Calcium dependence of the effects of abscisic acid on RNA synthesis during germination of Cicer arietinum seeds

The effect of abscisic acid (ABA) and CaCl₂ in the medium on RNA synthesis in embryonic axes of seeds of chick-pea (Cicer arietinum L. cv. Castellana) during the first hours of germination has been studied. ABA decreases the incorporation of ³H-uridine in the embryonic axes and modifies the mRNA populations by preventing the disappearance of three polypeptides of 25, 22 and 21.6 kDa and inducing the appearance of two polypeptides of 35 and 27 kDa absent in the control. Calcium increases the effect of ABA on the synthesis of these mRNAs and seems to be involved in the synthesis of at least the polypeptide of 25 kDa, as this polypeptide disappears in the treatments with EGTA and Verapamil. Moreover, cytosolic calcium seems to be necessary for the accumulation of these messengers since treatment with TMB-8 (chelating agent for endogenous calcium) decreases the intensity of the bands corresponding to these 5 polypeptides. The possible synergistic action of ABA and calcium in this process is discussed.     

Expression of three ABA-regulated clones and their relationship to maturation processes during the embryogenesis of chick-pea seeds

Water stress inhibits germination of chick-pea seeds and produces specific changes in gene expression. some of which are coincident with those induced by the exogenous application of abscisic acid (ABA). Three cDNA clones, GAB-8, GAB-9 and GAB-11, were previously identified as under the regulation of ABA and osmotic stress in embryonic axes of germinating chick-pea. Here we try to establish a relationship between the changes in gene expression induced by ABA and stress conditions during germination and those naturally occurring during the desiccation process that leads to seed maturation. Our results show that the germinative capacity of chick-pea is related to the water content of the organ. In vitro translation of the mRNAs from developing seed reveals that in the later stages of seed maturation some polypeptides appear that previously were found to be regulated by ABA and by water deficit in germinating seeds. Hybridization by northern blot of embryogenic mRNAs with GAB-8. GAB-9 and GAB-11 clones shows that the mRNAs corresponding to such clones only appear in the later phases of seed formation, coinciding with seed dehydration, and persisting until seeds became fully mature. The results suggest that these mRNAs are probably related to the response to dehydration that occurs during seed maturation, and that the pattern of expression of these ABA-regulated clones coincides with that of the established late embryogenesis-abundant (LEA) genes.     

Abscisic acid increases the content of free polyamines and delays mitotic activity induced by spermine in isolated embryonic axes of chick-pea seeds

The effects of abscisic acid (ABA) on the free polyamine: spermine (Spm), spermidine (Spd), putrescine (Put) and cadaverine (Cad) content, mitotic index (MI) and DNA synthesis have been studied in embryonic axes isolated from chick-pea ( Cicer ariennum L.) seeds. Spermine and spermidine decreased in controls during the first 24 h of germination, the former by 70% and the latter by 20%. This decrease was slowed down by ABA (5 μ M or 25 μ M ) in response to its concentration; at 24 h the values for Spm and Spd were 3-fold and 1.5-fold those of the controls, respectively. Exogenous ABA induced the synthesis of Put from 0 to 24 h, by which time the content of this polyamine had doubled. The controls at 24 h registered the same Put content as those with 25 μ M ABA, suggesting that ABA had ceased to induce Put synthesis. The pattern for cadaverine ABA-induced synthesis was similar to that of Put. From 18 to 24 h, however, the level of Cad in the controls was 3 times higher than in the treatments. The mitotic index in meristem cells reached a maximum at 30–36 h, when cell elongation stopped in the sub-apical region. This maximum was lowered by exogenous ABA in response to the concentration and occurred 12 h earlier in the presence of Spm (0.1–1.0 m M ), which induced high MI levels during the period studied. With 10 m M Spm, two complete waves of mitosis were observed, but when Spm and ABA were added together, the tetramine had no observable effect. Spermine stimulated ³H-thymidine uptake as well as DNA synthesis, with a maximum at 12 h, but did not counteract the inhibitory effect of ABA. Our results suggest that ABA causes changes in mitotic activity that Spm cannot overcome.     

