Conversion of Endogenous Indole-3-Butyric Acid to Indole-3-Acetic Acid Drives Cell Expansion in Arabidopsis Seedlings

Genetic evidence in Arabidopsis (Arabidopsis thaliana) suggests that the auxin precursor indole-3-butyric acid (IBA) is converted into active indole-3-acetic acid (IAA) by peroxisomal β-oxidation; however, direct evidence that Arabidopsis converts IBA to IAA is lacking, and the role of IBA-derived IAA is not well understood. In this work, we directly demonstrated that Arabidopsis seedlings convert IBA to IAA. Moreover, we found that several IBA-resistant, IAA-sensitive mutants were deficient in IBA-to-IAA conversion, including the indole-3-butyric acid response1 (ibr1) ibr3 ibr10 triple mutant, which is defective in three enzymes likely to be directly involved in peroxisomal IBA β-oxidation. In addition to IBA-to-IAA conversion defects, the ibr1 ibr3 ibr10 triple mutant displayed shorter root hairs and smaller cotyledons than wild type; these cell expansion defects are suggestive of low IAA levels in certain tissues. Consistent with this possibility, we could rescue the ibr1 ibr3 ibr10 short-root-hair phenotype with exogenous auxin. A triple mutant defective in hydrolysis of IAA-amino acid conjugates, a second class of IAA precursor, displayed reduced hypocotyl elongation but normal cotyledon size and only slightly reduced root hair lengths. Our data suggest that IBA β-oxidation and IAA-amino acid conjugate hydrolysis provide auxin for partially distinct developmental processes and that IBA-derived IAA plays a major role in driving root hair and cotyledon cell expansion during seedling development.The auxin indole-3-acetic acid (IAA) controls both cell division and cell expansion and thereby orchestrates many developmental events and environmental responses. For example, auxin regulates lateral root initiation, root and stem elongation, and leaf expansion (for review, see Davies, 2004). Normal plant morphogenesis and environmental responses require modulation of auxin levels by controlling biosynthesis, regulating transport, and managing storage forms (for review, see Woodward and Bartel, 2005a). In some storage forms, the carboxyl group of IAA is conjugated to amino acids or peptides or to sugars, and free IAA can be released by hydrolases when needed (Bartel et al., 2001; Woodward and Bartel, 2005a). A second potential auxin storage form is the side chain-lengthened compound indole-3-butyric acid (IBA), which can be synthesized from IAA (Epstein and Ludwig-Müller, 1993) and is suggested to be shortened into IAA by peroxisomal β-oxidation (Bartel et al., 2001; Woodward and Bartel, 2005a).Genetic evidence suggests that the auxin activity of both IAA-amino acid conjugates and IBA requires free IAA to be released from these precursors (Bartel and Fink, 1995; Zolman et al., 2000). Mutation of Arabidopsis (Arabidopsis thaliana) genes encoding IAA-amino acid hydrolases, including ILR1, IAR3, and ILL2, reduces plant sensitivity to the applied IAA-amino acid conjugates that are substrates of these enzymes, including IAA-Leu, IAA-Phe, and IAA-Ala (Bartel and Fink, 1995; Davies et al., 1999; LeClere et al., 2002; Rampey et al., 2004), which are present in Arabidopsis (Tam et al., 2000; Kowalczyk and Sandberg, 2001; Kai et al., 2007).Unlike the simple one-step release of free IAA from amino acid conjugates, release of IAA from IBA is suggested to require a multistep process (Zolman et al., 2007, 2008). Conversion of IBA to IAA has been demonstrated in a variety of plants (Fawcett et al., 1960; for review, see Epstein and