Quantification Of Abscisic Acid Research Articles

Engineering a highly sensitive biosensor for abscisic acid in mammalian cells

Abscisic acid (ABA) is a signalling molecule conserved in plants, bacteria, fungi, and animals. Recently, ABA has gained attention for its pharmacological activities and its potential as a biomarker for the severity of chronic obstructive pulmonary disease and glioma. This prompts the development of a reliable, sensitive, rapid, and cost-effective method to quantify ABA levels in mammalian cells and tissues. The previously described ABA biosensor system based on the ABA-dependent interaction between the plant ABA receptor PYL1 and co-receptor ABI1 is not sensitive enough for the low ABA levels seen in mammals. Therefore, we optimized this system by replacing PYL1 with other high-affinity plant PYL proteins. The optimized biosensor system engineered with the PYL8 receptor enabled the quantification of ABA at low concentrations in HEK293T cells.

Fine tuning of hormonal signaling is linked to dormancy status in sweet cherry flower buds.

In temperate trees, optimal timing and quality of flowering directly depend on adequate winter dormancy progression, regulated by a combination of chilling and warm temperatures. Physiological, genetic and functional genomic studies have shown that hormones play a key role in bud dormancy establishment, maintenance and release. We combined physiological and transcriptional analyses, quantification of abscisic acid (ABA) and gibberellins (GAs), and modeling to further investigate how these signaling pathways are associated with dormancy progression in the flower buds of two sweet cherry cultivars. Our results demonstrated that GA-associated pathways have distinct functions and may be differentially related with dormancy. In addition, ABA levels rise at the onset of dormancy, associated with enhanced expression of ABA biosynthesis PavNCED genes, and decreased prior to dormancy release. Following the observations that ABA levels are correlated with dormancy depth, we identified PavUG71B6, a sweet cherry UDP-GLYCOSYLTRANSFERASE gene that up-regulates active catabolism of ABA to ABA glucosyl ester (ABA-GE) and may be associated with low ABA content in the early cultivar. Subsequently, we modeled ABA content and dormancy behavior in three cultivars based on the expression of a small set of genes regulating ABA levels. These results strongly suggest the central role of ABA pathway in the control of dormancy progression and open up new perspectives for the development of molecular-based phenological modeling.

Interactions between brown planthopper (Nilaparvata lugens) and salinity stressed rice (Oryza sativa) plant are cultivar-specific

Salinity stress triggers changes in plant morphology, physiology and molecular responses which can subsequently influence plant-insect interactions; however, these consequences remain poorly understood. We analyzed plant biomass, insect population growth rates, feeding behaviors and plant gene expression to characterize the mechanisms of the underlying interactions between the rice plant and brown planthopper (BPH) under salinity stress. Plant bioassays showed that plant growth and vigor losses were higher in control and low salinity conditions compared to high salinity stressed TN1 (salt-planthopper susceptible cultivar) in response to BPH feeding. In contrast, the losses were higher in the high salinity treated TPX (salt-planthopper resistant cultivar). BPH population growth was reduced on TN1, but increased on TPX under high salinity condition compared to the control. This cultivar-specific effect was reflected in BPH feeding behaviors on the corresponding plants. Quantification of abscisic acid (ABA) and salicylic acid (SA) signaling transcripts indicated that salinity-induced down-regulation of ABA signaling increased SA-dependent defense in TN1. While, up-regulation of ABA related genes in salinity stressed TPX resulted in the decrease in SA-signaling genes. Thus, ABA and SA antagonism might be a key element in the interaction between BPH and salinity stress. Taken together, we concluded that plant-planthopper interactions are markedly shaped by salinity and might be cultivar specific.

