Pollination-Induced Ethylene in Carnation (Role of Stylar Ethylene in Corolla Senescence).

In carnation (Dianthus caryophyllus L. cv White Sim) cell to cell communication between the pollen and pistil induces ovary development and corolla senescence. The production of elevated ethylene by the style is the first measurable postpollination response. This is followed by a wave of ethylene production from the other floral organs. To investigate the regulation of ethylene biosynthesis in pollinated flowers we measured ethylene production and the expression of 1-aminocyclopropane-1-carboxylate synthase and 1-aminocyclopropane-1-carboxylate oxidase transcripts in individual floral organs after pollination. Ethylene production by pollinated styles can be defined temporally by three distinct peaks. By pollinating a single style from a multistyle gynoecium, it was determined that the unpollinated style produces ethylene that corresponds to the first and third peaks observed from a pollinated style. Inhibition of ethylene action in the pollinated style by diazocyclopentadiene treatment prevented both pollination-induced corolla senescence and ethylene production from the ovaries and petals. Treatment with diazocyclopentadiene decreased stylar ethylene production during the second peak and completely inhibited the third peak of ethylene in both pollinated and unpollinated styles. This later auto-catalytic ethylene in styles is likely responsible for pollination-induced corolla senescence and ovary development.

Temporal and Spatial Expression of 1-Aminocyclopropane-1-Carboxylate Oxidase mRNA following Pollination of Immature and Mature Petunia Flowers.

Pollination of petunia (Petunia hybrida) flowers induces a rapid increase in ethylene production by styles, which subsequently leads to increased ethylene production by the corolla, inducing senescence. We have investigated the temporal and spatial expression of 1-aminocyclopropane-1-carboxylate (ACC) oxidase transcripts in petunia styles in an attempt to elucidate its role in increased ethylene biosynthesis following pollination. Previously, we reported that the development of petunia flowers was associated with increased ACC oxidase mRNA localized specifically in the stigmatic regions of the style (X. Tang, A.M.T. Gomes, A. Bhatia, W.R. Woodson [1994] Plant Cell 6: 1227-1239). The rapid increase in ethylene production by styles within the 1st h following pollination was correlated with the expression of ACC oxidase mRNAs during development. Pollination of petunia flowers prior to anthesis and the expression of ACC oxidase mRNA led to a substantial increase in ethylene production, but this was delayed by several hours in comparison with flowers at anthesis. This delayed increase in ethylene production by pollinated styles from immature flowers was associated with an increased ACC oxidase transcript abundance. Treatment with the ethylene action inhibitor 2,5-norbornadiene did not affect the early increase in ethylene production or the expression of ACC oxidase mRNAs. No differences in the rate of pollen germination or tube growth were detected when applied to stigmas from immature or mature flowers, indicating that the delay in ethylene production was likely the result of limited ACC oxidase activity. Localization of ACC oxidase mRNAs following pollination by in situ hybridization revealed an abundance of transcripts in transmitting tract tissue within 4 h of pollination of both immature and mature styles, in contrast to their localization in stigmatic cells during development.

Cloning of a DNA-binding protein that interacts with the ethylene-responsive enhancer element of the carnation GST1 gene.

Ethylene transcriptionally activates a glutathione S-transferase gene (GST1) at the onset of the senescence program in carnation (Dianthus caryophyllus L.) flower petals. A 126 bp region of the GST1 promoter sequence has been identified as an ethylene-responsive enhancer element (ERE). In this paper, we demonstrate the ability of nuclear proteins from senescing petals to recognize a 22 bp sequence within the ERE (ERE oligonucleotide). Mutation of the ERE oligonucleotide sequence significantly alters the strength of this nuclear protein-DNA association. The wild-type ERE oligonucleotide sequence was used to isolate a cDNA clone encoding a sequence-specific DNA binding protein. Nucleotide sequencing and deduced amino acid sequence analysis of this cDNA predicted a 32 kDa protein which we have designated carnation ethylene-responsive element-binding protein-1 (CEBP-1). The mRNA expression pattern of CEBP-1 suggests that it is not transcriptionally regulated by ethylene. The amino acid sequence homology of CEBP-1 with other plant nucleic acid binding proteins indicates a conserved nucleic acid binding domain. Within this domain are two highly conserved RNA-binding motifs, RNP-1 and RNP-2. An acidic region and a putative nuclear localization signal are also identified.

Pollination-Induced Ethylene in Carnation (Role of Pollen Tube Growth and Sexual Compatibility).