Ludwig-Müller, 1993) and may involve β-oxidation of the four-carbon carboxyl side chain of IBA to the two-carbon side chain of IAA (Fawcett et al., 1960; Zolman et al., 2000, 2007). Mutation of genes encoding the apparent β-oxidation enzymes INDOLE-3-BUTYRIC ACID RESPONSE1 (IBR1), IBR3, or IBR10 results in IBA resistance, but does not alter IAA response or confer a dependence on exogenous carbon sources for growth following germination (Zolman et al., 2000, 2007, 2008), consistent with the possibility that these enzymes function in IBA β-oxidation but not fatty acid β-oxidation.Both conjugate hydrolysis and IBA β-oxidation appear to be compartmentalized. The IAA-amino acid hydrolases are predicted to be endoplasmic reticulum localized (Bartel and Fink, 1995; Davies et al., 1999) and enzymes required for IBA responses, including IBR1, IBR3, and IBR10, are peroxisomal (Zolman et al., 2007, 2008). Moreover, many peroxisome biogenesis mutants, such as peroxin5 (pex5) and pex7, are resistant to exogenous IBA, but remain IAA sensitive (Zolman et al., 2000; Woodward and Bartel, 2005b).Although the contributions of auxin transport to environmental and developmental auxin responses are well documented (for review, see Petrášek and Friml, 2009), the roles of various IAA precursors in these processes are less well understood. Expansion of root epidermal cells to control root architecture is an auxin-regulated process in which these roles can be dissected. Root epidermal cells provide soil contact and differentiate into files of either nonhair cells (atrichoblasts) or hair cells (trichoblasts). Root hairs emerge from trichoblasts as tube-shaped outgrowths that increase the root surface area, thus aiding in water and nutrient uptake (for review, see Grierson and Schiefelbein, 2002). Root hair length is determined by the duration of root hair tip growth, which is highly sensitive to auxin levels (for review, see Grierson and Schiefelbein, 2002). Mutants defective in the ABCG36/PDR8/PEN3 ABC transporter display lengthened root hairs and hyperaccumulate [³H]IBA, but not [³H]IAA, in root tip auxin transport assays (Strader and Bartel, 2009), suggesting that ABCG36 functions as an IBA effluxer and that IBA promotes root hair elongation. The related ABCG37/PDR9 transporter also can efflux IBA (Strader et al., 2008b; Růžička et al., 2010) and may have some functional overlap with ABCG36 (Růžička et al., 2010). In addition to lengthened root hairs, abcg36/pdr8/pen3 mutants display enlarged cotyledons, a second high-auxin phenotype. Both of these developmental phenotypes are suppressed by the mildly peroxisome-defective mutant pex5-1 (Strader and Bartel, 2009), suggesting that IBA contributes to cell expansion by serving as a precursor to IAA, which directly drives the increased cell expansion that underlies these phenotypes. However, whether IBA-derived IAA contributes to cell expansion events during development of wild-type plants is not known.Here, we directly demonstrate that peroxisome-defective mutants are defective in the conversion of IBA to IAA, consistent with previous reports that these genes are necessary for full response to applied IBA. We found that a mutant defective in three suggested IBA-to-IAA conversion enzymes displays low-auxin phenotypes, including decreased root hair expansion and decreased cotyledon size. We further found that these mutants suppress the long-root-hair and enlarged cotyledon phenotypes of an abcg36/pdr8 mutant, suggesting that endogenous IBA-derived IAA drives root hair and cotyledon expansion in wild-type seedlings.     

