Improvement of chilling resistance in rice by application of an abscisic acid analog in combination with the growth retardant tetcyclacis

The protective effect of a synthetic terpenoid Analog of the plant hormone abscisic acid (ABA) coded LAB 173711 applied alone or in combination with the growth retardant tetcyclacis on chilling injury of rice was studied under growth chamber, greenhouse, and field conditions. The compounds were applied as a foliar spray before and after the onset of chilling treatment, as a substrate via the root system, and as a medium for seed soaking. The ABA analog increased chilling resistance in a manner similar to ABA. Combination of the analog with tetcyclacis revealed additive effects. Increased chilling resistance involved several processes: stomatal closure which reduced water loss during chilling, stabilization of the membranes, stabilization of the chlorophyll level, and stabilization of the root system. Possibilities for practical use of the compounds in rice production are discussed.     

A comparative study of the effects of abscisic acid and methyl jasmonate on seedling growth of rice

The effects of abscisic acid (ABA) and methyl jasmonate (MJ) on growth of rice seedlings were compared. The lowest tested concentration of ABA and MJ that inhibited seedling growth was found to be 4.5 and 0.9 µM, respectively. Growth inhibition by ABA is reversible, whereas that by MJ is irreversible. GA₃ was found to be more effective in reversing inhibition of shoot growth by ABA than by MJ. KCl partially relieved MJ-inhibited, but not ABA-inhibited, growth of rice seedlings. The beneficial effect of K⁺ on growth of rice seedlings in MJ medium could not be replaced by Li⁺, Na⁺ or Cs⁺. MJ treatment caused a marked release of K⁺ into the medium. In order to understand whether cell wall-bound peroxidase activity was inversely related to rice seedling growth, effects of ABA and MJ on cell wall-bound peroxidase activity were also examined. Results indicated that both ABA and MJ increased cell wall-bound peroxidase activity in roots and shoots of rice seedlings. Although MJ (4.5 µM) was less effective in inhibiting root growth than ABA (9 µM), MJ was found to increase more cell wall-bound peroxidase activity in roots than ABA.     

Effects of water stress on cellular ultrastructure and on concentrations of endogenous abscisic acid and indole-3-acetic acid in Fatsia japonica leaves

Ultrastructural alterations in mesophyll cells as well as variations in bulk leaf endogenous ABA and IAA concentrations were studied in water-stressed field-grown plants of Fatsia japonica. Under water deficit cellular membranes were modified and an increase in vesicles was observed. The main damage to the chloroplasts included thylakoid swelling and disruption of the chloroplast envelope. Concomitant variations in abscisic acid and indole-3-acetic acid were observed. Despite the expected increased in endogenous ABA concentration in relation to water stress, after the highest concentration of ABA, observed at predawn in severely stressed plants (29-1), there was a sharp decline from 2768 pmol g fw^–1 to 145 pmol g fw^–1; thus in severely stressed plants ABA levels were not related to changes in bulk leaf ABA contents. Water stress did not influence the concentrations of indole-3-acetic acid, although the increase in the endogenous abscisic acid concentration could be related with the ultrastructural changes.Abbreviations ABA abscisic acid - IAA indole-3-acetic acid - leaf water potential     

Protein Synthesis in Bromegrass (Bromus inermis Leyss) Cultured Cells during the Induction of Frost Tolerance by Abscisic Acid or Low Temperature

Bromus inermis Leyss cell cultures treated with 75 micromolar abscisic acid (ABA) at both 23 and 3°C developed more freezing resistance than cells cultured at 3°C. Protein synthesis in cells induced to become freezing tolerant by ABA and low temperature was monitored by [¹⁴C]leucine incorporation. Protein synthesis continued at 3°C, but net cell growth was stopped. Most of the major proteins detected at 23°C were synthesized at 3°C. However, some proteins were synthesized only at low temperatures, whereas others were inhibited. ABA showed similar effects on protein synthesis at both 23 and 3°C. Comparative electrophoretic analysis of [¹⁴C]leucine labeled protein detected the synthesis of 19, 21 and 47 kilodalton proteins in less than 8 hours after exposure to exogenous ABA. Proteins in the 20 kilodalton range were also synthesized at 3°C. In addition, a 31 kilodalton protein band showed increased expression in freezing resistant ABA treated cultures after 36 hours growth at both 3 and 23°C. Quantitative analysis of [¹⁴C]leucine labeled polypeptides in two-dimensional gels confirmed the increased expression of the 31 kilodalton protein. Two-dimensional analysis also resolved a 72 kilodalton protein enriched in ABA treated cultures and identified three proteins (24.5, 47, and 48 kilodaltons) induced by low temperature growth.     

