A dominant mutant receptor from Arabidopsis confers ethylene insensitivity in heterologous plants.

Ethylene (C2H4) is a gaseous hormone that affects many aspects of plant growth and development. Ethylene perception requires specific receptors and a signal transduction pathway to coordinate downstream responses. The etr1-1 gene of Arabidopsis encodes a mutated receptor that confers dominant ethylene insensitivity. Evidence is presented here that etr1-1 also causes significant delays in fruit ripening, flower sensecence; and flower abscission when expressed in tomato and petunia plants. The ability of etr1-1 to function in heterologous plants suggests that this pathway of hormone recognition and response is highly conserved and can be manipulated.

A phosphonate monoester hydrolase from Burkholderia caryophilli PG2982 is useful as a conditional lethal gene in plants.

A bacterial phosphonate monoester hydrolase was evaluated in plants as a conditional lethal gene useful for cell ablation and negative selection. Glyphosate is a potent herbicide whereas its phosphonate monoester derivative, glyceryl glyphosate, is approximately 50-fold less active. A phosphonate monoesterase gene (pehA) encoding an enzyme that hydrolyzes phosphonate esters including glyceryl glyphosate to glyphosate and glycerol was cloned from the glyphosate metabolizing bacterium, Burkholderia caryophilli PG2982. Constitutive expression of the pehA gene in Escherichia coli and Arabidopsis thaliana RLD had no observable phenotypic effects on growth and development. However, cells and plants expressing the pehA gene were killed when treated with glyceryl glyphosate. The phytotoxicity resulted from the hydrolysis of glyceryl glyphosate to glyphosate and subsequent inhibition of aromatic amino acid biosynthesis. As an example of tissue-specific cell ablation, floral sterility without vegetative toxicity was demonstrated by expressing the pehA gene using a tapetal-specific promoter and treating the mature plants with glyceryl glyphosate. A chromogenic phosphonate ester substrate, 5-bromo-4-chloro-indolyl phenylphosphonate, was used to monitor in situ expression of the pehA gene. The general utility of the pehA gene as a heterologous conditional lethal gene in plants is discussed.

An ethylene-inducible component of signal transduction encoded by never-ripe.

The ripening-impaired tomato mutant Never-ripe (Nr) is insensitive to the plant hormone ethylene. The gene that cosegregates with the Nr locus encodes a protein with homology to the Arabidopsis ethylene receptor ETR1 but is lacking the response regulator domain found in ETR1 and related prokaryotic two-component signal transducers. A single amino acid change in the sensor domain confers ethylene insensitivity when expressed in transgenic tomato plants. Modulation of NR gene expression during fruit ripening controls response to the hormone ethylene.

The ethylene response mediator ETR1 from Arabidopsis forms a disulfide-linked dimer.

Mutations in the ETR1 gene of the higher plant Arabidopsis confer insensitivity to ethylene, indicating a role for the gene product in ethylene signal perception and transduction. The ETR1 gene product has an amino-terminal hydrophobic domain and a carboxyl-terminal domain showing homology to the two-component signal transduction proteins of bacteria. We report here that in both its native Arabidopsis and when transgenically expressed in yeast, the ETR1 protein is isolated from membranes as a dimer of 147 kDa. Treatment with the reducing agent dithiothreitol converted the dimer to a monomer of 79 kDa, indicative of a disulfide linkage between monomers. Expression of truncated versions of ETR1 in yeast confirmed that the high molecular mass form is a homodimer and demonstrated that the amino-terminal region of ETR1 is necessary and sufficient for this dimerization. Site-directed mutagenesis of two cysteines near the amino terminus of ETR1 prevented formation of the covalently linked dimer in yeast, consistent with a role in disulfide bond formation. These data indicate that ETR1 may use a dimeric mechanism of signal transduction in a manner similar to its bacterial counterparts but with the additional feature of a disulfide bond between monomers.

The tomato Never-ripe locus regulates ethylene-inducible gene expression and is linked to a homolog of the Arabidopsis ETR1 gene.

