Plant Physiol. 2007 February; 143(2): 745–758

Figure 7. Expression of ABA- and sugar-related genes in response to drought. Plants were grown in soil for 3 weeks, and then subjected to withholding water for 4 d (drought). Control samples were harvested at the same time as drought-treated samples. Three independent experiments were performed, except for control with two independent experiments, each with a duplicate and with consistent results. WT, Wild type; HXK, hexose kinase; Susy, Suc synthase; UBQ, ubiquitin. Plant Physiol. 2007 February; 143(2): 745–758.

Plant Physiol. 2007 November; 145(3): 853–862.

Figure 2. Localization of suberin and ABA in tumors. A, Four-week-old tumor, induced by A. tumefaciens (strain C58) at an inflorescence stalk of Arabidopsis. The arrow indicates the section plane of the cross sections shown in B to I. B to D, ABA immunolocalization in tumor cross sections using a primary mouse hybridoma monoclonal antibody against ABA and a secondary antibody labeled with the green Alexa 488 chromophore. Images were taken with a confocal laser scanning microscope and show overlays of images taken in a confocal and differential interference contrast mode. B, Fluorescence signals were strongest around vascular tissue (vascular, vs) of the tumor/host inflorescence stalk interface. C, ABA immunofluorescence at cellular resolution in thin cytoplasmic layers of cells near the xylem vessels (xy-ve). D, Control cross section of a tumor, treated with 1% (w/v) rabbit serum and the secondary Alexa 488 antibody conjugate in the absence of the primary antibody against ABA, revealed no significant fluorescence signal. E to G, Images from bright-field microscopy. H and I, UV illuminated (488 nm) cross sections. E, Cross section of a tumor (tu) with a disrupted epidermis attached to an inflorescence stalk (stalk). The reddish color of the outer tumor cell layers marks Sudan-III-stained cells indicating suberin. Note that the stalk is covered by an intact epidermis, containing a cuticle (arrows). F, Closeup of outer cell layers from the tumor, attached to the host inflorescence stalk as shown in E with suberized cell walls (red) and of outer cell layers (G) from the host inflorescence stalk shown in E with a cuticle (red). H, Strong autofluorescence indicates aromatic compounds of lignified xylem vessels and outer cell layers of the tumor. I, Closeup of outer cell layers from the border between the tumor and inflorescence stalk shown in H. Autofluorescence marks cell walls of cells at the tumor surface but not at the surface of the stalk (H, white arrows). Bars: A, 5 mm; B, 100 μm; C, D, F, G, and I, 50 μm; E and H, 200 μm. Plant Physiol. 2007 November; 145(3): 853–862.

Plant Physiol. 2006 January; 140(1): 302–310

ABA Extraction and Quantification by ELISASterilized seeds were immersed in deionized water or fluridone and kept in darkness at 4°C for 2 d and moved to plates with or without 6% Glc. After being cultured in light at 22°C for 5 d, the seeds and seedlings were lyophilized. Samples were ground in an ice-cooled mortar in 4 mL of 80% (v/v) methanol extraction medium containing 1 mm butylated hydroxytoluence as an antioxidant. The extract was incubated at 4°C for 24 h and centrifuged at 7,000 rpm for 15 min at the same temperature. The supernatant was passed through Chromosep C18 columns (C18 Sep-Park Cartridge; Waters) and prewashed with 10 mL of 100% (w/v) and 5 mL of 80% (v/v) methanol, respectively. Two milliliters of hormone fractions eluted from the columns were dried under N2 and dissolved in 0.5 mL phosphate-buffered saline (PBS) containing 0.1% (v/v) Tween 20 and 0.1% (w/v) gelatin (pH 7.5) for ABA analysis by competitive ELISA. The antigens (ABA hapten-carrier protein), mouse monoclonal antibodies against ABA, and IgG horseradish peroxidase used in ELISA were produced at the Phytohormones Research Institute (China Agricultural University). ELISA was performed on a 96-well microplate. Each well on the plate was coated with 100 μL coating buffer (1.5 g L−1 Na2CO3, 2.93 g L−1 NaHCO3, and 0.02 g L−1 NaN3, pH 9.6) containing 0.25 μg mL−1 antigens. The coated plates were incubated for 30 min at 37°C, and then kept at room temperature for 3 to 4 min. After washing three times with PBS-Tween 20 (0.1% [v/v]) buffer (pH 7.4), each well was filled with 50 μL of either extracts or ABA standards (0–2,000 ng mL−1 dilution range), and 50 μL of 20 μg mL−1 ABA antibodies. The plate was incubated for 30 min at 37°C, and then washed as above. One-hundred microliters of 1.25 μg mL−1 IgG horseradish peroxidase substrates was added to each well and incubated for 30 min at 37°C. The plate was rinsed four times with the above PBS-Tween 20 buffer, and 100 μL color-appearing solution containing 1.5 mg mL−1 orthophenylenediamine and 0.008% (v/v) hydrogen peroxide was added to each well. The reaction was stopped by adding 50 μL 4 m H2SO4 per well when the 2,000 ng mL−1 standard had a pale color and the 0 ng mL−1 standard had a deep color in the wells. Color development in each well was detected using a Microplate Reader (model EL310, Bio-TEK) at optical density A490. The results are the means ± se of at least three replicates.