Effects of different sterols on the inhibition of cell culture growth caused by the growth retardant tetcyclacis

Cell division in cell suspension cultures can be completely blocked by the growth retardant tetcyclacis at a concentration of 10^-4 mol l^-1. In rice cells it has been demonstrated that the growth inhibition can be completely overcome by application of cholesterol independent of the duration of pretreatment with tetcyclacis. In suspension cultures of maize and soybean, too, the effect of tetcyclacis on cell division was neutralized by adding cholesterol. Other plant sterols, stigmasterol, campesterol and sitosterol were active in a decreasing order. Modifications in the cholesterol perhydro-cyclopentanophenanthrene-ring system indicate that the hydroxyl group at C-3 and the double bond between C-5 and C-6 in ring B are required for the activity. In contrast, gibberellic acid as well as ent-kaurenoic acid could not compensate retardant effects. Likewise, tetcyclasis did not change the level of gibberellins in rice cells as shown by radioimmunoassay. Thus, it is concluded that in cell suspension cultures sterols play a more important role in cell division than gibberellins.Abbreviation GA_x gibberelin A_x     

Brassinolide Effect on Growth of Apical Meristems, Ethylene Production, and Abscisic Acid Content in Potato Tubers

Treatment of intact potato (Solanum tuberosum L., cv. Nevskii) tubers with 24-epibrassinolide (EB) resulted in prolonged deep dormancy, increased production of ethylene and higher contents of free and bound abscisic acid (ABA) in buds. EB at the most efficient concentration 0.021 mg dm^–3, applied immediately after tuber harvest, inhibited sprouting by 36 – 38 d, increased ethylene formation after 1 and 7 d of storage by almost 300 and 150%, respectively, and increased the content of both free and bound ABA during the whole period of storage (on average by about 80%). Electron microscopic and morphometric studies showed that EB brings about a decrease in cell volume in tunica and all types of meristems and an increase in the number of vacuoles, accompanied by a decrease in their volume.     

The role of the cotyledons in the photocontrol of hypocotyl extension in Cucumis sativus L.

Summary When pea seedlings lose about 5% of their water content the abscisic acid ((+)-ABA) level of the shoots increases ca. 20 times and the level of bound ABA, in all probability ABA-glucose, ca.7-10 times. After watering both ABA and bound ABA contents decrease within 24–48 h to the level in the control plants.After application of (±)-[2-¹⁴C] ABA to wilted pea shoots at the time of watering radioactive substances appear in the water-soluble, ether-insoluble fraction of ethanolic extracts and increase with time whereas radioactivity in the acidic ether fraction decreases. The neutral ether fraction remains free of radioactivity. Three radioactive zones, A, B, and C, are seen on chromatograms of the water-soluble fraction. A increases considerably within the entire experimental time, whereas B increases in the first 4–8 h after application and subsequently decreases. The third substance, C, which releases free ABA after hydrolytic treatment, does not change during the experiment. Chromatograms of the acidic ether fraction yield ABA and a substance staying at the origin, possibly phaseic acid and/or dihydrophaseic acid. Only the activity of ABA decreases during the experiment.     

Abscisic Acid Stimulated Osmotic Adjustment and Its Involvement in Adaptation of Tobacco Cells to NaCl

Osmotic adjustment of cultured tobacco (Nicotiana tabacum L. var Wisconsin 38) cells was stimulated by 10 micromolar (±) abscisic acid (ABA) during adaptation to water deficit imposed by various solutes including NaCl, KCl, K₂SO₄, Na₂SO₄, sucrose, mannitol, or glucose. The maximum difference in cell osmotic potential (Ψπ) caused by ABA treatment during adaptation to 171 millimolar NaCl was about 6 to 7 bar. The cell Ψπ differences elicited by ABA were not due to growth inhibition since ABA stimulated growth of cells in the presence of 171 millimolar NaCl. ABA caused a cell Ψπ difference of about 1 to 2 bar in medium without added NaCl. Intracellular concentrations of Na⁺, K⁺, Cl⁻, free amino acids, or organic acids could not account for the Ψπ differences induced by ABA in NaCl treated cells. However, since growth of NaCl treated cells is more rapid in the presence of ABA than in its absence, greater accumulation of Na⁺, K⁺, and Cl⁻ was necessary for ion pool maintenance. Higher intracellular sucrose and reducing sugar concentrations could account for the majority of the greater osmotic adjustment of ABA treated cells. More rapid accumulation of proline associated with ABA treatment was highly correlated with the effects of ABA on cell Ψπ. These and other data indicate that the role of ABA in accelerating salt adaptation is not mediated by simply stimulating osmotic adjustment.     

