Conformational dynamics underlie the activity of the auxin-binding protein, Nt-abp1.

The auxin-binding protein 1 (ABP1) has been proposed to be involved in the perception of the phytohormone at the plasma membrane. Site-directed mutagenesis was performed on highly conserved residues at the C terminus of ABP1 to investigate their relative importance in protein folding and activation of a functional response at the plasma membrane. Detailed analysis of the dynamic interaction of the wild-type ABP1 and mutated proteins with three distinct monoclonal antibodies recognizing conformation-dependent epitopes was performed by surface plasmon resonance. The influence of auxin on these interactions was also investigated. The Cys(177) as well as Asp(175) and Glu(176) were identified as critical residues for ABP1 folding and action at the plasma membrane. On the contrary, the C-terminal KDEL sequence was demonstrated not to be essential for auxin binding, interaction with the plasma membrane, or activation of the transduction cascade although it does appear to be involved in the stability of ABP1. Taken together, the results confirmed that ABP1 conformational change is the critical step for initiating the signal from the plasma membrane.

Characterization of a tobacco Bright Yellow 2 cell line expressing the tetracycline repressor at a high level for strict regulation of transgene expression.

Manipulating the expression of a transgene in transient and stable transformed cells is a requirement for many functional analyses. We have investigated the use of the tetracycline-dependent gene expression system developed by Gatz et al. (1992) in tobacco (Nicotiana tabacum L. cv Bright Yellow 2 [BY2]) cells, the most widely used plant cell culture. We have selected a BY2 cell line, named BY2-tetracycline repressor (tetR) 17, which expresses the tetR at a high level, and have evaluated the capacity of this cell line to suppress the expression of a green fluorescent protein reporter gene under the control of the "Triple-Op" promoter in the absence of tetracycline in a large number of independent transformants. The ability to induce the expression of green fluorescent protein after treatment by anhydrotetracycline in the same transformants was also analyzed. BY2-tetR17 cells were demonstrated to be excellent recipient cells for recovery of clonal cell lines with a highly controlled regulation of the introduced transgene.

Novel auxin transport inhibitors phenocopy the auxin influx carrier mutation aux1.

The hormone auxin is transported in plants through the combined actions of diffusion and specific auxin influx and efflux carriers. In contrast to auxin efflux, for which there are well documented inhibitors, understanding the developmental roles of carrier-mediated auxin influx has been hampered by the absence of specific competitive inhibitors. However, several molecules that inhibit auxin influx in cultured cells have been described recently. The physiological effects of two of these novel influx carrier inhibitors, 1-naphthoxyacetic acid (1-NOA) and 3-chloro-4-hydroxyphenylacetic acid (CHPAA), have been investigated in intact seedlings and tissue segments using classical and new auxin transport bioassays. Both molecules do disrupt root gravitropism, which is a developmental process requiring rapid auxin redistribution. Furthermore, the auxin-insensitive and agravitropic root-growth characteristics of aux1 plants were phenocopied by 1-NOA and CHPAA. Similarly, the agravitropic phenotype of inhibitor-treated seedlings was rescued by the auxin 1-naphthaleneacetic acid, but not by 2,4-dichlorophenoxyacetic acid, again resembling the relative abilities of these two auxins to rescue the phenotype of aux1. Further investigations have shown that none of these compounds block polar auxin transport, and that CHPAA exhibits some auxin-like activity at high concentrations. Whilst results indicate that 1-NOA and CHPAA represent useful tools for physiological studies addressing the role of auxin influx in planta, 1-NOA is likely to prove the more useful of the two compounds.

A novel immunological approach establishes that the auxin-binding protein, Nt-abp1, is an element involved in auxin signaling at the plasma membrane.

Interactions of a collection of monoclonal antibodies (mAbs) to the recombinant Nicotiana tabacum auxin-binding protein 1 (Nt-abp1) were extensively characterized using surface plasmon resonance. Dynamic interaction studies using combinations of Nt-abp1, synthetic peptides corresponding to conserved sequences within auxin-binding proteins, and the mAbs have shown that a number of the mAbs recognized discontinuous epitopes revealing the junction of distinct domains in the folded protein. In particular, the two putative auxin binding domains and the C terminus of the protein were shown to interact with each other in the folded protein. Using the auxin-induced electrical response of tobacco protoplasts as a functional assay, all the mAbs exhibited either auxin antagonist or hormonomimetic properties. These effects, measured for the first time in homologous conditions, confirm that Nt-abp1 is present at the plasma membrane and is involved in the activation of the auxin-dependent electrical response of tobacco protoplasts. Based on our surface plasmon resonance data, we propose that the key event leading to the activation of this auxin electrical response consists of a conformational change in Nt-abp1.

