Relationship of endogenous abscisic Acid to sucrose level and seed growth rate of soybeans

It has been proposed that abscisic acid (ABA) may stimulate sucrose transport into filling seeds of legumes, potentially regulating seed growth rate. The objective of this study was to determine whether the rate of dry matter accumulation in seeds of soybeans (Glycine max L.) is correlated with the endogenous levels of ABA and sucrose in those sinks. The levels of ABA and sucrose in seed tissues were compared in nine diverse Plant Introduction lines having seed growth rates ranging from 2.5 to 10.0 milligrams dry weight per seed per day. At 14 days after anthesis (DAA), seeds of all genotypes contained less than 2 micrograms of ABA per gram fresh weight. Levels of ABA increased rapidly, however, reaching maxima at 20 to 30 DAA, depending upon tissue type and genotype. ABA accumulated first in seed coats and then in embryos, and ABA maxima were higher in seed coats (8 to 20 micrograms per gram fresh weight) than in embryos (4 to 9 micrograms per gram fresh weight. From 30 to 50 DAA, ABA levels in both tissues decreased to less than 2 micrograms per gram fresh weight. Levels of sucrose were also low early in development, less than 10 milligrams per gram fresh weight at 14 DAA. However, by 30 DAA, sucrose levels in seed coats had increased to 20 milligrams per gram fresh weight and remained fairly constant for the remainder of the filling period. In contrast, sucrose accumulated in embryos throughout the filling period, reaching levels greater than 40 milligrams per gram fresh weight by 50 DAA. Correlation analyses indicated that the level of ABA in seed coats and embryos was not directly correlated to the level of sucrose measured in those tissues or to the rate of seed dry matter accumulation during the linear filling period. Rather, the ubiquitous pattern of ABA accumulation early in development appeared to coincide with water uptake and the rapid expansion of cotyledons occurring at that time. Whole tissue sucrose levels in embryos and seed coats, as well as sucrose levels in the embryo apoplast, were generally not correlated with the rate of dry matter accumulation. Thus, it appears that, in this set of diverse soybean genotypes, seed growth rate was not limited by endogenous concentrations of ABA or sucrose in reproductive tissues.     

Regulation of Embryo Dormancy by Manipulation of Abscisic Acid in Kernels and Associated Cob Tissue of Zea mays L. Cultured in Vitro

Sectors of Zea mays cobs, with and without kernels were cultured in vitro in the presence and absence of fluridone. Cultured kernels, cob tissue, and embryos developed similarly to those grown in the field. Abscisic acid (ABA) levels in the embryos were evaluated by enzyme-linked immunosorbant assay. ABA levels in intact embryos cultured in the presence of fluridone were extremely low and indicate an inhibition of ABA synthesis. ABA levels in isolated cob tissue indicate that ABA can be produced by cob tissue. Sections containing kernels cultured in the presence of fluridone were transferred to medium containing fluridone and ABA. Dormancy was induced in more than 50% of the kernels transferred from 13 to 15 days after pollination, but all of the kernels transferred at 16 days after pollination or later were viviparous. ABA recovered from kernels that were placed in medium containing fluridone and ABA suggest that ABA can be transported through the cob tissue into developing embryos and that ABA is required for induction of dormancy in intact embryos.     

Role of ABA in Maturation of Rapeseed Embryos

Development of Brassica napus L. cv Tower embryos of different ages cultured in vitro with and without abscisic acid (ABA) was compared with normal development in situ to investigate the role of ABA in embryo maturation. Endogenous ABA levels were measured by radioimmunoassay, and sensitivity to ABA was assayed in terms of its ability to suppress precocious germination and stimulate accumulation of storage protein and storage protein mRNA. During development in situ, the levels of endogenous ABA and 12S storage protein mRNA both reach their peaks just before the embryos begin to desiccate. The ABA levels during this phase of development also correlate with the time required in culture before germination is evident. Following these peaks, increasing concentrations of exogenous ABA are required to both suppress germination and continue storage protein accumulation in vitro. Thus, both endogenous ABA and ABA sensitivity decline during maturation. The concentrations of exogenous ABA required to suppress germination at these later stages result in abnormally high levels of endogenous ABA and appear to be toxic. These results are consistent with the hypothesis that in maturing rapeseeds, low water content rather than ABA prevents germination during the later stages of development.     

