Abscisic Acid In Seeds Research Articles

An in vivo GA- and ABA-responsive dual-luciferase reporter system for simultaneous detection of GA and ABA responses, hormone crosstalk and heat stress response in rice.

Gibberellic acid (GA) and abscisic acid (ABA) are the primary phytohormones that play antagonistic roles in the control of the transition from embryogenesis to germination in seeds (Finch-Savage and Leubner-Metzger, 2006). Given the low concentrations of GA and ABA in seeds and plants, biosynthesis inhibitors (Toh et al., 2008) or deficient mutants (Debeaujon and Koornneef, 2000) have been used to investigate the relative importance of their physiological functions. However, biosynthetic inhibitors may have pleiotropic effects in plants, and the use of GA- or ABA-deficient mutants does not permit simultaneous monitoring of dynamic changes in GA and ABA during seed development or germination (Dejonghe et al., 2018). Analytical determination provides accurate quantification of GA and ABA contents, but the extraction procedure is time-consuming, and the antagonism and dynamic changes between GA and ABA may not be detected.

The H3K27me3 Demethylase RELATIVE OF EARLY FLOWERING6 Suppresses Seed Dormancy by Inducing Abscisic Acid Catabolism.

Seed dormancy is an adaptive trait that is crucial to plant survival. Abscisic acid (ABA) is the primary phytohormone that induces seed dormancy. However, little is known about how the level of ABA in seeds is determined. Here we show that the Arabidopsis (Arabidopsis thaliana) H3K27me3 demethylase RELATIVE OF EARLY FLOWERING6 (REF6) suppresses seed dormancy by inducing ABA catabolism in seeds. Seeds of the ref6 loss-of-function mutants displayed enhanced dormancy that was associated with increased endogenous ABA content. We further show that the transcripts of two genes key to ABA catabolism, CYP707A1 and CYP707A3, but not genes involved in ABA biosynthesis, were significantly reduced in ref6 mutants during seed development and germination. In developing siliques, REF6 bound directly to CYP707A1 and CYP707A3, and was responsible for reducing their H3K27me3 levels. Genetic analysis demonstrated that the enhanced seed dormancy and ABA concentration in ref6 depended mainly on the reduced expression of CYP707A1 and CYP707A3 Conversely, overexpression of CYP707A1 could offset the enhanced seed dormancy of ref6 Taken together, our results revealed an epigenetic regulation mechanism that is involved in the regulation of ABA content in seeds.

Factors affecting the dormancy and germination of bleeding heart [Lamprocapnos spectabilis (L.) Fukuhara] seeds.

Information on the optimal conditions to promote the germination of Lamprocapnos spectabilis (L.) Fukuhara seeds is limited; consequently, this study was conducted to establish the requirements to break seed dormancy and promote germination. The selected seeds had morphophysiological dormancy and had not begun embryo development. To study the dormancy breaking and embryo development processes, seeds were subjected to constant or changing temperature treatments during moist stratification. High temperature and humidity resulted in vigorous embryo growth, with the longest embryos occurring after 1month of incubation at 20°C. At 4°C, the seeds required incubation period of at least 3months to germinate. Embryo growth and germination were higher with changing high and low temperatures than under a constant temperature, and changing temperatures also considerably changed the endogenous hormone levels, embryo development and germination. Bioactive gibberellin (GA) content was higher in seeds incubated at 20°C for 1month, then at 4°C for 2months. The content of endogenous abscisic acid in seeds subjected to the same treatment decreased by 97.6% compared with that of the untreated seeds. Embryo growth and seed germination require changing high and low temperatures; however, exogenous GA3 could substitute for high temperatures, as it also causes accelerated germination. In this study, the seeds of L.spectabilis were identified as an intermediate simple type, a sub-level of morphophysiologically dormant seeds.

Salt stress inhibits germination of Stylosanthes humilis seeds through abscisic acid accumulation and associated changes in ethylene production

In Stylosanthes humilis, salt stress tolerance is associated with ethylene production by the seeds, however, how salt stress controls seed germination and ethylene production is poorly understood. Here, we studied the hormonal and metabolic changes triggered by salt stress on germination of S. humilis seeds. Salt stress led to decreased seed germination and ethylene production, concomitantly with higher abscisic acid (ABA) production by seeds. Treatment with NaCl and ABA promoted distinct changes in energy metabolism, allowing seeds to adapt to salt stress conditions. Treatment with the ABA biosynthesis inhibitor fluridone or ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) reversed the effects of salt stress on seed germination and ethylene production. Moreover, ethylene concentration was decreased by increasing the pH of the salt solution. High pH, however, did not influence concentration of ABA in seeds under salt stress. We conclude that biosynthesis of ABA and ethylene in response to salt stress constitutes a point of convergence that provides flexibility to regulate energy metabolism and embryo growth potential of S. humilis seeds within a given pH condition.

