenhouse with approximately 16 h of supplemental light (700 pmol s-' m-', metal halide) daily. Seeds were planted in 15-cm diameter clay pots containing a soil mix, and the plants were thinned to two or three per pot 8 to 10 d after emergence. Soluble fertilizer (20 N-8.6 P-16.6 K; 1.0 g L-' Peters 20-20-20, W.R. Grace and Co., Fogelsville, PA) was applied once or twice after planting and again prior to treatment. The pots were labeled, randomized for treatments within blocks, and isolated from each other on the greenhouse bench. They were not disturbed for several hours prior to initiation of the treatments.Experiment...

The naturally occurring plant growth substance elicited by triacontanol was found to be 9-#-L(+) adenosine by physical and spectral methods . At picomolar concentrations, 9-J3-L(+) adenosine stimulated growth as determined by dry weight measurements of several plant species . Reaction of adenosine deaminase with adenosine from rice showed that small quantities of 9-f-L(+) adenosine exist in plants . We believe this is the first report of 9-R-L(+) adenosine as a natural product .

Triacontanol, a 30-carbon primary alcohol, applied in nutrient culture solutions to rice (Oryza sativa L.) seedlings at 2.3×10(-8) M (10 μg/l), caused an increase in dry weight and leaf area of the whole plants. The response could be observed as early as 3 h of treatment. It was observed at relatively high and low light intensities as well as in the dark where control plants lost but triacontanol-treated plants gained in dry weight. The dry weight gain in the dark was, however, eliminated by removing CO2 from the atmosphere. Triacontanol-treated plants also increased their content of Kjeldahl-N and contained 30% more total N per plant than controls after 6 h in the dark.

Colloidal dispersions of crystalline 1-triacontanol in water, upon foliar application to corn (Zea mays L.) seedlings, resulted in growth increases at femtomole dosages (spray concentrations as low as 1 nanogram per cubic decimeter). The maximum growth increase occurred at 100 nanograms per cubic decimeter; at both higher and lower concentrations lessened growth increase was observed. The dispersions were prepared by sonication, with control of temperature and composition. Selected surfactants, which facilitate the dispersion process, are effective at 1 percent of the 1-triacontanol composition and are nontoxic.

The effect of several analogs of 1-triacontanol (TRIA), differing in C-chain length (16-32), the position of the hydroxyl group and the terminal functional group, were tested alone and in combination with TRIA on the growth of rice (Oryza sativa L.), maize (Zea mays L.) and tomato (Lycopersicon esculentum Mill.) seedlings. Applied alone, none of the compounds caused an increase in growth; thus, chain length (30 C) and presence and position (terminal) of the hydroxyl group appear to be specific for the growth-promoting activity of TRIA. When applied simultaneously with TRIA, all analogs inhibited the response to the latter in all three test plants, whether applied in the nutrient solution, as foliar spray or by seed soaking. 1-Octacosanol inhibited the response of rice seedlings to 2.3 x 10(-8) M TRIA at concentrations as low as 2.4 x 10(-12) M. Thus preparations of TRIA and application equipment must be free from trace amounts of other long-chain compounds if they are to be used to increase plant growth.

(9). Triacontanol was first identified in alfalfa in 1933 (2), and it occurs in smali quantities in the waxes of many plant species (6) including wheat and triticale (1 1).Triacontanol applied in nutrient solutions to rice seedlings at 23 nm (10 ,ug/l) caused an increase in dry wt and leaf area of rice seedlings within 3 hr. The response was independent of light conditions. However, the response in the dark was eliminated by removing CO2 from the atmosphere. Triacontanol also increased Kjeldahl-N content up to 30%o after 6 hr in the dark (10).The present study considers the growth response of rice seedlings to triacontanol, in the dark, under various CO2 and 02 levels, different N sources, and the kinetics of the response using presentation experiments. Hoagland solution. The seedlings were supported in the solution by sponge rubber discs at the top of the cups.Prior to an experiment the seedlings were blocked by size, and a randomized complete block design with at least two blocks was used in each experiment. The experimental unit was one cup containing four seedlings. A random number table was used to assign treatments in all experiments, including those harvested when the test was initiated (zero time). Data were analyzed by analysis of variance or linear regression-correlation.Triacontanol dissolved in chloroform or chloroform alone (control), was applied to 2-cm2 filter paper squares, dried, and placed in nutrient solutions the night before an experiment. Unless otherwise specified, all treatments were made in 150 ml of 6 mM NO3-and 0.5-strength Hoagland solution with 10 ,ug/l triacontanol under dark conditions at 30 C for 6 hr.For gas treatments and respiration measurements, the seedlings were placed in 1.8-liter glass bottles with air-tight lids fitted with air inlets and outlets. Air flow was controlled by calibrated capillary flow meters and CO2 was measured with a Beckman IR-ISA CO2 analyzer. Flow rates of 100 ml min-' were used in the air versus COrfree air experiments, 50 ml min-' for the 02 concentration experiment, and 300 ml min-' for the CO2 concentration experiment. The higher flow rate was necessary to maintain similar influent and effluent CO2 levels in the atmosphere for the CO2 concentration experiment.Fixation of CO2 was further investigated by pulsing (160 ml min-' for 10 or 20 sec) radioactive "CO2 (322 ,tl/l, 0.53 ,uCi 1-') to intact leaves in a portable leaf chamber (1-cm diameter) under dark conditions after 4-hr exposure to triacontanol in the nutrient solution. After exposure to C02, the leaf sections (two/seedling) were removed, solubilized with NCS (Amersham/Searle), and the radioactivity determined by liquid scintillation procedures (7). For the NO3-versus NH4' experiment, plants were grown on NH4+ for at least I week prior to treatment. The pH of the NH4' nutrient solution was adjusted daily to that of the NO3-solution.Five ,ug/l 2-chloro-6-(trichloromethyl) pyridine was added to the NH4' and N03-nutrient solutions (N-serve, Dow Chemical Co.) to prevent nitrification of ...