This thesis examines the role of root-sourced abscisic acid in the regulation of stomatal closure and leaf expansion in response to drying approximately half of the roots of Ricinus communis L. plants. Drying part of the root system of Ricinus communis promoted stomatal closure and slowed leaf expansion in the absence of any disturbances in shoot water relations, implying the involvement of chemical rather than hydraulic signalling. Initially root-sourced ABA was believed to be responsible for these responses. Delivery rates (concentration x flow rate) of ABA out of the drying roots were calculated which took into account the effect of dilution on solutes in the well-watered and droughted plants due to different transpiration rates in these plants. The delivery term was further modified to account for the differences in sizes of their roots and shoots. ABA delivery out of the roots of plants with drying upper roots increased within the first 12 h and was maintained over the next 3 days. However, significant decline in stomatal apertures and leaf elongation occurred only 2 - 3 days after root drying began. During the early stages of drying upper roots (2-3 d) xylem sap pH, and delivery rate of nitrate and l-aminocyclopropane-l-carboxylic acid were little changed, while hydraulic conductivity of the root system as a whole was reduced approximately 25%, and ABA accumulation (synthesis?) in roots increased. Increased ABA levels in phloem sap was not found, suggesting no enhanced re-cycling of ABA between shoots and roots was taking place in the plants during this time. Antitranspirant activity in xylem sap of droughted plants that was not ABA was sought as a possible cause of stomatal closure. However, convincing evidence of such activity was not found. Examination of ABA output by roots into shoot compared to that entering lamina of the 5th leaf in the canopy showed the attenuation of the signal as transpiration fluid moved up the plant. These obs~f\ations indicate that ABA from roots is unlikely to be a highly active signal eliciting shoot responses to mild drought in Ricinus communis.

The biosynthetic origin of the plant growth regulator abscisic acid remains equivocal and almost nothing is known about the enzymes involved in this process. The present research programme describes the development of a cell-free system, capable of synthesizing abscisic acid and attempts to provide further information about the biochemistry and enzymology of this important biosynthetic pathway. Cell-free extracts were prepared either directly from the flavedo (crude) or from an acetone powder derived from flavedo, of mature coloured fruits of Citrus sinensis L. cv. Midknight and incubated with mevalonic acid, isopentenyl pyrophosphate, famesylpyrophosphate, geranylgeranyl pyrophosphate, ß-carotene and 1',4'-trans-abscisic acid diol. The neutral and acidic products formed were purified by thin-layer chromatography and high performance liquid chromatography, and quantified by high performance liquid chromatography, gas chromatography-electron capture and unequivocally identified by combined gas chromatography-mass spectrometry. Abscisic acid, 1',4'-trans-abscisic acid diol and phaseic acid were unequivocally identified as the major acidic products formed in this cell-free system. The acid fraction also contained xanthoxin acid. Labelled and unlabelled ß-carotene was converted into the neutral compounds xanthoxin and xanthoxin alcohol. In addition. high performance liquid chromatography-photodiode array analYSis of the oxy-carotenoid fraction revealed the complete spectrum of ß, ß-carotenoids induding zeaxanthin, antheraxanthin and violaxanthin with accumulation of an oxygenated carotenoid tentatively identified as 9- cis-violaxanthin. Identification of putative C₁₅ intermediates was achieved by either UV spectrophotometry and combined capillary gas chromatography-mass spectrometry or microchemical analYSis and co-chromatography. Refeeding studies using (±)-[2-¹⁴C]_ abscisic acid diol as substrate revealed that abscisic acid was not metabolized to abscisic acid diol, suggesting that it was/is produced as an intermediate rather than as a catabolite of ABA in this system. Stigmasterol, and to a lesser extent cholesterol reduced conversion of ß-carotene to abscisic acid but did not influence transformation of 1',4'-trans-abscisic acid diol to abscisic acid. AM01618 stimulated fonnation of abscisic acid and appeared to exert its effect at the level of conversion of 1' ,4'-trans-abscisic acid diol. Zeatin and the cytokinin analogue, ancymidol inhibited the biosynthesis of abscisic acid whereas dithiothreitol increased incorporation of label from ß-carotene into abscisic acid suggesting involvement of a cytochrome P450-type mixed function oxidase in this reaction sequence. Sodium dodecylsulphate polyacrylamide gel electrophoresis of the enzyme extract derived from Citrus flavedo revealed the presence of a 53 kD protein with peroxidase activity characteristic of a cytochrome P-450. Abscisic acid biosynthesizing activity was always greater in extracts from acetone powder and abscisic acid biosynthesis was enhanced in the presence of AMO 1618, NAD+, NADH, NADPH, MgCI₂ and Molybdate but was inhibited by FAD. Activity was further enhanced by the addition of (R,S)-abscisic acid as a cold-pool trap and by induding 0.1% w/v of either Tween 20 or Triton X 100 in the extraction buffer. When cis-ß-carotene was used as substrate, no abscisic acid was produced. Conversely when either all-trans-ß-carotene or a mixture of the two isomers was used, incorporation into abscisic acid occurred. Upoxygenase activity in cell-free extracts of Citrus flavedo increased with increasing protein concentration. As the ability of lipoxygenase to make xanthoxin from violaxanthin, had been reported, increased activity in the cell-free system implied that carotenoid deavage was being brought about by a non-haem oxygenase with lipoxygenase-like properties. Reports had implicated phoshorylation in the activation of many catalytic enzymes (Hanks et aI., 1985). Phosphorylation of the enzymes in this cell-free system proved unsuccessful. Further, it had been reported that in vitro phosphorylation of several membrane polypeptides and soluble polypeptides from com, had been promoted by the addition of Ca²₊ In this cell-free system Ca + did not have a stimulatory effect on protein phosphorylation. Dioxygenases generally occur as soluble enzymes, where they catalyse many oxygenation reactions in metabolic pathways. The addition of 2-oxo-glutarate, a requirement of most soluble oxidases, did not affect the activity of the cell-free system.

