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

Abstract

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., 1995Zou J. Abrams G.D. Barton D.L. Taylor D.C. Pomeroy M.K. Abrams S.R. Induction of lipid and oleosin biosynthesis by (+)-abscisic acid and its metabolites in microspore-derived embryos of Brassica napus L.cv Reston.Plant Physiol. 1995; 108: 563-571Google Scholar, Kepka et al., 2011Kepka M. Benson C.L. Gonugunta V.K. Nelson K.M. Christmann A. Grill E. Abrams S.R. Action of natural abscisic acid precursors and catabolites on abscisic acid receptor complexes.Plant Physiol. 2011; 157: 2108-2119Google Scholar). 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., 1998Krochko J.E. Abrams G.D. Loewen M.K. Abrams S.R. Cutler A.J. (+)-Abscisic acid 8′-hydroxylase is a cytochrome P450 monooxygenase.Plant Physiol. 1998; 118: 849-860Google Scholar, Kushiro et al., 2004Kushiro T. Okamoto M. Nakabayashi K. Yamagishi K. Kitamura S. Asami T. Hirai N. Koshiba T. Kamiya Y. Nambara E. The Arabidopsis cytochrome P450 CYP707A encodes ABA 8′-hydroxylases: key enzymes in ABA catabolism.EMBO J. 2004; 23: 1647-1656Google Scholar, Saito et al., 2004Saito S. Hirai N. Matsumoto C. Ohigashi H. Ohta D. Sakata K. Mizutani M. Arabidopsis CYP707As encode (+)-abscisic acid 8′-hydroxylase, a key enzyme in the oxidative catabolism of abscisic acid.Plant Physiol. 2004; 134: 1439-1449Google Scholar) (Figure 1). 8′-hydroxy ABA is isomerized spontaneously to phaseic acid (PA), involving internal cyclization of the 8′-hydroxyl moiety onto the enone at C-2′ position. This ring closure occurs in concert with the initial hydroxylation of ABA both in vitro and in vivo (Krochko et al., 1998Krochko J.E. Abrams G.D. Loewen M.K. Abrams S.R. Cutler A.J. (+)-Abscisic acid 8′-hydroxylase is a cytochrome P450 monooxygenase.Plant Physiol. 1998; 118: 849-860Google Scholar, Kushiro et al., 2004Kushiro T. Okamoto M. Nakabayashi K. Yamagishi K. Kitamura S. Asami T. Hirai N. Koshiba T. Kamiya Y. Nambara E. The Arabidopsis cytochrome P450 CYP707A encodes ABA 8′-hydroxylases: key enzymes in ABA catabolism.EMBO J. 2004; 23: 1647-1656Google Scholar, Saito et al., 2004Saito S. Hirai N. Matsumoto C. Ohigashi H. Ohta D. Sakata K. Mizutani M. Arabidopsis CYP707As encode (+)-abscisic acid 8′-hydroxylase, a key enzyme in the oxidative catabolism of abscisic acid.Plant Physiol. 2004; 134: 1439-1449Google Scholar); however, this reaction is reversible, and differential biological activities between 8′-hydroxy ABA and PA have been reported (Jadhav et al., 2008Jadhav A.S. Taylor D.C. Giblin M. Ferrie A.M. Ambrose S.J. Ross A.R. Nelson K.M. Irina Z.L. Sharma N. Anderson M. et al.Hormonal regulation of oil accumulation in Brassica seeds: metabolism and biological activity of ABA, 7′-, 8′- and 9′-hydroxy ABA in microspore derived embryos of B. napus.Phytochemistry. 2008; 69: 2678-2688Google Scholar). Finally, the subsequent conversion of PA to dihydrophaseic acid (DPA) is catalyzed by a soluble PA reductase (PAR) that reduces the 4′-ketone to a 4′-hydroxyl group. This PAR had remained elusive until the recent breakthrough of Weng et al., 2016Weng J.K. Ye M. Li B. Noel J.P. Co-evolution of hormone metabolism and signaling networks expands plant adaptive plasticity.Cell. 2016; 166: 881-893Google Scholar. Chemical reactions similar to PAR are catalyzed by dihydroflavonol 4-reductase (DFR)-like NAD(P)H-dependent reductases, and accordingly, Weng et al., 2016Weng J.K. Ye M. Li B. Noel J.P. Co-evolution of hormone metabolism and signaling networks expands plant adaptive plasticity.Cell. 2016; 166: 881-893Google Scholar screened for potential candidates through a phylogenetic analysis of the DFR-like reductase family. Only one candidate was found in seed plants, which was named ABH2, for ABA hypersensitive 2, upon phenotype analysis of abh2 T-DNA mutant lines. Catabolism of ABA is predicted to be impaired in abh2, which might explain the enhanced sensitivity to ABA during early seedling development. However, because PA levels are predicted to increase in abh2 mutants, Weng et al., 2016Weng J.K. Ye M. Li B. Noel J.P. Co-evolution of hormone metabolism and signaling networks expands plant