Triacontanol: a potent plant growth regulator in agriculture

Abstract

Triacontanol (TRIA) is a natural plant growth regulator found in epicuticular waxes. It is used to enhance the crop production in millions of hectares, particularly in Asia. Quite a number of researchers have reported the TRIA-mediated improvement in growth, yield, photosynthesis, protein synthesis, uptake of water and nutrients, nitrogen-fixation, enzymes activities and contents of free amino acids, reducing sugars, soluble protein, and active constituents of essential oil in various crops. Expectedly, TRIA enhances the physiological efficiency of the cells and, thus, exploits the genetic potential of plant to a large extent. In fact, TRIA increased free amino acids, reducing sugars, and soluble protein of rice (Oryza sativa L.) and maize (Zea mays L.) within 5 min. TRIA elicited the appearance of L(+)-adenosine within 1 min in the roots of plants, the shoots of which were sprayed with nanomolar concentrations of TRIA. TRIA and octacosanol (OCTA), the primary alcohols, are ubiquitous in the environment. OCTA was reported to inhibit the activity of TRIA in the seedlings of rice (Oryza sativa L.) at equimolar concentrations; and both TRIA and OCTA elicited a second messenger, known as OCTAM and triacontanol second messenger (TRIM), respectively. TRIA rapidly increases the ratio of L(+)- to D(–)-adenosine, probably at the tonoplast. However, it is to be resolved as to how TRIA elicits L(+)-adenosine and what is the source of L(+)-adenosine in plants. Based on known metabolic processes, de novo synthesis of L(+)-adenosine is unlikely, because of the rapidity of the response. TRIA-mediated increase in dry matter production could influence the inter-relationship between primary and secondary metabolism, leading to increased biosynthesis of secondary products. Various studies present strong evidences that application of TRIA applied either to the root medium or to leaves enhanced the growth and yield of vegetables and other crops, including agronomic and horticultural crops as well as medicinal and aromatic crop plants under normal and adverse conditions. However, further investigations are required to elucidate the possible role of TRIA on plant growth regulation, physiological activities and secondary metabolite biosynthesis regarding medicinal and aromatic plants subjected to abiotic stress. The present review covers the pivotal role of TRIA in plant growth and development, its mode of action and its significance in improving the crop productivity and quality of agricultural crops.     

Triacontanol and Its Second Messenger 9-beta-l(+)-Adenosine as Plant Growth Substances

Triacontanol (TRIA), a common constituent of plant waxes, was first shown in 1977 to be an active growth substance which at nanomolar concentrations increased the growth and yield of crops. TRIA is used to increase crop yields on millions of hectares, particularly in Asia. Many investigators have shown that it affects several basic metabolic processes including photosynthesis, nutrient uptake, and enzyme activity. However, the initial site of action has not been elucidated. TRIA rapidly elicits a second messenger (TRIM) in rice (Oryza sativa L.), which at nanomolar concentrations causes plants to respond in a manner similar to TRIA. TRIM has been identified as 9-β-l(+)-adenosine (9H-purin-6-amine, 9-β-l-ribofuranosyl). During the process of isolating and identifying 9-β-l(+)-adenosine, it was shown that this enantiomer, which previously has not been reported as occurring in nature, made up about 1% of the total adenosine pool in roots from untreated rice seedlings.     

Specificity of 1-triacontanol as a plant growth stimulator and inhibition of its effect by other long-chain compounds

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.Abbreviation TRIA 1-triacontanol     

Metabolic adaptation to flooding stress in upland and lowland rice seedlings

Ability of metabolic adaptation in upland and lowland rice (Oryza sativa L.) seedlings to flooding stress was compared. Flooding stress increased alcohol dehydrogenase (ADH) activity and ethanol concentration in shoots and roots of the upland and lowland rice seedlings. The difference in ADH activity and ethanol concentration in shoots between the upland and lowland rice was not apparent. However, both ADH activity and ethanol concentration in roots of the lowland rice were 2-fold greater than those in roots of the upland rice, suggesting that flooding-induction of ethanolic fermentation in lowland rice roots may be significantly greater than that in the upland rice roots. Since flooding often causes the anaerobic conditions in rooting zone than aerial part of plants and ethanolic fermentation is essential to survive in the anaerobic conditions, the ability of metabolic adaptation in lowland rice seedlings to flooding stress may be greater than that in upland rice seedlings.     