Fruit ripening represents a complex system of genetic and hormonal regulation of eukaryotic development unique to plants. We are using tomato ripening mutants as tools to elucidate genetic components of ripening regulation and have recently demonstrated that the Never-ripe (Nr) mutant is insensitive to the plant growth regulator ethylene (M.B. Lanahan, H.-C. Yen, J.J. Giovannoni, H.J. Klee [1994] Plant Cell 6:521-530). We report here ethylene sensitivity over a range of concentrations in normal and Nr tomato seedlings and show that the Nr mutant retains residual sensitivity to as little as 1 part per million of ethylene. Analysis of ripening-related gene expression in normal and mutant ethylene-treated fruit demonstrates that Nr exerts its influence on development at least in part at the level of ethylene-inducible gene expression. We have additionally used cloned tomato and Arabidopsis sequences known to influence ethylene perception as restriction fragment length polymorphism probes, and have identified a tomato locus linked to Nr that hybridizes to the Arabidopsis ETR1 gene at low stringency, suggesting the possibility that Nr may be homologous to ETR1.

Identification of mRNAs with enhanced expression in ripening strawberry fruit using polymerase chain reaction differential display.

Fruit ripening is a complex developmental process that involves specific changes in gene expression and cellular metabolism. In climateric fruits these events are coordinated by the gaseous hormone ethylene, which is synthesized autocatalytically in the early stages of ripening. Nonclimacteric fruits do not synthesize or respond to ethylene in this manner, yet undergo many of the same physiological and biochemical changes associated with the production of a ripe fruit. To gain insight into the molecular determinants associated with nonclimacteric fruit ripening, we examined mRNA populations in ripening strawberry fruit using polymerase chain reaction (PCR) differential display. Five mRNAs with ripening-enhanced expression were identified using this approach. Three of the mRNAs appear to be fruit-specific, with little or no expression detected in vegetative tissues. Sequence analysis of cDNA clones revealed positive identities for three of the five mRNAs based on homology to known proteins. These results indicate that the differential display technique can be a useful tool to study fruit ripening and other developmental processes in plants at the RNA level.

The cis-acting gibberellin response complex in high pI alpha-amylase gene promoters. Requirement of a coupling element for high-level transcription.

In cereal alpha-amylase gene promoters the cis-acting gibberellin response element (GARE) is required for increased transcription in the presence of gibberellin. In low-isoelectric point (pI) alpha-amylase gene promoters a second type of cis element, termed a coupling element, must also be present in a specific position near the GARE; otherwise, the level of transcription in the presence of gibberellin is only a few percent of maximum. The coupling element may help determine where and when in development high-level, hormonally regulated transcription will occur. Such coupling elements have not yet been shown to be necessary for high-level transcription from high-pI alpha-amylase gene promoters. Here we use quantitative transient expression assays to show that a high-pI promoter truncated to -300 is a weak promoter due to the absence of a functional coupling element in the vicinity of the GARE. Gibberellin-induced transcription increases substantially when coupling element function is provided, either by appending upstream regions normally attached to the promoter or by inserting a defined coupling element from a low-pI promoter. Thus, in a second type of gibberellin-regulated promoter coupling element function was found to be crucial for hormone regulation to result in high-level transcription.

The never ripe mutation blocks ethylene perception in tomato.

Seedlings of tomato fruit ripening mutants were screened for their ability to respond to ethylene. Ethylene induced the triple response in etiolated hypocotyls of all tomato ripening mutants tested except for one, Never ripe (Nr). Our results indicated that the lack of ripening in this mutant is caused by ethylene insensitivity. Segregation analysis indicated that Nr-associated ethylene insensitivity is a single codominant trait and is pleiotropic, blocking senescence and abscission of flowers and the epinastic response of petioles. In normal tomato flowers, petal abscission and senescence occur 4 to 5 days after the flower opens and precede fruit expansion. If fertilization does not occur, pedicel abscission occurs 5 to 8 days after petal senescence. If unfertilized, Nr flowers remained attached to the plant indefinitely, and petals remained viable and turgid more than four times longer than their normal counterparts. Fruit development in Nr plants was not preceded by petal senescence; petals and anthers remained attached until they were physically displaced by the expanding ovary. Analysis of engineered 1-aminocyclopropane-1-carboxylate (ACC) synthase-overexpressing plants indicated that they are phenotypic opposites of Nr plants. Constitutive expression of ACC synthase in tomato plants resulted in high rates of ethylene production by many tissues of the plant and induced petiole epinasty and premature senescence and abscission of flowers, usually before anthesis. There were no obvious effects on senescence in leaves of ACC synthase overexpressers, suggesting that although ethylene may be important, it is not sufficient to cause tomato leaf senescence; other signals are clearly involved.