Effects of plant-derived smoke on the growth dynamics of Barnyard Grass (Echinochloa crus-galli)

The stimulation of seed germination by smoke and aqueous smoke extracts from the combustion of plant material has recently received a great deal of attention. This study was conducted to assess the effects of smoke on the weed species Echinochloa crus-galli (barnyard grass). Plant-derived smoke was produced from Bauhinia variegata using a newly designed furnace and then applied to the weed species. The results showed that plant-derived smoke solution significantly increased the germination rate (70–92.3%), germination percentage (31–47%), fresh weight (30–69%), shoot and root length (10–14.9%, 35–44%), germination index (50–100%) and vigour index (114–157%) of barnyard grass relative to the control. Treatment with plant-derived smoke solution also significantly reduced the endogenous abscisic acid of seeds, while α-amylase activity increased significantly compared to the control. Overall, these findings indicate that stimulation and quick emergence from a dormant soil seed bank of barnyard grass can be induced by plant-derived smoke solution followed by chemical control through known weedicide/herbicide treatments as an eco-friendly alternative method of weed control.

(207) Sensitivity of Two Lettuce Genotypes to Abscisic Acid during Germination

In many lettuce (Lactuca sativa L.) cultivars, temperatures above 30 °C can inhibit germination completely. Lettuce seeds imbibed at supraoptimal temperature for 72 hours or more will not germinate even when the temperature is returned to the optimal range. Sung et al. (1998) reported that thermosensitive `Dark Green Boston' and thermotolerant `Everglades' responded to temperature at seed maturation by being able to show greatly enhanced germination at 36 °C when seeds were matured at 30/20 °C. Abscisic acid (ABA) plays an important role relative to both dormancy and germination of many seeds and may contribute to lettuce seed thermodormancy. Therefore, sensitivity of `Everglades' and `Dark Green Boston' to ABA of seeds maturated at 30/20 °C and 20/10 °C in light and dark was determined. Seeds were germinated at 20 °C and 36 °C in light and dark. All seeds of `Dark Green Boston'and `Everglades' matured at 30/20 °C and 20/10 °C germinated similarly regardless of maturation temperature and light/dark condition at 20 °C. At 36 °C in dark, both genotypes matured at 30/20 °C germinated more than those matured at 20/10 °C. `Dark Green Boston' genotypes were more sensitive to ABA (0.5, 1, 10, 50, 100 μM) during germination at 36°C in dark. When ABA was added, seeds matured at 30/20 °C germinated better than those matured at 20/10 °C, regardless of genotype. At 36 °C in light, 40% of both genotypes were inhibited at 0.5 μM ABA. At 36 °C in dark, germination of all seeds but `Everglades' matured at 30/20 °C were completely inhibited at all ABA concentrations. Thus, seed maturation temperature has an influence on lettuce seed germination at high temperature and sensitivity to ABA.

Rapid extraction of abscisic acid and its metabolites for liquid chromatography–tandem mass spectrometry

We have described a simple, reliable and rapid method of extracting and partially purifying the phytohormone (+)-abscisic acid and its catabolites for liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS–MS) analysis. Lyophilized tissue samples were powdered by high-speed agitation with ceramic beads for 5 s. Metabolites were extracted from the tissue powder using acetone–water–acetic acid (80:19:1, v/v) with the addition of deuterated internal standards for quantification. Essentially all endogenous hormones were recovered by three successive tissue extractions. However we demonstrated that, with the use of internal standards, one extraction with vigorous vortexing was sufficient to obtain accurate results (recovery 65–90%). Solvents were optimized for partial purification of abscisic acid and related compounds by solid-phase extraction using Oasis HLB cartridges. The eluted metabolites were then analyzed by LC–MS–MS. To illustrate the applicability of these techniques, we analyzed the levels of abscisic acid and metabolites in seeds and valves of Brassica napus siliques at two stages of development. We detected abscisic acid, phaseic acid, 7′-hydroxyabscisic acid, dihydrophaseic acid and abscisic acid glucose ester. In both tissues, dihydrophaseic acid was the major accumulating product, reaching 97 300 pmol/g dry mass in valves at 24 days after anthesis. The amount of abscisic acid in seeds was high at 24 days after anthesis (23 300 pmol/g dry mass), but low in the other samples (292–447 pmol/g dry mass).