Strigolactones (SLs) are a novel class of phytohormones that shape shoot and root architecture. Carlactone, the precursor of SLs, is synthesized from all-trans-β-carotene by the sequential action of three enzymes: the all-trans/9-cis-β-carotene isomerase DWARF27 (D27) that reversibly converts all-trans-β-carotene into 9-cis-β-carotene (shown only for the rice D27) and the carotenoid cleavage dioxygenases 7 and 8 (CCD7 and CCD8). Genomes of higher plants encode two D27 homologs (D27-like 1 and D27-like 2) with unknown function. Rice and Arabidopsis d27 mutants show less pronounced high tillering/more branching phenotype, compared to ccd7 or ccd8 mutants. This difference might be the result of functional redundancy caused by the presence of D27-like 1 and D27-like 2. In this study, we investigated the enzymatic and biological activity of the Arabidopsis D27 and the rice and Arabidopsis D27-like 1, using both in vivo and in vitro studies. Our results show that AtD27 is a true ortholog of the rice D27. Like SLs, the biosynthesis of ABA requires an all-trans/9-cis-isomerization reaction. However, the enzyme postulated for this activity is still elusive. Our enzymatic activity tests exclude a direct involvement of AtD27 in ABA biosynthesis. Nevertheless, Atd27 mutant shows low level of ABA, and analysis of AtD27 promoter

No description available.

No description available.

Abscisic acid (ABA) has been well defined as an important stress hormone in plants. The signaling pathway of ABA involves a family of pyrabactin resistant-like-1 PYR/PYL/RCAR receptors (PYL receptors) that bind ABA and form a complex with a protein phosphatase 2C (PP2C) family member resulting in downstream signaling events. The ABA receptor family has been well characterized in the model dicot Arabidopsis thaliana and more recently this characterization has branched out into cereals Oryza sativa (rice) and Hordeum vulgare (barley), as well as the monocot model plant Brachypodium distachyon and Fragaria vesca (strawberry). The analysis of these characterized ABA receptors and the use of online databases has allowed the identification of multiple putative ABA receptors in Triticum aestivum (wheat). ABA has been historically called a positive effector. Overexpression of proteins in the ABA signalling pathway or exogenous application of ABA is known to cause an increase in drought, cold, and salt tolerance. More recently ABA has been linked to increased fungal susceptibility in several plants. The role ABA plays in the biotic stress response is still largely unexplored. The focus of this project was to identify and characterize a putative wheat ABA receptor through bioinformatics and an in vitro enzyme activity assay, and use virus induced gene silencing (VIGS) to test what role this receptor plays in fugal susceptibility. A total of 13 putative ABA receptors were located, nine of which are unique between the wheat subgenomes. One receptor TaPYL5.1 was recombinantly expressed, purified, and confirmed as an ABA receptor through a phosphatase based enzyme activity assay. A receptor with high sequence identity to TaPYL5.1, TaPYL5.2A, was targeted for plant trials because the TaPYL5.1 plasmid sequence was codon optimized. A VIGS approach was used to knock down TaPYL5.2A in planta. The TaPYL5.2A knockdown plants were found to have an increased resistance to Fusarium Head Blight progression in the early stages of the disease. In conclusion, wheat ABA receptors were successfully identified and an important correlation between decreased receptor levels and increased early Fusarium Head Blight resistance was found. This correlation however was not easily reproducible due to the severity of coupling VIGS with Fusarium Head Blight, and should be followed up with additional studies looking at the broader family of wheat ABA receptors.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