The auxin-binding protein Nt-ERabp1 alone activates an auxin-like transduction pathway.

Hyperpolarization of tobacco protoplasts is amongst the earliest auxin responses described. It has been proposed that the auxin-binding protein, ABP1, or a related protein could be involved in the first step of auxin perception at the plasma membrane. Using for the first time homologous conditions for interaction between the protein Nt-ERabp1 or a synthetic peptide corresponding to the C-terminus and tobacco protoplasts, we have demonstrated that both can induce the hyperpolarization response. The results show that Nt-ERabp1 or the C-terminal peptide alone activates the auxin pathway from the outer face of the plasma membrane.

AUX1 regulates root gravitropism in Arabidopsis by facilitating auxin uptake within root apical tissues.

Plants employ a specialized transport system composed of separate influx and efflux carriers to mobilize the plant hormone auxin between its site(s) of synthesis and action. Mutations within the permease-like AUX1 protein significantly reduce the rate of carrier-mediated auxin uptake within Arabidopsis roots, conferring an agravitropic phenotype. We are able to bypass the defect within auxin uptake and restore the gravitropic root phenotype of aux1 by growing mutant seedlings in the presence of the membrane-permeable synthetic auxin, 1-naphthaleneacetic acid. We illustrate that AUX1 expression overlaps that previously described for the auxin efflux carrier, AtPIN2, using transgenic lines expressing an AUX1 promoter::uidA (GUS) gene. Finally, we demonstrate that AUX1 regulates gravitropic curvature by acting in unison with the auxin efflux carrier to co-ordinate the localized redistribution of auxin within the Arabidopsis root apex. Our results provide the first example of a developmental role for the auxin influx carrier within higher plants and supply new insight into the molecular basis of gravitropic signalling.

Identification of new early auxin markers in tobacco by mRNA differential display.

Differential display of mRNA has been improved by developing a two-step PCR amplification procedure. The modified differential display has been applied to identify early alterations of mRNA expression in response to auxin treatment of tobacco seedlings. This approach has led to the isolation of four fragments corresponding to auxin-up-regulated mRNAs. One, named GO15-13, shows significant homology with the 3' end of the coding region of the soybean SAUR X10A. The three other fragments present no homology with sequences available in the databases and constitute potential new early auxin markers.

Molecular cloning and expression of the early auxin-responsive Aux/IAA gene family in Nicotiana tabacum.

Early auxin-regulated tobacco cDNAs, belonging to the Aux/IAA gene family have been isolated by screening of a cDNA library prepared from auxin-treated suspension-grown etiolated seedlings of Nicotiana tabacum. The probes used were either RT-PCR fragments or an insert resulting from mRNA differential display selection. All of them possessed the structural features which characterize the Aux/IAA gene products. The auxin response of three distinct Nt-iaa subclasses has been characterized in terms of kinetics, dose-response and specificity as several plant hormones and chemicals have been tested for their ability to alter Nt-iaa mRNA accumulation. Differences of auxin responses have been observed between the Nt-iaa analysed, revealing significant differences of regulation. The effect of the protein synthesis inhibitor cycloheximide suggested that Nt-iaa2.3, Nt-iaa4.3 and strictly related genes can be classified as primary auxin-responsive genes and Nt-iaa28 as a late one. The steady-state mRNA level of these Nt-iaa has also been determined in organs of tobacco plants.

Characterization of two cDNAs encoding auxin-binding proteins in Nicotiana tabacum.

