Petunia Lines Research Articles

Petunia flower longevity: the role of sensitivity to ethylene

Two petunia (Petunia hybrida L.) lines, differing in their flower longevity, were studied, Similar tendencies were found in the changes of corolla fresh weight, electrolyte leakage and membrane microviscosity over the life spans of the two lines. Ethylene production by flowers of the two lines showed a similar pattern, peaking at 3 nl flower−1 h−1. However, in flowers of the short‐lived line, ethylene production peaked at 6 days of age, but in the long‐lived line, the peak appeared at 10 days of age. A large difference was found in the responsiveness of the flower to ethylene, Flowers of the short‐lived line responded to a similar ethylene by immediate wilting, while those of the long‐lived line responded to a similar ethylene treatment only after two days. Differences in sensitivity to ethylene were also, observed when the flowers were treated continuosly with (aminooxy)acetic acid, which blocks ethylene synthesis. Flowers of both lines responded to ethylene treatment by increased ethylene production to a similar rate. Differential sensitivity to ethylene, independent of ethylene production, seemingly governs flower longevity in the two petunia lines studied.

Differences in DNA-methylation are associated with a paramutation phenomenon in transgenic petunia.

The transgenic petunia line 17-R contains one copy of the maize A1 gene which mediates brick-red pelargonidin pigmentation of the flower. A white derivative, 17-W, was isolated from homozygous progeny of this line in which no pelargonidin pigmentation was observed. In 17-W the 35S promoter driving the A1 gene was hypermethylated, in contrast to its hypomethylated state in 17-R. Progeny plants carrying both the 17-R and 17-W allele did not show the expected A1 phenotype. Predominantly white progeny and variable plants were observed which showed a continuous change in pattern and intensity of pelargonidin pigmentation. This reduction of A1 activity argues for a semidominant effect of the 17-W allele which inhibits the activity of its homologue, 17-R. This system shows striking similarities to some paramutation phenomena in plants which represent a heritable change in gene function of a paramutable allele directed by a paramutagenic homologue. The analysis of the methylation patterns of the A1 alleles suggests that interactions between differentially methylated alleles are responsible for the paramutation-like effect which is mediated by somatic pairing. The analogy of this system to other phenomena based on homology-dependent interlocus trans-inactivation supports the assumption that those may be based on a related mechanism which includes an interaction between ectopic homologues.

Identification of a benzyladenine disaccharide conjugate produced during shoot organogenesis in Petunia leaf explants.

Prior studies of benzyladenine (BA) metabolism in Petunia hybrida Vilm. leaf explants during shoot organogenesis revealed the presence of an abundant unidentified BA conjugate. The BA conjugate, compound C, made up to 39% of the total pool of BA conjugates in two Petunia lines and was associated with an increased shoot organogenic response when compared with a third Petunia line that did not produce any compound C. Structural analysis of compound C utilizing fast atom bombardment mass spectrometry, two methods of carbohydrate analysis, ultraviolet absorption spectra, and Fourier transform infrared spectra identified it as a new cytokinin conjugate, 6-benzylamino-9-[O-glucopyranosyl-(1-->3)-ribofuranosyl]-purine. Based on our prior biological studies and the similarity of this compound to related cytokinin conjugates, this disaccharide cytokinin conjugate may be part of the interconvertible pool of cytokinins active in Petunia shoot organogenesis.

667 (PS 4) IDENTIFICATION OF A NEW BENZYL ADENINE COMPOUNDPRODUCED DURING PETUNIA SHOOT ORGANOGENESIS

Studies of benzyl adenine (BA) uptake and metabolism in Petunia leaf explants during shoot organogenesis revealed a novel BA conjugate. The new conjugate comprised up to 39% of the total pool of BA conjugates in two petunia lines and was associated with an increased shoot organogenic response when compared with a third petunia line which did not produce the conjugate. To identify the new compound, structural analysis was conducted using FAB mass spectrometry, two methods of carbohydrate analysis, UV absorption spectra and FTIR spectra. Experimental results identified the compound as 6-benzylamino-9-[O-glucopyranosyl-(1→3)-ribofuranosyl]-purine, a disaccharide conjugate of BA. Based on biological studies and similarity to other cytokinin conjugates, this disaccharide cytokinin conjugate may be part of the interconvertible pool of cytokinins active in shoot organogenesis.

