Phaseic Acid Levels Research Articles

Mycorrhizal symbiosis affects ABA metabolism during berry ripening in Vitis vinifera L. cv. Tempranillo grown under climate change scenarios

Arbuscular mycorrhizal symbiosis is a promising tool for improving the quality of grapes under changing environments. Therefore, the aim of this research was to determine if the ability of arbuscular mycorrhizal fungi (AMF) to enhance phenolic content (specifically, anthocyanins) in a climate change framework could be mediated by alterations in berry ABA metabolism during ripening. The study was carried out on fruit-bearing cuttings of cv. Tempranillo (CL-1048 and CL-1089) inoculated (+M) or not (−M) with AMF. Two experimental designs were implemented. In the first experiment +M and -M plants were subjected to two temperatures (24/14 °C or 28/18 °C (day/night)) from fruit set to berry maturity. In the second experiment, +M and -M plants were subjected to two temperatures (24/14 °C or 28/18 °C (day/night)) combined with two irrigation regimes (late water deficit (LD) and full irrigation (FI)). At 28/18 °C AMF contributed to an increase in berry anthocyanins and modulated ABA metabolism, leading to higher ABA-GE and 7′OH-ABA and lower phaseic acid (PA) in berries compared to –M plants. Under the most stressful scenario (LD and 28/18 °C), at harvest +M plants exhibited higher berry anthocyanins and 7´OH-ABA and lower PA and dihydrophaseic acid (DPA) levels than –M plants. These findings highlight the involvement of ABA metabolism into the ability of AMF to improve some traits involved in the quality of grapes under global warming scenarios.

Abscisic Acid Catabolism Generates Phaseic Acid, a Molecule Able to Activate a Subset of ABA Receptors

Abscisic acid (ABA) catabolic pathways are conventionally thought of as routes for ABA inactivation and, thus, reduction of the well-known ABA responses to cope with stress. However, this point of view was challenged by the finding that some forms of hydroxylated ABA generated by ABA catabolism still maintain biological activity (Zou et al., 1995; Kepka et al., 2011). Hydroxylation at C-8′ position is thought to be the predominant ABA catabolic pathway, and this reaction is catalyzed by the CYP707A family cytochrome P450 monooxygenases (P450s) (Krochko et al., 1998; Kushiro et al., 2004; Saito et al., 2004) (Figure 1).

Profiling ABA metabolites in Nicotiana tabacum L. leaves by ultra-performance liquid chromatography–electrospray tandem mass spectrometry

We have developed a simple method for extracting and purifying (+)-abscisic acid (ABA) and eight ABA metabolites – phaseic acid (PA), dihydrophaseic acid (DPA), neophaseic acid (neoPA), ABA-glucose ester (ABAGE), 7′-hydroxy-ABA (7′-OH-ABA), 9′-hydroxy-ABA (9′-OH-ABA), ABAaldehyde, and ABAalcohol – before analysis by a novel technique for these substances, ultra-performance liquid chromatography–electrospray ionisation tandem mass spectrometry (UPLC–ESI-MS/MS). The procedure includes addition of deuterium-labelled standards, extraction with methanol–water–acetic acid (10:89:1, v/v), simple purification by Oasis ® HLB cartridges, rapid chromatographic separation by UPLC, and sensitive, accurate quantification by MS/MS in multiple reaction monitoring modes. The detection limits of the technique ranged between 0.1 and 1 pmol for ABAGE and ABA acids in negative ion mode, and 0.01–0.50 pmol for ABAGE, ABAaldehyde, ABAalcohol and the methylated acids in positive ion mode. The fast liquid chromatographic separation and analysis of ABA and its eight measured derivatives by UPLC–ESI-MS/MS provide rapid, accurate and robust quantification of most of the substances, and the low detection limits allow small amounts of tissue (1–5 mg) to be used in quantitative analysis. To demonstrate the potential of the technique, we isolated ABA and its metabolites from control and water-stressed tobacco leaf tissues then analysed them by UPLC–ESI-MS/MS. Only ABA, PA, DPA, neoPA, and ABAGE were detected in the samples. PA was the most abundant analyte (ca. 1000 pmol/g f.w.) in both the control and water-stressed tissues, followed by ABAGE and DPA, which were both present at levels ca. 5-fold lower. ABA levels were at least 100-fold lower than PA concentrations, but they increased following the water stress treatment, while ABAGE, PA, and DPA levels decreased. Overall, the technique offers substantial improvements over previously described methods, enabling the detailed, direct study of diverse ABA metabolites in small amounts of plant tissue.