Gibberellin treatment stimulates nuclear factor binding to the gibberellin response complex in a barley alpha-amylase promoter.

The promoters of a majority of cereal alpha-amylase genes contain three highly conserved sequences (gibberellin response element, box I, and pyrimidine box). Recent studies have demonstrated the functional importance of four regions that either coincide with or are immediately proximal to these three conserved elements as well as an upstream Opaque-2 binding sequence. In this study, we describe the characterization of nuclear protein factors from barley aleurone layers whose binding activity toward gibberellin response complex sequences from the barley low-pl alpha-amylase gene (Amy32b) promoter is stimulated by gibberellin A3 (GA3) treatment. Barley proteins isolated from crude nuclear extracts prepared from aleurone layers incubated with or without GA3 were fractionated by anion exchange fast protein liquid chromatography and studied using band shift assays, sequence-specific competitions, and DNase I footprinting. A GA3-dependent binding activity eluting at 210 mM KCl was shown to bind specifically to the gibberellin response element and the closely associated box I. DNase I footprinting with the proteins in this fraction indicated interactions with sequences in the gibberellin response element and box I. A second DNA binding activity eluting at 310 mM KCl was present constitutively in extracts prepared from tissues incubated both in the absence and in the presence of hormone. Proteins in this fraction were able to bind to many DNA sequences and, in general, were largely nonspecific. DNase I footprinting with the proteins in this fraction indicated a large area of protection with a single unoccupied region located at the 3' end of box I. The possible function of such an activity in hormone regulation of the alpha-amylase genes is discussed.

A gibberellin response complex in cereal alpha-amylase gene promoters.

The Amy32b gene is a representative member of a closely related family of alpha-amylase genes expressed under hormonal control in aleurone layers of barley grains. Transcription of this gene is induced by gibberellin (GA) and suppressed by abscisic acid. In this study, we functionally defined the promoter elements of the Amy32b gene that govern the developmental and hormonal control of its expression in aleurone. Two functionally distinct yet physically associated elements are essential: a gibberellin response element mediates regulation by GA and abscisic acid, and an Opaque-2 binding sequence (O2S) is thought to interact with a barley homolog of the maize endosperm-specific transcriptional regulator Opaque-2. An additional element CCTTTT, which with the O2S forms part of a canonical "endosperm box," is important in modulating the absolute level of expression of the Amy32b promoter, as is another separate, highly conserved element TATCCATGCAGTG.

Slender barley: A constitutive gibberellin-response mutant.

In barley (Hordeum vulgare L. cv. Herta), slender (sln1) is a single-locus recessive mutation which causes a plant to appear as if it had been grown in sturating concentrations of gibberellin (GA). We have investigated two of the GA-mediated processes in slender barley, shoot elongation and the induction of hydrolytic enzymes in aleurone layers. Shoot elongation is severely retarded in normal (wild-type) barley if the biosynthesis of GA is blocked by an inhibitor, ancymidol (α-cyclopropyl-α-(p-methoxyphenyl)-5-pyrimidinemethanol). However, the slender mutant continues to elongate in the presence of ancymidol. In isolated normal aleurone layers, the synthesis and secretion of α-amylase (EC 3.2.1.1), protease (EC 3.4) and nuclease (EC 3.1.30.2) are induced by exogenously applied GA3. However, in the aleurone layers of the slender mutant these enzymes are produced even in the absence of GA but their synthesis is still susceptible to inhibition by abscisic acid. Bioassays of half-seeds of the slender mutant and their normal siblings show no detectable differences in endogenous levels of GA-like substances. We suggest that the slender mutation allows competent tissues to express fully, or over-express, appropriate GA-induced processes independent of GA. We also conclude that shoot elongation, and hydrolytic-enzyme secretion in aleurone layers, share a common regulatory element.