Detection of an open reading frame related to the CMS-associated urf-s in fertile Petunia lines and species and in other fertile Solanaceae species

In Petunia, a mitochondrial (mt) locus, S-Pcf, has been found to be strongly associated with cytoplasmic male sterility (CMS). The S-Pcf locus consists of three open reading frames (ORF) that are co-transcribed. The first ORF, termed Pcf, contains an unidentified reading frame urf-s that has been detected so far only in sterile Petunia lines and sterile somatic hybrids. In the study described here, a urf-s-related sequence was detected in seven different normal fertile Petunia lines and species as well as in additional members of the Solanaceae family by means of the polymerase chain reaction. The urf-s-related sequence identified in the fertile lines was termed orf152. In Petunia the nucleotide sequence of orf152 was found to be identical to the corresponding part of urf-s. However, the genome organization around orf152 was found to be different from that of urf-s. These results indicate that: (1) at least part of the urf-s sequence is present in fertile lines and species of Petunia and in other Solanaceae species; (2) the orf152 sequence of Petunia is not part of the Pcf ORF. The relevance of these findings to a better understanding of the evolution of the S-pcf locus in (S) cytoplasm in Petunia is discussed.

Phosphatase and ATPase activities in isonuclear lines of cytoplasmic male-sterile and male-fertile petunia

Soluble and membrane-bound fractions of plant leaves, cell suspension cultures and seedlings of petunia were examined for phosphohydrolase activity on p-nitrophenyl phosphate (pNPPase) and adenosine triphosphate (ATPase). One cytoplasmic male-sterile (CMS) and one fertile (F) line was examined for each tissue. Both pNPPase and ATPase exhibited a broad optimal activity between pH 5.5-7.0 for the membrane-bound fraction and between 4.5-7.0 for the soluble fractions. The activity of both were inhibited by divalent ions including Mg(2+). At pH 7.2, the activities on various triphosphonucleotides were similar and they were hydrolyzed by a rate of 20-50% of that of ATP. Significant differences between CMS and F extracts were: (a) higher activities in CMS membranes; (b) lower Ea (energy of activation) values for activities in CMS membrane functions; (c) seedling and cell-culture CMS extracts exhibited a higher sensitivity to high temperature denaturation; (d) the hydrolase activity on monoand triphospho-cytosine compounds was significantly higher in CMS than in F membranes.

The cloning and characterization of a cDNA encoding a cytochrome P450 from the flowers of Petunia hybrida

We have cloned and sequenced a cDNA encoding a P450 protein from petunia flowers. This cDNA is truncated, as it lacks the coding sequence for approximately 80 amino acids in the carboxy-terminal region, as determined by comparing our DNA sequence with that of other P450 genes listed in a genetic sequence database. This protein shows 82% homology with eggplant P450, thus indicating that these two genes form a new P450 gene family, CYP75. The expression of this gene in mutant petunia lines defective in flavonoid synthesis was investigated. Transcripts were detected only in delphinidin producing lines. These results suggest that this gene encodes a flavonoid 3′,5′-hydroxylase which produces delphinidin.

Interaction of the mitochondrial S-Pcf locus for cytoplasmic male sterility in Petunia with multiple fertility-restoration genes in somatic hybrid plants

The Cytoplasmic Male Sterility (CMS)-associated region in Petunia, the S-Pcf locus, was defined by the analysis of recombinant mitochondrial genomes of somatic hybrid plants resulting from a fusion of protoplasts from CMS and fertile lines. The presence of the S-Pcf locus was shown to correlate with the CMS trait in stable somatic hybrids and in other CMS Petunia lines. A small population of unstable, sterile somatic hybrids was also generated in this fusion, most of which underwent cytoplasmic segregation in subsequent generations. Stable revertants of such sterile somatic hybrids were shown to lose the S-Pcf locus. In this paper we present a molecular and genetic analysis of unstable progenies of an unstable, sterile somatic hybrid plant derived from the same fusion experiment. Both male-sterile and fertile progenies of this somatic hybrid plant have shown continuous segregation of fertile and male-sterile progenies. All segregants in this line contained, and transcribed, the S-Pcf locus. Genetic analysis indicated the presence of various levels of multiple nuclear fertility-restoration genes in this group of progenies. These findings consolidate the association between the S-Pcf locus and the CMS trait in Petunia. It also shows that the restoration of fertility by the multiple nuclear gene system does not affect the transcription of the S-Pcf locus and that the presence of an intact S-Pcf locus is necessary in order to maintain the potential sterility in the cytoplasm.