adaptive plasticity.Cell. 2016; 166: 881-893Google Scholar reasoned that PA directly inhibits germination in an ABA-like manner. In addition, the authors examined slow and fast response to drought stress in abh2-1 plants. They found that rapid control of leaf transpiration was not affected in abh2-1; however, after a 2-week drought stress period abh2-1 plants displayed improved drought tolerance. The levels of ABA and its catabolites, i.e., PA, DPA, and its 4′-O-β-glucoside (DPAG), were measured in 2-day-old wild-type (WT) and abh2-1 seedlings by high-performance liquid chromatography combined with mass spectrometry. First at all, they found that abh2-1 contains very little DPA compared with WT but much higher levels of PA, which is consistent with the hypothesis ABH2 being the PAR. Indeed, recombinant ABH2 reduces PA to DPA using NADPH as cofactor. Second, ABA levels in WT and abh2-1 were comparable, which discounts the hypothesis that elevated PA levels in abh2-1 might result in feedback inhibition of the CYP707A family P450s. Therefore, the enhanced sensitivity of abh2-1 to ABA during early seedling development might reflect an ABA-like effect of PA or PA-specific effects that are inhibitory for germination. Previous researchers have studied the capability of PA to inhibit germination and induce stomatal closure and ABA-responsive genes, but conclusive results have not yet been obtained to support a clear ABA-like effect of PA in different plant species. Among these studies, Walker-Simmons et al., 1997Walker-Simmons M.K. Holappa L.D. Abrams G.D. Abrams S.R. ABA metabolites induce group 3 LEA mRNA and inhibit germination in wheat.Physiol. Plant. 1997; 100: 474-480Google Scholar concluded that PA lacked inhibitory effect on wheat germination at concentrations 50-fold higher than ABA but induced an ABA-responsive LEA gene. Hill et al., 1992Hill R.D. Durnin D. Nelson L.A.K. Abrams G.D. Gusta L.V. Abrams S.R. Effects of (±)-phaseic acid on developing embryos of barley (Hordeum vulgare, L. cv. Bonanza) cultured in vitro.Seed Sci. Res. 1992; 2: 207-214Google Scholar found that PA was about 10% as effective as ABA as a germination inhibitor of barley immature embryo. Sharkey and Raschke, 1980Sharkey T.D. Raschke K. Effects of phaseic acid and dihydrophaseic acid on stomata and the photosynthetic apparatus.Plant Physiol. 1980; 65: 291-297Google Scholar found that PA can cause stomatal closure in some species but is barely effective in barley and maize. Moreover, rates of CO2 assimilation were not affected by PA or DPA but were reduced markedly by ABA. Weng et al., 2016Weng J.K. Ye M. Li B. Noel J.P. Co-evolution of hormone metabolism and signaling networks expands plant adaptive plasticity.Cell. 2016; 166: 881-893Google Scholar performed a transcriptomic profiling of Arabidopsis treated with 50 μM PA and found that most PA-responsive genes overlapped with ABA-responsive genes; however, differentially expressed genes in response to PA were about 10% of those in response to ABA and were less altered upon PA treatment than upon ABA treatment. Therefore, 50 μM PA possesses ABA-like activity on gene expression but PA's effect is markedly lower than the dramatic change induced by ABA treatment on plant transcriptome. To further increase PA levels in Arabidopsis, Weng et al., 2016Weng J.K. Ye M. Li B. Noel J.P. Co-evolution of hormone metabolism and signaling networks expands plant adaptive plasticity.Cell. 2016; 166: 881-893Google Scholar crossed the abh2-1 mutant with plants that overexpressed the biosynthetic PA enzyme CYP707A3. Thus, blocking PA degradation together with enhancing biosynthesis leads to high PA levels and reduced ABA content. These plants do not show retarded growth as observed in some ABA biosynthetic mutants, which suggests a compensatory effect of PA under conditions of low ABA. ABA metabolite profiling over a 20-day drought period revealed that PA levels increased about 10-fold at day 12 and then remained relatively constant until day 20, whereas ABA levels also increased markedly (at least two-fold over PA levels) at day 12 and then declined at the end of the drought period. Thus, PA might be able to extend or prolong