Rapid Growth and Apparent Total Nitrogen Increases in Rice and Corn Plants following Applications of Triacontanol

Triacontanol (TRIA) increased fresh and dry weight and total reducible nitrogen (total N) of rice (Oryza sativa L.) seedlings within 40 minutes. Increases in total N in the supernatants from homogenates of corn (Zea mays L.) and rice leaves treated with TRIA for one minute before grinding occurred within 30 and 80 minutes, respectively. The source for the increase was investigated utilizing atmospheric substitution and enrichment and depletion studies with ¹⁵N. The increase in total N in seedlings was shown to be independent of method of N analysis and the presence of nitrate in the plants. Automated Kjeldahl determinations showing apparent increases in N composition due to TRIA were shown to be correlated with hand Kjeldahl, elemental analysis, and chemiluminescent analysis in three independent laboratories. TRIA did not alter the nitrate uptake or endogenous levels of nitrate in corn and rice seedlings. Enrichment experiments revealed that the total N increases in rice seedlings, in vivo, and in supernatants of corn leaf homogenates, in vitro, are not due to atmospheric N₂. TRIA increased the soluble N pools of the plants, specifically the free amino acid and soluble protein fractions. No differences in depletion or enrichment of ¹⁵N incorporated into soluble and insoluble N fractions of rice seedlings could be detected on an atom per cent ¹⁵N basis. The apparent short-term total N increases cannot be explained by current knowledge of major N assimilation pathways. TRIA may stimulate a change in the chemical composition of the seedlings, resulting in interference with standard methods of N analysis.     

Influence of 9-β-l(+)-adenosine on malate dehydrogenase activity in rice

The activity of malate dehydrogenase (MDH, EC 1.1.1.37) in rice ( Oryza sativa L. cv. California M-201) roots was increased within 25 min by a foliar application of 37 n M of 9-β- l (+)-adenosine [ l (+)-adenosine]. A similar concentration of 9-β- d (-)-adenosine [ d (-)-adenosine] did not affect MDH. Triacontanol (2.7 n M ) which elicits l (+)-adenosine in plants also increased MDH activity in roots of rice seedlings. whereas neither octacosanol nor a mixture of equimolar concentrations of triacontanol and octacosanol had any effect on MDH activity. l (+)-Adenosine increased MDH activity of rice seedlings grown at 10.20 and 40°C. The effect was measurable 24 h after application at 10 and 20°C, but not 6 h after treatment at 40°C. Ninety minutes after l (+)-adenosine was applied to the foliage of plants grown at 10, 20 and 40°C, MDH activity in the roots was 40, 30 and 9% more, respectively, than in the untreated controls. The concentration of water soluble protein was also increased by l (+)-adenosine and was positively correlated with MDH activity. When measured 90 min after application of l (+)-adenosine at different times of day, MDH activity and dry weight were increased most when l (+)-adenosine was applied 9 and 12 h after the lights came on in a 16-h photoperiod. The optimum light intensity for the response of rice to l (+)-adenosine, as measured by MDH activity, was 450 μmol m⁻² s⁻¹.     

Effects of Capsaicin on Plant Growth

Rice (Oryza sativa L.) seedlings inhibited the growth of hypocotyls and roots of cress (Lepidium sativum L.) seedlings when both seedlings were grown together. Two growth inhibiting substances were found in the culture solution in which rice seedlings were hydroponically grown for 14 d. One growth inhibitor was further purified. This suggests that the rice seedlings may produce growth inhibiting substances, acting as allelochemicals to other plants, and release them from their roots into the environment.     

Organ-specific analysis of the anaerobic primary metabolism in rice and wheat seedlings. I: Dark ethanol production is dominated by the shoots

During anaerobiosis in darkness the main route for ATP production in plants is through glycolysis in combination with fermentation. We compared the organ-specific anaerobic fermentation of flooding-tolerant rice (Oryza sativa) and sensitive wheat (Triticum aestivum) seedlings. A sensitive laser-based photoacoustic trace gas detection system was used to monitor emission of ethanol and acetaldehyde by roots and shoots of intact seedlings. Dark-incubated rice seedlings released 3 times more acetaldehyde and 14 times more ethanol than wheat seedlings during anaerobiosis. Ninety percent of acetaldehyde originated from shoots of both species. In comparison to wheat shoots, the high ethanol production of rice shoots correlated with larger amounts of soluble carbohydrates, and higher activities of fermentative enzymes. After 24 h of anaerobiosis in darkness rice shoots still contained 30% of aerated ATP level, which enabled seedlings to survive this period. In contrast, ATP content declined almost to zero in wheat shoots and roots, which were irreversibly damaged after a 24-h anaerobic period. When plants were anaerobically and dark incubated for 4 h and subsequently transferred back to aeration, shoots showed a transient peak of acetaldehyde release indicating prompt re-oxidation of ethanol. Post-anoxic acetaldehyde production was lower in rice seedlings than in wheat. This observation accounts for a more effective acetaldehyde detoxification system in rice. Compared to wheat the greater tolerance of rice seedlings to transient anaerobic periods is explained by a faster fermentation rate of their shoots allowing a sufficient ATP production and an efficient suppression of toxic acetaldehyde formation in the early re-aeration period.Angelika Mustroph and Elena I. Boamfa contributed equally to the paper.     