Dormancy, ABA content and sensitivity of a barley mutant to ABA application during seed development and after ripening.

Assessment of dormancy inception, maintenance and release was studied for artificially dried immature seeds harvested throughout seed development in the barley cv. Triumph and its mutant line TL43. Each was grown in Spain and Scotland under low and high dormancy inducing conditions, respectively. Both TL43 and Triumph followed a similar pattern of release from dormancy across the seasons, although seeds of TL43 were able to germinate at an earlier seed development stage. Abscisic acid (ABA) content was also studied in immature grains throughout the seed development period. Total amount of ABA in seeds of Triumph and TL43 was higher in plants grown in Scotland than in Spain. However, no clear genotypic differences in ABA pattern in the course of grain development could be detected whilst significant genotypic differences were observed for germination percentage (GP). Endogenous ABA content alone throughout grain development did not explain genetic differences in GP within environments. Environmental and genetic differences in dormancy were also observed on mature seeds throughout the after-ripening period. The initial germination (GP(0)) played a key role in the sensitivity to ABA of post-harvest mature seeds. For the same after-ripening stage, TL43 was more insensitive to exogenous ABA than Triumph. However, ABA responses in seeds of the two genotypes with similar GP(0) at different after-ripening stages were comparable. Therefore, differences in exogenous ABA sensitivity of post-harvest mature grain of these two genotypes seemed to be determined by, or coincident with, the initial germination percentage.

An abscisic acid-induced protein kinase, PKABA1, mediates abscisic acid-suppressed gene expression in barley aleurone layers.

The phytohormone abscisic acid (ABA) induces genes-encoding proteins involved in desiccation tolerance and dormancy in seeds, but ABA also suppresses gibberellin (GA)-responsive genes encoding hydrolytic enzymes essential for postgermination growth. A unique serine/threonine protein kinase, PKABA1 mRNA, up-regulated by ABA in seeds, has been identified. In this report, the effect of PKABA1 on the signal transduction pathway mediating ABA induction and suppression of genes has been determined in aleurone layers of barley seeds. Two groups of gene constructs were introduced to barley aleurone layers by using particle bombardment: the reporter constructs containing the coding sequence of beta-glucuronidase gene linked to hormone-responsive promoters and the effector constructs containing the coding region of protein kinases linked to a constitutive promoter. Constitutive expression of PKABA1 drastically suppressed expression of low- and high-pI alpha-amylase and protease genes induced by GA. However, the presence of PKABA1 had only a small effect on the ABA induction of a gene encoding a late embryogenesis abundant protein, HVA1. Our results indicate that PKABA1 acts as a key intermediate in the signal transduction pathway leading to the suppression of GA-inducible gene expression in cereal aleurone layers.

Effect of Potassium Nutrition during Bell Pepper Seed Development on Vivipary and Endogenous Levels of Abscisic Acid (ABA)

Seeds in fruit of bell pepper (Capsicum annuum `California Wonder') plants grown in nutrient solutions deficient in potassium (<3 mmol·L-1) showed a higher incidence of sprouting (i.e., vivipary) than seeds in fruit from plants grown at adequate potassium levels (6 mmol·L-1). Tissue analysis showed a progressive drop in the leaf content of potassium with increasing plant maturation for all levels of potassium nutrition. However, potassium in fruit and seeds increased at later stages of maturity. ABA was extracted, isolated and identified from bell pepper seeds obtained from fruit grown under the potassium treatments (0.0, 0.6, 1.5, 3.0, and 6.0 mmol·L-1) at five fruit maturity stages (mature-green to overripe). At early fruit maturity stages, there were no significant differences in seed ABA content in the fruit from the different potassium treatments. However, differences in ABA content and vivipary among the potassium treatments became highly significant as the fruit matured. The concentration of ABA in seeds of potassium-deficient treatments was ≈14% of the control (0.4 versus 2.8 μg·g-1 dry mass). High concentrations of ABA in bell pepper seeds were associated with low incidence of vivipary and high potassium content in the leaves, fruit and nutrient solution.

Seed-specific immunomodulation of abscisic acid activity induces a developmental switch.