The pollen-pistil interactions that result in the stimulation of ethylene production and petal senescence in carnation (Dianthus caryophyllus L.) flowers were investigated. Pollination of White Sim flowers with Starlight pollen elicited an increase in ethylene production by styles, leading to increased petal ethylene and premature petal senescence. In contrast, pollination with 87-29G pollen led to an early increase in ethylene production, but this was not sustained, and did not lead to petal senescence. Both Starlight and 87-29G pollen germinated on White Sim stigmas and their tubes grew at similar rates, penetrating the length of the style. Crosses between Starlight and White Sim led to the production of viable seeds, whereas 87-29G pollen was infertile on White Sim flowers. Pollination of other carnations with 87-29G elicited ethylene production and petal senescence and led to the production of viable seeds. These results suggest that physical growth of pollen tubes is insufficient to elicit a sustained increase in ethylene production or to lead to the production of signals necessary for elicitation of petal ethylene production and senescence. Rather, the cell-cell recognition reactions leading to sexual compatibility in Dianthus appear to play a role in this interorgan signaling after pollination.

Ethylene-regulated expression of a carnation cysteine proteinase during flower petal senescence.

The senescence of carnation (Dianthus caryophyllus L.) flower petals is regulated by the phytohormone ethylene and is associated with considerable catabolic activity including the loss of protein. In this paper we present the molecular cloning of a cysteine proteinase and show that its expression is regulated by ethylene and associated with petal senescence. A 1600 bp cDNA was amplified by polymerase chain reaction using a 5'-specific primer and 3'-nonspecific primer designed to amplify a 1-aminocyclopropane-1-carboxylate synthase cDNA from reverse-transcribed stylar RNA. The nucleotide sequence of the cloned product (pDCCP1) was found to share significant homology to several cysteine proteinases rather than ACC synthase. A single open reading frame of 428 amino acids was shown to share significant homology with other plant cysteine proteinases including greater than 70% identity with a cysteine proteinase from Arabidopsis thaliana. Amino acids in the active site of cysteine proteinases were conserved in the pDCCP1 peptide. RNA gel blot analysis revealed that the expression of pDCCP1 increased substantially with the onset of ethylene production and senescence of petals. Increased pDCCP1 expression was also associated with ethylene production in other senescing floral organs including ovaries and styles. The pDCCP1 transcript accumulated in petals treated with exogenous ethylene within 3 h and treatment of flowers with 2,5-norbornadiene, an inhibitor of ethylene action, prevented the increase in pDCCP1 expression in petals. The temporal and spatial patterns of pDCCP1 expression suggests a role for cysteine proteinase in the loss of protein during floral senescence.

An ethylene-responsive enhancer element is involved in the senescence-related expression of the carnation glutathione-S-transferase (GST1) gene.

The increased production of ethylene during carnation petal senescence regulates the transcription of the GST1 gene encoding a subunit of glutathione-S-transferase. We have investigated the molecular basis for this ethylene-responsive transcription by examining the cis elements and trans-acting factors involved in the expression of the GST1 gene. Transient expression assays following delivery of GST1 5' flanking DNA fused to a beta-glucuronidase receptor gene were used to functionally define sequences responsible for ethylene-responsive expression. Deletion analysis of the 5' flanking sequences of GST1 identified a single positive regulatory element of 197 bp between -667 and -470 necessary for ethylene-responsive expression. The sequences within this ethylene-responsive region were further localized to 126 bp between -596 and -470. The ethylene-responsive element (ERE) within this region conferred ethylene-regulated expression upon a minimal cauliflower mosaic virus-35S TATA-box promoter in an orientation-independent manner. Gel electrophoresis mobility-shift assays and DNase I footprinting were used to identify proteins that bind to sequences within the ERE. Nuclear proteins from carnation petals were shown to specifically interact with the 126-bp ERE and the presence and binding of these proteins were independent of ethylene or petal senescence. DNase I footprinting defined DNA sequences between -510 and -488 within the ERE specifically protected by bound protein. An 8-bp sequence (ATTTCAAA) within the protected region shares significant homology with promoter sequences required for ethylene responsiveness from the tomato fruit-ripening E4 gene.

Pistil-Specific and Ethylene-Regulated Expression of 1-Aminocyclopropane-1-Carboxylate Oxidase Genes in Petunia Flowers.

