Insertion mutagenesis and study of transposable elements using a new unstable virescent seedling allele for isolation of haploid petunia lines

The new unstable virescent seedling ( vis* ) allele of a petunia mutant, that has green leaves but white cotyledons with green revertant spots, was used to identify spontaneously occurring haploid petunia lines with active transposable elements. Endogenous transposons were trapped into the single petunia nitrate reductase structural gene ( nia ) using chlorate selection on haploid protoplasts. In two mutant lines, the dTph1 -like transposable element dTph1–3 was inserted at almost the same position but in opposite orientations in the first exon of the nia gene. In a third mutant, a different transposable element was integrated into the fourth exon. This element, called dTph4 , is 787 bp long and has 13 bp terminal inverted repeats of which 12 bp are identical to those of dTph1 . Insertion of dTph1–3 and dTph4 results in an 8 bp duplication of the target site, as already described for dTph1 . In contrast to dTph1 -like elements, dTph4 is present at low copy number in the petunia genome. This can facilitate its use for gene tagging in petunia. The dTph1–3 and dTph4 elements excise frequently, as transposon footprints were found in most of the insertion mutants. The data demonstrate that haploid petunia is an excellent system for gene tagging and for the study of transposable elements.     

A general method to isolate genes tagged by a high copy number transposable element

The Petunia hybrida line W138 contains more than 200 copies of the transposable element dTph1. In W138 progeny these elements give rise to new unstable mutations at high frequency. With the aim of isolating these mutated genes a method was developed to isolate dTph1 flanking sequences unique for mutant plants. This method is based on differential screening of cloned inverse polymerase chain reaction (IPCR) products originating from the mutated plant. It directly yields a probe for the mutated gene which can be used to screen pre-existing cDNA and genomic libraries. This method may be generally applicable to isolate genes tagged by other high copy number transposable elements, like Mutator (Mu) or Dissociation (Ds) in Zea mays.     

Epigenetic interactions among three dTph1 transposons in two homologous chromosomes activate a new excision-repair mechanism in petunia.

Unstable anthocyanin3 (an3) alleles of petunia with insertions of the Activator/Dissociation-like transposon dTph1 fall into two classes that differ in their genetic behavior. Excision of the (single) dTph1 insertion from class 1 an3 alleles results in the formation of a footprint, similar to the "classical" mechanism observed for excisions of maize and snapdragon transposons. By contrast, dTph1 excision and gap repair in class 2 an3 alleles occurs via a newly discovered mechanism that does not generate a footprint at the empty donor site. This novel mechanism depends on the presence of two additional dTph1 elements: one located in cis, 30 bp upstream of the an3 translation start in the same an3 allele, and a homologous copy, which is located in trans in the homologous an3 allele. Absence of the latter dTph1 element causes a heritable suppression of dTph1 excision-repair from the homologous an3 allele by the novel mechanism, which to some extent resembles paramutation. Thus, an epigenetic interaction among three dTph1 copies activates a novel recombination mechanism that eliminates a transposon insertion.     

Stable two-element control of dTph1 transposition in mutator strains of Petunia by an inactive ACT1 introgression from a wild species

The high copy dTph1 transposon system of Petunia (Solanaceae) is one of the most powerful insertion mutagens in plants, but its activity cannot be controlled in the commonly used mutator strains. We analysed the regulation of dTph1 activity by QTL analysis in recombinant inbred lines of the mutator strain W138 and a wild species (P. integrifolia spp. inflata). Two genetic factors were identified that control dTph1 transposition. One corresponded to the ACT1 locus on chromosome I. A second, previously undescribed locus ACT2 mapped on chromosome V. As a 6-cM introgression in W138, the P. i. inflata act1(S6) allele behaved as a single recessive locus that fully eliminated transposition of all dTph1 elements in all stages of plant development and in a heritable fashion. Weak dTph1 activity was restored in act1S6/ACT2S6 double introgression lines, indicating that the P. i. inflata allele at ACT2 conferred a low level of transposition. Thus, the act1S6 allele is useful for simple and predictable control of transposition of the entire dTph1 family when introgressed into an ultra-high copy W138 mutator strain. We demonstrate the use of the ACT1W138/act1S6 allele pair in a two-element dTph1 transposition system by producing 10,000 unique and fixed dTph1 insertions in a population of 1250 co-isogenic lines. This Petunia system produces the highest per plant insertion number of any known two-element system, providing a powerful and logistically simple tool for transposon mutagenesis of qualitative as well as quantitative traits.     

A transgenic dTph1 insertional mutagenesis system for forward genetics in mycorrhizal phosphate transport of Petunia

The active endogenous dTph1 system of the Petunia hybrida mutator line W138 has been used in several forward-genetic mutant screens that were based on visible phenotypes such as flower morphology and color. In contrast, defective symbiotic phosphate (P_i) transport in mycorrhizal roots of Petunia is a hidden molecular phenotype as the symbiosis between plant roots and fungi takes place below ground, and, while fungal colonization can be visualized histochemically, P_i transport and the activity of P_i transporter proteins cannot be assessed visually. Here, we report on a molecular approach in which expression of a mycorrhiza-inducible bi-functional reporter transgene and insertional mutagenesis in Petunia are combined. Bi-directionalization of a mycorrhizal P_i transporter promoter controlling the expression of two reporter genes encoding firefly luciferase and GUS allows visualization of mycorrhiza-specific P_i transporter expression. A population of selectable transposon insertion mutants was established by crossing the transgenic reporter line with the mutator W138, from which the P _i transporter downregulated ( ptd1 ) mutant was identified, which exhibits strongly reduced expression of mycorrhiza-inducible P_i transporters in mycorrhizal roots.     

Molecular characterization of a nonautonomous transposable element (dTph1) of petunia.