Involvement of cytokinins in the germination of chick-pea seeds

Eight cytokinins detected in germinated chick-pea (Cicer arietinum L. var. Castellana) seeds were first present in the embryonic axes but appeared in the cotyledons after 12h of germination. The cytokinins detected in the cotyledons originate in the embryonic axes, but no passage of these substances from the cotyledons to the axes was detected, except when the seeds were treated with red light.It is concluded that the role played by the embryonic axis in mobilizating the main reserves of the cotyledons is mainly effected through these cytokinins. Both natural and synthetic cytokinins exert an important regulatory role in the hydrolysis of reserve proteins and calcium could be involved as an intermediate.Abbreviations BA benzyladenine - cot. cotyledon - (diH)Z dihydrozeatin - (diH)ZR dihydrozeatin riboside - GZR glycosyl zeatin riboside - 2iP 277-1 - iPA 277-2 riboside - Kin kinetin - Z zeatin - ZG zeatin glucoside - ZR zeatin riboside     

Abscisic acid and temperature modify the levels of calmodulin in embryonic axes of Cicer arietinum

Changes in calmodulin (CaM) levels in embryonic axes of Cicer arietinum L. cv. Castellana germinated under three different conditions were measured. Abscisic acid (ABA) and a temperature of 30°C, which delay chick-pea germination, respectively decreased and increased the concentration of CaM compared to the values obtained under normal germinating conditions (H₂O-25°C). The CaM concentration was higher in those zones of the axes undergoing an active cell division.
The compartmentalization of CaM in 36-h-old embryonic axes grown under these three conditions was also measured. Cytosolic and mitochondrial CaM was higher in axes where a delayed germination occurred as well as in the cell walls of normally grown ones. On the other hand, CaM was higher in nuclear and microsomal fractions extracted from H₂-O-25°C-treated axes. From these data we postulate that delayed germination could be an effect of altered CaM distribution in chick-pea embryonic axes.     

Dormancy in somatic embryos and seeds ofVitis: changes in endogenous abscisic acid during embryogeny and germination

Abscisic acid (ABA) in extracts of somatic embryos and seeds of Gloryvine (Vitis vinifera L.xV. rupestris Scheele) was measured by gas chromatography-mass spectrometry-selected ion monitoring using deuterated ABA, (±)-[C-3Me-²H₃]ABA, ([²H₃]ABA) as internal standard. The ABA content increased rapidly during embryogeny (0.035 ng/embryo at the globular stage to 0.22 ng/embryo at the mature stage). The level of ABA in the tissues of somatic embryos, expressed in ng/mg dry weight, decreased from the globular stage (0.76 ng/mg) to the mature stage (0.25 ng/mg). Chilling (4° C) induced normal germination of seeds and mature somatic embryos and precocious germination of globular, heart-shaped and torpedoshaped somatic embryos. In all cases chilling led to a marked reduction in endogenous ABA. Exogenous (±)-ABA inhibited the germination of chilled somatic embryos.Abbreviations ABA abscisic acid - [²H₃]ABA (±)-[C-3Me-²H₃]-abscisic acid - BHT 2,6-di-t-butyl-4-methylphenol - GC-MS gas chromatography-mass spectrometry - Me-ABA and Me-[²H₃]ABA methyl esters of ABA and [²H₃]ABA, respectively - SIM selected ion monitoring     

Regulation of cadaverine and putrescine levels in different organs of chick-pea seed and seedlings during germination