The differential expression of the petunia 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene family during flower development and senescence was investigated. ACC oxidase catalyzes the conversion of ACC to ethylene. The increase in ethylene production by petunia corollas during senescence was preceded by increased ACC oxidase mRNA and enzyme activity. Treatment of flowers with ethylene led to an increase in ethylene production, ACC oxidase mRNA, and ACC oxidase activity in corollas. In contrast, leaves did not exhibit increased ethylene production or ACC oxidase expression in response to ethylene. Gene-specific probes revealed that the ACO1 gene was expressed specifically in senescing corollas and in other floral organs following exposure to ethylene. The ACO3 and ACO4 genes were specifically expressed in developing pistil tissue. In situ hybridization experiments revealed that ACC oxidase mRNAs were specifically localized to the secretory cells of the stigma and the connective tissue of the receptacle, including the nectaries. Treatment of flower buds with ethylene led to patterns of ACC oxidase gene expression spatially distinct from the patterns observed during development. The timing and tissue specificity of ACC oxidase expression during pistil development were paralleled by physiological processes associated with reproduction, including nectar secretion, accumulation of stigmatic exudate, and development of the self-incompatible response.

Organization and structure of the 1-aminocyclopropane-1-carboxylate oxidase gene family from Petunia hybrida.

In this paper we present the structural analysis of the 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene family from Petunia hybrida. Southern blot analysis and restriction endonuclease mapping showed that two cloned regions of the petunia genome contained sequences highly homologous to a previously isolated ACC oxidase cDNA clone. Nucleotide sequencing of these two regions of the genome showed that each contained two tandemly arranged genes designated ACO1, ACO2, ACO3 and ACO4. Comparison of the nucleotide sequences of the cloned genomic regions with the cDNA clone pPHEFE indicated that ACO1 encoded the transcript in 4 exons interrupted by 3 introns. The other three members of the petunia ACC oxidase gene family shared identical intron numbers and positions with ACO1 and their exons were greater than 80% homologous. Nucleotide substitutions and deletions in the ACO2 gene indicate that it likely represents a pseudogene. Overall homology between ACO1 and ACO2 indicates that this gene cluster arose by a more recent duplication event than the gene duplication giving rise to the ACO3 and ACO4 cluster. The 5-flanking sequences share little overall homology between members of this gene family. However, sequences which likely make up the core promoter of these genes including the TATA box are highly homologous. RNA-based PCR amplification of ACC oxidase cDNAs from ethylene-treated corollas and wounded leaves revealed transcripts for ACO1, ACO3 and ACO4 indicating that a least three of these genes are transcriptionally active. The proteins encoded by ACO1, ACO3 and ACO4 share more than 90% identity with one another and more than 70% identity with ACC oxidases from other species. The ACC oxidase proteins share significant sequence homology with other enzymes that require Fe(II) and ascorbate for catalytic activity.

A flower senescence-related mRNA from carnation encodes a novel protein related to enzymes involved in phosphonate biosynthesis.

We have isolated a cDNA clone (pSR132) representing a mRNA which accumulates in senescing carnation flower petals in response to ethylene. In vitro translation of RNA selected by hybridization with pSR132 indicated the mRNA encoded a polypeptide of approximately 36 kDa. This was confirmed by DNA sequence analysis, which predicted a peptide composed of 318 amino acids with a calculated molecular weight of 34.1 kDa. Comparison of the predicted peptide sequence of pSR132 with other proteins compiled in the NBRF data base revealed significant homology with carboxyphosphonoenolpyruvate mutase and phosphoenolpyruvate mutase from Streptomyces hygroscopicus and Tetrahymena pyriformis, respectively. These enzymes are involved in the formation of C-P bonds in the biosynthesis of phosphonates. C-P bonds are found in a wide range of organisms, but their presence or formation in higher plants has not been investigated.

Characterization of an ethylene-responsive glutathione S-transferase gene cluster in carnation.

