Early genes and auxin action.
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Base Sequence↗
Biomedical subjects
A Theologis
Publications and source records attributed to A Theologis.
Characterization of two members (ACS1 and ACS3) of the 1-aminocyclopropane-1-carboxylate synthase gene family of Arabidopsis thaliana.
The nucleotide sequences of two highly homologous 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS; EC 4.4.1.14)-encoding genes, ACS1 and ACS3, of Arabidopsis thaliana (At) have been determined. The sequence analysis shows that ACS3 is a pseudogene representing a truncated version of ACS1. The missing region of ACS3 corresponding to the fourth exon of ACS1 has been shown by Southern analysis to be absent in the At genome. The chromosomal locations of the five members of the At ACS multigene family have been determined. The results show that each family member resides on a different chromosome. This observation suggests that the ACS3 pseudogene originated by a partial inter-chromosomal gene duplication. The ACS1 polypeptide contains all the conserved and characteristic domains found in the ACC synthase isoenzymes from various plant species, but is unable to express ACS activity in Escherichia coli and yeast. The predicted amino-acid sequence of ACS1 is missing the highly conserved tripeptide, Thr-Asn-Pro (TNP), between Ile204 and Ser205. Introduction of TNP into ACS1 restores the ACS activity, whereas its removal from the enzymatically active ACS2 results in a loss of activity. The results suggest that TNP is crucial for expression of ACS activity in E. coli.
Amino Acid Sequence↗
Ethylene sensors: how perceptive!
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Ethylenes↗
Transcriptional regulation of PS-IAA4/5 and PS-IAA6 early gene expression by indoleacetic acid and protein synthesis inhibitors in pea (Pisum sativum).
The transcription of two genes, PS-IAA4/5 and PS-IAA6, in pea is induced by indoleacetic acid (IAA) and protein synthesis inhibitors such as cycloheximide (CHX) and anisomycin (ANI). Induction by IAA is rapid, taking 5 and 7.5 minutes for PS-IAA4/5 and PS-IAA6, respectively, and is independent of IAA concentration and whether IAA has a free or esterified carboxyl group (ethyl-IAA). The rate of mRNA accumulation, however, is dependent on hormone concentration, and is greater with IAA than with ethyl-IAA. The turnover rates (t1/2) of the PS-IAA4/5 and PS-IAA6 mRNAs are 60 and 75 minutes, respectively, and are not affected by IAA. CHX or ANI induce the transcription of PS-IAA4/5 and PS-IAA6 more slowly than IAA (5 to 10 minutes for PS-IAA4/5 and 20 minutes for PS-IAA6). While protein synthesis inhibitors stabilize both mRNAs, the rapidity of induction by CHX and ANI cannot be accounted for solely by mRNA stabilization. The relationship between mRNA induction and protein synthesis inhibition does not obey Michaelis-Menten kinetics, but rather is best described by a hyperbolic curve, suggesting the release of transcriptional repression by the inhibition of protein synthesis. RNA expression experiments with transgenic tobacco seedlings or with transfected pea protoplasts using PS-IAA4/5 promoter GUS or CAT fusions reveal that CHX transcriptionally activates PS-IAA4/5 gene expression. Thus, protein synthesis inhibitors have a dual effect on PS-IAA4/5 and PS-IAA6. (1) They stabilize both mRNAs (possibly by a translational arrest-linked process or by preventing the synthesis of a labile nuclease(s)). (2) They activate transcription (possibly by preventing the synthesis or function of a repressor).
Anisomycin↗
The PS-IAA4/5-like family of early auxin-inducible mRNAs in Arabidopsis thaliana.
