Modulation of abscisic acid signal transduction and biosynthesis by an Sm-like protein in Arabidopsis.

The phytohormone abscisic acid (ABA) regulates plant growth and development as well as stress tolerance. The Arabidopsis sad1 (supersensitive to ABA and drought) mutation increases plant sensitivity to drought stress and ABA in seed germination, root growth, and the expression of some stress-responsive genes. sad1 plants are also defective in the positive feedback regulation of ABA biosynthesis genes by ABA and are impaired in drought stress induction of ABA biosynthesis. SAD1 encodes a polypeptide similar to multifunctional Sm-like snRNP proteins that are required for mRNA splicing, export, and degradation. These results suggest a critical role for mRNA metabolism in the control of ABA signaling as well as in the regulation of ABA homeostasis.

Quantitative analysis by flow cytometry of abscisic acid-inducible gene expression in transiently transformed rice protoplasts.

BACKGROUND: Quantifying plant gene expression by flow cytometry (FCM) would allow multidimensional cell-parameter analysis on a per-cell basis, thereby providing insight into the cellular mechanisms of plant gene regulation. Here we sought to establish quantitation by FCM of plant hormone (abscisic acid, ABA)-inducible green fluorescent protein (GFP) expression and to compare the method directly with traditional reporter enzyme assays. MATERIALS AND METHODS: GFP, beta-glucuronidase, and luciferase reporter genes driven by ABA-inducible or constitutive promoter constructs were expressed in transiently cotransformed rice protoplasts and reporter activities quantified by FCM (for GFP) or traditional enzyme assays. Treatments included cotransformations with specific ABA signaling effector cDNA constructs (encoding VIVIPAROUS-1, an ABA transcription factor, and ABA-INSENSITIVE1-1, a dominant-negative protein phosphatase regulator) and the ABA agonist lanthanum chloride. Dual-color FCM was also performed on GFP-expressing cells immunodecorated with an mAb recognizing a rice cell surface epitope. RESULTS: Quantitative analysis of ABA-inducible gene expression by FCM using GFP as reporter gave comparable results to traditional reporter enzyme assays, although the signal-to-noise ratio was less for FCM, which can be a limitation of the method at low promoter strengths. Multiparameter-correlated analysis of ABA-inducible GFP expression with a plasma membrane marker showed no apparent correlation between ABA sensitivity, marked by GFP, and presence of a cell surface arabinogalactan glycoprotein. CONCLUSIONS: Quantitative FCM of GFP-expressing plant cells is a rapid, robust, reproducible, and value-added method relative to traditional enzymatic reporter gene assays.

Functional interactions of lanthanum and phospholipase D with the abscisic acid signaling effectors VP1 and ABI1-1 in rice protoplasts.

cis,trans-Abscisic acid (ABA) plays an important role in plant growth and development, regulation of seed maturation, germination, and adaptation to environmental stresses. Knowledge of ABA mechanisms of action and the interactions of components required for ABA signal transduction is far from complete. Using transient gene expression in rice protoplasts, we observed additive and inhibitory effects between maize VP1 (Viviparous-1, a transcriptional activator) and a dominant-negative mutant protein phosphatase, ABI1-1 (ABA-insensitive-1-1), from Arabidopsis. Lanthanide ions were shown to be specific agonists of ABA-inducible gene expression and to interact synergistically with ABA and overexpressed VP1. Both VP1 and lanthanum activities could be antagonized by coexpression of ABI1-1, which demonstrates the specific ABA dependence of these effectors on ABA-regulated gene expression. We obtained pharmacological evidence that phospholipase D (PLD) functions in ABA-inducible gene expression in rice. Antagonism of ABA, VP1, and lanthanum synergy by 1-butanol, a specific inhibitor of PLD, was similar to the inhibition by coexpression of ABI1-1. These results demonstrate that ABA, VP1, lanthanum, PLD, and ABI1 are all involved in ABA-regulated gene expression and are consistent with an integrated model whereby La(3+) acts upstream of PLD.

Trivalent ions activate abscisic acid-inducible promoters through an ABI1-dependent pathway in rice protoplasts.