Expression of ABA 8′‐hydroxylases in relation to leaf water relations and seed development in bean

In plants, the level of abscisic acid (ABA) is determined by synthesis and catabolism. Hydroxylation of ABA at the 8' position is the key step in ABA catabolism. This reaction is catalyzed by ABA 8'-hydroxylase, a cytochrome P450 (CYP). The cDNAs of PvCYP707A1 and PvCYP707A2 were isolated from bean (Phaseolus vulgaris L.) axes treated with (+)-ABA and that of PvCYP707A3 from dehydrated bean leaves. The recombinant PvCYP707A proteins expressed in yeast were biochemically characterized. Yeast strains over-expressing any of the three PvCYP707As were able to convert ABA to phaseic acid (PA). The microsomal fractions from these yeast strains also exhibited ABA 8'-hydroxylase activity. Expression of PvCYP707A3 in primary leaves was strongly increased by water stress, whereas PvCYP707A1 and PvCYP707A2 mRNA levels were rapidly increased by rehydration of water-stressed leaves. Northern blot analysis of PvCYP707As in bean showed a high level of expression in the mature fruits, senescent leaves, roots, seed coats and axes. All three PvCYP707As were expressed at varying intensities throughout seed development. Imbibed seeds also had high PvCYP707A mRNA levels. Thus, expression of PvCYP707As is both environmentally and developmentally regulated. Transgenic Nicotiana sylvestris plants over-expressing PvCYP707As displayed a wilty phenotype, and had reduced ABA levels and increased PA levels. These results demonstrate that expression of PvCYP707As is the major mechanism by which ABA catabolism is regulated in bean.

Effects of Drought Stress on Abscisic Acid and Jasmonate Metabolism in Citrus

Drought stress was applied to ‘Shiranui’ trees [ (Citrus unshiu Marc. × Citrus unshiu Osbeck) × Citrus reticulata Blanco], and xanthoxin, abscisic acid (ABA), phaseic acid (PA), dihydrophaseic acid (DPA), epi-dihydrophaseic acid (epi-DPA), and jasmonates were monitored in fruit and leaf. They were quantified using the respective deuterium-labeled internal standards. Xanthoxin, ABA, and DPA concentrations in the pulp of the drought-stressed trees were found to be higher than those of the well-watered trees throughout development. Xanthoxin concentrations, however, were lower than those of ABA. These results may imply a rapid turnover from xanthoxin to ABA. In addition, ABA concentrations in the leaf were higher in drought-stressed trees than the well-watered trees, although PA and DPA levels between the drought-stressed and well-watered trees were not clear. DPA was found to be the primary metabolite in the pulp, skin, and leaf. Both jasmonic acid (JA) and ABA concentrations in the pulp, skin, and leaf showed high values in the drought-stressed trees. However, JA levels in the pulp decreased as the harvest approached. Sucrose concentrations in the pulp of the drought-stressed trees were higher than those of the well-watered trees. ABA and jasmonate applications induced stomatal closure in leaf The effect of ABA on stomatal closure was stronger than that of jasmonate. We discuss herein the relationship between ABA, jasmonates, and drought stress.

Dormancy termination of western white pine ( Pinus monticola Dougl. Ex D. Don) seeds is associated with changes in abscisic acid metabolism

Western white pine (Pinus monticola) seeds exhibit deep dormancy at maturity and seed populations require several months of moist chilling to reach their uppermost germination capacities. Abscisic acid (ABA) and its metabolites, phaseic acid (PA), dihydrophaseic acid (DPA), 7'-hydroxy ABA (7'OH ABA) and ABA-glucose ester (ABA-GE), were quantified in western white pine seeds during dormancy breakage (moist chilling) and germination using an HPLC-tandem mass spectrometry method with multiple reaction monitoring and internal standards incorporating deuterium-labeled analogs. In the seed coat, ABA and metabolite levels were high in dry seeds, but declined precipitously during the pre-moist-chilling water soak to relatively low levels thereafter. In the embryo and megagametophyte, ABA levels decreased significantly during moist chilling, coincident with an increase in the germination capacity of seeds. ABA catabolism occurred via several routes, depending on the stage and the seed tissue. Moist chilling of seeds led to increases in PA and DPA levels in both the embryo and megagametophyte. Within the embryo, 7'OH ABA and ABA-GE also accumulated during moist chilling; however, 7'OH ABA peaked early in germination. Changes in ABA flux, i.e. shifts in the ratio between biosynthesis and catabolism, occurred at three distinct stages during the transition from dormant seed to seedling. During moist chilling, the relative rate of ABA catabolism exceeded ABA biosynthesis. This trend became even more pronounced during germination, and germination was also accompanied by a decrease in the ABA catabolites DPA and PA, presumably as a result of their further metabolism and/or leaching/transport. The transition from germination to post-germinative growth was accompanied by a shift toward ABA biosynthesis. Dormant imbibed seeds, kept in warm moist conditions for 30 days (after an initial 13 days of soaking), maintained high ABA levels, while the amounts of PA, 7'OH ABA, and DPA decreased or remained at steady-state levels. Thus, in the absence of conditions required to break dormancy there were no net changes in ABA biosynthesis and catabolism.