Abscisic acid and the antioxidant system are involved in germination of Butia capitata seeds

ABSTRACT Seed germination is an important step for plants without vegetative propagation and is a physiological process that begins with specific environmental cues resulting in biochemical responses. Breaking-dormancy is necessary to study germination in dormant seeds with asynchronous germination. We investigated the processes of breaking dormancy and germination of Butia capitata (Arecaceae) seeds, in which germination is slow and asynchronous, by operculum removal. This treatment increased germination of B. capitata to 90 %. Embryos of dry, imbibed, 24-hours post-operculum removal and early-germinated seeds were collected for biochemical analysis of the following: quantification of abscisic acid (ABA) and hydrogen peroxide (H2O2), activities of antioxidant enzymes (catalase - CAT, superoxide dismutase - SOD, glutathione reductase - GR) and histolocalization of superoxide anion (O2 -). Decreases in H2O2 and ABA were recorded 24 hours post-operculum removal. Increased GR and SOD activities during imbibition, and CAT upon germination, indicate a role in controlling reactive oxygen species. Interestingly, the accumulation of O2 - on the haustorium upon imbibition seems to be involved in germination, instead of H2O2. For B. capitata seeds, signaling from the removal of the operculum probably resulted in ABA catabolism mediated by O2 -, which thus promoted seed germination.

Sensitive and high throughput quantification of abscisic acid based on quantitative real time immuno-PCR

BackgroundAbscisic acid (ABA) functions as a stress phytohormone in many growth and developmental processes in plants. The ultra-sensitive determination of ABA would help to better understand its vital roles and action mechanisms.ResultsWe report a new sensitive and high throughput quantitative real time immuno-PCR (qIPCR) method based on biotin–avidin linkage system for ABA determination in plants. ABA monoclonal antibody (McAb) coated on the inner surface of PCR well pretreated with glutaraldehyde. The pre-prepared probe complex, including biotinylated McAb, biotinylated DNA and streptavidin linker, was convenient for high throughput operations. Finally, probe DNA was quantified by real-time PCR. The detectable ranges were from 10 to 40 ng/L with a limit of detection (LOD) of 2.5 fg. ABA contents in plant sample were simultaneously analyzed using LC–MS/MS to validate the qIPCR method. The results showed that qIPCR method has good specificity and repeatability with a recovery rate of 96.9%.ConclusionThe qIPCR method is highly sensitive for ABA quantification for actual plant samples with an advantage of using crude extracts instead of intensively purified samples.

Abscysic acid and compatibility of atemoya (Annona x atemoya Mabb.) grafted onto native species

Abstract Grafting is an effective technique used in the cultivation of commercial fruit species given the necessity to guarantee the genetic characteristics of productive species using selected clones. Although grafting is a common and widespread technique and phytohormones play a key role in the formation of tissues, the relationship between phytohormones, such as abscisic acid, and mechanisms of incompatibility is not yet well elucidated. Thus, the objective of this study was to establish whether a correlation exists between variations in abscisic acid and the compatibility of the atemoya (Annona x atemoya Mabb.) cultivar ‘Thompson’ grafted onto biribá [Annona mucosa (Bail.) H. Rainer], araticum-mirim [Annona emarginata (Schltdl.) H. Rainer ‘var. mirim’] and araticum-de-terra-fria [Annona emarginata (Schltdl.) H. Rainer ‘var. terra-fria’]. Plant cultivation was carried out at the Botany Department of Instituto de Biociências (IB), Unesp, Botucatu, São Paulo, Brazil. The plant material of grafted plants (stem above the grafted area, stem containing the grafted region, and stem below the grafted region) and ungrafted plants (stem 20 cm above ground) was collected 500 days after grafting (DAG) for the extraction and quantification of abscisic acid. The results of this study show that ungrafted Annona plants exhibit variations in the concentration of abscisic acid among the native rootstock species. When grafted, the most commonly used grafting combinations, araticum-de-terra-fria and araticum-mirim, present the same concentrations of abscisic acid in the graft region as self-grafted atemoya. It was concluded that the observed variations in the concentrations of abscisic acid in the graft region did not cause incompatibility in the combinations of atemoya grafted onto different native species.

Spatio-temporal profiling of abscisic acid, indoleacetic acid and jasmonic acid in single rice seed during seed germination