Plant Physiol. 2006 January; 140(1): 302–310

Hormonal and Sugar Treatment The effects of ABA on seed germination were studied by determining the germination rates of 70 to 100 seeds pretreated with deionized water or 100 μm fluridone and planted in triplicate on medium containing ABA (mixed isomers; Sigma). The effects of Glc, Suc, mannitol, and sorbitol were studied in a similar manner. Sterilized seeds were stratified at 4°C for 48 h and sown on plates containing different concentrations or types of sugars (d-Glc, Suc, d-mannitol, and d-sorbitol) in triplicate. For direct comparisons of germination rates, each plate was subdivided and all seed lines were planted on the same plate.

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Determination of ABA ContentFree ABA and ABA released from its conjugates was analyzed by ELISA as described earlier (Jiang et al., 2004). Recoveries of ABA during the purification procedures were checked routinely using radioactive ABA and found to be more than 95%. The immunochemicals were generously supplied by Professor Weiler, Ruhr Universit鋞 Bochum (Germany).

Plant Physiol. 2007 November; 145(3): 853–862.

Tissue Preparation and Immunolocalization of ABAFour-week-old Arabidopsis tumors were infiltrated under vacuum and fixed for at least 24 h and further on treated as described in Schraut et al. (2004). For immunolocalization the green Alexa conjugate 488 (488 goat anti-mouse IgG, H+L, Molecular Probes; excitation 488 nm, emission 506–512 nm) was used as a secondary antibody as described previously (Langhans et al., 2001; Veselov et al., 2003; Schraut et al., 2004). Control cross sections, demonstrating the specificity of ABA-derived signals, were performed in the same way as shown in Schraut et al. (2004). Sections were stained with toluidine blue to quench the autofluorescence of the lignified cell walls and inspected with a confocal laser-scanning microscope (LEICA-TCS-SP).

Plant Physiol. 2007 November; 145(3): 853–862.

Arabidopsis Root Cultures and ABA Treatment Surface-sterilized Col-0 seeds were transferred into an Erlenmeyer flask containing 100 mL of sterile Murashige and Skoog growth solution (Murashige and Skoog salts [Sigma] supplemented with Gamborg's B5 vitamins [Sigma] and 1% Suc) and shaken at 50 rpm. Seedlings that developed a sufficient amount of root material within 2 weeks were taken and ABA was added to a final concentration of 10 μm to induce suberization. Roots were harvested 4 d later. Control roots were treated with water.

Plant Physiol. 2007 November; 145(3): 853?62.

