The hormonal action of abscisic acid (ABA) in plants is controlled by the precise balance between its biosynthesis and catabolism. In plants, ABA 8′-hydroxylation is thought to play a predominant role in ABA catabolism. ABA 8′-hydroxylase was shown to be a cytochrome P450 (P450); however, its corresponding gene had not been identified. Through phylogenetic and DNA microarray analyses during seed imbibition, the candidate genes for this enzyme were narrowed down from 272 Arabidopsis P450 genes. These candidate genes were functionally expressed in yeast to reveal that members of the CYP707A family, CYP707A1–CYP707A4, encode ABA 8′-hydroxylases. Expression analyses revealed that CYP707A2 is responsible for the rapid decrease in ABA level during seed imbibition. During drought stress conditions, all CYP707A genes were upregulated, and upon rehydration a significant increase in mRNA level was observed. Consistent with the expression analyses, cyp707a2 mutants exhibited hyperdormancy in seeds and accumulated six-fold greater ABA content than wild type. These results demonstrate that CYP707A family genes play a major regulatory role in controlling the level of ABA in plants.

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The Arabidopsis cytochrome P450 CYP707A encodes ABA 8′‐hydroxylases: key enzymes in ABA catabolism

INTRODUCTION 454 TECHNIQUES ....... 454 Quantitation of ABA ... 454 Synthesis of Tritiated ABA 456 BIOCHEMISTRY 456 ABA Biosynthesis .... 456 ABA Metabolism 457 Regulation 0/ Enzyme Synthesis 460 PHYSIOLOGY 462 Water Stress 462 The stimulus fOT Ttlising ABA levels ..... ........ ........ .... ..... ..•.. .... 462 Localization and regulation of ABA. synthesis •••••••••••••••••••. ......... .... .... ........ .... 463 Stomatal complexes and A.lJA. ...... ......... ....... .... ..... ........ .... 466 Mode of action 467 A.lJA. levels and stomatal aperture ........ ....... ....... ......... ..... 469 Drought mistance ..... 470 Root Geotropism 470 Seed Development and Germination 472 Seed development 472 Seed gennil/Qtion 473 Dormancy 474 Induction of dormancy 474 Maintenance of dormancy 475 Correlative Inhibition 0/ Buds 475 Ion Movement 477 ABA tran.spoTf 478 CONCLUDING REMARKS 479

Biochemistry and physiology of abscisic acid.

Mature seeds of the Cape Verde Islands (Cvi) ecotype of Arabidopsis thaliana (L.) Heynh. show a very marked dormancy. Dormant (D) seeds completely fail to germinate in conditions that are favourable for germination whereas non-dormant (ND) seeds germinate easily. Cvi seed dormancy is alleviated by after-ripening, stratification, and also by nitrate or fluridone treatment. Addition of gibberellins to D seeds does not suppress dormancy efficiently, suggesting that gibberellins are not directly involved in the breaking of dormancy. Dormancy expression of Cvi seeds is strongly dependent on temperature: D seeds do not germinate at warm temperatures (20–27°C) but do so easily at a low temperature (13°C) or when a fluridone treatment is given to D seeds sown at high temperature. To investigate the role of abscisic acid (ABA) in dormancy release and maintenance, we measured the ABA content in both ND and D seeds imbibed using various dormancy-breaking conditions. It was found that dry D seeds contained higher amounts of ABA than dry ND after-ripened seeds. During early imbibition in standard conditions, there was a decrease in ABA content in both seeds, the rate of which was slower in D seeds. Three days after sowing, the ABA content in D seeds increased specifically and then remained at a high level. When imbibed with fluridone, nitrate or stratified, the ABA content of D seeds decreased and reached a level very near to that of ND seeds. In contrast, gibberellic acid (GA3) treatment caused a transient increase in ABA content. When D seeds were sown at low optimal temperature their ABA content also decreased to the level observed in ND seeds. The present study indicates that Cvi D and ND seeds can be easily distinguished by their ability to synthesize ABA following imbibition. Treatments used here to break dormancy reduced the ABA level in imbibed D seeds to the level observed in ND seeds, with the exception of GA3 treatment, which was active in promoting germination only when ABA synthesis was inhibited.