Assessment of Allelopathic Potential of Root Exudate of Rice Seedlings

To determine the allelopathic potential of root exudate from early developmental stage of rice (Oryza sativa L), 6-d-old seedlings of eight cultivars were grown with 3-d-old alfalfa (Medicago sativa L.), cress (Lepidium sativum L.) or lettuce (Lactuca sativa L.) seedlings in Petri dishes under controlled condition. All rice cultivars (cv. Norin 8, Kamenoo, Nipponbare, Kinuhikari, Koshihikari, Sasanishiki, Yukihikari and Hinohikari) inhibited growth of roots, shoots and fresh mass of alfalfa, cress and lettuce seedlings. Effectiveness of cv. Koshihikari was the greatest and more than 60% inhibition was recorded in all bioassays, followed by that of cv. Norin 8 of which effectiveness was more than 40%.     

Stimulation of ATPase activity in barley (Hordeum vulgare) root plasma membrane after treatment of intact tissues and cell free extracts with triacontanol

Ca²⁺- and Mg²⁺-dependent ATPase activity (EC 3.6.1.3) in a plasma membrane-enriched fraction increased rapidly after in vivo application of physiologically active concentrations of triacontanol (TRIA) to the roots of barley ( Hordeum vulgare L. cv. Conquest) seedlings. Ca²⁺- and Mg²⁺-dependent ATPase activity was 64 and 85% higher, respectively, in the roots of seedlings germinated in the presence of growth-promoting concentrations of TRIA compared to controls. The increase in vivo was concentration dependent, with the greatest increase obtained at 2.3 n M TRIA. Maximal stimulation of ATPase activity of excised tissue treated with TRIA coincided with the temperature at which the barley was grown. At this temperature the plasma membrane is primarily in a mixed gel/liquid crystalline state. Pretreatment of barley roots with cyclohexamide did not alter ATPase stimulation by TRIA. Two to three times more [¹⁴C]-TRIA (mg membrane protein)⁻¹ was found associated with plasma membrane-enriched vesicles treated with TRIA than with vesicles enriched for mitochondrial membranes or for vesicles enriched for tonoplast, Golgi and rough endoplasmic reticulum. Both Ca²⁺- and Mg²⁺-dependent ATPase activity increased by 40–60% within 30 min of the addition of 2.3 n M TRIA to cell-free extracts of barley roots. The addition of octacosanol, the C₂₈ analogue of TRIA, to cell-free extracts did not affect metal-dependent ATPase activity. Consistent with many studies in the green-house, simultaneous additions of equimolar amounts of TRIA and octacosanol to cell-free extracts resulted in inhibition of ATPase stimulation by TRIA. TRIA may directly affect plasma membrane function in barley roots.     

Isolation and characterization of triacontanol-regulated genes in rice (Oryza sativa L.): possible role of triacontanol as a plant growth stimulator

Triacontanol (TRIA) is a saturated long-chain alcohol that is known to have a growth promoting activity when exogenously supplied to a number of plants. In this study, dry weight, protein and chlorophyll contents of rice seedlings were increased by foliar application of TRIA. Leaf net photosynthesis rate (Pn) was increased very quickly and persistently at a given photon flux density (PFD). The TRIA-regulated genes in rice were isolated from cDNA library by differential screening with probes generated from the forward- and reverse-suppression subtractive hybridization (SSH) populations and confirmed by Northern blot. Sequence analysis revealed that most of the up-regulated genes encoded the photosynthetic and photorespiratory proteins. Two down-regulated genes were identified as those encoding an ABA- and stress-related protein and a wounding-related protein. These results suggested that TRIA up-regulated the photosynthesis process and suppressed stresses in rice plants. Time-course profiles of expression of rbcS isogenes suggested the complex mechanisms involved in the regulation of photosynthesis promoted by TRIA.     

Growth responses of rice seedlings to triacontanol in light and dark

Triacontanol, a 30-carbon primary alcohol, applied in nutrient culture solutions to rice (Oryza sativa L.) seedlings at 2.3×10^-8 M (10 g/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.     