Premature dissolution of the microsporocyte callose wall causes male sterility in transgenic tobacco.

Male sterility in a petunia cytoplasmic male sterile line has been attributed to the early appearance of active callase, a beta-1,3-glucanase, in the anther locule. This leads to premature dissolution of the callose walls surrounding the microsporogenous cells. We have mimicked this aspect of the petunia line in transgenic tobacco by engineering the secretion of a modified pathogenesis-related vacuolar beta-1,3-glucanase from the tapetum prior to the appearance of callase activity in the locule. Plants expressing the modified glucanase from tapetum-specific promoters exhibited reduced male fertility, ranging from complete to partial male sterility. Callose appearance and distribution are normal in the male sterile transgenic plants up to prophase I, whereupon callose is prematurely degraded. Meiosis and cell division occur normally. The resultant microspores have an abnormally thin cell wall that lacks sculpturing. The tapetum shows hypertrophy. Male sterility is probably caused by bursting of the aberrant microspores at a time corresponding to microspore release. These results demonstrate that premature callose degradation is sufficient to cause male sterility and suggest that callose is essential for the formation of a normal microspore cell wall.

Differences in amino acid transport in isonuclear lines of cytoplasmic male-sterile and male-fertile petunia

Two pairs of isonuclear lines of cytoplasmic male-sterile (CMS) and fertile (F) petunia cells grown in suspension culture in the presence or absence of amino acid sources were examined for uptake of 11 amino acids and adenosine. Cells from CMS lines exhibited a significant lower rate of uptake than F cells. These differences, for various amino acids, are a result of lower affinity (high Km) values and of lower maximal velocities. Although the uptake of most of the amino acids examined was affected by the availability of energy in the cell, the differences in uptake seem to be less dependent on the energy status of the cell.

Petunia plants escape from negative selection against a transgene by silencing the foreign DNA via methylation.

Transgenic Petunia hybrida clones harbouring the T-DNA gene 2 of Agrobacterium tumefaciens were used to test a strategy for the trapping of plant transposable elements. In the Petunia line used, floral variegation is due to the presence of the non-autonomous transposable element dTph1 at the An1 locus. The gene 2 product converts the auxin precursor indole-3-acetamide and its analogue 1-naphthalene acetamide into the active auxins indole-3-acetic acid and 1-naphthalene acetic acid. Plant cells that express gene 2 can use a low concentration of the precursors as auxins and become sensitive to the toxicity of high concentrations of these compounds. By selecting protoplast-derived microcalli or seedlings able to grow on medium with high precursor concentrations, variant plants were obtained in which gene 2 was no longer expressed. Southern analysis, using gene 2-specific probes, revealed that in one variant the T-DNA was deleted. For 30 other variants no alteration in gene 2 structure was observed, indicating that transposable element insertion was not responsible for the inactivation of gene 2. Analysis with restriction enzymes allowing discrimination between methylated or non-methylated DNA sequences showed that the inactivated gene 2 sequences were methylated. Addition of the in vivo methylation inhibitor 5-azacytidine to the medium led to reactivation of gene 2 expression in some of the variants. These observations demonstrated that reversible DNA methylation was the main cause of silencing of gene 2 in this system.