Perturbation of Indole-3-Butyric Acid Homeostasis by the UDP-Glucosyltransferase UGT74E2 Modulates Arabidopsis Architecture and Water Stress Tolerance

Reactive oxygen species and redox signaling undergo synergistic and antagonistic interactions with phytohormones to regulate protective responses of plants against biotic and abiotic stresses. However, molecular insight into the nature of this crosstalk remains scarce. We demonstrate that the hydrogen peroxide–responsive UDP-glucosyltransferase UGT74E2 of Arabidopsis thaliana is involved in the modulation of plant architecture and water stress response through its activity toward the auxin indole-3-butyric acid (IBA). Biochemical characterization of recombinant UGT74E2 demonstrated that it strongly favors IBA as a substrate. Assessment of indole-3-acetic acid (IAA), IBA, and their conjugates in transgenic plants ectopically expressing UGT74E2 indicated that the catalytic specificity was maintained in planta. In these transgenic plants, not only were IBA-Glc concentrations increased, but also free IBA levels were elevated and the conjugated IAA pattern was modified. This perturbed IBA and IAA homeostasis was associated with architectural changes, including increased shoot branching and altered rosette shape, and resulted in significantly improved survival during drought and salt stress treatments. Hence, our results reveal that IBA and IBA-Glc are important regulators of morphological and physiological stress adaptation mechanisms and provide molecular evidence for the interplay between hydrogen peroxide and auxin homeostasis through the action of an IBA UGT.     

The Role of Arabidopsis ABCG9 and ABCG31 ATP Binding Cassette Transporters in Pollen Fitness and the Deposition of Steryl Glycosides on the Pollen Coat

The pollen coat protects pollen grains from harmful environmental stresses such as drought and cold. Many compounds in the pollen coat are synthesized in the tapetum. However, the pathway by which they are transferred to the pollen surface remains obscure. We found that two Arabidopsis thaliana ATP binding cassette transporters, ABCG9 and ABCG31, were highly expressed in the tapetum and are involved in pollen coat deposition. Upon exposure to dry air, many abcg9 abcg31 pollen grains shriveled up and collapsed, and this phenotype was restored by complementation with ABCG9_pro:GFP:ABCG9. GFP-tagged ABCG9 or ABCG31 localized to the plasma membrane. Electron microscopy revealed that the mutant pollen coat resembled the immature coat of the wild type, which contained many electron-lucent structures. Steryl glycosides were reduced to about half of wild-type levels in the abcg9 abcg31 pollen, but no differences in free sterols or steryl esters were observed. A mutant deficient in steryl glycoside biosynthesis, ugt80A2 ugt80B1, exhibited a similar phenotype. Together, these results indicate that steryl glycosides are critical for pollen fitness, by supporting pollen coat maturation, and that ABCG9 and ABCG31 contribute to the accumulation of this sterol on the surface of pollen.     

A DTX/MATE-Type Transporter Facilitates Abscisic Acid Efflux and Modulates ABA Sensitivity and Drought Tolerance in Arabidopsis

Abscisic acid （ABA） regulates numerous physiological and developmental processes in plants. Recent studies identify intracellular ABA receptors, implicating the transport of ABA across cell membranes as crucial for ABA sensing and response. Here, we report that a DTX/Multidrug and Toxic Compound Extrusion （MATE） family member in Arabidopsis thaliana, AtDTX50, functions as an ABA efflux transporter. When expressed heterologously in both an Escherichia coli strain and Xenopus oocyte cells, AtDTX50 was found to facilitate ABA efflux. Furthermore, dtx50 mutant mesophyll cells preloaded with ABA released less ABA compared with the wild-type （WT）. The AtDTX50 gene was expressed mainly in the vascular tissues and guard ceils and its expression was strongly up-regulated by exogenous ABA. The AtDTX50：：GFP fusion protein was localized predominantly to the plasma membrane. The dtx50 mutant plants were observed to be more sensitive to ABA in growth inhibition. In addition, compared with the WT, dtx50 mutant plants were more tolerant to drought with lower stomatal conductance, consistent with its function as an ABA efflux carrier in guard cells.     