Abscisic acid (ABA), indoleacetic acid (IAA) and jasmonic acid (JA) are plant hormones that were reported to play indispensable roles during seed germination. However, the interactions between these plant hormones during rice seed germination have still not been explored clearly. A sensitive method for determination of these plant hormones would be beneficial for the exploration of such interactions. Herein, we present a liquid chromatography coupled with mass spectrometry (LC-MS) method for the quantification of ABA, IAA and JA in a single tissue of rice seed to investigate the spatio-temporal distribution of these plant hormones during rice seed germination. To this end, an in silico strategy was developed in order to select a derivatization reagent with an ideal sensitivity of MS detection. This strategy was confirmed with experimental studies on three reagents N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC), N,N-dimethylethylenediamine (DMED), and N-(acridin-9-yl)-2-bromoacetamide (AYBA) and their formic acid derivatives. Our results from the in silico and LC-MS experiments show that AYBA is a good derivatization reagent for ABA, IAA and JA due to its reasonable ionization efficiency in electrospray ionization mass spectrometry (ESI-MS) and excellent hydrophobicity. Finally, a sensitive LC-MS method upon AYBA was established for the determination of ABA, IAA and JA in germinated seeds. Good linearities for ABA, IAA, and JA were obtained with correlation coefficients greater than 0.99. The limits of detection (LODs) were in the range of 0.14–0.16 pg mL−1. The method exhibits good precisions with RSD 1.5%–13.8% (intra-day) and 1.2%–7.3% (inter-day) and acceptable recoveries (88.6%–102.9%, n = 6). Finally, the method was successfully employed in the spatio-temporal profiling of ABA, IAA and JA in a single tissue of rice seed during rice seed germination.

Extraction and Quantification of Abscisic Acid and Derivatives in Strawberry by LC-MS

Phytohormones are important plant components that are involved in a signaling cascade in plant development. In strawberry, the influence of abscisic acid (ABA) and its different forms [phaseic acid (PA), dihydrophaseic acid (DPA), and ABA glucose ester (ABA-GE)] on the process of fruit maturation is not yet completely understood. Quantification of phytohormones is currently performed by liquid chromatography coupled to mass spectrometry (LC-MS) due to its sensitivity and specificity. However, the sample matrix and the extraction procedure will influence the analysis. Thus, this study aimed to optimize a simple extraction method and validate the LC-MS quantification of ABA as well as the identification and quantification of ABA derivatives (PA, DPA, and ABA-GE) in strawberry fruit. Hormone extraction was performed using either methanol (80% v/v—S1) or acetone:water:acetic acid (80:19:1 v/v—S2) solutions with or without the use of sonication. The most efficient extraction was obtained using S1 without sonication, and LC-MS validation parameters for ABA were within acceptable scores.

Embryo and hip development in hybrid roses

Roses are known to produce seeds with high concentrations of abscisic acid (ABA), both in the pericarp and in the testa tissues of the seed coat. No studies on roses have documented embryo morphological differentiation or the concentration of ABA in the embryo, which is known to inhibit premature germination. In this study, hip and seed growth of two hybrid roses were characterised from 3 to 60 days after pollination (DAP). An increase of about five times the hip mass at 3 DAP was necessary to obtain fully developed seeds. Fully developed embryos were found at 15 DAP and completely developed seeds at 30 DAP. The same pattern of hip mass increase was shown in both genotypes. In parallel, quantification of ABA in the developing embryos was carried out by ELISA. An exponential decay in ABA concentration was found in embryos of both genotypes, with basal levels (<0.5 pmol mg−1) registered at 30 DAP. These changes in ABA, during the embryo development, could be used to formulate time points for embryo rescue and understanding of the pollination to seedling stage.

The role of matrix effects on the quantification of abscisic acid and its metabolites in the leaves of Bauhinia variegata L. using liquid chromatography combined with tandem mass spectrometry

Phytohormone analysis using liquid chromatography combined with tandem mass spectrometry is a very important tool for studies involving plant metabolism; however, this analysis can be affected by matrix composition. Although it is necessary to understand this effect to produce reliable results, it has been widely neglected in analyses of plant hormones. Leaf extracts from Bauhinia variegata were obtained by solvent extraction followed by solid-phase cleanup. The extract was analyzed with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) in negative ionization mode, using the multiple reaction monitoring mode with two or three transitions to enhance analytical quality control. Although deuterated standards were used as surrogates, pronounced matrix effects were detected for phaseic acid (PA), abscisic acid (ABA) -glycosyl ester (ABA-GE) and dihydrophaseic acid (DPA), whereas ABA, neoPA and 7'-hydroxy-ABA (7'OHABA) showed negligible matrix effects. The method was validated using spiked samples and was applied to monitor ABA, PA, DPA, neoPA, 7´OHABA and ABA-GE on a daily basis. Analyte recoveries from spiked samples ranged from 67% to 87%. The highest leaf concentration of phytohormones was found at 2:00 pm and 5:00 pm, which represent typical day times related to the decrease of stomatal conductance. Despite the use of deuterated phytohormones as internal standards, we showed that the use of a calibration curve constructed with a matrix extract is mandatory for the reliable quantification of ABA and its metabolites, especially PA, ABA-GE and DPA. Daily variations in endogenous ABA leaf concentration were discussed.

