Article Figures & Data
Figures
- Figure 1.
Domain Structure of B3 and bZIP Domain Transcription Factors Affecting ABA Response.
Percentages shown beneath the conserved domains in B3 domain family proteins indicate the range of similarity to maize VP1 among orthologs from Arabidopsis, rice, oat, carrot, bean, resurrection plant, and poplar and with the related FUS3 protein. Comparisons among the bZIP domain family represent percentage of similarity to ABI5 within the conserved domains among homologous genes in Arabidopsis, sunflower, and rice. The EmBP-1 protein used for comparison was from wheat.
- Figure 2.
Scheme of Signaling Pathways in Seed Development.
Arrows represent promotion of processes or expression of the regulators. Bars represent inhibitors of the indicated processes. The positions of loci do not imply the order of gene action.
- Figure 3.
Regulation of ABA-Responsive Promoter Activity in a Rice Embryonic Protoplast Transient Expression System.
(A) Fold effect of transiently expressed bZIP transcription factors on the ABA activation of the wheat Em promoter. Rice protoplasts were transformed with Em-GUS and/or the effector construct 35S-EmBP1, Ubi-ABI5, Ubi-ABF1, Ubi-ABF3, or 35S-VP1 and treated with or without 100 μM ABA for 16 hr. Fold activation of Em-GUS was calculated relative to control (no ABA, no effector) and normalized to the non-ABA-inducible Ubi-LUC reporter, which was cotransformed as an internal control. Asterisks indicate values that are significantly different from the control (P < 0.002; two-sided Student's t test, equal variance assumed). Daggers indicate that treatments with effectors resulted in a significant difference in Em-GUS activity from ABA treatment alone (P < 0.007; two-sided Student's t test, equal variance assumed). “Dummy” DNA was transformed along with Em-GUS to balance the total amount of input plasmid DNA between various treatments. Comparisons were done between paired samples from parallel experiments and plotted to obtain the meta-analysis. Error bars represent ±sem, with three or four replicates per sample.
(B) ABI5 interacts synergistically with ABA and VP1 to transactivate the wheat Em promoter. Protoplasts were transformed with either Em-GUS alone or in combination with Ubi-ABI5 and/or 35S-VP1 constructs. Numbers in parentheses indicate average fold activation compared with ABA induction alone. Asterisks indicate values that are significantly different from the control (P < 0.003; two-sided paired Student's t test). The dagger indicates that ABI5/VP1 synergy is significantly different from activation by any of the effectors alone (P < 1 × 10−7; two-sided paired Student's t test). Error bars represent ±sem, with three replicates per sample. (Figure modified from Gampala et al. [2002]; reprinted with permission.)
(C) 1-Butanol (1-But) antagonizes to the same extent the ABA induction of Em-GUS and ABI5 and VP1 synergy with ABA. Experimental treatments and fold induction calculations were as described in (A). Numbers in parentheses indicate the relative percentage inhibition of Em-GUS expression compared with control samples (no 1-butanol added). Asterisks indicate that ABI5 and VP1 synergy with ABA is significantly different from ABA activation alone (P < 0.01; two-sided Student's t test, equal variance assumed). Daggers indicate significant difference from the no butanol treatment (P < 0.008; two-sided Student's t test, equal variance assumed). Values are averages (±sem) of three replicate transformations.
In (A) to (C), the y axis indicates fold induction in Em-GUS activity per unit of Ubi-LUC.
- Figure 4.
Scheme of Signaling Pathways That Interact with the ABA Regulation of Germination.
Arrows represent promotion of processes or expression of the regulators. Bars represent inhibitors of the indicated processes. The positions of loci do not imply the order of gene action.
- Figure 5.
Sensitivity of Seedlings of Wild-Type, abi, and ABI Overexpression Lines to Glc.
(A) Growth and anthocyanin accumulation in seedlings exposed to Glc. Seed of the indicated genotypes were incubated on minimal mineral salt media supplemented with 0, 4, or 6% Glc for 7 days before scoring individuals with either expanded true leaves or pink cotyledons caused by anthocyanin accumulation. The low percentages of either characteristic observed for the ABI overexpression lines on all media, and even for the wild-type lines on high Glc, reflect the inhibition of both germination and seedling growth.
(B) Seedlings of the indicated genotypes after 7 days of growth on 4% Glc. The 35S::ABI3 line is isolate C7A19, described by Parcy et al. (1994); the 35S::ABI4 line is isolate 114, described by Söderman et al. (2000); the 35S::ABI5 transgene contains an ABI5 cDNA, extending 23 bp 5′ to the initiating codon, fused downstream of the 35S promoter of Cauliflower mosaic virus in pGA643 (T. Lynch and R.R. Finkelstein, unpublished data).