Induction of alpha-amylase inhibitor synthesis in barley embryos and young seedlings by abscisic Acid and dehydration stress

An endogenous α-amylase inhibitor was found to be synthesized in embryos of developing barley grain (Hordeum vulgare cv Bonanza). Accumulation of this protein occurred late in development (stage IV), at the same time that endogenous abscisic acid (ABA) showed a large increase. The inhibitor could be induced up to 23-fold in isolated immature embryos (stage III) by culture in ABA. Precocious germination was also blocked in stage III embryos by ABA. Dehydration stress on the isolated immature embryos also induced higher levels of the inhibitor and ABA. An even greater response to dehydration stress was observed in young seedlings, where inhibitor content increased 20-fold and ABA increased 80-fold during water stress. The high degree of correlation between ABA and inhibitor contents in in situ embryos, dehydrated embryos and young seedlings, as well as the increase in inhibitor caused by exogenously applied ABA to isolated embryos, suggests that increased α-amylase inhibitor synthesis in response to dehydration stress is mediated by ABA.     

In Situ Abscisic Acid Synthesis : A Requirement for Induction of Embryo Dormancy in Helianthus annuus

When applied to young nondormant embryos of sunflower (Hellanthus annus) (7-10 day[s] after pollination [DAP]), abscisic acid (ABA) inhibited germination as long as it was present. However, whatever the dose used and the duration of its application, ABA was unable to induce dormancy because after transfer of treated embryos to control (without ABA) medium, germination occurred. Thereafter, exogenous ABA became effective and allowed the dormancy to develop in 13 and 17 DAP embryos, i.e. in embryos which after isolation were still able to germinate in high percentage. After embryo dormancy was well established (21 DAP), application of fluridone allowed the germination to occur very quickly on control medium. Isolated dormant axes were also induced to germinate by an application of fluridone. Radioimmunological analysis showed that 24 hours after these treatments, endogenous ABA levels were drastically reduced in the axes. When these fluridone-treated embryos were cultured on ABA medium, germination was again inhibited as long as exogenous ABA was present but germination occurred as soon as embryos were transferred to control medium. Such behavior suggested that in situ ABA synthesis is necessary to impose and maintain the embryo dormancy.     

Abscisic Acid and the maturation of cacao embryos in vitro

Abscisic acid (ABA) was tested for its ability to affect development of immature zygotic embryos of cacao (Theobroma cacao) in vitro, by adding exogenous ABA, fluridone, or mefluidide to cultured embryos. Endogenous ABA levels, measured by enzyme-linked immunosorbent assay, were increased by exogenous ABA or by culture on sucrose increasing to 21%, and were decreased by fluridone and, to a lesser extent, by mefluidide. The effects of these on maturation were measured as effects on anthocyanins, lipids, and fatty acid saturation, all of which increase with maturation of the cacao embryo. Maturation was stimulated by increasing sucrose and, to a lesser degree, the addition of ABA, but decreasing endogenous ABA by treating with fluridone significantly inhibited all maturation parameters. Although desiccation tolerance does not develop in cacao embryos, these results suggest that ABA and sucrose are both needed for the initiation of events associated with maturation in vitro.     

Rapeseed embryo development in culture on high osmoticum is similar to that in seeds

The development of Brassica napus L. cv Tower embryos of different ages cultured in vitro with and without high osmoticum (0.48 and 0.69 molar sorbitol) was compared with normal development in situ to investigate the role of a drying environment in embryo maturation. Sensitivity to osmoticum was assayed in terms of its ability to mimic normal development, i.e. to both suppress germination and maintain 12 S storage protein (cruciferin) synthesis at levels comparable to those seen in the developing seed. The osmotic conditions used block germination of predesiccation stage embryos but were not sufficient to prevent desiccation stage embryos from taking up water and germinating. At all stages tested, the osmotically treated embryos had approximately normal levels of cruciferin mRNA. Measurements of endogenous abscisic acid (ABA) levels by radioimmunoassay indicated that the osmotic effects on germination and gene expression were not mediated by elevated embryonic ABA. Comparison of the kinetics of osmotic and ABA effects on gene expression showed that the osmotic effect is more rapid. These results are consistent with the hypothesis that ABA acts by inhibiting water uptake, which mechanically prevents germination and affects gene expression in some unknown manner.     