The isolation and the characterization of two tobacco cDNAs, Nt-ERabp1 and Nt-ERabp2, homologous to Zm-ERabp1, encoding the major auxin-binding protein from maize coleoptiles, are described. Their predicted amino acid sequences correspond to proteins of ca. 21 kDa, in which the characteristic regions common to ABP1-related polypeptides are well-conserved. Southern analysis indicates that the genes corresponding to Nt-ERabp1 cDNA and Nt-ERabp2 cDNA derive respectively from Nicotiana tomentosiformis and Nicotiana sylvestris, the diploid progenitors of Nicotiana tabacum. Analysis of mRNA distribution in tobacco plants indicates that these two genes are preferentially expressed in flowers and growing seedlings. Whatever the tissue tested, Nt-ERabp1 mRNA is more abundant than Nt-ERabp2 mRNA. Furthermore, RT-PCR reveals developmental and organ-specific expression of these two genes in flower parts of tobacco plants. In particular, regulation of Nt-ERabp1 mRNA accumulation appears to be correlated with elongation growth of each floral organ. Recombinant Nt-ERabp1, produced in Escherichia coli, is recognized by antibodies raised against Zm-ERabp1.

Isolation by differential display and characterization of a tobacco auxin-responsive cDNA Nt-gh3, related to GH3.

By use of differential display, we have isolated a new early auxin-responsive cDNA in Nicotiana tabacum. Nt-gh3 had 70% identity with the unique GH3 sequence isolated in soybean by Hagen et al. [Planta 162 (1984) 147-153] and is thus the first reported cDNA related to this gene until now. Nt-gh3 mRNA accumulates within a short time after auxin treatment, responds to very low concentrations of NAA (as little as 10[-9] M) and specifically to active auxins. Nt-gh3 mRNA is demonstrated to be one of the most relevant early molecular markers of primary auxin response.

Alterations of auxin perception in rolB-transformed tobacco protoplasts. Time course of rolB mRNA expression and increase in auxin sensitivity reveal multiple control by auxin.

Expression and physiological effects of the root-inducing rolB gene of Agrobacterium rhizogenes T-DNA were studied simultaneously in tobacco (Nicotiana tabacum) mesophyll protoplasts. The kinetic study of the expression of rolB mRNA following exogenous auxin application showed that auxin transiently stimulated rolB expression, with mRNA levels starting to accumulate 6 to 9 h after auxin was supplied and increasing 300-fold after 12 to 18 h. The parallel study of the auxin sensitivity of rolB-transformed protoplasts, as assayed by their electrical response to the hormone, showed that the auxin treatment generated an increase in sensitivity by a factor of up to 100,000, whereas in untransformed protoplasts the same auxin treatment induced an increase in auxin sensitivity that never exceeded 30- to 50-fold. This reflects a strong cooperative effect of auxin and rolB in transformed protoplasts. Surprisingly, the maximal increase in sensitivity was observed several hours before the maximal accumulation of rolB mRNA, suggesting that the dramatic control of auxin sensitivity by auxin in rolB-transformed protoplasts requires only low levels of rolB expression. Antibodies directed against ZmER-abp1, the major auxin-binding protein from maize, differentially altered the auxin sensitivity of the electrical response of rolB-transformed and normal protoplasts. This suggests that alterations of the auxin reception-transduction pathway at the plasma membrane of rolB-transformed protoplasts may account for their increased auxin sensitivity.

The rolB Gene of Agrobacterium rhizogenes Does Not Increase the Auxin Sensitivity of Tobacco Protoplasts by Modifying the Intracellular Auxin Concentration.

Phenotypical alterations observed in rolB-transformed plants have been proposed to result from a rise in intracellular free auxin due to a RolB-catalyzed hydrolysis of auxin conjugates(J.J. Estruch, J. Schell, A. Spena [1991] EMBO J 10: 3125-3128).We have investigated this hypothesis in detail using tobacco (Nicotiana tabacum) mesophyll protoplasts isolated from plants transformed with the rolB gene under the control of its own promoter (BBGUS 6 clone) or the cauliflower mosaic virus 35S promoter (CaMVBT 3 clone). Protoplasts expressing rolB showed an increased sensitivity to the auxin-induced hyperpolarization of the plasma membrane when triggered with exogenous auxin. Because this phenotypical trait was homogeneously displayed over the entire population, protoplasts were judged to be a more reliable test system than the tissue fragments used in previous studies to monitor rolB gene effects on cellular auxin levels. Accumulation of free 1-[3H]-naphthaleneacetic acid (NAA) was equivalent in CaMVBT 3, BBGUS 6, and wild-type protoplasts, Naphthyl-[beta]-glucose ester, the major NAA metabolite in protoplasts, reached similar levels in CaMVBT 3 protoplasts, reached similar levels in CaMVBT 3 and normal protoplasts and was hydrolyzed at the same rate in BBGUS 6 and normal protoplasts. Furthermore, NAA accumulation and metabolism in BBGUS 6 protoplasts were independent of the rolB gene expression level. Essentially similar results were obtained with indoleacetic acid. Thus, it was concluded that the rolB-dependent behavior of transgenic tobacco protoplasts is not a consequence of modifying the intracellular auxin concentration but likely results from changes in the auxin perception pathway.