The plant hormone auxin transcriptionally activates early genes. We have isolated a 14-member family of DNA sequences complementary to indoleacetic acid (IAA)-inducible transcripts in Arabidopsis thaliana. The corresponding genes, IAA1 to IAA14, are homologs of PS-IAA4/5 and PS-IAA6 from pea, Aux22 and Aux28 from soybean, ARG3 and ARG4 from mungbean, and AtAux2-11 and AtAux2-27 from Arabidopsis. The members of the family are differentially expressed in mature Arabidopsis plants. Characterization of IAA gene expression in etiolated seedlings demonstrates specificity for auxin inducibility. The response of most family members to IAA is rapid (within 4 to 30 minutes) and insensitive to cycloheximide. Cycloheximide alone induces all the early genes. Auxin-induction of two late genes, IAA7 and IAA8, is inhibited by cycloheximide, indicating requirement of protein synthesis for their activation. All IAA genes display a biphasic dose response that is optimal at 10 microM IAA. However, individual genes respond differentially between 10 nM and 5 microM IAA. Expression of all genes is defective in the Arabidopsis auxin-resistant mutant lines axr1, axr2 and aux1. The encoded polypeptides share four conserved domains, and seven invariant residues in the intervening regions. The spacers vary considerably in length, rendering the calculated molecular mass of IAA proteins to range from 19 kDa to 36 kDa. Overall sequence identity between members of the family is highly variable (36 to 87%). Their most significant structural features are functional nuclear transport signals, and a putative beta alpha alpha-fold whose modeled three dimensional structure appears to be compatible with the prokaryotic beta-ribbon DNA recognition motif. The data suggest that auxin induces in a differential and hierarchical fashion a large family of early genes that encode a structurally diverse class of nuclear proteins. These proteins are proposed to mediate tissue-specific and cell-type restricted responses to the hormone during plant growth and development.
Amino Acid Sequence↗
ACS4, a primary indoleacetic acid-responsive gene encoding 1-aminocyclopropane-1-carboxylate synthase in Arabidopsis thaliana. Structural characterization, expression in Escherichia coli, and expression characteristics in response to auxin [corrected].
1-Aminocyclopropane-1-carboxylic acid (ACC) synthase is the key regulatory enzyme in the biosynthetic pathway of the plant hormone ethylene. The enzyme is encoded by a divergent multigene family in Arabidopsis thaliana, comprising at least five genes, ACS1-5 (Liang, X., Abel, S., Keller, J.A., Shen,N. N.F., and Theologis, A. (1992) Poc. Natl. Acad. Sci. U.S.A. 89, 11046-11050). In etiolated seedlings, ACS4 is specifically induced by indoleacetic acid (IAA). The response to IAA is rapid (within 25 min) and insensitive to protein synthesis inhibition, suggesting that the ACS4 gene expression is a primary response to IAA. The ACS4 mRNA accumulation displays a biphasic dose-response curve which is optimal at 10 microM of IAA. However, IAA concentrations as low as 100 microM are sufficient to enhance the basal level of ACS4 mRNA. The expression of ACS4 is defective in the Arabidopsis auxin-resistant mutant lines axr1-12, axr2-1, and aux1-7. ACS4 mRNA levels are severely reduced in axr1-12 and axr2-1 but are only 1.5-fold lower in aux1-7. IAA inducibility is abolished in axr2-1. The ACS4 gene was isolated and structurally characterized. The promoter contains four sequence motifs reminiscent of functionally defined auxin-responsive cis-elements in the early auxin-inducible genes PS-IAA4/5 from pea and GH3 from soybean. Conceptual translation of the coding region predicts a protein with a molecular mass of 53,795 Da and a theoretical isoelectric point of 8.2. The ACS4 polypeptide contains the 11 invariant amino acid residues conserved between aminotransferases and ACC synthases from various plant species. An ACS4 cDNA was generated by reverse transcriptase-polymerase chain reaction, and the authenticity was confirmed by expression of ACC synthase activity in Escherichia coli.
Amino Acid Sequence↗
Two auxin-responsive domains interact positively to induce expression of the early indoleacetic acid-inducible gene PS-IAA4/5.