Stimulation of ATPase activity in barley (Hordeum vulgare) root plasma membranes after treatment with triacontanol and calmodulin

Triacontanol (TRIA) treatment of plasma membrane-enriched vesicles from barley ( Hordeum vulgare L., cv. Conquest) roots resulted in stimulation of membrane-associated, divalent cation-dependent ATPase activity (EC 3.6.1.3). The stimulation at physiologically active concentrations of TRIA (10⁻¹¹–10⁻⁹ M ) occurred only when the vesicles were treated with TRIA in the presence of calmodulin. Octacosanol, the C₂₈-analogue of TRIA, had no effect on divalent cation-dependent ATPase activity. Consistent with in vivo studies, simultaneous treatment of vesicles with weight equivalents of TRIA and octacosanol reduced the stimulation of ATPase activity. The effect of calmodulin on the stimulation of ATPase activity was diminished by calmidazolium, a specific inhibitor of calmodulin. Circular dichroism studies did not show a change in the α-helix content of calmodulin in the presence of TRIA. TRIA also had no apparent effect on soluble calcium-calmodulin 3',5'-cyclic nucleotide phosphodiesterase activity. Removal of excess TRIA from the medium after treatment still resulted in stimulation of divalent cation-dependent ATPase activity in the presence of calmodulin was comparable to treated vesicles from which excess TRIA had not been removed. These data further support the contention that TRIA affects membrane structure and function.     

Preferential Induction of Alcohol Dehydrogenase in Coleoptiles of Rice Seedlings Germinated in Submergence Condition

Difference in the growth response to submergence between coleoptiles and roots of rice (Oryza sativa L.) was investigated in 9-d-old rice seedlings. The coleoptile length in the submergence condition was much greater than that in aerobic condition, whereas the root length in the submergence condition was less than that in the aerobic condition. Alcohol dehydrogenase (ADH) activity in the coleoptiles in the submergence condition was much greater than that in the aerobic condition, but ADH activity in the roots in the submergence condition increased slightly. These results suggest that the preferential ADH induction in rice seedlings may contribute to the difference in the growth response between the coleoptiles and roots under low oxygen conditions.     

Effect of low temperature on ethanolic fermentation in rice seedlings

Rice seedlings (Oryza sativa L.) were incubated at 5-30 degrees C for 48 h and the effect of temperature on ethanolic fermentation in the seedlings was investigated in terms of low-temperature adaptation. Activities of alcohol dehydrogenase (ADH, EC 1.1.1.1) and pyruvate decarboxylase (PDC, EC 4.1.1.1) in roots and shoots of the seedlings were low at temperatures of 20-30 degrees C, whereas temperatures of 5, 7.5 and 10 degrees C significantly increased ADH and PDC activities in the roots and shoots. Temperatures of 5-10 degrees C also increased ethanol concentrations in the roots and shoots. The ethanol concentrations in the roots and shoots at 7.5 degrees C were 16- and 12-times greater than those in the roots and shoots at 25 degrees C, respectively. These results indicate that low temperatures (5-10 degrees C) induced ethanolic fermentation in the roots and shoots of the seedlings. Ethanol is known to prevent lipid degradation in plant membrane, and increased membrane-lipid fluidization. In addition, an ADH inhibitor, 4-methylpyrazole, decreased low-temperature tolerance in roots and shoots of rice seedlings and this reduction in the tolerance was recovered by exogenous applied ethanol. Therefore, production of ethanol by ethanolic fermentation may lead to low-temperature adaptation in rice plants by altering the physical properties of membrane lipids.     

Disturbed ammonium assimilation is associated with growth inhibition of roots in rice seedlings caused by NaCl

The effects of NaCl on changes in ammonium level and enzyme activities of ammonium assimilation in roots growth of rice (Oryza sativa L.) seedlings were investigated. NaCl was effective in inhibiting root growth and stimulated the accumulation of ammonium in roots. Accumulation of ammonium in roots preceded inhibition of root growth caused by NaCl. Both effects caused by NaCl are reversible. Exogenous ammonium chloride and methionine sulfoximine (MSO), which caused ammonium accumulation in roots, inhibited root growth of rice seedlings. NaCl decreased glutamine synthetase and glutamate synthase activities in roots, but increased glutamate dehydrogenase activity. The growth inhibition of roots by NaCl or MSO could be reversed by the addition of L-glutamic acid or L-glutamine. The current results suggest that disturbance of ammonium assimilation in roots may be involved in regulating root growth reduction caused by NaCl.Abbreviations GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase - MSO methionine sulfoximine     

A cytosolic NADP-malic enzyme gene from rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) confers salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