In chick-pea ( Cicer arietinum L.) seed germinated in the presence of ¹⁴C-lysine, the latter is taken up and partly metabolised to cadaverine and TCA-precipitable molecules. Labelled cadaverine is detectable in seedlings only after 3 days, on a labelled lysine-containing medium, as confirmed also by the presence of lysine decarboxylase (LDC) activity, measured in the embryo axis and cotyledons of the seed and in the epicotyl, cotyledons, hypocotyl and roots of the seedling on the basis of ¹⁴CO₂ evolution from the labelled precursor. Putrescine biosynthesis occurred only via arginine decarboxylase (ADC) activities in soaked seeds and via both ADC and ornithine decarboxylase (ODC) activities in seedlings. Both putrescine and cadaverine were present in soaked seed, and accumulated in very large amounts in the different portions of both 3- and 8-day-old seedlings, while spermidine and spermine titers were maintained at similar levels with respect to the seed. Diamine oxidase activity, measured by evaluating oxygen consumption in the presence of putrescine, was absent in ungerminated seed and appeared in 3- and 8-day-old seedlings. In order to clarify the metabolic relationships between cadaverine and the more common polyamines, gradients of biosynthesis, accumulation and degradation of putrescine and cadaverine along the seedling axis were compared, indicating that the two diamines behave similarly during seed germination and seedling development. Their conspicuous accumulation (up to 6 m M for putrescine) seems to be regulated mainly via oxidation rather than biosynthesis.     

Endogenous abscisic acid and wheat germ agglutinin content in wheat grains during development

Abscisic acid (ABA) and wheat germ agglutinin content of immature wheat grains and embryos was determined by immunoassay throughout the development of a field-grown wheat crop ( Triticum aestivum cv. Timmo). Wheat germ agglutinin accumulation in the embryo was not preceded by an increase in endogenous abscisic acid amount or concentration in either embryos or grains. At a later stage in development the endogenous concentration of abscisic acid in both embryos and grains was found to be two orders of magnitude lower than the endogenous levels required to inhibit precocious germination and promote wheat germ agglutinin accumulation in excised embryos cultured in vitro. These findings are discussed in the context of the control of embryo development in vivo by both ABA and the water status of the grain and embryo.     

Endogenous levels of polyamines and abscisic acid in pepper fruits during growth and ripening

The levels of polyamines (PA) and abscisic acid (ABA) in the pericarp of California variety pepper fruit ( Capsicum annuum L.) were analyzed during development and ripening. Putrescine level was 2.75 μmol g⁻¹ fresh weight 7 days after fruit set and fell during the exponential stage of growth to 1.05 μmol g⁻¹ fresh weight. During the second growth stage. PA and ABA levels remained stable and fell sharply at the beginning of maturation. The levels of spermidine and spermine decreased throughout fruit development and maturation from 0.61 to 0.05 and 0.31 to 0.02 μmol g⁻¹ fresh weight, respectively, but no changes were associated with the onset of maturation. ABA levels remained high (0.70-0.80 μg g⁻¹ fresh weight) during the stages of fruit growth and fell at the beginning of maturation to 0.12 μg g⁻¹ fresh weight, before rising again during the last stages of maturation and senescence. The decrease in putrescine and ABA levels and the subsequent increase in the latter may be responsible for controlling the processes of ripening in pepper fruit.     

Synthesis and biological activity of abscisic acid esters

16 ABA esters including 11 new compounds were prepared by two different esterification routes. All the structures of ABA esters were confirmed by ¹H NMR, ¹³C NMR and HRMS. Their biological activity and hydrolysis stability were investigated. Fortunately, there were 15 and 9 compounds which displayed much better or nearly the same inhibition activity for rice seedling growth and Arabidopsis thaliana seed germination compared to ABA, respectively. Especially, compounds 2d and 2g showed better biological activities than ABA in the three tests. Moreover, we found that chemical hydrolysis ability of the esters in vitro had little relationship to their biological activity.     

Changes during light and dark osmotic treatment independently modulating germination and ribonucleic acid synthesis in Chenopodium bonus-henricus seeds

The content and synthesis of RNA in Chenopodium bonus-henricus L. seeds in different physiological states were evaluated. A moist-chilling treatment at 4°C removed the primary dormancy of seeds. A pretreatment of chilled seed in low osmotic potential (–8.6 bars) polyethylene glycol-6000 solution at 15°C in light led to an advancement in subsequent germination time in water while a treatment in darkness induced a secondary dormancy. The synthesis of total RNA and poly A (+) RNA was correlated with the capacity of seeds to germinate. Cordycepin or α-amanitin failed to inhibit the germination of unchilled seeds, chilled seeds or chilled seeds given a prior light osmotic treatment. Also, germination of chilled seeds was not affected by cordycepin applied during light osmotic treatment. However, cordycepin effectively depressed the synthesis of both total RNA and poly A (+) RNA in chilled seeds with or without a prior light osmotic treatment. These data suggest that germination per se may depend upon the activation of pre-existing mRNA, which might be functionally different from the newly synthesized mRNA including poly A (+) RNA.     

