William R. Woodson

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.

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.

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 [15] 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 carantion 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 [5]. 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.

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.

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 at 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.

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 pb sufficient to encode a protein of 82.8 kDa. No significant homology at the DNA or protein levels was found with other known gene. 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.

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.

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.

Transformation of carnation by microprojectile bombardment

Abstract Transgenic carnation (Dianthus caryophyllus L.) plants were produced by microprojectile bombardment of highly regenerative stem segments. A two-step regeneration procedure based on the use of two different cytokinins—6-benzylaminopurine and thidiazuron—was employed for the production of adventitious shoots from stem segments. The size of the original stem was found to affect the regeneration efficiency of stem segments: the highest efficiency of adventitious shoot regeneration was obtained with segments originating from stems with two mature leaves, as compared to those with four, six or eight mature leaves. The tissue culture procedure was shown to be suitable for a number of standard and spray cultivars. Bombardment of cultivar “White Sim” stem segments was performed with a plasmid containing uidA and bar genes encoding β-glucuronidase and phosphinothricin-acetyltransferase, respectively. Transformation frequency was determined, based on the transient expression of uidA in stem segments. Following selection in the presence of the herbicide bialaphos, about 70 plantlets per 100 stem segments were recovered. Upon analysis, about 3% of these recovered plantlets exhibited strong stable uidA expression throughout the plant. Presence of the bar gene in plants stably expressing uidA was confirmed by Southern blot analysis.