Col, Columbia; Ws, Wassilewskija.
Tables
Species Mutation Selection/Screen Phenotype Alleles or Orthologs Gene product References Arabidopsis aba1 Suppressors of non-germinating GA-deficient lines ABA deficient; wilty; decreased stress or ABA induction of gene expression; sugar-resistant seedling growth los6 npq2 Zeaxanthin epoxidase Koornneef et al., 1982; Ishitani et al., 1997; Niyogi et al. 1998; Xiong et al., 2001b aba2 Reduced dormancy ABA deficient; wilty; decreased stress or ABA induction of gene expression; sugar-resistant seedling growth gin1 isi4 sis4 Leon-Kloosterziel et al., 1996; Laby et al., 2000; Rook et al., 2001 aba3 Reduced dormancy ABA deficient; wilty; decreased stress or ABA induction of gene expression; freezing sensitive; sugar-resistant seedling growth frs1 los5 Aldehyde oxidase Moco Leon-Kloosterziel et al., 1996; Ishitani et al., 1997; Llorente et al., 2000; Rook et al., 2001; Xiong et al., 2001c aao3 Wilty phenotype ABA-deficient leaves; wilty, but near-normal seed dormancy Tomato sitiens? Aldehyde oxidase Seo et al., 2000 abi1-1 ABA-resistant germination Nondormant seed; pleiotropic defects in vegetative ABA response Protein phosphatase 2C Koornneef et al., 1984; Leung et al., 1994; Meyer et al., 1994 abi2-1 ABA-resistant germination Similar to abi1-1 Protein phosphatase 2C Koornneef et al., 1984; Leung et al., 1997; Rodriguez et al., 1998 abi3 ABA-resistant germination Pleiotropic defects in seed maturation; vegetative effects on plastid differentiation Cereal VP1 B3 domain transcription factor Koornneef et al., 1984; Giraudat et al., 1992 abi4 ABA-resistant germination Sugar- and salt-resistant germination and seedling growth gin6 isi3 san5 sis5 sun6 APETALA2 domain transcription factor Finkelstein, 1994; Finkelstein et al., 1998; Arenas-Huertero et al., 2000; Huijser et al., 2000; Laby et al., 2000; Rook et al., 2001 abi5 ABA-resistant germination Slightly sugar-resistant germination and seedling growth AtDPBF1 bZIP domain transcription factor Finkelstein, 1994; Finkelstein and Lynch, 2000a; Lopez-Molina and Chua, 2000 abi8 ABA-resistant germination Stunted growth, defective stomatal regulation; male sterile Finkelstein and Lynch, 1997 abh1 ABA-hypersensitive germination and guard cell response Pleiotropic; also enhanced drought tolerance mRNA CAP binding protein Hugouvieux et al., 2001 era1 Enhanced response to ABA at germination Enhanced stomatal response/drought tolerance, meristem defect wiggum Farnesyl transferase, β-subunit Cutler et al., 1996 era3 Enhanced response to ABA at germination Allelic to ein2; ethylene insensitive ein2 Membrane-bound metal sensor? Alonso et al., 1999; Ghassemian et al., 2000 ctr1 Enhance ABA resistance of abi1-1 Reduced dormancy, constitutive triple response to ethylene Protein kinase (Raf family) Kieber et al., 1993; Beaudoin et al., 2000 gca1 gca2 ABA-resistant root growth Pleiotropic effects on growth, stomatal regulation, and germination Himmelbach et al., 1998; Pei et al., 2000 gca3-gca8 ABA-resistant root growth Himmelbach et al., 1998 ade1 Deregulation of KIN2:: LUC expression No growth phenotype Foster and Chua, 1999 hlq sbr Aberrant patterns of Dc3::GUS expression Pleiotropic, seedling lethal Rock, 2000; Subramanian et al., 2002 hos1 Hypersensitive to osmotic stress induction of RD29::LUC Hypersensitive to ABA-induced gene expression RING finger protein (Ishitani et al., 1997; Lee et al., 2001 hos2 hos5 Hypersensitive to osmotic stress induction of RD29::LUC Hypersensitive to ABA-induced gene expression Ishitani et al., 