The plant hormone abscisic acid (ABA) mediates many vital processes in plant growth and development, including seed dormancy, cell division, water use efficiency, and adaptation to drought, salinity, chilling, pathogen attack, and UV light. Our understanding of ABA signal transduction is fragmentary and would benefit from specific and facile probes of the process. Protoplasts from rice (Oryza sativa L. cv IR54) embryonic suspension cultures cotransformed with effector plasmids encoding the maize (Zea mays) VIVIPAROUS1 cDNA and/or the Arabidopsis dominant negative mutant (abi1-1) ABA-insensitive cDNA demonstrated genetic interactions of VIVIPAROUS1 and abi1-1 in transactivation of the ABA-inducible HVA1 promoter from barley (Hordeum vulgare), suggesting the mechanisms of these effectors are conserved among monocots and dicots. Trivalent ions have been shown to act as an effector of gene expression in plants and animals, although the mechanism of action is unknown. We show in two complementary transient ABA-inducible gene expression assays (beta-glucuronidase and luciferase enzymatic activities and quantitative flow cytometry of green fluorescent protein) that trivalent ions specifically interact with an ABI1-dependent ABA-signaling pathway leading to gene expression. Trivalent ions mimic ABA effects on gene expression and may be a useful tool to study ABA signaling.

The genes ABI1 and ABI2 are involved in abscisic acid- and drought-inducible expression of the Daucus carota L. Dc3 promoter in guard cells of transgenic Arabidopsis thaliana (L.) Heynh.

The ABA INSENSITIVE1 (ABI1) and ABI2 genes encode homologous type-2C protein phosphatases with redundant yet distinct functions in abscisic acid (ABA) responses. Results from Northern blot analysis showed that ABA- and mannitol-inducible expression of the COR47 and COR78/LTI78/RD29A (COR78) genes was more impaired in the abi2 mutant of Arabidopsis thaliana (L.) Heynh than in the abi1 mutant. Furthermore, ABA-plus-mannitol treatments were additive towards COR47 gene expression; however, the ABA-deficient aba1 mutant showed reduced COR expression relative to the wild type in response to mannitol and ABA-plus-mannitol treatments. These results support the notion that drought- and ABA-signalling pathways are separate yet overlapping. To facilitate quantitative analysis of the genetic control of tissue-specific ABA- and desiccation-response pathways, we analyzed ABA- and mannitol-inducible expression of a carrot (Daucus carota L.) Dc3 promoter:uidA (beta-glucuronidase; GUS) chimaeric reporter (Dc3-GUS) in transgenic wild-type, ABA-deficient aba1, and ABA-insensitive abi1 and abi2 mutants. The Dc3 promoter directed ABA- and mannitol-inducible GUS expression in Arabidopsis guard cells and the two treatments were additive. The aba1, abi1, and abi2 mutant genotypes had reduced GUS expression in guard cells of cotyledons in response to mannitol, whereas abi1 and abi2 mutants were reduced in ABA-inducible GUS expression, consistent with overlapping ABA- and drought-response pathways. Quantitative fluorometric GUS assays of leaf extracts showed that abi2 mutants responded less to exogenous ABA than did abi1 mutants, and abi2 mutants responded more to mannitol than did abi1 mutants. We conclude that Dc3-GUS Arabidopsis is a tractable system in which to study tissue-specific ABA and drought signalling and suggest that ABI2 functions predominantly over ABI1 in COR78 and COR47 gene expression and guard-cell Dc3-GUS expression.

Dominant Wilty mutants of Zea mays (Poaceae) are not impaired in abscisic acidperception or metabolism.

Abscisic acid (ABA) is a plant hormone involved in growth, development, and stress adaptation. It acts via multiple pathways including rapid closure of stomatal pores by ion efflux from guard cells (thereby decreasing water loss) and by slower changes in gene expression. The Wilty2, Wilty3, and Wilty-2445 mutants are nonallelic members of a class of dominant mutants whose top leaves wilt when plants are subjected to drought conditions. We investigated the ABA responses of the Wi2 mutant by analysis of leaf transpiration rates and RAB17 (Responsive to ABA) gene expression. Wi2 leaves transpired less than those of wild-type siblings, but the slopes of Wi2 and wild-type ABA dose-leaf transpiration curves were identical, suggesting that Wi2 guard cell sensitivity to ABA is normal. Based on total RNA blot analysis, RAB17 transcripts in unstressed and drought-stressed Wi2 leaves were elevated relative to wild-type tissue. Wi2 ABA concentrations were also elevated relative to wild type in both unstressed and drought-stressed leaves. Similar to the Wi2 phenotype, Wilty3 and Wi-2445 mutants transpired less than their wild-type siblings and had normal ABA and ABA-conjugate levels, as measured by gas chromatography-mass spectrometry. Despite lower leaf transpiration rates, Wi2 mutants lost a greater percentage of fresh mass over time compared to the wild type. The previously characterized recessive mutant wilty1, which has a defect in vascular element development, also had reduced transpiration rates. It is concluded that the dominant Wi2, Wi3, and Wi-2445 mutants are not impaired in ABA metabolism or signaling. It is hypothesized, based on preliminary data, that the dominant mutants described here are impaired in vascular element development, analogous to the wilty1 mutant.