Changes of abscisic acid and its metabolite during development of apple fruit

SummaryFree and conjugated abscisic acid (ABA) and phaseic acid (PA) concentrations in the seed and pulp of apples (Malus pumila Mill. cv. Tsugaru) and ABA metabolism in the fruit after cis-(+)-ABA application were investigated. In the seed, the levels of cis- and trans-ABA and conjugated cis- and trans-ABA showed a general increase towards harvest, except for the decrease of cis-ABA at 131 d after full bloom (DAFB) (harvest). PA levels reached a peak at 41 DAFB, then decreased until harvest. The levels of free ABA in the seed were higher than those in the pulp. In the pulp, the levels of cis-ABA increased gradually from 20 DAFB and rapidly from 111 DAFB towards harvest. The levels of trans-ABA and conjugated trans-ABA also showed similar changes to cis-ABA, i.e. increased steeply from 111 DAFB. In contrast, the level of conjugated cis-ABA was the highest at 20 DAFB, then fell rapidly until 111 DAFB and then increased again towards harvest. PA showed the highest level at 20 DAFB, then decreased gradually until 111 DAFB and then increased again towards harvest. It is therefore assumed that not only cis-ABA but also its metabolite may be associated with the ripening of apple fruit. Cis- and trans-ABA and conjugated cis- and trans-ABA concentrations in cis-(+)-ABA treated fruit increased significantly on 11 d after ABA application (122 DAFB). However, at 20 d after cis-(+)-ABA application, only trans-ABA and conjugated trans-ABA showed higher levels than those of the untreated control, while the levels of cis-ABA and conjugated cis-ABA fell to the same levels as the untreated control. PA levels did not show significant increase on 11 and 20 d after cis-(+)-ABA application. These results suggest that cis-(+)-ABA treated exogenously to apple fruit may eventually metabolize to trans-ABA, conjugated trans-ABA and further metabolites.

DROUGHT RESISTANCE OF Sorghum bicolor. 4. HORMAL CHANGES IN RELATION TO DROUGHT STRESS IN FIELD-GROWN PLANTS

This study was undertaken to investigate the nature of hormonal changes in relation to drought stress in two cultivars of Sorghum bicolor L. Moench. Two cultivars, M–35 and NK300, were grown in a field plot protected by a rain shelter. Plants in one soil compartment were stressed by withholding water while those in another (controls) were irrigated frequently. Levels of the plant hormones abscisic acid (ABA), phaseic acid (PA) and indole-3-acetic acid (IAA) measured by high-performance liquid chromatography (HPLC) were determined in the youngest leaves of control and stressed plants at intervals throughout the growth cycle. Plant height, senescence, and leaf water status were also determined. Leaf water potential (ψw) and solute potential (ψs) were reduced in both cultivars by drought stress; values for M–35 plants were lower than NK300. Leaf senescence was higher in M–35 plants and was promoted by stress in both cultivars. Cultivar M–35 behaved as a drought-tolerant plant whereas cultivar NK300 behaved more like a drought avoider. ABA levels were higher in M–35 control plants than in corresponding NK300 plants and levels in both cultivars followed seasonal changes in leaf water potential. Under drought stress, ABA levels increased between 1.5 and 2 times in both cultivars with the largest increases occurring during the vegetative stage in M–35 and during the flowering stage for NK300. PA levels in both cultivars were higher in stressed than in control plants. PA levels in M–35 plants were relatively low and constant throughout the life cycle, whereas in NK300, levels were high until shortly before flowering. IAA levels were higher in NK300 than in M–35 plants, particularly during the vegetative stage. Under drought stress, IAA levels were reduced in both cultivars with a more pronounced reduction in NK300. The high level of ABA in the more drought-tolerant cultivar M–35 was associated with low leaf ψw and ψs and high leaf senescence. On the other hand, in the drought avoider, NK300, high levels of IAA and PA were associated with high leaf ψw and ψs. It is concluded that these cultivars, which differ in their response to drought stress, can be distinguished by their leaf hormone levels.