Use of the growth retardant tetcyclacis for potato tuber formation in vitro

The effects of the plant growth retardant tetcyclacis on in vitro tuber formation in potatoes was studied, using two different approaches: 1. tuber formation in various lines that did not or hardly form tubers under control conditions, and 2. tuber formation by the variety Bintje, which readily forms tubers. The ABA-deficient (droopy) lines of S. phureja hardly formed tubers without the addition of tetcyclacis. In the presence of this growth retardant tuberization was nearly 100%, within three weeks of in vitro culture, even in the absence of cytokinin. A series of somatic hybrids between S. tuberosum and S. brevidens, that did not form tubers in field and pot experiments, were tested. They all formed tubers in vitro in the presence of tetcyclacis. Stoloniferous shoots formed on single-node cuttings from in vitro grown Solanum tuberosum var Bintje plantlets were transferred to media containing a high level of sucrose. In the presence of tetcyclacis, tuber formation started after 4 days, reaching a maximum level of 80% at day 7. Tubers formed in the presence of tetcyclacis, accumulated starch and expressed several tuber-specific genes. These effects were fully antagonized by gibberellic acid. It is concluded that the growth retardant tetcyclacis is a potent tool in the study of tuber formation in potatoes.Abbreviations ABA abscisic acid - BAP benzylaminopurine - GA₃ gibberellic acid - STS silver thiosulphate - TET tetcyclacis     

Relative importance of Na+, Cl−, and abscisic acid in NaCl induced inhibition of root growth of rice seedlings

The relative importance of endogenous abscisic acid (ABA), as well as Na⁺ and Cl^– in NaCl-induced responses related to growth in roots of rice seedlings were investigated. The increase in ammonium, proline and H₂O₂ levels, and cell wall peroxidase (POD) activity has been shown to be related to NaCl-inhibited root growth of rice seedlings. Increasing concentrations of NaCl from 50 to 150 mM progressively decreased root growth and increased both Na⁺ and Cl^–. Treatment with NaCl in the presence of 4,4-diisothiocyano-2,2-disulfonic acid (DIDS, a nonpermeating amino-reactive disulfonic acid known to inhibit the uptake of Cl^–) had less Cl^– level in roots than that in the absence of DIDS, but did not affect the levels of Na⁺, and responses related to growth in roots. Treatment with 50 mM Na-gluconate (the anion of which is not permeable to membrane) had similar Na⁺ level in roots as that with 100 mM NaCl. It was found that treatment with 50 mM Na-gluconate effected growth reduction and growth-related responses in roots in the same way as 100 mM NaCl. All these results suggest that Cl^– is not required for NaCl-induced responses in root of rice seedlings. Endogenous ABA level showed no increase in roots of rice seedlings exposed to 150 mM NaCl. It is unlikely that ABA is associated with NaCl-inhibited root growth of rice seedlings.     

Induction of freezing tolerance in alfalfa cell suspension cultures

The effects of ABA, 2,4-D, kinetin and cold exposure on the cold hardiness of Medicago sativa L. cell suspensions were investigated. Cultures treated with 5×10^–5 M ABA at 2°C for 4 weeks in the absence of kinetin showed a 50% survival after freezing to –12.5°C, whereas cultures grown at 25°C under normal conditions tolerated freezing to only –3°C. The optimum ABA treatment of 5×10^–5 M for 4 weeks was effective only in combination with cold exposure. Of six cell lines tested, all showed different degrees of induced cold hardiness. The results suggest that ABA alone cannot induce freezing tolerance on alfalfa cell suspension cultures and that the deletion of kinetin and combination of low temperature and ABA is critical for the induction of cold hardiness in alfalfa cell suspension cultures.Abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - LT₅₀ 50% killing temperature     

Abscisic acid in developing wheat grains and its relationship to grain growth and maturation