Flow cytometry and surface plasmon resonance analyses demonstrate that the monoclonal antibody JIM19 interacts with a rice cell surface component involved in abscisic acid signalling in protoplasts.

Abscisic acid (ABA) is a plant hormone involved in many developmental and physiological processes, but as yet, no ABA receptor has been identified. Flow cytometry of rice protoplasts and immunoblotting of purified plasma membranes (PMs) have been used to demonstrate that the monoclonal antibody JIM19 recognizes carbohydrate epitopes of cell surface glycoproteins. Using surface plasmon resonance technology specific binding of PMs to JIM19 was observed. Such interaction was antagonized significantly by ABA, but not by the biologically inactive ABA catabolite phaseic acid. These in vitro interactions were correlated with the biological activities of JIM19, ABA and phaseic acid on activation of the ABA-inducible Em promoter using two different transient reporter gene assays, beta-glucuronidase/luciferase and quantitative flow cytometry of Aequoria green fluorescent protein. Pre-treatment with JIM19 resulted in significant inhibition of ABA-inducible gene expression. Taken together, these data suggest that JIM19 interacts with a functional PM complex involved in ABA signalling.

A conserved domain of the viviparous-1 gene product enhances the DNA binding activity of the bZIP protein EmBP-1 and other transcription factors.

The maize VP1 protein is a seed-specific regulator of gene expression that effects the expression of a subset of abscisic acid (ABA)-regulated genes that are expressed during the maturation program of the seed. In addition, VP1 has pleiotropic effects on seed development that are not related to ABA. In transient expression assays, VP1 has been shown to transactivate gene expression through at least two distinct promoter elements: the G boxes from the ABA-inducible wheat Em gene and the SphI box from the maize C1 gene. We have investigated how VP1 can transactivate gene expression through diverse promoter elements by analyzing its association in vitro with EmBP-1, a factor that binds the Em promoter. We demonstrate that VP1 can greatly enhance the DNA binding activity of EmBP-1 in a gel retardation assay. This enhancing activity has also been observed on transcription factors as diverse as Opaque-2, Max, Sp1, and NF-kappaB. Deletion of a small but highly conserved region (BR2) in VP1 eliminates the enhancement in vitro as well as the ability of VP1 to transactivate Em gene expression in a transient expression assay. A 40-amino acid fragment from VP1 sandwiched between the maltose-binding protein and LacZ can confer the enhancement function to this fusion protein in vitro. A weak and relatively nonspecific interaction between BR2 and DNA is demonstrated by UV cross-linking. The in vitro properties we observe for VP1 might explain the regulatory effects of VP1 on a diverse set of genes and why mutations in the vp1 locus have pleiotropic effects.

Lanthanide ions are agonists of transient gene expression in rice protoplasts and act in synergy with ABA to increase Em gene expression.

Previous work has shown that in rice suspension cells, NaCl at 0.4 M can induce Em gene expression and act synergistically with ABA, possibly by potentiating the ABA response pathway through a rate-limiting intermediate (R.M. Bostock and R.S. Quatrano (1992) Plant Physiol., 98, 1356-1363). Since calcium is an intermediate in ABA regulation of stomatal closure, we tested the effect of calcium changes on ABA-inducible Em gene expression in transiently transformed rice protoplasts. We show that calcium is required for ABA-inducible Em-GUS expression and can act in synergy with ABA. The trivalent ions lanthanum, gadolinium, and aluminum, which are known to interact with calcium- and other signaling pathways, can act at sub-millimolar concentrations to increase GUS reporter gene expression driven by several promoters in transiently transformed rice protoplasts. This effect is not specific for the ABA-inducible Em promoter, but is synergistic with ABA. The lanthanum synergy with ABA does not require calcium. In rice suspension cells, lanthanum alone does not induce Em gene expression, in contrast to transiently transformed protoplasts, yet can act synergistically with ABA to effectively increase the sensitivity to ABA greater than tenfold. Trivalent ions may be a useful tool to study the regulation of gene expression. The possible effects of trivalent ions on ABA signal transduction and gene expression are discussed.