Changes in endogenous abscisic acid levels during dormancy release and maintenance of mature seeds: studies with the Cape Verde Islands ecotype, the dormant model of Arabidopsis thaliana

Physiologically dormant seeds, like those of Arabidopsis, will cycle through dormant states as seasons change until the environment is favourable for seedling establishment. This phenomenon is widespread in the plant kingdom, but has not been studied at the molecular level. Full-genome microarrays were used for a global transcript analysis of Arabidopsis thaliana (accession Cvi) seeds in a range of dormant and dry after-ripened states during cycling. Principal component analysis of the expression patterns observed showed that they differed in newly imbibed primary dormant seeds, as commonly used in experimental studies, compared with those in the maintained primary and secondary dormant states that exist during cycling. Dormant and after-ripened seeds appear to have equally active although distinct gene expression programmes, dormant seeds having greatly reduced gene expression associated with protein synthesis, potentially controlling the completion of germination. A core set of 442 genes were identified that had higher expression in all dormant states compared with after-ripened states. Abscisic acid (ABA) responsive elements were significantly over-represented in this set of genes the expression of which was enhanced when multiple copies of the elements were present. ABA regulation of dormancy was further supported by expression patterns of key genes in ABA synthesis/catabolism, and dormancy loss in the presence of fluridone. The data support an ABA-gibberelic acid hormone balance mechanism controlling cycling through dormant states that depends on synthetic and catabolic pathways of both hormones. Many of the most highly expressed genes in dormant states were stress-related even in the absence of abiotic stress, indicating that ABA, stress and dormancy responses overlap significantly at the transcriptome level.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-313X.2006.02738.x

Gene expression profiles of Arabidopsis Cvi seeds during dormancy cycling indicate a common underlying dormancy control mechanism

Biochemistry and Physiology oj Plant Hormones is intended primarily as a textbook or major reference for a one-term intermediate-level or advanced course dealing with hormonal regulation of growth and development of seed plants for students majoring in biology, botany, and applied botany fields such as agronomy, forestry, and horticulture. Additionally, it should be useful to others who wish to become familiar with the topic in relation to their principal student or professional interests in related fields. It is assumed that readers will have a background in fundamental biology, plant physiology, and biochemistry. The dominant objective of Biochemistry and Physiology oj Plant Hor mones is to summarize, in a reasonably balanced and comprehensive way, the current state of our fundamental knowledge regarding the major kinds of hormones and the phytochrome pigment system. Written primarily for students rather than researchers, the book is purposely brief. Biochemical aspects have been given priority intentionally, somewhat at the expense of physiological considerations. There are extensive citations of the literature-both old and recent-but, it is hoped, not so much documentation as to make the book difficult to read. The specific choices of publications to cite and illustrations to present were made for different reasons, often to illustrate historical develop ment, sometimes to illustrate ideas that later proved invalid, occasionally to exemplify conflicting hypotheses, and most often to illustrate the current state of our knowledge about hormonal phenomena."

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Biochemistry and Physiology of Plant Hormones

Embryos from dormant and stratified Fraxinus americana seed were incubated with (S)-2-(14)C-abscisic acid (ABA) under a variety of conditions. Both dormant and stratified embryos rapidly metabolize abscisic acid to phaseic acid, dihydrophaseic acid, and an unidentified polar metabolite apparently derived from dihydrophaseic acid. Although the stratified embryos may have an increased capacity to metabolize abscisic acid, our calculations suggest that such an increased capacity would probably not be physiologically significant.Dormant intact seeds also metabolize (S)-2-(14)C-abscisic acid during stratification at 5 C or incubation at 25 C. The metabolites appear to be similar to those observed in excised embryos although by 12 days of stratification a fourth metabolite is observed. More than 90% of the (14)C-abscisic acid was metabolized after 26 days of stratification at 5 C or after 12 days of incubation at 25 C. Stratification at 5 C leads to the breaking of dormancy while incubation at 25 C does not.

The Metabolism of Hormones during Seed Germination and Dormancy: IV. The Metabolism of (S)-2-C-Abscisic Acid in Ash Seed.