Summary During the later stages of growth of grains of wheat (Triticum aestivum L. cvs. WW15 and Gabo) there is a dramatic increase (up to 40fold) in the content of abscisic acid (ABA) to 4–6 ng per grain. This level remains high from 25 to 40 days after anthesis. Then, in association with natural or forced drying of the grain, there is a rapid drop (5–10 fold) in the ABA content and a brief increase in the content of bound ABA. The bulk of ABA in an ear was in the grain (95%) and although the embryo contributed 19% of this ABA it was less than 5% of the grain by weight. There was no clear relationship between ABA content and the growth of grains in various spikelet or floret positions. Application of (±)-ABA to the ear had no effect on grain growth rate but led to an earlier cessation of grain growth and hastened the drying of the grain. Isolated embryos and whole grains were capable of germinating during the mid grain growth period (15–25 days), but germination capacity declined subsequently as ABA accumulated. Later, still, with grain drying and loss of ABA, embryo and grain became germinable again. At this time there was also a dramatic increase in the ability of the grain to synthesize -amylase. It is suggested that the accumulation of ABA at the later stages of grain growth prevents precocious germination and premature hydrolysis of starch reserves of the morphologically mature but still unripe grain. An inevitable consequence of such action may be in triggering grain maturation.     

A simple bioassay for abscisic acid using cucumber hypocotyls

A simple bioassay based on the inhibition by abscisic acid (ABA) of cucumber (Cucumis sativus L., cv. National Pickling) hypocotyl elongation was developed. Sections of 3-day-old dark-grown cucumber hypocotyl taken from 0–5 mm immediately below the cotyledon were used for the assay. A dark incubation period of 20 h was followed by an exposure to light for 24 h. Under these conditions, the inhibition of hypocotyl elongation is proportional to the abscisic acid applied. The minimum detectable level of abscisic acid was 10^–9 M, and the range of linear response to abscisic acid was between 10^–7 and 10^–3 M. This assay is 10 times more sensitive than the cucumber cotyledon greening bioassay for abscisic acid.     

Effect of drought stress,abscisic acid,and abscisic acid analogues on the efficacy of diclofop-methyl and tralkoxydim

The effects of drought stress, abscisic acid (ABA), and four ABA analogues on diclofop-methyl and tralkoxydim efficacy were investigated in oat (Avenu sativa). Drought stress conditions (6% soil moisture content) reduced the efficacy of diclofop-methyl at 350 g ha^–1, but not at 700 g ha^–1. Similarly, tralkoxydim efficacy was reduced by drought stress at 62.5 and 125 g ha^–1, but not at 250 g ha^–1. ABA (100 m), applied as a root drench 2 days before the herbicide, protected oat plants against all rates of diclofop-methyl and against low rates of tralkoxydim. Two ABA analogues protected oat plants from diclofop-methyl injury, whereas two others had no effect. Foliage applications of ABA were much less effective than root applications in protecting against herbicide injury. Protection by ABA and the two active analogues was dependent on the relative time of application with respect to the herbicides. Optimal protection by ABA and analogue I was obtained when they were applied between 2 days before and 1 day after diclofop-methyl application. Analogue IV protected plants when applied between 3 days before and 1 day after diclofop-methyl application. Partial protection against tralkoxydim activity by ABA was observed when it was applied between 1 day before and 1 day after herbicide application. Analogue I did not afford any protection against tralkoxydim, and analogue IV afforded partial protection when applied the same day or 1 day after tralkoxydim. The results indicate that protection against these postemergence herbicides, similar to that conferred by water stress, can be induced by ABA and structural analogues that apparently mimic the action of ABA.Abbreviations SMC soil moisture content - ABA abscisic acid     

Factors Influencing the Induction of Freezing Tolerance by Abscisic Acid in Cell Suspension Cultures of Bromus inermis Leyss and Medicago sativa L

A 2-gram fresh weight inoculum of bromegrass (Bromus inermis Leyss. culture BG970) cell suspension culture treated with 7.5 × 10⁻⁵ molar abscisic acid (ABA) for 7 days at 25°C survived slow cooling to −60°C. Over 80% of the cells in ABA treated cultures survived immersion in liquid N₂ after slow cooling to −40 or −60°C. In contrast, a 6-gram fresh weight inoculum only attained a hardiness level of −28°C after 5 days of ABA treatment. Ethanol (2 × 10⁻² molar) added to the culture medium at the time of ABA addition, inhibited the freezing tolerance of bromegrass cells by 25°C. A 6-gram inoculum of both control and ABA treated bromegrass cells altered the pH of the medium more than a 2-gram inoculum. ABA inhibited the increase in fresh weight of bromegrass by 20% after 4 days. Both control and ABA (10⁻⁴ molar) treated alfalfa cells (Medicago sativa L.) grown at 25°C hardened from an initial LT₅₀ of −5°C to an LT₅₀ of −23°C by the third to fifth day after subculture. Thereafter, the cells dehardened but the ABA treated cells did not deharden to the same level as the control cells. ABA inhibited the increase in fresh weight of alfalfa by 50% after 5 days.     