some ABA effects; however, simultaneous ABA action is also required to cope with drought stress because high PA levels combined with reduced ABA content did not upregulate the ABA-responsive genes RD29B and HIS1-3 nor improve water content in leaves after drought treatment (Weng et al., 2016Weng J.K. Ye M. Li B. Noel J.P. Co-evolution of hormone metabolism and signaling networks expands plant adaptive plasticity.Cell. 2016; 166: 881-893Google Scholar). Weng et al., 2016Weng J.K. Ye M. Li B. Noel J.P. Co-evolution of hormone metabolism and signaling networks expands plant adaptive plasticity.Cell. 2016; 166: 881-893Google Scholar determined whether the PYR/PYL/RCAR family of ABA receptors recognized PA as an effective ligand using the PP2C inhibition assay. ABA was more effective than PA in inhibiting PP2C activity for all the 11 recombinant receptors tested. This result is in agreement with previous results from Kepka et al., 2011Kepka M. Benson C.L. Gonugunta V.K. Nelson K.M. Christmann A. Grill E. Abrams S.R. Action of natural abscisic acid precursors and catabolites on abscisic acid receptor complexes.Plant Physiol. 2011; 157: 2108-2119Google Scholar, who found a moderate effect of PA in regulating ABI2 activity in the presence of RCAR1/PYL9, RCAR3/PYL8, and RCAR11/PYR1. However, Kepka et al., 2011Kepka M. Benson C.L. Gonugunta V.K. Nelson K.M. Christmann A. Grill E. Abrams S.R. Action of natural abscisic acid precursors and catabolites on abscisic acid receptor complexes.Plant Physiol. 2011; 157: 2108-2119Google Scholar did not find a significant effect of 3 μM PA in inhibiting germination, inducing stomatal closure, or inhibiting root growth of Arabidopsis Ler. Weng et al., 2016Weng J.K. Ye M. Li B. Noel J.P. Co-evolution of hormone metabolism and signaling networks expands plant adaptive plasticity.Cell. 2016; 166: 881-893Google Scholar found that some ABA receptors, namely PYL8, PYL9, and PYL11, are barely sensitive to PA. Other receptors, namely PYR1, PYL1, and PYL10, are ∼40-fold less sensitive to PA than ABA, and in the case of PYL2, PYL4, PYL5, and PYL6 were between 10-and 20-fold less sensitive. Finally, PYL3 has an IC50 value only four-fold higher for PA than ABA. PYL3 has a very specific expression in the chalazal seed coat and might sense PA during regulation of seed development or germination; however, PYL3 is barely expressed in the remainder of plant tissues (http://jsp.weigelworld.org/expviz/expviz.jsp). To further pinpoint potential receptors able to recognize PA in vivo, the abh2-1 p35S:CYP707A3 plants were crossed with individual pyr/pyl mutants. The authors found that pyl2-1 (Ler) abh2-1 p35S:CYP707A3 plants showed stunted growth and decreased drought stress tolerance, which suggests that PYL2 is an important receptor for PA. Intriguingly, pyl5-1 abh2-1 p35S:CYP707A3 does not seem to show this phenotype, even though PYL5 was eight-fold more effective than PYL2 (IC50 0.56 and 4.5 μM, respectively) in inhibiting PP2C activity in response to PA. Weng et al., 2016Weng J.K. Ye M. Li B. Noel J.P. Co-evolution of hormone metabolism and signaling networks expands plant adaptive plasticity.Cell. 2016; 166: 881-893Google Scholar then obtained crystal structures for the PYL2-PA complex and PYL3-PA-HAB1 ternary complex. PA induces a conformational change in PYL2 that might promote disassociation of the dimeric receptor as well as closure of one gating loop around the ligand. The bulkier head group of PA pivots ∼60° relative to the PYL2-ABA corresponding structure; however, both PA and ABA adopt similar conformations when comparing PYL3-PA-HAB1 and PYL3-ABA-HAB1 complexes. In summary, the results from Weng et al., 2016Weng J.K. Ye M. Li B. Noel J.P. Co-evolution of hormone metabolism and signaling networks expands plant adaptive plasticity.Cell. 2016; 166: 881-893Google Scholar provide a key advance toward our understanding of ABA catabolism and the ability of PA to activate a subset of ABA receptors. Further evaluation of the biological effects of PA and 8′-hydroxy ABA in different plant species will be required to obtain a comprehensive picture of the role of these ABA catabolites as general hormonal regulators in plants.