CHANGES IN ABA LEVELS IN VEGETATIVE AND FLOWER BUDS DURING DORMANCY IN CAMELLIA

Camellia japonica L. is an evergreen acidophilic perennial, member of Theaceae Mirb. (Theales), that includes more than 3,000 named cultivars of great ornamental value. Bud dormancy is a key adaptive trait during the annual cycle of overwintering temperate woody plants and is present in Camellia. There is sufficient evidence that abscisic acid (ABA) is responsible for the initiation and, in a number of species, also for the maintenance of endodormancy. Increase of endogenous ABA is generally observed at the onset of endodormancy, while loss of bud dormancy is linked to declining ABA levels. To deepen our understanding of the role of ABA in C. japonica during dormancy, both vegetative and flower buds were studied. In a first experiment, we assessed the seasonal dynamics of ABA in vegetative buds from the onset of dormancy to its release. In this case, the results described a complex type of response, with several peaks of ABA in the middle of dormancy, and a major peak followed by a drastic reduction right before dormancy release of the vegetative bud. The second experiment concerned the quantification of ABA in flower buds during a cold treatment (7°C) performed after dormancy onset. Here, the results indicated a clear downward trend of flower buds ABA levels in response to increasing periods at constant low temperatures. Overall, morphological and physiological data confirmed the critical role of cold in the release of endodormancy in camellia. Future studies concerning the release of bud dormancy and correlated ABA levels in response to cold should focus on a wide range of C. japonica cultivars. This could allow selection of genotypes that bloom later in spring on the one hand (garden camellias) or genotypes that can be easily forced in flowering (flowering pot plants) on the other hand. This knowledge could also be exploited in breeding programs.

Quantification of abscisic acid in grapevine leaf (Vitis vinifera) by isotope-dilution liquid chromatography–mass spectrometry

A specific, sensitive, precise, and accurate method for the determination of abscisic acid (ABA) in grapevine leaf tissues is described. The method employs high-performance liquid chromatography and electrospray ionization-mass spectrometry (LC-ESI-MS) in selected ion monitoring mode (SIM) to analyze ABA using a stable isotope-labeled ABA as an internal standard. Absolute recoveries ranged from 72% to 79% using methanol/water pH 5.5 (50:50 v/v) as an extraction solvent. The best efficiency was obtained when the chromatographic separation was carried out by using a porous graphitic carbon (PGC) column. The statistical evaluation of the method was satisfactory in the work range. A relative standard deviation (RDS) of < 5.5% and < 6.0% was obtained for intra-batch and inter-batch comparisons, respectively. As for accuracy, the relative error (%Er) was between -2.7 and 4.3%, and the relative recovery ranged from 95% to 107%.

Quantification of ABA and its metabolites in sweet cherries usingdeuterium-labeled internal standards

Abscisic acid (ABA), phaseic acid (PA), dihydrophaseic acid (DPA), and epi-dihydrophaseic acid (epi-DPA) were quantified in developing fruit and seeds of sweet cherry using each deuterium-labeled internal standard. ABA concentrations in the pulp were low at the early stage of fruit development, reached to the maximum before maturation, and subsequently declined during maturation. The significant increase of ABA after 29 days after full bloom (DAFB) coincides with the softening suggests that ABA may play a role to induce fruit maturation in sweet cherries. ABA metabolite levels were high at the immature stage and decreased with fruit maturation. This fact suggests that fruit may not need ABA in the early stage of fruit development. It was considered that DPA may be the major metabolite of ABA since the concentrations were higher than PA and epi-DPA at all stages of fruit development. ABA concentrations increased at the beginning of seed maturation and then decreased toward harvest. This decrease may be necessary to end seed dormancy. DPA in seeds changed similarly with ABA but its concentrations were always higher than those of ABA.