The plant growth hormone indole-3-acetic acid (IAA) transcriptionally activates expression of several genes in plants. We have previously identified a 164-bp promoter region (-318 to -154) in the PS-IAA4/5 gene that confers IAA inducibility. Linker-scanning mutagenesis across the region has identified two positive domains: domain A (48 bp; -203 to -156) and domain B (44 bp; -299 to -256), responsible for transcriptional activation of PS-IAA4/5 by IAA. Domain A contains the highly conserved sequence 5'-TGTCCCAT-3' found among various IAA-inducible genes and behaves as the major auxin-responsive element. Domain B functions as an enhancer element which may also contain a less efficient auxin-responsive element. The two domains act cooperatively to stimulate transcription; however, tetramerization of domain A or B compensates for the loss of A or B function. The two domains can also mediate IAA-induced transcription from the heterologous cauliflower mosaic virus 35S promoter (-73 to +1). In vivo competition experiments with icosamers of domain A or B show that the domains interact specifically and with different affinities to low abundance, positive transcription factor(s). A model for transcriptional activation of PS-IAA4/5 by IAA is discussed.
Base Sequence↗
Induction kinetics of the nuclear proteins encoded by the early indoleacetic acid-inducible genes, PS-IAA4/5 and PS-IAA6, in pea (Pisum sativum L.).
The plant hormone indoleacetic acid (IAA) rapidly induces transcription of two genes, PS-IAA4/5 and PS-IAA6, in pea that encode nuclear proteins. The proteins were expressed in Escherichia coli and polyclonal antibodies were raised. The proteins can neither be detected on immunoblots of pea extracts from IAA-treated epicotyls nor subcellularly localized by immunofluorescence, suggesting that they are of low abundance. However, they can be immunoprecipitated as 35S-methionine-labeled proteins synthesized in vivo from control and IAA-treated tissue segments. Short-term time-course experiments indicate that the amounts of PS-IAA4/5 and PS-IAA6 proteins decrease dramatically in non-IAA-treated tissue. However, the hormone slightly increases the PS-IAA4/5 and significantly enhances the PS-IAA6 proteins compared with the initial amounts present in the tissue, despite a large induction of both mRNAs. A net increase in the amount of the in vivo synthesized PS-IAA6 is observed after a lag period of 30 min after addition of IAA. Little or no PS-IAA4/5 or PS-IAA6 protein is detected after 6 h of induction, even though PS-IAA4/5 and PS-IAA6 mRNAs remain detectable. Immunoprecipitation of in vitro translated polypeptides with mRNAs from various auxin-treated and untreated mono- and dicotyledonous plants reveals that similar proteins are encoded by constitutive or IAA-induced mRNAs. Phylogenetic analysis of 10 PS-IAA4-like proteins from various plant species reveals that the PS-IAA4 and PS-IAA6 proteins belong to different lineages, suggesting that they may have distinct functions. The data suggest that as a primary response to IAA plant tissues produce short-lived nuclear proteins whose synthesis is regulated at the transcriptional and post-transcriptional levels.
Amino Acid Sequence↗
A polymorphic bipartite motif signals nuclear targeting of early auxin-inducible proteins related to PS-IAA4 from pea (Pisum sativum).
The plant hormone, indoleacetic acid (IAA), transcriptionally activates two early genes in pea, PS-IAA4/5 and PS-IAA6 that encode short-lived nuclear proteins. The identification of the nuclear localization signals (NLS) in PS-IAA4 and PS-IAA6 using progressive deletion analysis and site-directed mutagenesis is reported. A C-terminal SV40-type NLS is sufficient to direct the beta-glucuronidase reporter to the nucleus of transiently transformed tobacco protoplasts, but is dispensible for nuclear localization of both proteins. The dominant and essential NLS in PS-IAA4 and PS-IAA6 overlap with a bipartite basic motif which is polymorphic and conserved in related proteins from other plant species, having the consensus sequence (KKNEK)KR-X(24-71)-(RSXRK)/(RK/RK). Both basic elements of this motif in PS-IAA4, (KR-X41-RSYRK), function interdependently as a bipartite NLS. However, in PS-IAA6 (KKNEKKR-X36-RKK) the upstream element of the corresponding motif contains additional basic residues which allow its autonomous function as an SV40-type monopartite NLS. The spacer-length polymorphism, X(24-70), in respective bipartite NLS peptides of several PS-IAA4-like proteins from Arabidopsis thaliana does not affect nuclear targeting function. The structural and functional variation of the bipartite basic motif in PS-IAA4-like proteins supports the proposed integrated consensus of NLS.