Effects of growth regulators on inflorescence proliferation of Bambusa edulis

The effects of various growth regulators in Bambusa edulis inflorescence proliferation were studied. Cytokinin is essential for inflorescence proliferation. Thidiazuron (TDZ) had been the most efficient cytokinin to induce inflorescence proliferation. The optimal TDZ concentration was 0.01–0.1 mg l^–1. Inflorescences did not proliferate in media containing auxin, gibberellic acid (GA₃), abscisic acid (ABA), or 1-amino- cyclopropane-1-carboxylic acid (ACC) alone. In TDZ-containing medium, the proliferation ratio decreased when the naphthaleneacetic acid (NAA) concentration higher than 5 mg l^–1.     

Role of ABA in stamen and pistil development in the normal and solanifolia mutant of tomato (Lycopersicon esculentum)

Summary The role of abscisic acid (ABA) in stamen and pistil development of the normal and solanifolia (sf/sf) mutant of tomato (Lycopersicon esculentum Mill.) was analyzed. The solanifolia mutant produces flowers with separate floral organs, unlike the fused organs of normal flowers, and has greater number of carpels and locules per ovary than the normal. Applications of 10^–5 M ABA to normal floral buds produced flowers with separate stamens, but higher concentrations (10^–4 M ABA) resulted in the complete suppression of stamen growth or stamens that were devoid of anthers. ABA at both 10^–4 and 10^–5 M also induced an increase in the number of carpels and locules in normal flowers, but not in mutant ones. Analysis of endogenous ABA by a radioimmunoassay revealed that the pistils of mutant flowers contained a significantly higher level of ABA than those of normal flowers, but there was no difference in the ABA content of the stamens. The non-fusion of the stamens and the high number of carpels and locules in solanifolia mutant flowers may be explained by the high level of ABA in the floral apex during the initiation and development of carpels.     

Hormonal Control of Parthenocarpic Ovary Growth by the Apical Shoot in Pea

The role of the apical shoot as a source of inhibitors preventing fruit growth in the absence of a stimulus (e.g. pollination or application of gibberellic acid) has been investigated in pea (Pisum sativum L.). Plant decapitation stimulated parthenocarpic growth, even in derooted plants, and this effect was counteracted by the application of indole acetic acid (IAA) or abscisic acid (ABA) in agar blocks to the severed stump. The treatment of unpollinated ovaries with gibberellic acid blocked the effect of IAA or ABA applied to the stump. [³H]IAA and [³H]ABA applied to the stump were transported basipetally, and [³H]ABA but not [³H]IAA was also detected in unpollinated ovaries. The concentration of ABA in unpollinated ovaries increased significantly in the absence of a promotive stimulus. The application of IAA to the stump enhanced by 2- to 5-fold the concentration of ABA in the inhibited ovary, whereas the inhibition of IAA transport from the apical shoot by triiodobenzoic acid decreased the ovary content of ABA (to approximately one-half). Triiodobenzoic acid alone, however, was unable to stimulate ovary growth. Thus, in addition to removing IAA transport from the apical shoot, the accumulation of a promotive factor is also necessary to induce parthenocarpic growth in decapitated plants.     

The effect of abscisic acid on cell turgor pressures,solute content and growth of wheat roots

Abscisic acid (ABA) was shown to influence turgor pressure and growth in wheat (Triticum aestivum L.) roots. At a concentrations of 25 mmol·m^-3, ABA increased the turgor pressure of cells located within 1 cm of the tip by up to 450 kPa. At 4 to 5 cm from the root tip this concentration of ABA reduced the turgor pressure of peripheral cells (epidermis and the first few cortical cell layers) to zero or close to zero while that of the inner cells was increased. Increases in sap osmolality were dependent on the concentration of ABA and the effect saturated at 5 mmol·m^-3 ABA. The increase in osmolality took about 4 h and was partly the result of reducing-sugar accumulation. Levels of inorganic cations were not affected by ABA. Root growth was inhibited at ABA concentrations that caused a turgor-pressure increase. The results show that while ABA can affect root cell turgor pressures, this effect does not result in increased root growth.Abbreviation ABA abscisic acid