Diurnal Changes of Leaf Water Potential, Abscisic Acid, Phaseic Acid and Indole-3-Acetic Acid in Field Grown Sorghum bicolor L. Moench

Summary Diurnal changes of leaf water potential, abscisic acid, phaseic acid and indole-3-acetic acid were studied in field grown sorghum. Leaf water potential fluctuated over a range of -0.84 to -1.82 MPa with two distinct low value peaks at 10.00 and 16.00 h. Abscisic acid exhibited a distinct diurnal variation and fluctuated over a range of 45–110 ng g-1 fresh wt. with two distinct peaks at 08.00 and 18.00 h. Abscisic acid levels, except those from 10.00 to 17.00 h were, negatively correlated with leaf water potential and began to increase at leaf water potential values around -1.0 MPa. Phaseic acid levels were low compared to abscisic acid levels, but fluctuated in a manner similar to abscisic acid with peaks at 10.00 and 18.00 h. Fluctuations in indole-3-acetic acid levels were very small.

The effect of fruit-removal on cytokinins and gibberellin-like substances in grape leaves

Removal of fruit from potted cuttings of Vitis vinifera L. increased the concentration of a cytokininglucoside in leaf tissue extracts and decreased the level of extractable gibberellin-like substances. The glucoside (of zeatin riboside) is not present in xylem exudate of V. vinifera L., and appears to be synthesized in the leaves. Berry extracts contain zeatin-riboside and smaller amounts of cytokinin-glucoside. The changes in the level of these hormones are discussed in relation to previous results on abscisic acid and phaseic acid levels in grape leaves.

Abscisic Acid Metabolism in Water-stressed Bean Leaves

Phaseic acid (PA) and dihydrophaseic acid (DPA) are the major metabolites observed when (S)-2-(14)C-abscisic acid (ABA) is fed to 14-day excised primary bean leaves (Phaseolus vulgaris L. cv. Red Kidney). The distribution of (14)C in leaves which were wilted after feeding ABA appears to be the same as that observed in unwilted leaves. A reduction in the relative specific radioactivities of the two metabolites after wilting, compared with the specific radioactivities measured in unwilted plants, indicated that these metabolites continue to be formed endogenously after wilting. Estimates of the endogenous ABA levels showed that they rose from 0.04 mug to approximately 0.5 mug/g fresh weight within 4 hours after the beginning of a 10% wilt and remained at that level during a subsequent 20 hours of wilt. In unwilted leaves, the levels of PA and DPA were 5 times and 20 times higher than that of ABA, respectively. Both PA and DPA levels rose throughout the wilt period. PA rose from 0.20 mug to 1.0 mug and DPA from 0.8 mug to over 3 mug/g fresh weight. From these data, we calculated the rate of ABA synthesis to be at least 0.15 mug/hr.g fresh weight during this period. We have interpreted these results to mean that in wilted leaves an elevated level of ABA is maintained because the rate of synthesis and metabolism are both elevated and approximately equal.

Internal Control of Stomatal Physiology and Photosynthesis. I. Stomatal Regulation and Associated Changes in Endogenous Levels of Abscisic and Phaseic Acids

Both environmental factors and manipulative treatments (such as fruit excision or stem cincturing) were found to alter gaseous diffusion resistances in grape vine foliage. These responses have been analysed in terms of the hormonal physiology of Vitis vinifera L. leaf tissue. Environmental factors such as moisture stress or photoperiod alterations which contributed towards an increase in stomatal resistance (rs) were correlated with increased levels of endogenous abscisic acid (ABA) and phaseic acid (PA) in mature foliage. Conversely, treatments which elicited a decrease in rs were associated with lower levels of both ABA and PA. For example, rs had increased from 1.41 to 7.14 s cm-1 7 days after fruit removal and stem cincturing, while ABA and PA levels rose by 50 and 370 % respectively. This increase in endogenous ABA was not a consequence of decreased leaf water potential; moisture status was actually improved after treatment. It is proposed that changes in endogenous levels of ABA, and possibly PA, constitute a mechanism for regulating gas exchange in these perennial plants.