Amino Acid Sequence↗
Early auxin-induced genes encode short-lived nuclear proteins.
The plant growth hormone indoleacetic acid (IAA) transcriptionally activates gene expression in plants. Some of the genes whose expression is induced by IAA encode a family of proteins in pea (PS-IAA4 and PS-IAA6) and Arabidopsis (IAA1 and IAA2) that contain putative nuclear localization signals that direct a beta-glucuronidase reporter protein into the nucleus. Pulse-chase and immunoprecipitation experiments have defined the t1/2 of the PS-IAA4 and PS-IAA6 proteins to be 8 and 6 min, respectively. Their most prominent feature is the presence of a beta alpha alpha motif similar to the beta-sheet DNA-binding domain found in prokaryotic repressors of the Arc family. Based on these data, we suggest that plant tissues express short-lived nuclear proteins as a primary response to IAA. We propose that these proteins act as activators or repressors of genes responsible for mediating the various auxin responses.
Amino Acid Sequence↗
Ethylene biosynthesis and action: a case of conservation.
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Amino Acid Sequence↗
Transient transformation of Arabidopsis leaf protoplasts: a versatile experimental system to study gene expression.
An improved protocol is reported to isolate and transiently transform mesophyll protoplasts of Arabidopsis thaliana. Transfected leaf protoplasts support high levels of expression of the bacterial reporter gene coding for beta-glucuronidase (GUS), under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Transient expression of GUS activity was monitored spectrophotometrically and reached a maximum between 18 and 48 h after polyethylene glycol (PEG)-mediated DNA uptake. Histochemical staining for GUS activity revealed reproducible transformation frequencies between 40 and 60%, based on the number of protoplasts survived. To demonstrate the applicability of the transient expression system, the subcellular localization of GUS proteins tagged with different nuclear polypeptides was studied in transfected mesophyll protoplasts, revealing nuclear compartmentalization of the chimeric GUS enzymes. Furthermore, Arabidopsis mesophyll protoplasts support auxin-mediated induction of chloramphenicol acetyl-transferase (CAT) activity when transfected with a transcriptional fusion between the CAT reporter gene and the early auxin-inducible PS-IAA4/5 promoter. Hence, the method allows in vivo analysis of promoter activity and subcellular localization of fusion proteins in a homologous transformation system.
Arabidopsis↗
Identification of the auxin-responsive element, AuxRE, in the primary indoleacetic acid-inducible gene, PS-IAA4/5, of pea (Pisum sativum).