A member of the PLEIOTROPIC DRUG RESISTANCE family of ATP binding cassette transporters is required for the formation of a functional cuticle in Arabidopsis

Although the multilayered structure of the plant cuticle was discovered many years ago, the molecular basis of its formation and the functional relevance of the layers are not understood. Here, we present the permeable cuticle1 (pec1) mutant of Arabidopsis thaliana, which displays features associated with a highly permeable cuticle in several organs. In pec1 flowers, typical cutin monomers, such as ω-hydroxylated fatty acids and 10,16-dihydroxypalmitate, are reduced to 40% of wild-type levels and are accompanied by the appearance of lipidic inclusions within the epidermal cell. The cuticular layer of the cell wall, rather than the cuticle proper, is structurally altered in pec1 petals. Therefore, a significant role for the formation of the diffusion barrier in petals can be attributed to this layer. Thus, pec1 defines a new class of mutants. The phenotypes of the pec1 mutant are caused by the knockout of ATP BINDING CASSETTEG32 (ABCG32), an ABC transporter from the PLEIOTROPIC DRUG RESISTANCE family that is localized at the plasma membrane of epidermal cells in a polar manner toward the surface of the organs. Our results suggest that ABCG32 is involved in the formation of the cuticular layer of the cell wall, most likely by exporting particular cutin precursors from the epidermal cell.     

The Allosteric Regulatory Mechanism of the Escherichia coli MetNI Methionine ATP Binding Cassette (ABC) Transporter

The MetNI methionine importer of Escherichia coli, an ATP binding cassette (ABC) transporter, uses the energy of ATP binding and hydrolysis to catalyze the high affinity uptake of d- and l-methionine. Early in vivo studies showed that the uptake of external methionine is repressed by the level of the internal methionine pool, a phenomenon termed transinhibition. Our understanding of the MetNI mechanism has thus far been limited to a series of crystal structures in an inward-facing conformation. To understand the molecular mechanism of transinhibition, we studied the kinetics of ATP hydrolysis using detergent-solubilized MetNI. We find that transinhibition is due to noncompetitive inhibition by l-methionine, much like a negative feedback loop. Thermodynamic analyses revealed two allosteric methionine binding sites per transporter. This quantitative analysis of transinhibition, the first to our knowledge for a structurally defined transporter, builds upon the previously proposed structurally based model for regulation. This mechanism of regulation at the transporter activity level could be applicable to not only ABC transporters but other types of membrane transporters as well.     

The Binding of Indole-3-acetic Acid and 3-Methyleneoxindole to Plant Macromolecules

Homogenates of pea (Pisum sativum L., var. Alaska) seedlings exposed to ¹⁴C-indole-3-acetic acid or ¹⁴C-3-methyleneoxindole, an oxidation product of indole-3-acetic acid, were extracted with phenol. In both cases 90% of the bound radioactivity was found associated with the protein fraction and 10% with the water-soluble, ethanol-insoluble fraction. The binding of radioactivity from ¹⁴C-indole-3-acetic acid is greatly reduced by the addition of unlabeled 3-methyleneoxindole as well as by chlorogenic acid, an inhibitor of the oxidation of indole-3-acetic acid to 3-methyleneoxindole. Chlorogenic acid does not inhibit the binding of ¹⁴C-3-methyleneoxindole. The labeled protein and water-soluble, ethanol-insoluble fractions of the phenol extract were treated with an excess of 2-mercaptoethanol. Independently of whether the seedlings had been exposed to ¹⁴C-indole-3-acetic acid or ¹⁴C-3-methyleneoxindole, the radioactivity was recovered from both fractions in the form of a 2-mercaptoethanol-3-methyleneoxindole adduct. These findings indicate that 3-methyleneoxindole is an intermediate in the binding of indole-3-acetic acid to macromolecules.     

The Timing of Growth Promotion and Conversion to Indole-3-acetic Acid for Auxin Precursors

Using a high resolution continuous recording technique, the length of the latent period preceding the growth response to two suspected precursors of indole-3-acetic acid (IAA) indole-3-acetonitrile (IAN), and indole-3-ethanol was studied in corn (Zea mays L.), wheat (Triticum aestivum L.), and peas (Pisum sativum L.). The timing of the conversion in vivo of these presumed precursors to IAA was also examined. In wheat the rate of conversion of IAN to IAA is rapid, and the latent period in the growth response to IAN is correspondingly short. In corn the rate of conversion in vivo of IAN to IAA is slow, and there is a long latent period in the growth response. In peas there is no conversion of IAN to IAA, and there is no measurable growth response to IAN. These data indicate that IAN is active in promoting growth only after conversion to IAA. This conclusion is strengthened by transport studies. Using a similar approach, we have also obtained data indicating that indole-3-ethanol is active only after conversion to IAA.     