Abscisic acid and osmoticum prevent germination of developing alfalfa embryos,but only osmoticum maintains the synthesis of developmental proteins

Developing seeds of alfalfa (Medicago sativa L.) acquire the ability to germinate during the latter stages of development, the maturation drying phase. Isolated embryos placed on Murashige and Skoog medium germinate well during early and late development, but poorly during mid-development; however, when placed on water they germinate well only during the latter stage of development. Germination of isolated embryos is very slow and poor when they are incubated in the presence of surrounding seed structures (the endosperm or seed coat) taken from the mid-development stages. This inhibitory effect is also achieved by incubating embryos in 10^?5 M abscisic acid (ABA). Endogenous ABA attains a high level during mid-development, especially in the endosperm. Seeds developing in pods treated with fluridone (1-methyl-3-phenyl-5[3-(trifluoromethyl)-phenyl]-4(1H)-pyridinone) contain low levels of ABA during mid-development, and the endosperm and seed coat only weakly inhibit the germination of isolated embryos. However, intact seeds from fluridone-treated pods do not germinate viviparously, which is indicative that ABA alone is not responsible for maintaining seeds in a developing state. Application of osmoticum (e.g. 0.35 M sucrose) to isolated developing embryos prevents their germination. Also, in the developing seed in situ the osmotic potential is high. Thus internal levels of osmoticum may play a role in preventing germination of the embryo and maintaining development. Abscisic acid and osmoticum impart distinctly different metabolic responses on developing embryos, as demonstrated by their protein-synthetic capacity. Only in the presence of osmoticum do embryos synthesize proteins which are distinctly recognizable as those synthesized by developing embryos in situ, i.e. when inside the pod. Abscisic acid induces the synthesis of a few unique proteins, but these arise even in mature embryos treated with ABA. Thus while both osmoticum and ABA prevent precocious germination, their effects on the synthetic capacity of the developing embryo are quite distinct. Since seeds with low endogenous ABA do not germinate, osmotic regulation may be the more important of these two factors in controlling seed development.     

Seed development and vivipary in Zea mays L.

Seed development was investigated in kernels of developing wild-type and viviparous (vp-1) Zea mays L. Embryos and endosperm of wild-type kernels began to dehydrate at approx. 35 d after pollination (DAP); viviparous embryos did not desiccate but accumulated fresh weight via coleoptile growth in the caryopses. Concentrations of endogenous abscisic acid (ABA) in the embryo were relatively high early in development, being approx. 150 ng·g^-1 fresh weight at 20 DAP. The ABA content declined thereafter, falling to approx. 50 ng·g^-1 at 30 DAP. Endosperm ABA content was always low, being less than 20 ng·g^-1. There were no differences between wild-type and vp-1 tissues. Immature kernels did not germinate when removed from the ear until late in development. The ability to germinate was correlated with decreasing moisture content in the endosperm at the time of removal; premature drying of immature kernels resulted in greatly increased germination following imbibition. Excised embryos germinated precociously when removed from the endosperm as early as 25 DAP. Such germination could be prevented by treatment with 10^-5 M ABA or by lowering the solute potential (_s) of the medium with 0.3 M mannitol. Treatment of excised embryos with ABA led to internal ABA concentrations comparable to those in embryos in which germination was inhibited in situ. Mannitol treatment did not have this effect, although water-deficit stress of excised embryos resulted in substantial ABA production. Germinated vp-1 embryos were less sensitive to growth inhibition by ABA or mannitol than germinating wild-type embryos. The vp-1 seedlings were not wilty and their transpiration rates were reduced in response to ABA or water shortage.Abbreviations and symbols ABA abscisic acid - DAP days after pollination - FW fresh weight - vp-1 viviparous genotype - _s solute potential     

Abscisic Acid Regulation of DC8, A Carrot Embryonic Gene

DC8 encodes a hydrophylic 66 kilodalton protein located in the cytoplasm and cell walls of carrot (Daucus carota) embryo and endosperm. During somatic embryogenesis, the levels of DC8 mRNA and protein begin to increase 5 days after removal of auxin. To study the role of abscisic acid (ABA) in the regulation of DC8 gene, fluridone, 1-methyl-3-phenyl,-5(3-trifluoro-methyl-phenyl)-4(1H)-pyridinone, was used to inhibit the endogenous ABA content of the embryos. Fluridone, 50 micrograms per milliliter, effectively inhibits the accumulation of ABA in globular-tage enbryos. Western and Northern analysis show that when fluridone is added to the culture medium DC8 protein and mRNA decrease to very low levels. ABA added to fluridone supplemented culture media restores the DC8 protein and mRNA to control levels. Globular-stage embryos contain 0.9 to 1.4 × 10⁻⁷ molar ABA while 10⁻⁶ molar exogenously supplied ABA is the optimal concentration for restoration of DC8 protein accumulation in fluridone-treated embryos. The mRNA level is increased after 15 minutes of ABA addition and reaches maximal levels by 60 minutes. Evidence is presented that, unlike other ABA-regulated genes, DC8 is not induced in nonembryonic tissues via desiccation nor addition of ABA.     