1997; Xiong et al., 1999 sad1 Supersensitive to ABA and drought induction of RD29::LUC Reduced ABA biosynthesis, hypersensitive to ABA inhibition of germination and root growth U6-related Sm-like small ribonucleopro- tein Xiong et al., 2001a cla1 Chloroplasts altered ABA deficient 1-Deoxy-d-xylulose-5-phosphate synthase Estévez et al., 2001 hyl1 Hyponastic leaves Hypersensitive to ABA Double-stranded RNA binding protein Lu and Fedoroff, 2000 axr2 Auxin-resistant root growth Resistant to ABA and ethylene; dominant negative IAA7 transcription regulation Wilson et al., 1990; Nagpal et al., 2000 bri1 Brassinosteroid insensitive ABA hypersensitive Ser/Thr protein kinase Li and Chory, 1997; Steber and McCourt, 2001 det2 De-etiolated ABA hypersensitive Steroid reductase Steber and McCourt, 2001 fry1 Constitutive expression of RD29::LUC ABA hypersensitive Inositol polyphos-phate-1-phos-phatase Xiong et al., 2001b jar1 jin4 Jasmonic acid resistant Hypersensitive to ABA-inhibition of germination Staswick et al., 1992; Berger et al., 1996 lec1 Leafy cotyledons, seed lethal Slightly ABA-resistant germination CCAAT-box binding, HAP3 homolog Meinke et al., 1994; Parcy et al., 1997; Lotan et al., 1998 los1 Low sensitivity to osmotic stress induction of RD29::LUC Low sensitivity to ABA induction of gene expression prl1 Hypersensitivity to Glc and Suc Hypersensitive to ABA (also to cytokinin, ethylene, and auxin) Nuclear WD40 domain protein Németh et al., 1998; Bhalerao et al., 1999 sax1 Hypersensitive to auxin ABA hypersensitive, BR deficient Ephritikhine et al., 1999 uvs66 UV light sensitivity Hypersensitive to ABA inhibition of root growth Albinsky et al., 1999 Resurrection plant cdt-1 ABA-independent desiccation tolerance Constitutive ABA response in callus cultures Regulatory RNA or small peptide Furini et al., 1997 Barley cool Decreased leaf temperature ABA insensitivity in guard cells Raskin and Ladyman, 1988 Maize vp1 Viviparous ABA-insensitive seeds ABI3 B3-domain transcription factor Robertson, 1955; McCarty et al., 1991 vp2- vp14 Viviparous ABA deficient Robertson, 1955; Neill et al., 1986; Schwartz et al., 1997 rea Defective germination Red embryonic axis caused by anthocyanins; ABA-resistant germination; occasional vivipary Sturaro et al., 1996 Binding Site/Factor Class Genus Factor References ABA response elements (ABREs)/bZIPs Arabidopsis ABI5/AtDPBF1 AtDPBF2 AtDPBF3/AREB3 AtDPBF4 AtDPBF5/ABF3 ABF1 ABF2/AREB1 ABF4/AREB2 Choi et al., 2000; Finkelstein and Lynch, 2000a; Lopez-Molina and Chua, 2000; Uno et al., 2000 GBF3 Lu et al., 1996 Helianthus DPBF1, -2 and -3 Kim et al., 1997; Kim and Thomas, 1998 Oryza TRAB1 Hobo et al., 1999 Phaseolus PvZIP6 AF369792a ROM2 (repressor) Chern et al., 1996 Triticum EmBP-1 Guiltinan et al., 1990 RY/Sph elements/B3 domain proteins Arabidopsis ABI3 Giraudat et al., 1992 Avena AfVP1 Jones et al., 1997 Craterostigma CpVP1 Chandler and Bartels, 1997 Daucus C-ABI3 Shiota et al., 1998 Phaseolus PvALF Bobb et al., 1995 Populus PtABI3 Rohde et al., 1998 Oryza OsVP1 Hattori et al., 1994 Triticum TaVP1 Bailey et al., 1999 Zea mays VP1 McCarty et al., 1991 MYB Arabidopsis AtMYB2 Abe et al., 1997 MYC Arabidopsis AtMYC Abe et al., 1997 Unknown/HD-Zip Arabidopsis ATHB6 ATHB7 ATHB12 Söderman et al., 1996; Lee and Chun, 1998; Söderman et al., 1999 Unknown/AP2 Arabidopsis ABI4 Finkelstein et al., 1998; Söderman et al., 2000 ↵a Member of the ABI5 homologous subfamily, but no direct evidence for a role in ABA signaling; isolated from ethylene-treated leaf abscission zones of bean.