Regulation of Auxin Homeostasis and Gradients in Arabidopsis Roots through the Formation of the Indole-3-Acetic Acid Catabolite 2-Oxindole-3-Acetic Acid

The native auxin, indole-3-acetic acid (IAA), is a major regulator of plant growth and development. Its nonuniform distribution between cells and tissues underlies the spatiotemporal coordination of many developmental events and responses to environmental stimuli. The regulation of auxin gradients and the formation of auxin maxima/minima most likely involve the regulation of both metabolic and transport processes. In this article, we have demonstrated that 2-oxindole-3-acetic acid (oxIAA) is a major primary IAA catabolite formed in Arabidopsis thaliana root tissues. OxIAA had little biological activity and was formed rapidly and irreversibly in response to increases in auxin levels. We further showed that there is cell type–specific regulation of oxIAA levels in the Arabidopsis root apex. We propose that oxIAA is an important element in the regulation of output from auxin gradients and, therefore, in the regulation of auxin homeostasis and response mechanisms.     

The Effect of Indole-3-Butyric Acid and Riboflavin on the Morphogenesis of Adventitious Roots of Eucalyptus ficifolia F. Muell. Grown In Vitro

Shoot explants from seedling-derived culture of Eucalyptus ficifoliaF. Muell. cultured on a rooting medium free from indole-3-butyricacid (IBA) develop a root system (Type I) consisting of a fewcomparatively long roots and only small amounts of callus. IBAat 5.0 µM in a rooting medium free from riboflavin inducesthe development, on the shoot explants, of a compact root system(Type II) consisting of callus and many short roots. Riboflavinwhen exposed to light, is able to photo-oxidize IBA; the degreeof photo-oxidation depends on the photon fluence of the lightreceived. The rooting response of the cultures reflects thedegree of photo-oxidation of IBA: concentrations of IBA fromabout 10^–4M to 10^–6M in the rooting medium induceformation of the Type II root system whilst photo-oxidationof the auxin to concentrations of about 10^–8M or lowerinduces the formation of the Type I root system. Thus, exogenousriboflavin and exogenous IBA are linked in a distinct light-induced,riboflavin-mediated change in root morphogenesis. The anatomyof root development in the Type I and Type II root systems wasstudied and factors affecting the development were defined.Characteristics of riboflavin and IBA breakdown in various lightregimes were determined and related to root morphogenesis. Theresults and their implications are discussed. Key words: Auxin photo-oxidation, Riboflavin, Root morphogenesis, Tissue culture     

Binding of [3H]Hemicholinium-3 to the High-Affinity Choline Transporter in Electric Organ Synaptosomal Membranes

Sodium-dependent binding of [3H]hemicholinium-3 was observed to be 10-fold higher with presynaptic membranes from the electric organ than with electroplaque membranes and this binding site copurified with synaptosomal membranes. The KD for specific [3H]hemicholinium-3 binding was found to be 31 +/- 4 nM and the Bmax, 5.0 +/- 0.2 pmol/mg protein; a Ki of 16 nM was estimated for hemicholinium-3 as a competitive inhibitor of high-affinity choline transport in electric organ synaptosomes. Choline and choline analogues were equally potent as inhibitors of [3H]choline uptake and [3H]hemicholinium-3 binding. Tubocurarine and oxotremorine also inhibited uptake and binding, but carbachol was without effect in both tests. These findings suggest that [3H]hemicholinium binds to the high-affinity choline transporter present at the cholinergic nerve terminal membrane. A comparison of maximal velocities for choline transport and the maximal number of hemicholinium-3 binding sites indicated that the high-affinity choline transporter has an apparent turnover number of about 3s-1 at 20 degrees C under resting conditions. The high transport rates observed in electric organ synaptosomes are likely due to the high density of high-affinity choline transporters in this tissue, estimated on the basis of [3H]hemicholinium-3 binding to be of the order of 100/micron2 of synaptosomal membrane.     