Effects of abscisic acid,gibberellic acid and fusicoccin on the transmembrane potential during the early phases of germination in radish (Raphanus sativus L.) seeds

During germination, the transmembrane electric potential (PD) of cortical cells of the embryonal axis of radish seeds (Raphanus sativus L.) rises from-120 mV initially to a maximum of-150 mV after 5 h incubation, then falls again to stable values of around-120 mV. Treatments inhibiting germination block the transitory PD increase. Administration of uncoupling agents or low temperatures, during the process of germination, produces a marked fall of the PD transitory increase. Abscisic Acid has a parallel inhibitory effect on PD and germination, while fusicoccin produces a rise in both; administration of abscisic acid with fusicoccin inhibits germination, while the PD remains at the high levels given by fusicoccin. These results are discussed in relation to ion exchange at membrane level.Abbreviations ABA abscisic acid - FC fusicoccin - GA₃ gibberellic acid - PD electric potential difference (between the vacuole and the external medium) - CH cycloheximide - DNP dinitrophenol - FCCP (p-trifluormethoxy)-carbonylcyanide-phenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide     

Control of macaw palm seed germination by the gibberellin/abscisic acid balance

The hormonal mechanisms involved in palm seed germination are not fully understood. To better understand how germination is regulated in Arecaceae, we used macaw palm (Acrocomia aculeata (Jacq.) Lodd. Ex Mart.) seed as a model. Endogenous hormone concentrations, tocopherol and tocotrienol and lipid peroxidation during germination were studied separately in the embryo and endosperm. Evaluations were performed in dry (D), imbibed (I), germinated (G) and non‐germinated (NG) seeds treated (+GA₃) or not treated (control) with gibberellins (GA). With GA₃ treatment, seeds germinated faster and to a higher percentage than control seeds. The +GA₃ treatment increased total bioactive GA in the embryo during germination relative to the control. Abscisic acid (ABA) concentrations decreased gradually from D to G in both tissues. Embryos of G seeds had a lower ABA content than NG seeds in both treatments. The GA/ABA ratio in the embryo was significantly higher in G than NG seeds. The +GA₃ treatment did not significantly affect the GA/ABA ratio in either treatment. Cytokinin content increased from dry to germinated seeds. Jasmonic acid (JA) increased and 1‐aminocyclopropane‐1‐carboylic acid (ACC) decreased after imbibition. In addition, α‐tocopherol and α‐tocotrienol decreased, while lipid peroxidation increased in the embryo during germination. We conclude that germination in macaw palm seed involves reductions in ABA content and, consequently, increased GA/ABA in the embryo. Furthermore, the imbibition process generates oxidative stress (as observed by changes in vitamin E and MDA).     

Antagonistic effects of abscisic acid and gibberellic acid on the breaking of dormancy of Fagus sylvatica seeds

Study of the factors involved in the dormancy of Fagus sylvatica seeds shows that such dormancy is due partly to the seed coats and partly to endogenous factors. Seed coat removal accelerates both the release from dormancy and the effects of the other treatments that abolish it. The dormancy of these seeds is eliminated by cold treatment at 4°C over a period longer than 8 weeks, and exogenous application of abscisic acid (ABA) reverses the effects of low temperature, the seeds remaining in an ungerminated state. Additionally, ABA reduces protein synthesis but slightly increases RNA synthesis, which suggests its involvement in the synthesis of RNAs related to this process. In vitro translation of the RNAs isolated from these seeds shows that ABA delays the disappearance of at least 2 polypeptides (of ca 22 and 24 kDa), which are abundant in dormant seeds and under conditions that prevent the release from dormancy, but which disappear under treatments that abolish it. Exogenous application of gibberellic acid (GA₃) proved to be efficient in breaking the dormancy of these seeds and in substituting for cold treatment as well as in antagonizing the effects of ABA on the synthesis of both DNA and proteins. GA₃ also accelerates the disappearance of the two polypeptides abundant in dormant seeds and in ABA-treated seeds. These findings suggest that both ABA and GA₃ could be involved in the regulation of nucleic acid and protein metabolism during dormancy, acting antagonistically in these processes and, specifically, in the regulation of the synthesis of the two proteins that appear to play a role in the maintenance of dormancy in these seeds.     