Plant regulators. Insensitivity is in the genes.

The cloning of loci determining abscisic acid insensitivity in Arabidopsis has identified a phosphatase and a transcriptional activator that mediate responses to abscisic acid and so regulate plant growth and development.

The aba Mutant of Arabidopsis thaliana (L.) Heynh. Has Reduced Chlorophyll Fluorescence Yields and Reduced Thylakoid Stacking.

It has been shown that the aba mutant of Arabidopsis thaliana (L.) Heynh. is impaired in epoxy-carotenoid biosynthesis and accumulates the epoxy-carotenoid precursor, zeaxanthin (C.D. Rock, J.A.D. Zeevaart [1991] Proc Natl Acad Sci USA 88: 7496-7499). In addition to providing conclusive evidence for the indirect pathway of abscisic acid biosynthesis from epoxy-carotenoids, the aba mutation offers a powerful means to study the function of xanthophylls (oxygenated carotenoids) in photosynthesis. We measured in vivo the chlorophyll (Chl) fluorescence parameters F(o) (initial), F(m) (maximum), F(v) (variable = F(m) - F(o)), and t((1/2)) (half-rise time of fluorescence induction) of wild-type (WT) and three allelic aba mutants. The mutant genotypes had significantly lower F(o) and F(m) values relative to those of WT. The F(v)/F(m) ratio and t((1/2)), which are parameters affected by photochemical efficiency, photosystem II (PSII), and plastoquinone pool sizes, were similar in the aba alleles and WT. Because the aba genotypes accumulate high levels of zeaxanthin, which is involved in nonphotochemical quenching of Chl fluorescence, we propose that the reduced fluorescence yields in the aba genotypes are a consequence of the accumulated zeaxanthin. Measurement of PSII oxygen evolution rates in isolated thylakoid membranes of WT and aba-4 confirmed that quantum efficiency was not altered in aba-4 but indicated that the mutant had reduced PSII activity in vitro. Electron microscopy revealed an abnormal chloroplast ultrastructure in the aba plants: the mutants had significantly fewer thylakoid lamellae per granum stack but significantly more grana per chloroplast, as well as more chloroplasts per cell than WT. Immunoblot analysis established that aba-4 had normal levels of the Chl a/b-binding core polypeptide of PSII (CP29) and the PSII light-harvesting Chl a/b-binding complex. These results provide evidence for the role of zeaxanthin in nonphotochemical fluorescence quenching and suggest involvement of epoxy-carotenoids and/or zeaxanthin in thylakoid stacking and PSII activity.

Abscisic alcohol is an intermediate in abscisic Acid biosynthesis in a shunt pathway from abscisic aldehyde.

It has previously been shown that the abscisic acid (ABA)-deficient flacca and sitiens mutants of tomato are impaired in ABA-aldehyde oxidation and accumulate trans-ABA-alcohol as a result of the biosynthetic block (IB Taylor, RST Linforth, RJ Al-Naieb, WR Bowman, BA Marples [1988] Plant Cell Environ 11: 739-745). Here we report that the flacca and sitiens mutants accumulate trans-ABA and trans-ABA glucose ester and that this accumulation is due to trans-ABA biosynthesis. (18)O labeling of water-stressed wild-type and mutant tomato leaves and analysis of [(18)O]ABA by tandem mass spectrometry show that the tomato mutants synthesize a significant percentage of their ABA and trans-ABA as [(18)O]ABA with two (18)O atoms in the carboxyl group. We further show, by feeding experiments with [(2)H(6)]ABA-alcohol and (18)O(2), that this doubly-carboxyl-labeled ABA is synthesized from [(18)O]ABA-alcohol with incorporation of molecular oxygen. In vivo inhibition of [(2)H(6)]ABA-alcohol oxidation by carbon monoxide establishes the involvement of a P-450 monooxygenase. Likewise, carbon monoxide inhibits the synthesis of doubly-carboxyl-labeled ABA in (18)O-labeling experiments. This minor shunt pathway from ABA-aldehyde to ABA-alcohol to ABA operates in all plants examined. For the ABA-deficient mutants impaired in ABA-aldehyde oxidation, this shunt pathway is an important source of ABA and is physiologically significant.