Ethylene-Induced Increase in Glutamine Synthetase Activity and mRNA Levels in Hevea brasiliensis Latex Cells.

Ethylene, used as a stimulant of latex production in Hevea brasiliensis, significantly activates the regenerating metabolism within the laticiferous cells. In this context, attention was focused on glutamine synthetase (GS; EC 6.3.1.2), a key enzyme in nitrogen metabolism. A specific and significant activation of the cytosolic glutamine synthetase (GScyt) in the laticiferous cells after ethylene treatment parallels the increase of latex yield. A marked accumulation of the corresponding mRNA was found, but in contrast, a slight and variable increase of the polypeptide level is at the limit of detection by western blotting. The GS response to ethylene might be mediated by ammonia that increases in latex cytosol following ethylene treatment. The physiological significance for such a regulation by ethylene of the GScyt is discussed in terms of the nitrogen requirement for protein synthesis associated with latex regeneration.

Antibodies to a peptide from the maize auxin-binding protein have auxin agonist activity.

The major auxin-binding protein in maize membranes is thought to function as a physiological receptor. From earlier information, including the use of site-directed irreversible inhibitors, several of the amino acids likely to form part of the active auxin-binding site were provisionally assigned. Inspection of the amino acid sequence of the auxin-binding protein showed a short region containing all but one of these amino acids. We find that antisera raised against a synthetic peptide encompassing this region recognize all isoforms of the maize auxin-binding protein together with homologous polypeptides in other species. We further find that the antibodies hyperpolarize protoplast transmembrane potential in an auxin-like manner. We conclude that these antibodies display auxin agonist activity and that we have identified an essential portion of the auxin-binding site.

Detection of phosphoenolpyruvate and ribulose 1,5-bisphosphate carboxylase transcripts in maize leaves by in situ hybridization with sulfonated cDNA probes.

This report outlines an efficient in situ hybridization method for locating specific mRNAs in tissue cryosections using sulfonated cDNA probes. The method involves chemical modification of DNA probes by insertion of a sulfone radical on cytosine residues, which generates a specific epitope. Sulfonated DNA is then detected by using indirect immunochemical procedure. Alternatively, antibodies conjugated to fluorescein or to alkaline phosphatase were used for mRNA detection. In situ hybridization was developed to study aspects of mesophyll and bundle sheath cell differentiation in maize leaves. Our results indicate that phosphoenolpyruvate carboxylase (PEP C) mRNA is restricted to mesophyll cells, and the nucleus-encoded mRNA of the small subunit (SSU) ribulose 1,5-bisphosphate carboxylase (RuBP C) is limited to the cytosol of bundle sheath cells. Thus, using in situ hybridization, we have demonstrated that the differential distribution of PEP C and RuBP C proteins in the two cell types also reflects the location of their mRNAs. These data imply either a tissue-specific transcriptional regulation or a selective mRNA degradation.

Phosphoenolpyruvate carboxylase in soybean root nodules: An immunochemical study.

The activity of phosphoenolpyruvate carboxylase (E.C. 4.1.1.31) strongly increased during the maturation of soybean (Glycine max L. Weber) root-nodules. By using a specific immune serum it was shown that this increase was the consequence of an elevated population of enzyme molecules whose appearance preceded the emergence of nitrogen fixing capacity. Whether or not the phenomenon could be ascribed to the formation of a specific isoenzyme is not known. The location of the enzyme was also investigated. Immunocyto-fluorescence experiments established that phosphoenolpyruvate carboxylase was present in the cytoplasmic compartment of both infected and uninfected cells of nodules.