In this paper we present the structural analysis of two tightly linked genes from the glutathione S-transferase (GST) gene family in carnation (Dianthus caryophyllus). Southern blot analysis and restriction endonuclease mapping revealed a single cloned region of the carnation genome was highly homologous to the previously characterized ethylene-responsive GST mRNA expressed in flower petals during senescence. Nucleotide sequencing of this region revealed the presence of two tandemly arranged genes designated GST1 and GST2. Comparison of the nucleotide sequences of the cloned genomic region with the previously characterized GST cDNA clone pSR8 revealed that GST1 contains the entire transcription unit in 10 exons interrupted by 9 introns. The transcription unit of GST2 was found to be very similar to GST1 with complete conservation of intron position. In addition, the length and nucleotide sequences of the two genes' introns were highly conserved. GST2 was not completely represented by the cloned genomic region, missing the 3' portion of the transcription unit. Primer extension analysis indicated a single transcriptional start site for transcripts which accumulate in senescing carnation petals. The 5'-flanking sequences of GST1 and GST2 were compared and regions of homology and divergence identified. These upstream sequences were compared with other plant ethylene-responsive genes and GST genes and several sequence motifs of potential importance in the regulation of GST expression were identified. A chimeric gene constructed between -1457 bp of the 5'-flanking DNA of GST1 and the coding region of beta-glucuronidase was found to confer ethylene-inducible expression in flower petals following delivery of the construct into tissue by particle bombardment.

Expression of ethylene biosynthetic pathway transcripts in senescing carnation flowers.

We have examined the expression of mRNAs for S-adenosylmethionine synthetase (EC 2.5.1.6), 1-aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14), and the ethylene-forming enzyme (EFE) in various floral organs of carnation (Dianthus caryophyllus) during the increase in ethylene biosynthesis associated with petal senescence. The abundance of ACC synthase and EFE mRNAs increased and S-adenosylmethionine synthetase transcripts decreased concomitantly with the ethylene climacteric in senescing petals. The increase in abundance of ACC synthase and EFE mRNAs in aging flowers was prevented by treatment with the ethylene action inhibitor 2,5-norbornadiene. Furthermore, an increase in ACC synthase and EFE transcripts was detected in petals from presenescent flowers within 3 to 6 hours of exposure to 2 microliters per liter of ethylene. The increase in ethylene production by senescing petals was associated with a concomitant increase in ethylene biosynthesis in styles, ovary, and receptacle tissues. In all tissues, this increase was associated with increased activities of ACC synthase and EFE. The increase in EFE activities by all floral organs examined was correlated with increased abundance of EFE transcripts. In contrast, the level of ACC synthase mRNA, as detected by the cDNA probe pCARACC3, did not always reflect enzyme activity. The combined tissues of the pistil exhibited high rates of ACC synthase activity but contained low levels of ACC synthase mRNAs homologous to pCARACC3. In addition, pollinated styles exhibited a rapid increase in ethylene production and ACC synthase activity but did not accumulate detectable levels of ACC synthase mRNA until several hours after the initiation of ethylene production. These results suggest that transcripts for ACC synthase leading to the early postpollination increase in ACC synthase activity and ethylene production are substantially different from the mRNA for the ethylene-responsive gene represented by pCARACC3.

Molecular cloning of an 1-aminocyclopropane-1-carboxylate synthase from senescing carnation flower petals.

Synthetic oligonucleotides based on the sequence of 1-aminocyclopropane-1-carboxylate (ACC) synthase from tomato were used to prime the synthesis and amplification of a 337 bp tomato ACC synthase cDNA by polymerase chain reaction (PCR). This PCR product was used to screen a cDNA library prepared from mRNA isolated from senescing carnation flower petals. Two cDNA clones were isolated which represented the same mRNA. The longer of the two clones (CARACC3) contained a 1950 bp insert with a single open reading frame of 516 amino acids encoding a protein of 58 kDa. The predicted protein from the carnation ACC synthase cDNA was 61%, 61%, 64%, and 51% identical to the deduced proteins from zucchini squash, winter squash, tomato, and apple, respectively. Genomic DNA gel blot analysis indicated the presence of at least a second gene in carnation which hybridized to CARACC3 under conditions of low stringency. ACC synthase mRNA accumulates during senescence of carnation flower petals concomitant with the increase in ethylene production and ACC synthase enzyme activity. Ethylene induced the accumulation of ACC synthase mRNA in presenescent petals. Wound-induced ethylene production in leaves was not associated with an increase in ACC synthase mRNA represented by CARACC3. These results indicate that CARACC3 represents an ACC synthase transcript involved in autocatalytic ethylene production in senescing flower petals.

An ethylene-responsive flower senescence-related gene from carnation encodes a protein homologous to glutathione S-transferases.