NADP-malic enzyme (NADP-ME, EC 1.1.1.40) functions in many different pathways in plant and may be involved in plant defense such as wound and UV-B radiation. Here, expression of the gene encoding cytosolic NADP-ME (cytoNADP-ME, GenBank Accession No. AY444338) in rice (Oryza sativa L.) seedlings was induced by salt stress (NaCl). NADP-ME activities in leaves and roots of rice also increased in response to NaCl. Transgenic Arabidopsis plants over-expressing rice cytoNADP-ME had a greater salt tolerance at the seedling stage than wild-type plants in MS medium-supplemented with different levels of NaCl. Cytosolic NADPH/NADP⁺ concentration ratio of transgenic plants was higher than those of wild-type plants. These results suggest that rice cytoNADP-ME confers salt tolerance in transgenic Arabidopsis seedlings.     

Levels of endogenous polyamines and NaCl-inhibited growth of rice seedlings

The role of endogenous polyamines in the control of NaCl-inhibited growth of rice seedlings was investigated. Putrescine, spermidine and spermine were all present in shoots and roots of rice seedlings. NaCl treatment did not affect spermine levels in shoots and roots. Spermidine levels in shoots and roots were increased with increasing concentrations of applied NaCl. NaCl at a concentration of 50 mM, which caused only slight growth inhibition, drastically lowered the level of putrescine in shoots and roots. Addition of precursors of putrescine biosynthesis (L-arginine and L-ornithine) resulted in an increase in putrescine levels in NaCl-treated shoots and roots, but did not allow recovery of the growth inhibition of rice seedlings induced by NaCl. Pretreatment of rice seeds with putrescine caused an increase in putrescine level in shoots, but could not alleviate the inhibition effect of NaCl on seedling growth. The current results suggest that endogenous polyamines may not play a significant role in the control of NaCl-inhibited growth of rice seedlings.Abbreviations PUT putrescine - SPD spermidine - SPM spermine     

Nickel-induced oxidative stress and the role of antioxidant defence in rice seedlings

Seedlings of rice (Oryza sativa L.) cv. Pant-12 grown in sand cultures containing 200 and 400 μM NiSO₄, showed a decrease in length and fresh weight of roots and shoots. Nickel was readily taken up by rice seedlings and the concentration was higher in roots than shoots. Nickel-treated seedlings showed increased rates of superoxide anion (O₂ ^•− ) production, elevated levels of H₂O₂ and thiobarbituric acid reactive substances (TBARS) demonstrating enhanced lipid peroxidation, and a decline in protein thiol levels indicative of increased protein oxidation compared to controls. With progressively higher Ni concentrations, non-protein thiol and ascorbate (AsA) increased, whereas the level of low-molecular-weight thiols (such as glutathione and hydroxyl-methyl glutathione), the ratio of these thiols to their corresponding disulphides, and the ratio of AsA to dehydroascorbic acid declined in the seedlings. Among the antioxidant enzymes studied, the activities of all isoforms of superoxide dismutase (Cu-Zn SOD, Mn SOD and Fe SOD), guaiacol peroxidases (GPX) and ascorbate peroxidase (APX) increased in Ni-treated seedlings, while no clear alteration in catalase activity was evident. Activity of the ascorbate-glutathione cycle enzymes monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR)—significantly increased in Ni-treated seedlings. However such increase was apparently insufficient to maintain the intracellular redox balance. Results suggest that Ni induces oxidative stress in rice plants, resulting in enhanced lipid peroxidation and decline in protein thiol levels, and that (hydroxyl-methyl) glutathione and AsA in conjunction with Cu-Zn SOD, GPX and APX are involved in stress response.     

Effects of lemon balm (Melissa officinalis L.) extract on germination and seedling growth of six plants

The n-hexane-, acetone- and water-soluble fractions obtained from an aqueous acetone extract of lemon balm (Melissa officinalis L.) shoots inhibited the germination and the growth of roots and shoots of cockscomb (Amaranthus caudatus L.), cress (Lepidium sativum L.), crabgrass (Digitaria sanguinalis L.), timothy (Phleum pratense L.), lettuce (Lactuca sativa L.) and ryegrass (Lolium multiflorum Lam.). The inhibitory activity of the water-soluble fraction was the greatest, followed by that of acetone- and n-hexane-soluble fractions in all bioassays. The effectiveness of these fractions on the roots was greater than that of the shoots of the test plants. Significant reductions in the germination and growth of the roots and shoots were observed as the extract concentration increased. Such rate-dependent responses of the test plants to the fractions suggest that each fraction might contain allelochemical(s), but that the greatest potential was in the water-soluble fraction.