Immunochemical studies on xanthine dehydrogenase of soybean root nodules : Ontogenic changes in the level of enzyme and immunocytochemical localization.

Xanthine dehydrogenase (XDH, EC 1.2.1.37) was purified from root nodules of soybean (Glycine max) and used to prepare a polyclonal rabbit antiserum. Monospecificity of this antiserum was ascertained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipate. During root nodule development of soybean, only one form of XDH was detected on an immunological basis. Titration of XDH by immunoelectrophoresis showed that a remarkable increase in the amount of XDH occurred between two and four weeks after inoculation, in parallel with the increase in enzyme activity. Localization of XDH by immunofluorescence indicated that the enzyme was present exclusively in uninfected cells where it appeared to be associated with discrete organellels.

In-situ immunofluorescent localization of phosphoenolpyruvate and ribulose 1,5-bisphosphate carboxylases in leaves of C3, C 4, and C 3-C 4 intermediatePanicum species.

The in-situ inter- and intracellular localization patterns of phosphoenolpyruvate (PEP) and ribulose 1,5-bisphosphate (RuBP) carboxylases in green leaves of severalPanicum species were investigated using an indirect immunofluorescence technique. Four species were examined and compared:P. miliaceum (C4),P. bisulcatum (C3), andP. decipiens andP. milioides (C3-C4 intermediates which have Kranz-like leaf anatomy and reduced photorespiration). In the C4 Panicum, PEP carboxylase was located in the cytosol of the mesophyll cells and RuBP carboxylase was restricted to the bundle-sheath chloroplasts. In contrast, in the C3 Panicum species, PEP carboxylase was found throughout the leaf chlorenchyma, in both the cytosol and chloroplasts, and RuBP carboxylase was located in the chloroplasts. For the C3-C4 intermediate plants, the patterns depended on the species examined. ForP. decipiens, the in-situ localization of both carboxylases was similar to that described forP. bisulcatum and other C3 plants. However, inP. milioides, PEP carboxylase was found exclusively in the cytosol of the mesophyll cells, as inP. miliaceum and other C4 species, whereas RuBP carboxylase was distributed in both the mesophyll and bundle-sheath chloroplasts.

Glutamine Synthetase in Spinach Leaves : IMMUNOLOGICAL STUDIES AND IMMUNOCYTOCHEMICAL LOCALIZATION.

By polyacrylamide gel electrophoresis, DEAE Sephacel, and hydroxyapatite chromatography, one form of glutamine synthetase has been identified in spinach (Spinacia oleracea L. cv. Monstrueux de Viroflay) leaves. It is localized only inside the chloroplast. The enzyme was purified to homogeneity and specific antibodies against the protein were raised by immunization of rabbits. The intracellular localization of glutamine synthetase in spinach leaves was studied by indirect immunofluorescence microscopy on thin-sectioned spinach leaves. It has been demonstrated that the enzyme is specifically associated with the chloroplasts of parenchymatous cells.

A comparative immunocytochemical localization study of phosphoenolpyruvate carboxylase in leaves of higher plants.

The intracellular localization of phosphoenolpyruvate (PEP) carboxylase in plants belonging to the C4, Crassulacean acid metabolism (CAM) and C3 types was invetigated using an immunocytochemical method with an immune serum raised against the sorghum leaf enzyme. The plants studied were sorghum, maize (C4 type), kalanchoe (CAM type), french bean, and spinach (C3 type). In the green leaves of C4 plants, it was shown that the carboxylase was located in the mesophyll and stomatic cells, being largely cytosolic in the mesophyll cells. Similarly, in CAM plants, the enzyme was found mainly outside the chloroplasts. In contrast, in C3 plants, the PEP carboxylase appeared to be distributed between the cytosol and the chloroplasts of foliar parenchyma. Examination of sections from etiolated leaves showed fluorescence emission from etioplasts and cytosol for the parenchyma of french bean as well as for the bundle sheath and mesophyll of sorghum leaves. This data indicated that during the greening process photoregulation and evolution of PEP carboxylase is dependent on the tissue and on the metabolic type of the plant considered.