H3K36me2/3 Binding and DNA Binding of the DNA Methyltransferase DNMT3A PWWP Domain Both Contribute to its Chromatin Interaction

The PWWP domain of DNMT3 DNA methyltransferases binds to histone H3 tails containing methylated K36, and this activity is important for heterochromatic targeting. Here, we show that the PWWP domain of mouse DNMT3A binds to H3K36me2 and H3K36me3 with a slight preference for H3K36me2. PWWP domains have also been reported to bind to DNA, and the close proximity of H3K36 and nucleosomal DNA suggests a combined binding to H3K36me2/3 and DNA. We show here that the DNMT3A PWWP domain binds to DNA with a weak preference for AT-rich sequences and that the designed charge reversal R362E mutation disrupts DNA binding. The K295E mutation, as well as K295I recently identified in paraganglioma, a rare neuroendocrine neoplasm, disrupts both DNA and H3K36me2/3 binding, which is in agreement with the proximity of K295 to residues involved in K36me2/3 methyllysine binding. Nucleosome pulldown experiments show that DNA binding and H3K36me2/3 binding are important for the interaction of the DNMT3A PWWP domain with nucleosomes. Localization studies of transiently transfected fluorescently-tagged wild-type and PWWP-mutated full-length DNMT3A indicate that both interactions contribute to the subnuclear localization of DNMT3A in mouse cells. In summary, our data demonstrate that the combined binding of the DNMT3A PWWP domain to the H3 tail containing K36me2/3 and to the nucleosomal or linker DNA is important for its chromatin interaction and subnuclear targeting of DNMT3A in living cells.     

ATP Binding to the C Terminus of the Arabidopsis thaliana Nitrate/Proton Antiporter, AtCLCa, Regulates Nitrate Transport into Plant Vacuoles

Nitrate, one of the major nitrogen sources for plants, is stored in the vacuole. Nitrate accumulation within the vacuole is primarily mediated by the NO₃⁻/H⁺ exchanger AtCLCa, which belongs to the chloride channel (CLC) family. Crystallography analysis of hCLC5 suggested that the C-terminal domain, composed by two cystathionine β-synthetase motifs in all eukaryotic members of the CLC family is able to interact with ATP. However, interaction of nucleotides with a functional CLC protein has not been unambiguously demonstrated. Here we show that ATP reversibly inhibits AtCLCa by interacting with the C-terminal domain. Applying the patch clamp technique to isolated Arabidopsis thaliana vacuoles, we demonstrate that ATP reduces AtCLCa activity with a maximum inhibition of 60%. ATP inhibition of nitrate influx into the vacuole at cytosolic physiological nitrate concentrations suggests that ATP modulation is physiologically relevant. ADP and AMP do not decrease the AtCLCa transport activity; nonetheless, AMP (but not ADP) competes with ATP, preventing inhibition. A molecular model of the C terminus of AtCLCa was built by homology to hCLC5 C terminus. The model predicted the effects of mutations of the ATP binding site on the interaction energy between ATP and AtCLCa that were further confirmed by functional expression of site-directed mutated AtCLCa.Nitrate is among the major nitrogen sources for plants in aerobic soils. It is taken up by root cells through plasma membrane transporters of nitrate-nitrite transporter and peptide transporter families. Once in the cytoplasm it can enter the amino acid biosynthesis pathway (1) or be accumulated in the vacuolar lumen via tonoplast transporters (2).The vacuolar nitrate transporter of the model plant Arabidopsis thaliana, AtCLCa, has been shown to work as an anion/proton antiporter (3, 4), similarly to the bacterial CLCec-1 (5) and human hCLC-4 (6) as well as hCLC-5 (7). However, whereas bacterial and animal CLCs² transport chloride ions, the AtCLCa antiporter is more selective for nitrate, and therefore, it is able to mediate the accumulation of nitrate into the plant vacuole.Little is known on the modulation of CLC-proteins by nucleotides. The effects of ATP on the ion channel hCLC-1 are a matter of debate (8). Indeed, some reports have shown that ATP inhibits hCLC-1 currents, probably interacting with the C terminus of the protein (9–11). Conversely, other reports indicate that ATP does not modify the properties of hCLC-1 current (12). This discrepancy has been attributed to the oxidation state of the channel, as ATP would be effective only in the presence of reducing agents (13).The C terminus domain of all eukaryotic CLC proteins has two cystathionine β-synthetase motifs (CBS (14, 15)), each one characterized by a βαββα topology (16, 17). A structural and biochemical study of the hCLC-5 C-terminal part demonstrates that this region binds nucleotides (14). However, the effect of ATP binding on the transport activity of hCLC-5 is still unknown.The presence of analogous CBS domains in the C terminus of the AtCLCa antiporter suggested the hypothesis that ATP binds to this plant transporter and modulates its transport activity. Hence, we undertook a functional analysis of the effect of adenosine nucleotides on AtCLCa and found that ATP inhibits the AtCLCa-mediated transport. Based on a homology model of the C terminus of the channel, we identified two residues that would be putatively involved in the protein-nucleotide interaction.     