No description available.

Part I Four-week old tomato (Lycopersicon esculentum Mill.) seedlings were exposed to different constant temperatures of 15, 25, 35, or 45 c. To determine the effect of temperature on Abscisic Acid (ABA) young and old leaves were harvested at 0, 6, 12, 24, or 48 hand free, hydrolyzable and total ABA were measured using gas liquid chromatographic methods. Temperature had a significant effect on free , hydrolyzable and total ABA in both young and old leaves. Time had a significant effect as a cubic function on all ABA measurements in old leaves but in young leaves a significant effect was observed only on the hydrolyzable ABA. Interactions between temperature and time were observed in both young and old leaves. Young leaves had significantly higher amounts of free and hydrolyzable ABA than did old leaves . The total ABA levels were not significantly different in young and old leaves . Suboptimal temperatures increased ABA levels in the plants, in a manner similar to that of other kinds of stresses. Part II Five- week old tomato plants (Lycopersicon esculentum Mill.) were exposed to day-night temperatures of 10-5, 15-10, 25 - 15, 35-25, or 45-35 C. The day length was 16 h with a light intensity of approximately 400 ~ E m- 2s-1 . Plant tops were sampled at 12, 24 , 48, 68, or 72 h. Free, hydrolyzable and total absciscis acid (ABA) quantities were measured using standard gas chromatographic techniques. Stressful temperatures significantly increased both free and hydrolyzable ABA levels . The highest ABA levels were observed in plants grown at 10-5 C. Although time significantly affected ABA levels, its role needs more precise definition . Due to the similar involvements of ABA in temperature-induced and other stresses, ABA may be hypothesized to be a common mediator for different stresses. Part III Warm season crops: bean (Phaseolus vulgaris) CV . Burpee Stringless Green Pod; corn (Zea mays) CV. Golden ,Jubilee; Cantaloupe (Cucumis melo) cv. Hales Best; eggplant (Solunum melongena) CV. Ichiban; and okra (Hibiscus esculentus) CV . Dwarf Green and cool season crops: beet (Beta vulgari s) CV. Early wonder; lettuce (Lactuca sativa) •CV. Great Lakes; cabbage (Brassica oleracea) cv. Savoy; radish (Raphanus sativus) CV. Scarlet Globe; and pea (Pisum sativum) cv. Little Marvel were exposed to 10, 25 , or 40 C. After 24 h free and hydrolyzable ABA and DPA were measured in the plant tops using standard gas chromatographic methods. Warm season crops exhibited elevated levels of PABA, HABA and DPA under 10 C, compared to those at 25 or 40 c. Cool season crops showed similar PABA , HABA and DPA contents under all temperatures, except pea which had higher PABA and HABA levels at 40 C and beet which showed lower levels of HABA at 25 C as compared to 10 and 40 C. DPA existed at much higher concentrations than ABA in all plants of the two groups. It is concluded that while 1 0 C is stressful to warm season crops and thus the ABA and DPA increases, it is not so favorable to cool season crops. A temperature of 40 C would be stressful to both groups, but as long as water supply to the plant is not limiting the plants would not actively produce ABA . Higher levels of DPA under stressful conditions would warrant consideration and taking into account the role of metabolism in regulation of total ABA in the plant.

Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.