The plant hormone auxin transcriptionally activates early genes in pea. We have developed a transient assay system using protoplasts of auxin-responsive pea seedling cells to define the auxin-responsive element, AuxRE, of the early auxin-induced PS-IAA4/5 gene. The auxin responsive protoplasts show an authentic hormonal response identical to that observed in intact pea tissue, with respect to rapidity, specificity and cycloheximide (CHX) inducibility of the PS-IAA4/5 transcript. The hormone also mediates rapid and specific induction of chloramphenicol acetyltransferase (CAT) activity in protoplasts transfected with a chimeric IAA4/5-CAT gene. The IAA-induced CAT activity is developmentally regulated and is observed only in protoplasts derived from auxin-responsive regions of the pea seedling. Extensive deletion analysis of the PS-IAA4/5 promoter defined a promoter region between -318 and -154 that confers auxin inducibility. This AuxRE mediates auxin-inducible CAT activity in pea cells driven by the non auxin-responsive CaMV 35S minimal promoter. The functionality of this promoter region as an AuxRE was further verified in tobacco plants using IAA4/5-GUS gene fusions. The AuxRE contains two domains: Domain A acts as an auxin switch; domain B has an enhancer-like activity. The A and B domains contain the highly conserved sequences found in various auxin-regulated genes (T/GGT-CCCAT (domain A) and C/AACATGGNC/AA/GTGTT/CT/CC/A (domain B)). DNase I footprinting reveals binding of nuclear proteins to the highly conserved sequence found in A and B domains. The sequence of the A domain does not correspond to any known regulatory elements found in other eukaryotic genes, and the data suggest that this conserved motif functions as an AuxRE. A model for the early transcriptional activation of the PS-IAA4/5 gene by IAA is discussed.
Base Sequence↗
Structural characterization of the early indoleacetic acid-inducible genes, PS-IAA4/5 and PS-IAA6, of pea (Pisum sativum L.).
Two early auxin-inducible genes (PS-IAA4/5 and PSIAA6) from pea were cloned using previously isolated complementary DNA sequences. They are present in single copy per haploid genome, and are members of a large divergent multigene family that encodes similar proteins. The genes were structurally characterized and sequence analysis of their 5'-flanking regions revealed the presence of several highly conserved sequences found in various auxin-regulated genes from other plant species. Their coding regions are interrupted by three and two introns, respectively. Introns two and three of PS-IAA4/5 and introns one and two of PS-IAA6 are located in identical positions. These genes encode proteins of 189 (21,036 Da) and 179 (20,330 Da) residues that are 46% identical. They also share a significant degree of identity (42 to 80%) with other proteins encoded by auxin regulated genes in soybean, mungbean and Arabidopsis thaliana. All proteins contain four conserved domains ranging in size from 9 to 43 amino acids. Their most prominent feature is the presence of a highly charged N terminus consisting of two clusters of acidic residues separated by a cluster of basic amino acids.
Amino Acid Sequence↗
LE-ACS4, a fruit ripening and wound-induced 1-aminocyclopropane-1-carboxylate synthase gene of tomato (Lycopersicon esculentum). Expression in Escherichia coli, structural characterization, expression characteristics, and phylogenetic analysis.
ACC (1-aminocyclopropane-1-carboxylic acid) synthase is the key regulatory enzyme in the biosynthetic pathway of the plant hormone ethylene and is encoded by a highly divergent multigene family in tomato (Rottmann, W. H., Peter, G. F., Oeller, P. W., Keller, J. A., Shen, N. F., Nagy, B. P., Taylor, L. P., Campbell, A. D., and Theologis, A. (1991) J. Mol. Biol. 222, 937-961). Two members of the family, LE-ACS2 and LE-ACS4, are induced during fruit ripening and upon treatment of mature green fruits with exogenous ethylene (C2H4) in a dose-dependent manner. Both genes are superinduced by wounding of pericarp tissue during various stages of ripening. The wound-induced accumulation of LE-ACS2 mRNA is more rapid and greater than that of LE-ACS4. Both mRNAs accumulate in the absence of protein synthesis, suggesting that their induction is a primary response to the inducer. The LE-ACS4 gene was isolated and structurally characterized. The function of the LE-ACS4 protein (53,509 Da, pI 5.4) was verified by expression experiments in Escherichia coli. The promoters of LE-ACS2 and LE-ACS4 contain potential cis-acting regulatory elements responsible for induction by ethylene, wounding, and anaerobiosis. In addition, elements for binding the transcriptional factors EmBP1, GBF-1, and OCSBF-1 are also present. Phylogenetic analysis of 20 ACC synthases from dicots and monocots indicate that the LE-ACS2 and LE-ACS4 proteins belong to an unique sublineage that includes an additional member of the tobacco family, NT-ACS1. The divergence of this sublineage is a relatively recent event in the evolution of ACC synthase protein.