Arabidopsis PEN3/PDR8, an ATP binding cassette transporter, contributes to nonhost resistance to inappropriate pathogens that enter by direct penetration

Arabidopsis thaliana is a host to the powdery mildew Erysiphe cichoracearum and nonhost to Blumeria graminis f. sp hordei, the powdery mildew pathogenic on barley (Hordeum vulgare). Screening for Arabidopsis mutants deficient in resistance to barley powdery mildew identified PENETRATION3 (PEN3). pen3 plants permitted both increased invasion into epidermal cells and initiation of hyphae by B. g. hordei, suggesting that PEN3 contributes to defenses at the cell wall and intracellularly. pen3 mutants were compromised in resistance to the necrotroph Plectosphaerella cucumerina and to two additional inappropriate biotrophs, pea powdery mildew (Erysiphe pisi) and potato late blight (Phytophthora infestans). Unexpectedly, pen3 mutants were resistant to E. cichoracearum. This resistance was salicylic acid-dependent and correlated with chlorotic patches. Consistent with this observation, salicylic acid pathway genes were hyperinduced in pen3 relative to the wild type. The phenotypes conferred by pen3 result from the loss of function of PLEIOTROPIC DRUG RESISTANCE8 (PDR8), a highly expressed putative ATP binding cassette transporter. PEN3/PDR8 tagged with green fluorescent protein localized to the plasma membrane in uninfected cells. In infected leaves, the protein concentrated at infection sites. PEN3/PDR8 may be involved in exporting toxic materials to attempted invasion sites, and intracellular accumulation of these toxins in pen3 may secondarily activate the salicylic acid pathway.     

The Relationship between Levels of Endogenous Gibberellins and the Response of Vigna Hypocotyls to Exogenous Indole-3-Acetic Acid