Locus/Gene Engineered Effect Phenotype Response to ABA Gene Product References AAPK (from Vicia faba) Dominant negative No ABA-induced stomatal closure; no ABA activation of plasma membrane anion channels Activated Ser/Thr protein kinase Li and Assmann, 1996; Li et al., 2000 AMBP kinase (from Pisum sativum) Activation; correlated with stomatal closure & dehydrin expression ABA-activated myelin basic protein kinase Burnett et al., 2000 ARSK1 Induced by ABA or NaCl Root specific ser/thr kinase Hwang and Goodman, 1995 ATCDPK1 ATCDPK1a Constitutively active mutants expressed transiently in maize leaf protoplasts Constitutive activation of an ABA responsive reporter gene (HVA1-LUC) Calcium-dependent protein kinases Sheen, 1996 AtIP5PII Overexpression ABA-insensitive germination, growth and gene expression Inositol polyphosphate 5-phosphatase II Sanchez and Chua, 2001 AtPLC1 Antisense suppression ABA-insensitive germination, growth and gene expression Phosphoinositide specific phospholipase C Sanchez and Chua, 2001 AtPP2C Wild-type and mutant proteins expressed transiently in maize leaf protoplasts; antisense suppression Over-expression blocked ABA-inducible transcription; null mutation had little effect; dominant interfering mutant strongly repressed ABA responses. Antisense conferred increased sensitivity to ABA-induced cold-tolerance and growth inhibition Protein phosphatase 2C Sheen, 1998; Tahtiharju and Palva, 2001 AtRac1/Rop6 Dominant-positive and dominant-negative Dominant positive blocked ABA-mediated effects on actin cytoskeleton and stomatal closure; dominant negative induced closure in absence of ABA Small GTPase Lemichez et al., 2001 Repressed; correlated with cell division activity Cyclin-dependent kinase Hemerly et al., 1993 GPA1 T-DNA insertion Increased leaf transpiration; no ABA inhibition of guard cell K+in channels and pH-independent ABA-activation of anion channels G protein α-subunit Wang et al., 2001 Induced by ABA and NaCl; may function in stress response GSK3/shaggy-like protein kinase Piao et al., 1999 ABA effect unknown; induced by osmotic stress His kinase osmosensor Urao et al., 1999 ICK1 Induced; may suppress cell division Inhibitor of cyclin- dependent kinase Wang et al., 1998 MAPKKK ABA effect unknown; induced by abiotic stresses Mitogen-activated protein kinase kinase kinase Mizoguchi et al., 1996 PIP5K Induced by ABA and abiotic stresses Phosphatidylinosi-tol-4-phosphate 5-kinase Mikami et al., 1998 PKABA1 Constitutive expression Suppression of GA-inducible gene expression in aleurone; small effect on the ABA induction of a LEA gene Induced Ser/Thr-protein kinase (from barley) Gómez-Cadenas et al., 1999 PLDα Antisense Decreased ABA and ethylene promotion of senescence Phospholipase Dα Fan et al., 1997 ROP2 Dominant negative constitutive Altered sensitivity to germination inhibition by ABA Rho-type small GTPase Li et al., 200 RPK1 Induced by ABA and abiotic stresses; abiotic stress-induction not ABA-dependent Receptor-like protein kinase Hong et al., 1997 Unknown; induced by abiotic stresses Ribosomal S6 kinase-like Mizoguchi et al., 1996 WAPK (from tobacco) Induced Wounding-induced protein kinase Lee et al., 1998 - Table 4.
Comparison of Vivipary and ABA Sensitivity in abi, fus3, and lec1 Monogenic and Digenic Mutants
Percent Germination on ABAb Genotype Percent Viviparya 3 μM 100 μM Wild type (Wasilewskija) 0 0 0 abi4 0 70 (56–88) 0 abi5 0 39 (18–60) 0 lec1 3.5 (0–7) 32 (0–55) 0 abi4, lec1 0.6 (0–1.4) 100 97 (94–100) abi5, lec1 12 (2–32) 49 (39–58) 4 (3–4) fus3 0 5 (0–16) 0 abi4, fus3 19 (9–34) 100 96 (88–100) abi5, fus3 18 (5–28) 100 74 (53–94) ↵a The presence of germinated seeds in mature dry siliques was scored in at least four batches of 60 to 400 seed; high variability partly reflects the dependence on RH.
↵b Germination of seeds excised from siliques in late embryogenesis, just before desiccation, was scored after 3 days of incubation at 4°C followed by 7 days at 22°C in continuous light on minimal mineral salt medium supplemented with the indicated concentrations of ABA; variability partly reflects differences in viabilities among excised seed lots.
Percentages shown are means of multiple assay values; ranges are presented in parentheses.