Amino Acid Sequence↗
Plant hormones: what a gas!
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Journal Article↗
Use of a tomato mutant constructed with reverse genetics to study fruit ripening, a complex developmental process.
Fruit ripening is one of the most dramatic developmental transitions associated with extensive alteration in gene expression. The plant hormone ethylene is considered to be the causative ripening agent. Transgenic tomato plants were constructed expressing antisense or sense RNA to the key enzyme in the ethylene (C2H4) biosynthetic pathway, 1-aminocyclopropane-1-carboxylate (ACC) synthase using the constitutive CaMV 35S and fruit specific E8 promoters. Fruits expressing antisense LE-ACS2 RNA produce less ethylene and fail to ripen only when ethylene production is suppressed by more than 99% (> 0.1 nl/g fresh weight). Ethylene production is considerably inhibited (50%) in fruits expressing sense LE-ACS2 RNA. Antisense fruits accumulate normal levels of polygalacturonase (PG), ACC oxidase (pTOM13), E8, E17, J49, and phytoene desaturase (D2) mRNAs which were previously thought to be ethylene-inducible. E4 gene expression is inhibited in antisense fruits and its expression is not restored by treatment with exogenous propylene (C3H6). Antisense fruits accumulate PG mRNA, but it is not translated. Immunoblotting experiments indicate that the PG protein is not expressed in antisense fruits but its accumulation is restored by propylene (C3H6) treatment. The results suggest that at least two signal-transduction pathways are operating during tomato fruit ripening. The independent (developmental) pathway is responsible for the transcriptional activation of genes such as PG, ACC oxidase, E8, E17, D2, and J49. The ethylene-dependent pathway is responsible for the transcriptional and posttranscriptional regulation of genes involved in lycopene, aroma biosynthesis, and the translatability of developmentally regulated genes such as PG.
Enzyme Induction↗
Anaerobiosis and plant growth hormones induce two genes encoding 1-aminocyclopropane-1-carboxylate synthase in rice (Oryza sativa L.).
The plant hormone ethylene is believed to be responsible for the ability of rice to grow in the deepwater regions of Southeast Asia. Ethylene production is induced by hypoxia, which is caused by flooding, because of enhanced activity of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, the key enzyme in the ethylene biosynthetic pathway. We have cloned three divergent members, (OS-ACS1, OS-ACS2, and OS-ACS3), of a multigene family encoding ACC synthase in rice. OS-ACS1 resides on chromosome 3 and OS-ACS3 on chromosome 5 in the rice genome. The OS-ACS1 and OS-ACS3 genes are induced by anaerobiosis and indoleacetic acid (IAA) + benzyladenine (BA) + LiCl treatment. The anaerobic induction is differential and tissue specific; OS-ACS1 is induced in the shoots, whereas OS-ACS3 is induced in the roots. These inductions are insensitive to protein synthesis inhibitors, suggesting that they are primary responses to the inducers. All three genes are actually induced when protein synthesis is inhibited, indicating that they may be under negative control or that their mRNAs are unstable. The OS-ACS1 gene was structurally characterized, and the function of its encoded protein (M(r) = 53 112 Da, pI 8.2) was confirmed by expression experiments in Escherichia coli. The protein contains all eleven invariant amino acid residues that are conserved between aminotransferases and ACC synthases cloned from various dicotyledonous plants. The amino acid sequence shares significant identity to other ACC synthases (69-34%) and is more similar to sequences in other plant species (69% with the tomato LE-ACS3) than to other rice ACC synthases (50-44%). The data suggest that the extraordinary degree of divergence among ACC synthase isoenzymes within each species arose early in plant evolution and before the divergence of monocotyledonous and dicotyledonous plants.
Adenine↗