Carnation flower petal senescence is associated with the expression of specific senescence-related mRNAs, several of which were previously cloned. The cDNA clone pSR8 represents a transcript which accumulates specifically in senescing flower petals in response to ethylene. Here we report the structural characterization of this cDNA. A second cDNA clone was isolated based on shared sequence homology with pSR8. This clone, pSR8.4, exhibited an overlapping restriction endonuclease map with pSR8 and contained an additional 300 nucleotides. Primer extension analysis revealed the combined cDNAs to be near full-length and the transcript to accumulate in senescing petals. Analysis of the nucleotide sequence of SR8 cDNAs revealed an open reading frame of 220 amino acids sufficient to encode a 25 kDa polypeptide. Comparison of the deduced polypeptide sequence of pSR8 with other peptide sequences revealed significant similarity with glutathione s-transferases from a variety of organisms. The predicted polypeptide sequence shared 44%, 53% and 52% homology with GSTs from maize, Drosophila and man, respectively. We discuss our results in relation to the biochemistry of flower petal senescence and the possible role of glutathione s-transferase in this developmental process.

Characterization of an ethylene-regulated flower senescence-related gene from carnation.

The programmed senescence of carnation (Dianthus caryophyllus L.) petals requires active gene expression and is associated with the expression of several senescence-related mRNAs. Expression of the mRNA represented by the cDNA clone pSR12 has previously been shown to be transcriptionally activated by ethylene specifically in senescing flowers. We report in this paper the structural analysis of this cDNA and its corresponding gene. One cloned genomic DNA fragment, SR12-B, contained the entire transcription unit in 17 exons, interrupted by 16 introns. A second gene, SR12-A, was highly homologous to SR12-B with several nucleotide substitutions and a 489 bp deletion in the 5' flanking DNA sequence. The SR12 transcript has an open reading frame of 2193 bp sufficient to encode a protein of 82.8 kDa. No significant homology at the DNA or protein levels was found with other known genes. We have identified a DNA-binding factor which specifically interacts with two upstream fragments (-149 to -337 and -688 to -1055) of SR12-B. Both fragments apparently compete for the same binding factor. The DNA-binding activity was present in nuclear extracts from both presenescent and senescing carnation petals. The upstream DNA fragments that bind this factor have sequence homology with promoter sequences of other ethylene-regulated genes.

Regulation of senescence-related gene expression in carnation flower petals by ethylene.

Ethylene plays a regulatory role in carnation (Dianthus caryophyllus L.) flower senescence. Petal senescence coincides with a burst of ethylene production, is induced prematurely in response to exogenous ethylene, and is delayed by inhibitors of ethylene biosynthesis or action. We have investigated the role of ethylene in the regulation of three senescence-related cDNA clones isolated from a senescent carnation petal library (KA Lawton et al. [1989] Plant Physiol 90: 690-696). Expression of two of the cloned mRNAs in response to ethylene is floral specific, while the expression of another mRNA can be induced in both leaves and flowers exposed to ethylene. Although ethylene induces expression of these mRNAs in petals, message abundance decreases when flowers are removed from ethylene unless an autoenhancement of ethylene production is induced. This indicates continued perception of ethylene is required for their expression. Interruption of ethylene action following the onset of natural senescence results in a substantial decrease in transcript abundance of two of these mRNAs. However, the abundance of another mRNA remains unaffected, indicating this gene responds to temporal cues as well as to ethylene. As flowers age the dosage of exogenous ethylene required to induce expression of the cloned mRNAs decreases, indicating sensitivity to ethylene changes as the tissue matures. Nuclear run-on transcription experiments indicate that relative transcription rates of cloned mRNAs increase in response to exogenous ethylene.

Molecular cloning and characterization of senescence-related genes from carnation flower petals.

The senescence of carnation (Dianthus caryophyllus L.) flower petals is associated with increased production of ethylene which plays an important role in regulating this developmental event. Three senescence-related cDNA clones were isolated from a cDNA library prepared from mRNA isolated from senescing petals. These cDNAs are representative of two classes of mRNAs which increase in abundance in senescing petal tissue. The mRNA for one class is present at low levels during the early stages of development and begins to accumulate in mature petals prior to the increase in ethylene production. The accumulation of this mRNA is reduced, but not eliminated, in petals treated with aminooxyacetic acid, an inhibitor of ethylene biosynthesis, or silver thiosulfate, an ethylene action inhibitor. In contrast, expression of the second class of mRNAs appears to be highly regulated by ethylene. These mRNAs are not detectable prior to the rise in ethylene production and increase in abundance in parallel with the ethylene climacteric. Furthermore, expression of these mRNAs is significantly inhibited by both aminooxyacetic acid and silver thiosulfate. Expression of these mRNAs in vegetative and floral organs was limited to floral tissue, and predominantly to senescing petals.