The aba mutant of Arabidopsis thaliana is impaired in epoxy-carotenoid biosynthesis.

The three mutant alleles of the ABA locus of Arabidopsis thaliana result in plants that are deficient in the plant growth regulator abscisic acid (ABA). We have used 18O2 to label ABA in water-stressed leaves of mutant and wild-type Arabidopsis. Analysis by selected ion monitoring and tandem mass spectrometry of [18O]ABA and its catabolites, phaseic acid and ABA-glucose ester (beta-D-glucopyranosyl abscisate), indicates that the aba genotypes are impaired in ABA biosynthesis and have a small ABA precursor pool of compounds that contain oxygens on the ring, presumably oxygenated carotenoids (xanthophylls). Quantitation of the carotenoids from mutant and wild-type leaves establishes that the aba alleles cause a deficiency of the epoxy-carotenoids violaxanthin and neoxanthin and an accumulation of their biosynthetic precursor, zeaxanthin. These results provide evidence that ABA is synthesized by oxidative cleavage of epoxy-carotenoids (the "indirect pathway"). Furthermore the carotenoid mutant we describe undergoes normal greening. Thus the aba alleles provide an opportunity to study the physiological roles of epoxy-carotenoids in photosynthesis in a higher plant.

Abscisic (ABA)-Aldehyde Is a Precursor to, and 1',4'-trans-ABA-Diol a Catabolite of, ABA in Apple.

Previous (18)O labeling studies of abscisic acid (ABA) have shown that apple (Malus domestica Borkh. cv Granny Smith) fruits synthesize a majority of [(18)O]ABA with the label incorporated in the 1'-hydroxyl position and unlabeled in the carboxyl group (JAD Zeevaart, TG Heath, DA Gage [1989] Plant Physiol 91: 1594-1601). It was proposed that exchange of (18)O in the side chain with the medium occurred at an aldehyde intermediate stage of ABA biosynthesis. We have isolated ABA-aldehyde and 1'-4'-trans-ABA-diol (ABA-trans-diol) from (18)O-labeled apple fruit tissue and measured the extent and position of (18)O incorporation by tandem mass spectrometry. (18)O-Labeling patterns of ABA-aldehyde, ABA-trans-diol, and ABA indicate that ABA-aldehyde is a precursor to, and ABA-trans-diol a catabolite of, ABA. Exchange of (18)O in the carbonyl of ABA-aldehyde can be the cause of loss of (18)O from the side chain of [(18)O]ABA. Results of feeding experiments with deuterated substrates provide further support for the precursor-product relationship of ABA-aldehyde --> ABA --> ABA-trans-diol. The ABA-aldehyde and ABA-trans-diol contents of fruits and leaves were low, approximately 1 and 0.02 nanograms per gram fresh weight for ABA-aldehyde and ABA-trans-diol, respectively, while ABA levels in fruits ranged from 10 to 200 nanograms per gram fresh weight. ABA biosynthesis was about 10-fold lower in fruits than in leaves. In fruits, the majority of ABA was conjugated to beta-d-glucopyranosyl abscisate, whereas in leaves ABA was mainly hydroxylated to phaseic acid. Parallel pathways for ABA and trans-ABA biosynthesis and conjugation in fruits and leaves are proposed.

The maize plastid psbB-psbF-petB-petD gene cluster: spliced and unspliced petB and petD RNAs encode alternative products.

The chloroplast psbB, psbF, petB, and petD genes are cotranscribed and give rise to many overlapping RNAs. The mechanism and significance of this mode of expression are of interest, particularly because the accumulation of the psb and pet gene products respond differently to both light and, in C4 species such as maize, developmental signals. We present an analysis of the maize psbB, psbF, petB, and petD genes and intergenic regions. The genes are organized similarly in maize (a C4 species) and in several C3 species. Functional class II-like introns interrupt the 5' ends of petB and petD. Both spliced and unspliced RNAs accumulate; these encode alternative forms of the petB and petD proteins, differing at their N-termini. Promoter-like elements between psbF and petB, and biased codon usage suggest that the differential regulation of the psb and pet genes might be achieved at both the transcriptional and translational levels.