Many seeds wvill not germinate uinless they are first stratified, that is, held in cold, moist storage for several months. WVhile these effects are not well understood at a physiological or biochemical level, XVilliers and Wareinig (8) showed that in ash, Fraxinnis excelsior L. both dormant and nondormant seeds have growth-inhibitory activity. However, in the nondormant seeds a growth accelerator was detected which antagonized the activity of the inhibitor. Recently, abscisin II, a new plant growvth regttlator isolated from cotton fruit (5) and driecl leaves of sycamore maple (3) has been identified as 3-methyl-5(1 -hydroxy-4-oxo-2, 6, 6-trimethyl-2-cyclohexene-1-yl)-cis, trans 2,4-pentadienoic acid and shown to bring abouit abscission of young cotton frulit and to possibly play a part in the reguilation of bud dormancv. The term dormin has also been suggested for abscisin II (3). Abscisin II has no activity as an atlxin or gibberellin, but can counteract the action of these hormones in Arena coleoptiles, in the barley endosperm assay and in the growth of dwarf maize or dwarf peas (1, 7). DLAbscisin II and the DL-trans, trans isomer have been synthesized (2). It seemed of interest to determine whether abscisin II woutld act as a germination inhibitor in those seeds that require cold treatment for germination. We now report the effects obtained with DL-abscisin II and other plant growth regulators on excised Ash embryos from Fraximus orntus L. and Fraxinuis anericana L. Depericarped seeds purchased from F. WV. Schumacher, Sandwich, Massachusetts, were imbibed for 24 hours in water, the embryos excised and placed onl WVhatman No. 1 filter paper in 5 cm petri dishes. Ten embryos per dish and 1 ml solution consisting of test substances dissolved in 0.01 m sodium phosphate, pH 6.0, were used. Triplicate plates were held at 220 tinder normal laboratory light and the formation of a geotropic bend at the basal end was interpreted as a positive germination response. Following the recommendations of Timson (6) we are reporting ouir germination (lata as the suim of the dlaily percentage of germiinatioin for a 10 clay period starting at the dav of excision. Thus, z 10 = 1000 would mean 100 % germination during the first day and , 10 = 0, Ino germination during the 10 day period. Depericarped seeds were stratified at 50 on moist filter paper in petri dishes for 3 months. Chlorophyll contenit was dletermineid spectrophotometrically from acetonie extracts by the proceduire of Maclachlan and( Zalick (4). DL,-Abscisin II and the DL-trans, trans-isomer were prepared by the proceduire of Cornforth et al (2). Excised F. ornuis embryos germinated well even without stratification (table I). The small enhancement obtained with gibberellin A. (gibberellic acid) is duie primarily to earlier germination. Embryos from F. amtericana gave good germination only when stratified, or treated with gibberellins A3 or A1. Gibberellin A, may be somewhat less effective than gibberellin A2, buit the differences are small. Contrary to Vlilliers and W;\\areing (8, 9) who worked with F. excelsior, we foulnd no enhancement of germination on soaking of the excised F. orutlis and F. amiiericanaia embryos in water.

/pdf/effects-of-abscisin-ii-and-other-plant-growth-substances-on-24b8nzlwrd.pdf

Effects of Abscisin II and Other Plant Growth Substances on Germination of Seeds with Stratification Requirements.

Zeatin and zeatin-9, beta-ribonucleoside enhance the germination of dormant ash embryos. While the first macroscopic signs of germination appear only after about 72 hours, 12 hours of exposure to 50 mum zeatin is as effective as continuous incubation. There must be barriers against transport out of the embryos since 8-(14)C-zeatin and its metabolites, zeatin-9, beta-ribonucleoside, the 5'-mono and the suspected di- and triphosphates, accumulate against a concentration gradient. Zeatin ribonucleoside is about as effective as zeatin in enhancing embryo germination, yet the internal 8-(14)C-zeatin level is lower by a factor of about 50 when the ribonucleoside is fed. The physiological effects of zeatin and abscisic acid on the germination of ash embryos are antagonistic. There is, however, no evidence that abscisic acid has a significant effect on 8-(14)C-zeatin uptake or conversions.

The Metabolism of Hormones during Seed Germination and Release from Dormancy: III. The Effects and Metabolism of Zeatin in Dormant and Nondormant Ash Embryos.

In excised embryos of Fraxinus ornus, the first macroscopic sign of germination is a curvature near the root apex. Histological studies show that this curvature is due to cell elongation on the convex side and is accompanied by cell division. Metabolic changes, manifested by the disintegration of protein bodies and tissue differentiation, also occur during this time. In embryos treated with phleomycin cell division is completely inhibited but elongation, root hair formation, disintegration of protein bodies, and differentiation are still detectable. Phleomycin also drastically inhibits the formation of chlorophylls and the fresh weight increase of embryos.

Eva C. Galson

Papers

The Metabolism of Hormones during Seed Germination and Dormancy: IV. The Metabolism of (S)-2-C-Abscisic Acid in Ash Seed.

Effects of Abscisin II and Other Plant Growth Substances on Germination of Seeds with Stratification Requirements.

The Metabolism of Hormones during Seed Germination and Release from Dormancy: III. The Effects and Metabolism of Zeatin in Dormant and Nondormant Ash Embryos.

Germination of Excised Fraxinus ornus Embryos With and Without Phleomycin