A single-chain Fv antibody (scFv) gene, which has previously been used to immunomodulate abscisic acid (ABA) activity in transgenic tobacco to create a 'wilty' phenotype, was put under control of the seed-specific USP promoter from Vicia faba and used to transform tobacco. Transformants were phenotypically similar to wild-type plants apart from their seeds. Anti-ABA scFv embryo development differed markedly from wild-type embryo development. Seeds which accumulated similar levels of a scFv that binds to oxazolone, a hapten absent from plants, developed like wild-type embryos. Anti-ABA scFv embryos developed green cotyledons containing chloroplasts and accumulated photosynthetic pigments but produced less seed storage protein and oil bodies. Anti-ABA scFv seeds germinated precociously if removed from seed capsules during development but were incapable of germination after drying. Total ABA levels were higher than in wild-type seeds but calculated free ABA levels were near-zero until 21 days after pollination. We show for the first time seed-specific immunomodulation and the resulting switch from the seed maturation programme to a germination programme. We conclude that the immunomodulation of hormones can alter the development programme of target organs, allowing the study of the directly blocked endogenous molecules and manipulation of the system concerned.

Effects of abscisic acid and temperature on erucic acid accumulation in oilseed rape ( Brassica napus L.)

The qualitative effect of growth temperature on the level of erucic acid (22:1) is similar in seeds and microspore-derived embryos (MDEs) of oilseed rape, as we have previously shown (Wilmer et al., 1996), but absolute levels of 22:1 are lower in MDEs. In this paper we studied whether this temperature effect is mediated by changes in the level of or sensitivity to abscisic acid (ABA). For this purpose we measured endogenous levels of ABA in developing seeds and MDEs. In addition, 22:1 accumulation was determined in MDEs exposed to a range of concentrations of ABA. The endogenous level of ABA in seeds, about 5 to 10 nmol/g FW, was twentyfold higher than the concentration at which maximum sensitivity in MDEs occurred. Further evaluation using dose-response curves showed that at both 15 and 25°C the half-maximum increase in 22: 1 level in MDEs was observed at about 0.3 umol/L ABA, though throughout the concentration range tested levels of 22:1 were about 10 mol % higher at 15°C than at 25°C. This indicates that the sensitivity to ABA remains the same in this temperature range. Independent from the temperature effect, exogenously applied ABA induces an increase of about 10 mol % in 22:1 within the range of 10 to 25°C. In addition to this effect on 22:1 level, ABA stimulates the production of triglycerides in MDEs, whereas temperature has no effect on oil accumulation. In combination, our data indicate that growth temperature and ABA are independent and additive stimuli in regulating the level of 22:1 in oilseed rape.

Evidence that ethylene, light and abscisic acid interact to inhibit germination in the recalcitrant seeds ofQuercus roburL.

The effects of ethylene, light and abscisic acid (ABA) on the germination of prematurely-harvested and mature Q. robur seeds were studied. There was evidence of active biosynthesis of ethylene, but not of ABA in seeds from both harvests. Addition of ABA, the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC) or the ethylene-releasing compound Ethephon (E) to germination media and exposure to light individually inhibited germination and their effects were additive. The ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG) tended to promote germination of premature seeds only

Effect of Altered Intraraceme Competition on Carbon‐14‐Labeled Assimilate and Abscisic Acid in Soybean1

Abscission probability varies among floral positions within soybean [Glycine max (L.) Merr.] racemes. Field (1982, Beltsville silt loam, Typic Fragiudult; 1983, Mattapeake silt loam, Typic Hapludult) grown soybean plants (‘Williams’) were used to determinee the distribution of translocated l4C‐labeled photosynthate and abscisic acid (ABA) among reproductive structures differing for abscission probability. The effect of proximal pods on translocation of 14C‐labeled photosynthate to and ABA concentration in distal pods (floral positions 3 and above) was also evaluated. Treatments consisted of removing or not removing the two proximal pods (floral positions 1 and 2) from the major racemes at nodes 8 and 10 in 1982 and 8, 10, and 12 in 1983. Six days after treatment in 1982 and 12 days after treatment in 1983, 14C‐translocation from the leaf at the node of interest was determined after exposure to 14CO2. On the same day, distal pods were collected for determination of ABA by gas chromatography. For untreated plants, proximal and distal pods accounted for 50 and 7% of the translocated 14C, respectively. Proximal pod removal had no effect on 14C retained by the source leaf (72%) or the concentration of ABA in seeds. Concentration of ABA in pod walls of distal pods was increased by proximal pod removal in only one instance. Amount of 14C translocated to distal pods and distal pod specific activity were not altered by proximal pod removal in 1982 but were increased in 1983. The different treatment effect between years may indicate that distal pods undergo a period of adjustment after proximal pod removal. An understanding of processes that occur during the adjustment period may be necessary before the mechanism of soybean reproductive abscission is known.