ABA Induces Drought Adaptations and Regulates Water Flow into TumorsAlthough water loss is minimized in tumors, the tissue is still endangered by desiccation. Like in other species, tumors of Arabidopsis accumulate high levels of the stress hormone ABA (Mistrik et al., 2000; Veselov et al., 2003; W鋍hter et al., 2003). Accordingly, we identified ABA-dependent adaptive mechanisms on the level of gene regulation, as well as accumulation of osmoprotectants. A pattern of ABA-inducible genes that is different from the classical ABA-induced genes so far studied in seedlings, leaves, or roots was found to be up-regulated in tumors. This specific pattern includes a set of LEA and dehydrin genes. LEA proteins have been shown to be strongly accumulated in maturating seeds, but they can also be found in vegetative tissues. Their functional implication in drought protection has been worked out by Tolleter and colleagues (2007), prompting us to suggest this function for the tumor as well. During dehydration, these proteins can maintain the structure of endomembranes and other proteins by sequestration of ions, such as calcium. Additionally, these proteins have been shown to bind or replace water and act as molecular chaperones (Grelet et al., 2005; Mahajan and Tuteja, 2005; Tolleter et al., 2007). Thus, water stress in tumors is counteracted by an ABA-dependent up-regulation of stress genes. Both Arabidopsis and Ricinus tumors accumulate high amounts of the osmoprotectant Pro. This amino acid is involved in elevation of the cell turgor and hence in maintaining the osmotic balance under drought stress conditions (W鋍hter et al., 2003). This role of Pro and other osmoprotectants in drought adaptation requires ABA signaling, since ABA-deficient mutants accumulate Pro to much lower levels when raised under drought or salt stress conditions (Xiong et al., 2001; Verslues and Bray, 2006). Analysis of tumor growth on mutant plants, exhibiting defects in ABA signaling, showed that phosphatases of the type 2C class affected in abi1 and abi2 plants, as well as the APETALA2 domain transcription factor affected in abi4 mutant plants, play a pivotal role during tumorigenesis (Leung et al., 1997; Finkelstein et al., 1998). The reduced tumor growth of these mutants, however, was not due to a reduction in T-DNA transformation efficiency. ABI1 and ABI2 are involved in adaptation of vegetative tissues to drought stress and have been shown to regulate Pro accumulation, together with ABI4, in response to a low water potential at reduced water availability (Verslues and Bray, 2006). In contrast, the transcription factor ABI3 has no effect on tumor development. As ABI3 has been shown to act downstream of the phosphatases ABI1 and ABI2, it may define a separate signaling pathway different from that controlled by ABI4 (Brady et al., 2003). Taken together, we propose the following working model on how ABA might support tumor development (Fig. 7). While the physiological effects of ABA have already been discussed in previous paragraphs and are well in line with the effects discussed in the literature, our microarray analysis did not reveal any evidence for key enzymes of ABA metabolism being up-regulated in tumor tissue. Rather, immunolocalization of ABA suggests that this stress phytohormone might be translocated via the transpiration stream of the host plant into the tumor. This observation corresponds to earlier findings on the distribution of nitrate reductase activity, which was also found to be repressed in tumors; hence, amino acids have to be imported from the leaves as well (Deeken et al., 2006). From tumors of castor bean (Ricinus communis) it was proposed that the synthesis of auxin and cytokinin promotes an increased production of ethylene. Communication between the tumor and the host plant might well be provided by this gaseous factor, which in turn triggers synthesis of high ABA content in leaves of the host plant (Aloni et al., 1998; W鋍hter et al., 1999; Veselov et al., 2003). Since Arabidopsis tumors produce increased levels of the ethylene precursor ACC and develop very close to rosette leaves, ethylene diffusion seems very likely. High levels of ABA levels are found in Arabidopsis stalks above the rosette, as well as in tumor tissue close to vascular bundles. Thus, ethylene-induced ABA production in leaves might result in its transport via the phloem from leaves into tumors (Mistrik et al., 2000).

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ABA AssayFor ABA extraction, seedlings harvested at appropriate stages or after stress treatments were treated with extraction buffer (80% methanol and 2% glacial acetic acid) for 24 h under darkness, followed by centrifugation for 10 min at 2,000g. Supernatants were taken up and dried in a speedvac, then resuspended in 100% methanol plus 0.2 m NH4H2PO4 (pH 6.8) for 10 min. To avoid plant pigment and other nonpolar compound effects on the immunoassay, the extracts were first passed through a polyvinylpolypyrrolidone column and then C18 cartridges. Elutes were concentrated to dryness in a speedvac and resuspended in Tris-buffered saline for immunoassay (Hsu and Kao, 2003). For ABA determination, ABA was quantified by ELISA (Phytodetek ABA kit; Agdia) according to the manufacturer's protocol.

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RT-PCRTotal RNA was extracted from wild type, aba2, and ABA2 overexpression lines using TRIzol reagent (Invitrogen). Six micrograms of total RNA of each genotype with 1 μg oligo(dT) primer (Invitrogen) were heated at 70°C for 5 min and then chilled on ice immediately. RNA was then subjected to RT with reverse-transcriptase Avian myeloblastosis virus (Roche) at 42°C for 1 h according to the manufacturer's protocol. Synthesized cDNA was used as a template for PCR.