Quantification of abscisic acid using liquid chromatography/thermospray ionization tandem mass spectrometry for selected reaction monitoring

AbstractA liquid chromatography/thermospray ionization tandem mass spectrometry (LC/TSP‐MS/MS) method was developed for the quantification of abscisic acid (ABA) in crude plant extracts without derivatization. By this technique interfering chemical background ions are eliminated and a detection limit for ABA olf 0.2 ng is achieved. Reproducibility for quantification is attained by parallel reaction monitoring of ABA and added ABA‐d6 used as an internal standard. The standard curve is linear over the range 200 pg to 10 ng of injected ABA. As examples of the application of the methodology, the quantification of ABA in some plant tissues is given.

Quantitative analysis of abscisic acid in needles of Abies alba Mill. by electron capture gas chromatography

The amount of abscisic acid (ABA) in needles of silver fir from a natural location was investigated with regard to position in the crown, damage, seasonal variation, and needle age. Because of problems of quantification of ABA in coniferous needles, which contain numerous secondary plant products, a method for reliable determination of both isomers cis-trans-ABA (c-ABA) and transtrans-ABA (t-ABA) was developed. By means of gas chromatography (GC) using an electron capture detector (BCD) and a programmed temperature vaporizer (PTV) injector complete separation of both compounds was achieved. Two different pairs of fir were investigated — in each case a damaged and a healthy tree. Needles from both trees from the first and the second pair collected in September contained 500–1100 ng c-ABA/g fresh weight (FW), and the concentrations of t-ABA varied from 400 to 700 ng/g FW. Investigations from the second pair show highest amounts of 2900 ng/g Fw c-ABA and 1800 ng/g FW of t-ABA in May and June. For the first pair a higher c-ABA content was found in needles from the top of the crown than in those from the middle and the base. This difference could not be confirmed in the analysis of the second pair. Because of the strong natural deviation no statistically significant difference between the healthy and the damaged tree was found. The first pair of firs examined showed a higher t-ABA concentration than the second one. In this case the highest amount was found in the top of the crown. Methodical mistakes during the clean-up procedure and in quantification by gas chromatography could be excluded. The presence of c- and t-ABA in the purified extract was corroborated by mass spectrometry. With regard to the seasonal variation both isomers of ABA show an unequivocal trend. The maximum concentration is achieved in May to June, whereas the content is minimal in August/September. In any case the level of t-ABA is lower than that of c-ABA. No correlation between the amount of ABA and the needle age could be established.

Identification of Gibberellins and Abscisic Acid in Bulbs of Lilium elegans Thunb. and Their Quantitative Changes during Cold Treatment and the Subsequent Cultivation.

Abscisic acid (ABA), gibberellin Al (GA1), GA4, GA9, GA12, GA15, GA19, GA20, GA24, GA34, GA44, GA51 and 3-epi-GA4 were identified by full-scan gas chromatography/mass spectrometry (GC/MS) and/or GC/selected ion monitoring (GC/SIM) in purified extracts of dormant bulbs of Litium elegans Thunb. cv. Connecticut King. The findings suggest that two independent GA biosynthetic pathways exist, the early-non-hydroxylation pathway leading to GA4 (active GA) and the early-13-hydroxylation pathway leading to GA1 (active GA). To investigate the physiological roles of ABA and GAs in breaking the dormancy of lily bulbs by cold treatment, quantification of ABA and GAs in the lily bulbs during the cold treatment and the subsequent growth was carried out by GC/SIM, using the corresponding deuterium-labeled compounds as internal standards. The levels of ABA and GA4 increased during cold treatment, reaching a maximum level at the end of the treatment; thereafter the levels decreased rapidly. The level of GA24, a precursor of GA4, also increased during cold treatment and remained elevated, whereas levels of 13-hydroxylated GAs such as GA1, GA19 and GA20 decreased during the cold treatment. The above results suggest that GA4 plays a principal role in breaking dormancy in lily bulbs. The physiological roles of ABA in lily bulbs are also discussed.