Comparison of Benzyl Adenine Metabolism in Two Petunia hybrida Lines Differing in Shoot Organogenesis

The uptake and metabolism of the cytokinin benzyl adenine (BA) was compared in two lines of Petunia hybrida Vilm. differing in their shoot organogenic response. Leaf transfer experiments using shoot induction medium containing 4.4 micromolar BA showed that leaf explants from petunia line St40 required a shoot induction period of 6 to 10 days for commitment to shoot organogenesis; whereas leaf explants from petunia TLV1 required 12 to 28 days. The short induction period of petunia St40 and the higher organogenic response was positively associated with a threefold higher absorption of BA from the medium, an increased BA ribotide metabolite pool, the presence of BA within the explant during the shoot induction period, and the production of an unidentified metabolite C. However, the study of petunia TLV1 leaf explants showed that neither BA nor metabolite C are required during the shoot induction period for eventual shoot development. The longer shoot induction period of TLV1 was associated with low BA uptake during 24 days, a decreasing ribotide metabolite pool, the absence of benzyl adenosine triphosphate and metabolite C throughout the study, and the absence of BA within the explant during the shoot induction period. Differences in the shoot organogenic response of these related plant lines have been shown to be associated with differences in exogenous cytokinin uptake and the subsequent metabolism of that hormone.

A single nuclear gene specifies the abundance and extent of RNA editing of a plant mitochondrial transcript.

A number of cytosines are altered to be recognized as uridines in transcripts of the NADH-dehydrogenase subunit 3 (nad3) gene in the mitochondria of the higher plant Petunia hybrida. Here we show that the extent of editing for three of the edit sites, all of which change the encoded amino acid, varies between different Petunia lines. Genetic analysis indicates that a single nuclear gene is responsible for this variation. Interestingly, according to RNA blot hybridization analysis, RNA editing extent and transcript abundance are correlated. This observation is consistent with the hypothesis that RNA editing is a post-transcriptional event.

The male sterility-associated pcf gene and the normal atp9-1 gene in Petunia are located on different mitochondrial DNA molecules.

A mitochondrial DNA (mtDNA) region termed the S-pcf locus has previously been correlated with cytoplasmic male sterility (CMS) in Petunia. In order to understand the relationship of the S-pcf locus to homologous sequences found elsewhere in mtDNAs of both CMS and fertile lines, the structure of the mitochondrial genome of CMS Petunia line 3688 was determined by cosmid walking. The S-pcf locus, which includes the only copies of genes for NADH dehydrogenase subunit 3 (nad3) and small ribosomal subunit protein 12 (rps12) was found to be located on a circular map of 396 kb, while a second almost identical circular map of 407 kb carries the only copies of the genes for 18S and 5S rRNA (rrn18 and rrn5), the only copy of a conserved unidentified gene (orf25), and the only known functional copy of atp9. Three different copies of a recombination repeat were found in six genomic environments, predicting sub-genomic circles of 277, 266 and 130 kb. The ratio of atp9 to S-pcf mtDNA sequences was approximately 1.5 to 1, indicating that sub-genomic molecules carrying these genes differ in abundance. Comparison of the mtDNA organization of the CMS line with that of the master circle of fertile Petunia line 3704 reveals numerous changes in order and orientation of ten different sectors.

Transcription of the Petunia mitochondrial CMS-associated Pcf locus in male sterile and fertility-restored lines

Transcripts of the Petunia mitochondrial cytoplasmic male sterility (CMS)-associated S-Pcf locus, which consists of three co-transcribed genes (pcf, NADH dehydrogenase subunit 3, and ribosomal protein S12), have been characterized in reproductive tissues of CMS and fertility restored (Rf) Petunia lines by nuclease protection experiments and by RNA blot hybridization. Three 5' transcript termini have been previously described. Two 3' transcript termini and an additional S-Pcf locus transcript have now been identified. The relative abundance of the three 5' transcript termini is influenced by the presence of the nuclear Rf gene. A decrease in the abundance of the -121 5' transcript terminus relative to the -266 and -522 termini is consistently seen in Petunia lines which are restored to fertility by the Rf gene, compared to CMS Petunia lines. An additional transcript with a 5' terminus within the urf-s region of pcf is much more abundant in immature bud and anther tissue than in leaf or suspension cells. The total abundance of pcf transcripts varies greatly between plants of different nuclear backgrounds which lack the nuclear Rf allele, indicating that other nuclear genes also influence expression of the S-Pcf locus.

Regulation and manipulation of flavonoid gene expression in anthers of petunia: the molecular basis of the Po mutation.