Segments excised from the upper and the lower parts of cowpea(Vigna unguiculata L.) hypocotyls were compared in terms oftheir responses to exogenous indole-3-acetic acid (IAA) in relationto their endogenous levels of gibberellin. Growth of the segmentswas measured continuously during xylem perfusion with a lineardifferential transformer. IAA induced a burst of elongationin the upper segments but only slight promotion of growth inthe lower segments. Treatment with uniconazole, a potent inhibitorof the biosynthesis of gibberellins, reduced the responsivenessof the upper segments to exogenous IAA to about one half ofthe control value. Pre-perfusion with GA₃ of such segments fortwo hours prior to application of IAA, partially restored theresponsiveness to IAA. Analysis by GC/MS identified GA₁, GA₄,GA₉ GA₂₀ and GA₅₁ as native gibberellins in the hypocotyls ofcowpea seedlings. Analysis by GC/SIM also showed that the physiologicallyactive gibberellins (GA₁ and GA₄) were located mainly in theupper part of the hypocotyl and the treatment with uniconazolemarketly reduced the endogenous level of gibberellins thereto less than 11% of the control level. These results suggestthat levels of endogenous gibberellins possibly control theresponse to IAA in these segments. (Received May 12, 1994; Accepted November 15, 1994)     

The eta7/csn3-3 Auxin Response Mutant of Arabidopsis Defines a Novel Function for the CSN3 Subunit of the COP9 Signalosome

The COP9 signalosome (CSN) is an eight subunit protein complex conserved in all higher eukaryotes. In Arabidopsis thaliana, the CSN regulates auxin response by removing the ubiquitin-like protein NEDD8/RUB1 from the CUL1 subunit of the SCF^TIR1/AFB ubiquitin-ligase (deneddylation). Previously described null mutations in any CSN subunit result in the pleiotropic cop/det/fus phenotype and cause seedling lethality, hampering the study of CSN functions in plant development. In a genetic screen to identify enhancers of the auxin response defects conferred by the tir1-1 mutation, we identified a viable csn mutant of subunit 3 (CSN3), designated eta7/csn3-3. In addition to enhancing tir1-1 mutant phenotypes, the csn3-3 mutation alone confers several phenotypes indicative of impaired auxin signaling including auxin resistant root growth and diminished auxin responsive gene expression. Unexpectedly however, csn3-3 plants are not defective in either the CSN-mediated deneddylation of CUL1 or in SCF^TIR1-mediated degradation of Aux/IAA proteins. These findings suggest that csn3-3 is an atypical csn mutant that defines a novel CSN or CSN3-specific function. Consistent with this possibility, we observe dramatic differences in double mutant interactions between csn3-3 and other auxin signaling mutants compared to another weak csn mutant, csn1-10. Lastly, unlike other csn mutants, assembly of the CSN holocomplex is unaffected in csn3-3 plants. However, we detected a small CSN3-containing protein complex that is altered in csn3-3 plants. We hypothesize that in addition to its role in the CSN as a cullin deneddylase, CSN3 functions in a distinct protein complex that is required for proper auxin signaling.     

在吲哚乙酸不同位点偶联载体蛋白对其抗体特异性的影响

分别选择吲哚乙醇分子上的Ｃ１位羧基和吲哚环上的Ｎ位作为偶联载体蛋白的位点,用混合酸酐法和甲醛搭桥法分别合成了两种免疫原ＩＡＡ—ＣＯ—ＮＨ一ＨＳＡ和ＩＡＡ－Ｎ－ＢＳＡ,并进而制得了对吲哚乙酸侧链识别能力不同的两种多克隆抗体,分别可特异识别甲酯化ＩＡＡ和游离态ＩＡＡ;用碳化二亚胺法和甲醛搭桥法分别合成ＩＡＡ—ＣＯ—ＮＨ－ＢＳＡｂＩＡＡ—Ｎ—ＯＶＡ两种复合物,以之为包被物,建立了两种ＩＡＡＥＬＩＳＡ。其灵敏度分别为０．３５ｐｍｏｌ和１．８０ｐｐｍｏｌ;检测范围分别为０．７８～８００ｐｍｏｌ和１．９５～２０００ｐｍｏｌ;批内变异系数分别为４．４５％和４．７９％;批间变异系数分别为１．１５％和１．５０％。笔者用这两种ＥＬＩＳＡ检测了兰花气生根和桑树苗样品中ＩＡＡ的含量,发现两种检测结果相当一致。