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GUS Activity AssayCold-pretreated transgenic seeds harboring the ABA2GUS transgene were grown on agar plates or in soil for different time periods, depending on experiments, and then subjected to various stresses (cold, drought, or salinity). The treated plants were harvested and ground with extraction buffer (50 mm NaHPO4 [pH 7.0], 10 mm β-mercaptoethanol, 10 mm Na2EDTA, 0.1% sodium lauryl sarcosine, 0.1% Triton X-100). After centrifugation at 13K rpm for 10 min at 4°C, the supernatants were removed for further GUS assay, essentially according to the protocol of Jefferson et al. (1987), using a DyNA Quant 200 fluorometer (Amersham-Pharmacia Biotech).

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NaCl and Dehydration TreatmentsWild type, aba2, and ABA2 overexpression seeds with cold pretreatment were grown on agar plates with various NaCl concentrations for 18 or 28 d. The ratios of bleached to total plants were counted to define tolerance to salinity. Seeds with cold pretreatment were also grown in soil for 3 weeks; then plants were subjected to watering with solutions containing four gradually increased concentrations of NaCl (50, 100, 150, and 200 mm), each for 4 d for a total of 16 d (Shi et al., 2003). For dehydration, seeds with cold pretreatment were grown in soil for 21 d and then plants were excised. The aerial parts of tissues were placed on plastic weighing boats and kept in an electronic dry box (model-DX-76; Taiwan Dry Tech Corp.) with approximately 40% relative humidity. The fresh weights of tissues were measured at 0-, 1.5-, and 3-h time points.

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Preparation of Low-Water-Potential Medium Plates One-half-strength modified Murashige and Skoog medium (pH 5.7) with 0.7% Phyto agar (Duchefa Biochemie B.V.) was autoclaved. The sterilized medium was cooled to 50°C to 60°C and then aliquoted into petri dishes (100- × 20-mm depth), 20 mL each, for solidification. PEG-infused plates were made by dissolving PEG-8000 (Sigma) powder into one-half-strength Murashige and Skoog solution (pH 5.7) with the above-mentioned components, except phyto agar, and then filter sterilized; this PEG solution was then overlaid on agar-solidified medium at a ratio of 3:2 (v/v) and equilibrated overnight (≥12 h). The excess PEG solution was then removed. The procedure essentially followed the protocol of Verslues and Bray (2004).

Plant Physiol. 2007 February; 143(2): 745–758

Germination and Root Elongation Tests For germination tests, seeds harvested from the same batches were cold pretreated and then grown on modified Murashige and Skoog medium supplemented with Glc, Suc, NaCl, ABA, glufosinate ammonium, or fluridone at various concentrations listed in the “Results.” The medium was autoclaved and cooled to 50°C to 60°C prior to the addition of filter-sterilized ABA, glufosinate ammonium, or fluridone. For root elongation experiments, cold-pretreated seeds from different genotypes were first grown on agar plates with 1% Suc for 4 or 5 d, then uniform seedlings of similar size and primary root length were transferred to appropriate fresh medium and grown vertically for another 6 or 7 d.

Plant Physiol. 2007 February; 143(2): 745–758

Transgene Constructs and Transgenic Plant Isolation For overexpression, ABA2 (GIN1) full-length cDNA was amplified by RT-PCR and cloned into pGEM-T Easy vector (Promega), followed by subcloning into the binary vector (pSMAB704) driven by a constitutive 35S promoter (35SABA2). For tissue-specific expression and GUS assay, the ABA2 promoter, approximately 2.9 kb upstream of the ATG start codon, was amplified by PCR and fused to a GUS coding region to generate ABA2GUS in the pSMAB704 binary vector. Transgene constructs were confirmed by sequencing and subsequently transformed into wild type or the aba2 mutant (T0) by use of the floral-dip method (Clough and Bent, 1998). T1 seeds with cold pretreatment were screened on 1% Suc agar plates containing 25 mg/L herbicide, glufosinate ammonium. At least 10 homozygous lines resistant to herbicide were obtained at the T3 or T4 generation. Three homozygous lines were randomly chosen for further study.