Molecular mechanisms governing development of the male reproductive organs of flowers, the anthers, are largely unknown. In this article, we report on the investigation of the molecular basis of a mutation involving the expression of a gene encoding the flavonoid biosynthesis enzyme chalcone flavanone isomerase (CHI) in anthers of petunia. In petunia, the gene Po regulates the expression of CHI in anthers: PoPo petunia lines contain CHI enzyme activity in petals and anthers, whereas popo lines contain the CHI enzyme only in petals but not in anthers. As a result of the Po mutation, the substrate of CHI accumulates and therefore the pollen of a popo line are yellow or greenish. The genome of petunia contains two chi genes, chiA and chiB. In a restriction fragment length polymorphism analysis, a 100% linkage was observed between Po and chiA. This result suggested that Po is identical to chiA and that Po is not a regulatory gene of chiA. Introduction of a chiA gene isolated from a PoPo line into a popo line resulted in a complementation of the mutation that was directly visible because the pollen color shifted from yellow to white. This proved that chiA and Po are identical. Because chiA encodes a functional CHI enzyme in flower petals of a popo line, we propose that the Po mutation is a mutation in the regulatory region of chiA abolishing chiA promoter activity in anthers but not in corollas. This change in anther color is a fine illustration of how floral pigmentation can be manipulated in a predictable way and suggests the use of CHI as a visible marker.

Sequence Requirements of the 5-Enolpyruvylshikimate-3-phosphate Synthase 5[prime]-Upstream Region for Tissue-Specific Expression in Flowers and Seedlings.

We have analyzed expression from deletion derivatives of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) 5[prime]-upstream region in transgenic petunia flowers and seedlings. In seedlings, expression was strongest in root cortex cells and in trichomes. High-level expression in petals and in seedling roots was conferred by large (>500 base-pair) stretches of sequence, but was lost when smaller fragments were analyzed individually. This apparent requirement for extensive sequence suggests that combinations of cis-elements that are widely separated control tissue-specific expression from the EPSPS promoter. We have also used the high-level, petal-specific expression of the EPSPS promoter to change petal color in two mutant petunia lines.

Attempts to detect extra genomial factors in cytoplasmic male-sterile petunia lines

In the present study we examined the possibility that viruses, viroids or dsRNA are associated with cytoplasmic male sterile (cms) petunia. The assumption was made that if viruses or viroids were present, the treatments for elimination of viruses and viroids would produce "healthy" fertile plants. Male sterile plants were subjected to heat and cold treatments for 10 weeks and/ or for 5 months, after which apical meristems were isolated and cultured with the addition of antiviral factors. The mother plants, the regenerated plants and their progeny were sterile. These treatments did not affect sterility in sterile plants or the fertility of fertile plants. No dsRNA was found in cms petunia by gel electrophoresis. Thus, our data suggest that there are no viruses, viroids or dsRNA associated with cms petunia. Our data are in agreement with recent data, which suggests that the mitochondrial DNA is the site of the cytoplasmic male sterile gene in petunia.

A functional mitochondrial ATP synthase proteolipid gene produced by recombination of parental genes in a petunia somatic hybrid.

A novel ATP synthase subunit 9 gene (atp9) was identified in the mitochondrial genome of a Petunia somatic hybrid line (13-133) which was produced from a fusion between Petunia lines 3688 and 3704. The novel gene was generated by intergenomic recombination between atp9 genes from the two parental plant lines. The entire atp9 coding region is represented on the recombinant gene. Comparison of gene sequences indicate that the 5' transcribed region is contributed by an atp9 gene from 3704 and the 3' transcribed region is contributed by an atp9 gene from 3688. The recombinant atp9 gene is transcriptionally active. The location of the 5' and 3' transcript termini are conserved with respect to the parental genes, resulting in the production of hybrid transcripts.

Recombination between parental mitochondrial DNA following protoplast fusion can occur in a region which normally does not undergo intragenomic recombination in parental plants

A novel restriction fragment, which was not present in either parent's mitochondrial DNA, was cloned from the mitochondrial genome of a somatic hybrid Petunia line. This fragment resulted from interspecific recombination between homologous mitochondrial DNA regions of the parental plants, Petunia line 3704 and line 3688. Hybridization with a cloned Petunia atp9 gene revealed that the regions involved in recombination carry atp9 coding sequences. Intragenomic recombination within the parental mitochondrial genomes was not detected between the atp9 regions which recombined following protoplast fusion.