Solid Phase Radioimmunoassay for the Quantitation of Abscisic Acid in Plant Crude Extracts Using a New Monoclonal Antibody

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.Growth of the peach fruit occurs in three stages, as shown by Connors (4): rapid growth (stage I), delayed growth (stage II), and second rapid growth (stage III). The growth of the nucellus and integuments during stage I is parallel to the rapid growth ofthe pericarp, while embryo development is arrested. At the beginning of stage II (delayed development of the pericarp), the nucellus and integuments enter a period of delayed development, and the endosperm begins a rapid enlargement. During stage III, the nucellus degenerates and the embryo begins its development; depending on the variety, it reaches maximum size during this period.The endogenous systems that regulate this growth pattern are not known, but several scientists have described a regulating role played by hormones. A high content of gibberellins has been observed during peach seed development (6,17,18). Ethylene production also occurs at relatively high rates and, at the same time, 1 -aminocyclopropane-1 -carboxylic acid concentration peaks in the fruit (8, 10). Auxin level is also high during seed development, as shown by Miller et al. (1 1). Few data are available on ABA content in peach seeds (2) and particularly on ABA quantification in the various seed components during development.The availability of immunological methods for the quantification of ABA allows accurate analysis of ABA content over a great number of samples in much less time compared with the more traditional gas-chromatographic analysis. In fact, ABA content in the various tissues during seed development has been thoroughly measured in recent investigations carried out on Sechium edule (15) and Phaseolus coccineus (12).In this paper, we report on the quantitation of ABA in integuments, nucellus, endosperms, and embryos of Prunus persica cv Springcrest during seed development, with the aim of extending information on the hormonal relation between these seed components. MATERIALS AND METHODS Plant MaterialDeveloping fruits of peach (Prunus persica cv Springcrest), were harvested in the open field from May to June 1990. Care was taken to spread the sampling over the trees so that it would not drastically alter the crop load of any tree. Fruits were open...

Section: Resultsmentioning

confidence: 99%

“…The amount of ABA in the HPLC fractions was determined by a solid-phase RIA based on the use of a monoclonal antibody (DBPA1) raised against free (S)-ABA (14).…”

Section: Aba Determinationmentioning

confidence: 99%

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

Piaggesi¹,

Perata²,

Vitagliano³

et al. 1991

“…Water extraction efficiency was checked and found to be over 95% (22). Extraction and analysis of ABA were repeated five times with seed tissues from collections taken at different times.…”

Section: Plant Materialsmentioning

confidence: 99%

“…The amount of ABA in the tissues was determined by means of a solid-phase RIA based on the use of a monoclonal antibody (DBPA 1) raised against free (S)-ABA (22). …”

Section: Aba Determinationmentioning

confidence: 99%

Pattern of Variations in Abscisic Acid Content in Suspensors, Embryos, and Integuments of Developing Phaseolus coccineus Seeds

Perata¹,

Picciarelli²,

Alpi³

1990

Free abscisic acid (ABA) content in suspensors, embryos, and integuments was determined during seed development of Phaseolus coccineus. A highly specific and sensitive solid-phase radioimmunoassay based on a monoclonal antibody raised against free (S)-ABA was used for ABA quantification. Very small amounts of ABA were detected in the suspensor during initial stages of development; later two peaks of ABA occurred. Levels of ABA in the embryo and integument show a coincident triphasic distribution: two maxima in ABA content occurred when the embryo was 11 to 12 and 15 to 16 millimeters in length; later, when the embryo was 19 to 20 millimeters long, a further increase was observed. The role of ABA in runner bean seeds is discussed in relation to the development of the different seed tissues.

“…Endogenous levels of ABA in the same seeds have been also investigated (4), and absence of hormone accumulation in coincidence with the maximal seed growth, as observed in other species (1,10) (18) and found to be more than 95%.…”

mentioning

confidence: 99%

Abscisic Acid Levels during Early Seed Development in Sechium edule Sw

Vernieri¹,

Perata²,

Lorenzi³

et al. 1989

The time-course growth of single tissues in pollinated and unpollinated ovules of Sechium edule Sw. is described in relation to the endogenous levels of abscisic acid. Quantitation of abscisic acid (ABA) in the minute amounts of material obtained after ovule dissection has been performed by using a highly specific and sensitive solid-phase radioimmunoassay based on a monoclonal antibody raised against free (S)-ABA. While the absolute amount of ABA rises in both types of ovules, only in unpollinated ones does this leads to an increase in the hormone concentration. Infact in pollinated ovules the rapid growth following pollination prevents, through a dilution effect, the increase in ABA concentration. Growth patterns and endogenous ABA levels are similar for integuments and nucellus tissues either in pollinated or unpollinated ovules. It is suggested that the growth inhibition induced by the increase in ABA concentration after anthesis could be counteracted by the pollination triggered fast ovule growth.Accumulation of ABA during seed development has been reported in many species (10,14,17), and it is generally considered to be implicated in the regulation of seed dormancy and germination (19,20). While the role of ABA as a unique factor in inducing seed dormancy has been questioned in recent years (20,22), evidence has been reported in support of a specific role of ABA in the prevention of "in vitro" and "in vivo" precocious germination of seeds through specific genome regulation (1,2,9,16 MATERIALS AND METHODS Plant MaterialPlants of Sechium edule Sw. (Fam. Cucurbitaceae, tribe: Sicyoideae, common name: "chayote") were grown in the field during June to October 1988 and flowers were singularly tagged at anthesis. Ovaries were dissected and single tissues measured and weighed throughout seed development in order to characterize the growth patterns.The material for the experiments on ABA content in pollinated and unpollinated ovules was obtained as follows. During September a sufficient amount of female flowers was selected on the basis of synchronous flower opening and divided in two groups. The first group was hand pollinated, whereas flowers of the second group were capped with cheesecloth in order to prevent pollination. Ovaries harvested at anthesis in coincidence with hand pollination and flower capping were referred to as time 0. Ovaries from the two groups were collected and dissected every 2 d and processed as follows. ExtractionFruits were opened, ovules were dissected on ice under a microscope, and the material was weighed and immediately frozen in liquid nitrogen. Isolated ovule tissues (nucellus and integuments) were then separately extracted in distilled water (v/w = 20:1) for 16 h at 4°C in the dark. Extracts were centrifuged (1 3,000g, 10 min) and the supernatants, opportunely diluted, used for RIA analysis. Water extraction efficiency for ABA in Sechium tissues was previously checked (18) and found to be more than 95%. ABA DeterminationQuantitation of ABA in the tissues was accomplished em...