Level of Abscisic Acid in Integuments, Nucellus, Endosperm, and Embryo of Peach Seeds (Prunus persica L. cv Springcrest) during Development

Free abscisic acid (ABA) in integuments, nucellus, endosperm, and embryo was determined throughout seed development of peach (Prunus persica L. cv Springcrest). Quantification of ABA was performed using combined high performance liquid chromatography-radioimmunoassay based on a monoclonal antibody raised against free (S)-ABA. In the integuments and endosperm, ABA concentration remained constant during the first 100 days after anthesis and rose in the following days when fresh weight was rapidly decreasing. In the nucellus, the ABA concentration variation pattern paralleled that of tissue growth. ABA concentration in the embryo increased constantly with the growth of the tissues to reach a maximum at the last growth stage. The role of ABA in peach seeds is discussed in relation to the development of the different seed tissues.

Quantification of Abscisic Acid in Wheat Leaf Tissue by Direct Enzyme Immunoassay

The plant growth hormone abscisic acid (ABA) modulates plant responses and adaptations to environmental stress. The objective of this study was to develop a procedure which would facilitate broad‐scale screening of wheat (Triticum aestivum L. em. Thell.) germplasm in which ABA content or rate of accumulation in leaf tissue is the selection criterion. The effects of extraction time, C18 reverse‐phase chromatography, solvent volatilization, and dilution of sample extract on quantification of ABA by direct enzyme immunoassay (EIA) were investigated. Results showed that complete volatilization of extraction solvent did not affect ABA determination. Extraction of ABA from wheat leaves, using 800 mL L−1 methanol (MeOH), was complete after 24 h. Interference to EIA due to MeOH was eliminated when MeOH was diluted to ≤80 mL L−1. No apparent inhibitors to EIA were detected by a parallelism test of dilution curves or by partial purification of leaf extract using C18 reverse‐phase chromatography. Simplified and effective procedures are proposed which facilitate the preparation of a large number of leaf samples at minimal expense and labor without sacrificing accuracy of ABA estimation.

Uniconazole-induced changes in abscisic acid, total amino acids, and proline in Phaseolus vulgaris

Fourteen-day-old bean ( Phaseolus vulgaris) plants were soil treated with 100 ml of either 10 mg liter −1 uniconazole (( E)-( p-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)-1-penten-3-ol) or 3 mg liter −1 abscisic acid (ABA). Over the next 10 days, the concentration of uniconazole, ABA, total amino acids, and proline in the primary leaves and their stomatal resistance were monitored. In plants treated with exogenous ABA, there was a single transient increase in stomatal resistance with the endogeous concentrations of ABA, proline, and total amino acids increasing to a maximum approximately 2 days after treatment. In the uniconazole-treated plants, the accumulation of uniconazole in the primary leaves was not linear but was biphasic over the duration of the experiments. This correlated very closely to changes in the stomatal resistance. The concentrations of ABA, proline, and total amino acids followed a similar pattern with the first and second peaks occurring 3–4 and 8–9 days after treatment, respectively. These results suggest that the accumulation of ABA may be the direct result of the presence of uniconazole in the primary leaf tissue. Furthermore, the accumulation of proline and other amino acids that have been associated with environmental stress resistance is likely mediated through the uniconazole-induced accumulation of ABA. A radioimmunoassay for the rapid and sensitive quantification of ABA is also outlined.

Identification and Quantification of Abscisic Acid in Zoysiagrass Seeds and Its Inhibitory Effect on Germination

Seed dormancy has been a major limiting factor in the use of zoysiagrass (Zoysia japonica Steud.) for turf. The identification of the major dormancy factors and their mode of action would increase the potential for developing seed treatments to break dormancy. Abscisic acid (ABA), a well‐known seed dormancy‐inducing substance found in the dormant seeds of many temperate perennial weeds and grasses, was confirmed and quantified in Zoysiagrass seed. High performance liquid chromatography was used to purify the seed extract, gas chromatography/electron capture was used to quantitate the amount of ABA present in the seeds via the methyl ester, and further verification of the compound in the seed as ABA was made using masspectrometry. The quantity of the cis, trans isomer found, 3.44 mg ABA kg1 seed, is the highest level of ABA reported for any seed. There was no trans, trans isomer found in zoysiagrass seed. The high level of ABA may be the reason that dormancy is so difficult to break in zoysiagrass seed. Exogenous ABA applied to nondormant (KOH and light treated) seed inhibited germination. Germination after 21 d was reduced 69 and 95% by 0.4 and 40.0 μM of ABA, respectively. The promotive effects of light on germination were reversed by ABA applications. A positive relationship was found between ABA concentration and the duration of light required for germination.