A radioimmunoassay for abscisic acid

We have developed a radioimmunoassay (RIA) for abscisic acid (ABA) in the 0.1 ng to 2.5 ng range. Antibodies were obtained from rabbits immunized with ABA bound via its carboxyl group to bovine serum albumin. Cross-reactivity studies indicate that ABA esters are completely cross-reactive with ABA, while trans, trans abscisic acid (t-ABA) phaseic acid (PA) and dihydrophaseic acid (DPA) have much lower but significant cross-reactivities. Purification methods which reduce the levels of cross-reacting substances are described.Abbreviations RIA radioimmunoassay - DPA 4-dihydrophaseic acid - PA phaseic acid - GC gas chromatography - HPLC high performance liquid chromatography - TLC thin-layer chromatography - BSA bovine serum albumin - ABA abscisic acid - t-ABA trans, trans abscisic acid - IAA indoleacetic acid     

Indoleacetic acid, abscisic acid and phenolic substances during development of hazel leaves

The levels of indoleacetic acid (IAA), abscisic acid (ABA) and phenolic substances were measured monthly in leaves of hazel ( Corylus avellana L. cv. Negreta) in different stages of development from April till abscission in November. IAA and ABA were quantified by flame ionization detector gas-chromatography (GLC), and the total phenolic content by a colorimetric technique. During spring, IAA levels were the highest, coinciding with the greatest expansion of the leaves; after which IAA gradually decreased as the leaves became older, although minor increases were observed in July-August and November. The ABA level in hazel leaves was low throughout the period of leaf existence, with a maximum in July and a second increase in November, coinciding with the last month in which hazel bears leaves. The phenols of the neutral fraction showed a minimum in May and a pronounced increase in June, remaining at this level during summer and decreasing in November. The acid and alkaline fractions showed a maximum in August, which could be related to the decrease in the growth rate of leaves. The residual fraction remained at a relatively low level until September, increasing rapidly in October and November.     

Effect of ABA, fusicoccin and thiourea on germination and K+ and glucose uptake in chick-pea seeds at different temperatures

Germination and growth of seeds of Cicer arietinum L. are retarded or inhibited either by temperatures above 30°C or by the presence of abscisic acid (ABA). Both treatments affect the uptake of K⁺ and glucose. ABA may affect the properties of the cell membrane or the kinetics of the enzymes bound to it. Fusicoccin (FC) and thiourea (TU) are to a greater or lesser extent able to counteract the inhibition produced by ABA and high temperatures. Both FC and TU increase the uptake of K⁺ by the embryonic axis. FC seems to act directly on H⁺ extrusion, but the mode of action of TU appears to be different. The results seem to indicate the importance of the activation of the ion transport process for the growth of the embryonic axis during early germination, and suggest a control of the growth of the embryo by part of the cotyledons. Physiological inhibitors such as ABA might be implied in the control mechanism.     

The relationships between ethylene production and cell elongation during the initial growth period of chick-pea seeds (Cicer arietinum)

Ethylene production by isolated chick-pea embryonic axes during the initial stages of germination has been studied. Maximum production of ethylene occurs when growth is entirely due to cell elongation and before mitotic activity begins. This peak increases three-fold in the presence of calcium, but it is diminished by osmotic inhibitors, polyamines and abscisic acid (ABA), with a parallel fall in growth rate. Fusicoccin stimulates ethylene production and counteracts the effects of polyethylene glycol; thiourea, which breaks thermodormancy in chick-pea seeds, reduces ethylene production but does not counteract the effects of osmotic inhibitors. Of the polyamines studied, spermine (in low concentrations, 0.1 to 1.0 m M ) is the only one to stimulate ethylene production and cell elongation. It is concluded that there is a close relationship between ethylene production and cell elongation.