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 [¹⁴C] 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.

     

Effect of exogenous ABA on embryo maturation and quantification of endogenous levels of ABA and IAA in <Emphasis Type="Italic">Quercus suber</Emphasis> somatic embryos

Knowledge of the relationship between indole-3-acetic acid (IAA) and abscisic acid (ABA) is relevant to control the development and the maturation of cork oak (Quercus suber L.) somatic embryos. The addition of 1 M ABA to the culture medium significantly promoted somatic embryo maturation and increased both fresh and dry matter without affecting the relative water content. This effect was parallel to the pattern of variation observed in the endogenous ABA level, which increased from the immature to the mature stage. Endogenous ABA content during the occurrence of secondary embryogenesis was similar to that of the immature stage, showing that embryos with lower ABA levels produced secondary embryos. In contrast, IAA showed the highest concentration during early embryo development and decreased afterwards. Only in somatic embryos subjected to 1-week desiccation followed by stratification at 4 °C for 2 weeks, was a moderate increment of endogenous IAA content observed. IAA and ABA showed opposite levels during the development and maturation of cork oak somatic embryos and characterised specific stages of the embryonic development.     

A hormonal misunderstanding in Acca sellowiana embryogenesis: levels of zygotic embryogenesis do not match those of somatic embryogenesis

Endogenous levels of IAA, ABA and four types of CKs were analyzed in zygotic and indirect (ISE) and direct somatic embryogenesis of Acca sellowiana. Zygotic and somatic embryos at different developmental stages were sampled for morphological and hormonal analysis. Both embryo types showed substantial asymmetry in hormone levels. Zygotic embryos displayed a conspicuous peak of IAA in early developmental stages. The results outlined the hormonal variations occurring during zygotic and somatic embryogenesis regarding the timing, nature and hormonal status involved in both processes. The short transient pulse of IAA observed on the 3rd day in culture was suggested to be involved with the signaling for the induction of somatic embryogenesis. Fertilized ovule development was associated with increased IAA levels 21?C24?days after pollination, followed by a sharp decrease in the cotyledonary stage, both in zygotic and somatic embryos. There was a prominent increase in ABA levels in cultures which generated ISE 24?C30?days after pollination, a period that corresponds to the heart and torpedo stages. The levels of total CKs (Z, [9R]Z, iP and [9R]iP) were also always higher in zygotic than in somatic embryogenesis. While zygotic embryogenesis was dominated by the presence of zeatin, the somatic process, contrarily, was characterized by a large variation of the other cytokinin forms and amounts studied. The above results, when taken together, could be related to the previously observed high frequency formation of anomalous somatic embryos formed in A. sellowiana, as well as to their low germination ability.     

The transition of proembryogenic masses to somatic embryos in Araucaria angustifolia (Bertol.) Kuntze is related to the endogenous contents of IAA,ABA and polyamines

In somatic embryogenesis (SE) of conifers, the inability of many embryogenic cell lines to form well-developed somatic embryos may results from failure and constraints during the transition of proembryogenic masses (PEMs) to early somatic embryos. In the present work, we propose the inclusion of a preculture and prematuration steps looking at enhancing PEM III-to-early somatic embryos transition. It was further hypothesized that these results would correlate with the contents of endogenous indole-3-acetic acid (IAA), abscisic acid (ABA) and polyamines (PA). To test these hypotheses, the embryogenic culture was subjected to preculture with fluridone (FLD) and prematuration treatments with different combinations of carbon source and polyethylene glycol (PEG). The frequency of PEM III was increased after FLD preculture and the contents of IAA and ABA decreased, while the contents of PA increased. Putrescine (Put) was the most abundant PA present at this stage, followed by spermidine (Spd) and spermine (Spm). In early embryogenesis, prematuration treatments supplemented with maltose or lactose plus PEG enhanced the PEM III-to-early somatic embryos transition. IAA and ABA contents increased at this stage, while a decrease of the total free PA levels was observed. Put was the most abundant PA, followed by Spd and Spm, mainly in the treatment supplemented with PEG. This resulted in a decrease of PA ratio (Put/Spd + Spm) and, hence, PEM III-to-early somatic embryos transition. It was concluded that the preculture with FLD and prematuration treatments promote the PEM III-to-early somatic embryos transition throughout the whole early developmental process in Araucaria angustifolia.