Abscisic Acid and Its Relationship to Seed Filling in Soybeans

The effect of exogenous abscisic acid (ABA) on the rate of sucrose uptake by soybean (Glycine max L. Merr.) embryos was evaluated in an in vitro system. In addition, the concentrations of endogenous ABA in seeds of three soybean Plant Introduction (PI) lines, differing in seed size, were commpared to their seed growth rates. ABA (10(-7) molar) stimulated in vitro sucrose uptake in soybean (cv ;Clay') embryos removed from plants grown in a controlled environment chamber, but not in embryos removed from field-grown plants of the three PI lines. However, the concentration of ABA in seeds of the three field-grown PI lines correlated well with their in situ seed growth rates and in vitro [(14)C] sucrose uptake rates.Across genotypes, the concentration of ABA in seeds peaked at 8.5 micrograms per gram fresh weight, corresponding to the time of most rapid seed growth rate, and declined to 1.2 micrograms per gram at physiological maturity. Seeds of the large-seeded genotype maintained an ABA concentration at least 50% greater than that of the small-seeded genotype throughout the latter half of seed filling. A higher concentration of ABA was found in seed coats and cotyledons than in embryonic axes. Seed coats of the large-seeded genotype always had a higher concentration of ABA than seed coats of the small-seeded line. It is suggested that this higher concentration of ABA in seed coats of the large-seeded genotype stimulates sucrose unloading into the seed coat apoplast and that ABA in cotyledons may enhance sucrose uptake by the cotyledons.

Temperature and Photoperiod Effects on Abscisic Acid Content in ‘Earliwax’ Snapbean Seed1

Abstract Seeds from plants of ‘Earliwax’ snapbean (Phaseolus vulgaris L.) stressed at 46°C during the reproductive cycle had higher abscisic acid (ABA) levels (285 ng/g) than those of plants stressed at lower temperatures (257 ng/g at 41°C, 249 ng/g at 35°). Higher levels of ABA in seeds also resulted when more mature plants were stressed. A trend towards higher levels of ABA/g seed was observed in plants grown under 16 hour days (219 ng/g) as opposed to 10 hour days (139 ng/g).

Changes in the Concentrations of Abscisic Acid in Fruits of Normal and Nr, rin and nor Mutant Tomatoes During Growth, Maturation and Senescence

The concentrations of free and base-hydrolysable (bound) abscisic acid (ABA) were measured in fruits of cv. Rutgers (normal) and of the mutants Nr, rin and nor during growth, maturation and senescence. Measurements were made also of postharvest changes in free ABA in immature Rutgers fruits. Free ABA began to accumulate rapidly in the pericarp of developing fruits of Rutgers, Nr and rin during the period of most rapid growth but accumulation in nor was delayed and slower. Peak concentrations in Rutgers, Nr and rin were similar but the maximum level in nor was about 50% lower. Peak concentrations of free ABA coincided with the completion of growth in Rutgers and rin but peak levels in Nr and nor were not reached until several days later. Colouring in all strains occurred at approximately the same time as the accumulation of peak concentrations of free ABA. Changes in bound ABA paralleled those in free ABA in pericarp tissue of all strains but the levels were about one-seventh of those of free ABA. Free and bound ABA were measured in seeds and associated mucilaginous tissue only in 50% developed and fully grown fruits. In the younger fruits of Rutgers, Nr and rin, this fraction contained a higher concentration of free ABA than the pericarp tissue. In fully grown fruits, the level of ABA in the seeds and associated tissue was much less than in this fraction of younger fruits and less than half that in the pericarp tissue. Free ABA in seeds and associated tissues remained low in nor fruits of both ages. The ratios of bound and free ABA in seeds and associated tissues in all strains were generally similar to those found in pericarp tissue. In Rutgers fruits, free ABA increased after harvest. It is suggested that ABA is produced in both pericarp and seeds plus associated mucilaginous tissue. Accumulation of ABA does not seem to be a result of increased ethylene production but conversely may be involved in the increased ethylene evolution which accompanies ripening in normal strains. Since the pattern of changes in ABA and the accumulation of peak concentrations in pericarp tissue was not consistently related to growth but was closely related to the onset of symptoms of ripening or senescence, ABA may be a regulator of ripening in the tomato.