An insertion sequence of 283 base pairs has been isolated from the DFR-C gene (dihydroflavonol-4-reductase) of petunia. This insert was found only in a line unstable for the An1 locus (anthocyanin 1, located on chromosome VI) and not in fully pigmented progenitor and revertant lines or in stable white derivative lines. This implies that the An1 locus encodes the DFR-C gene. The unstable An1 system in the line W138 is known to be a two-element system, the autonomous element being located on chromosome I. In the presence of the autonomous element, W138 flowers exhibit a characteristic pattern of red revertant spots and sectors on a white background. In the absence of the autonomous element, the W138 allele gives rise to a stable recessive (white) phenotype. Sequence analysis of progenitor, unstable, and revertant alleles revealed dTph1 to contain perfect terminal inverted repeats of 12 base pairs. In DFR-C, it is flanked by an 8-base pair target site duplication. Sequences homologous to dTph1 are present in at least 50 copies in the line W138. Sequence analysis of An1 revertant alleles indicated that excision, including removal of the target site duplication, is required for reversion to the wild-type phenotype. Derivative stable recessive alleles showed excision of dTph1 and a rearrangement of the target site duplication. dTph1 is the smallest transposable element described to date that is still capable of transposition. The use of dTph1 in tagging experiments and subsequent gene isolation is discussed.     

Promoter activity of a putative pollen monosaccharide transporter in Petunia hybrida and characterisation of a transposon insertion mutant

Summary. For the growth of the male reproductive cells of plants, the pollen, the presence of sufficient sucrose or monosaccharides is of vital importance. From Petunia hybrida a pollen-specific putative monosaccharide transporter designated PMT1 (for petunia monosaccharide transporter) has been identified previously. The present work provides an in-depth analysis and characterisation of PMT1 in the context of pollen development with the GUS reporter gene and an insertion mutant. The promoter of the pollen-specific putative PMT1 gene has been isolated by inverse PCR and sequenced. Analysis of plants transformed with the promoter-GUS fusion confirmed the specificity of this gene, belonging to the late pollen-specific expressed genes. GUS activity was detected even after 24 h of in vitro pollen germination, at the pollen tube tip. To elucidate the importance of PMT1 for gametophyte development and fertilisation, we isolated a mutant plant containing a transposon insertion in the PMT1 gene by the dTph1 transposon-tagging PCR-based assay. The PMT1 mutant contained a dTph1 insertion in position 1474 bp of the transcribing part of the gene, before the last two transmembrane-spanning domains. Analysis of the progeny of the heterozygous mutant after selfing revealed no alterations in pollen viability and fertility. Mature pollen grains of a plant homozygous for the transposon insertion were able to germinate in vitro in a medium containing sucrose, glucose, or fructose, which indicates that PMT1 is not essential for pollen survival. Several explanations for these results are discussed in the present work. Correspondence and reprints (present address): Department of Plant Biology, University of Granada. Fuentenueva s/n, 18001 Granada, Spain. Present address: Swammerdam Institute for Life Sciences, Amsterdam, the Netherlands.     

Analysis by Transposon Display of the behavior of the dTph1 element family during ontogeny and inbreeding of Petunia hybrida

Transposon Display is a high-resolution method that was used here to visualize simultaneously individual members of the dTph1 transposable element family of Petunia hybrida. The method provides a tool for accurate analyses of copy numbers and insertion frequencies, and a means to study the behavior of a family of elements as a whole. Somatic insertion events can be identified and insertion events in a cell of the L2 apical lineage can be distinguished unequivocally from those in a cell outside this lineage. In sublines of the high-copy-number line W137, an average insertion rate equivalent to transposition of 10% of the total number of element copies in each generation was measured, copy number increases of over 20% in four generations were recorded, and element position turnover was analyzed. Insertion events are detected essentially randomly both in time and space. The general applicability of the technique for the analysis of the transpositional behavior of element systems is discussed.     

Genetic characterisation of Act1, the activator of a non-autonomous transposable element from Petunia hybrida

The line W138 of Petunia hybrida has variegated flowers because it is homozygous for the mutable an1-W138 allele. Excision of the element, causing instability, depends on the presence of the activatorAct1. The previously characterised non-autonomous element dTph1 excises from the dfrC gene in response to Act1. This implies that both non-autonomous elements belong to the same transposable element family. In a range of distantly related cultivars we could detect a single functional Act1 element. Linkage analysis for 11 of these lines showed that Act1 was located on chromosome I in all cases, indicating that the element might be fixed in the genome. A group of cultivars that did not exhibit Act1 activity could be traced back to a recent common origin (Rose of Heaven). Cultivars within this group presumably harbour the same inactivated Act1 element. Among the lines tested were 7 lines representing the two species (P. axillaris and P. integrifolia) from which P. hybrida originated. None of these exhibited Act1 activity. We assume that Act1 is present in an inactive state in these lines and that it was activated upon interspecific crossing. In general, lines representing the two parental species and P. hybrida cultivars contain between 5 and 25 dTph1 elements. The lines R27 and W138, however, contain significantly more dTph1 elements (> 50) than all other lines.     

Partial silencing of the NEC1 gene results in early opening of anthers in Petunia hybrida

The NEC1 gene, previously isolated from Petunia hybrida, is expressed at high levels in nectaries, and in a very localized fashion in stamens, particularly in the anther stomium cells and the upper part of the filament. To elucidate the function of the NEC1 gene, co-suppression was employed for down-regulation of NEC1 expression, and transposon insertion mutagenesis was used to knock out the NEC1 function. Among the transgenic plants and plants carrying dTph1 inserted in the NEC1 gene, an "early open anther" phenotype was observed. In this mutant phenotype, the anthers already open in young flower buds (1.8 cm) that still contain immature pollen, resulting in poor pollen quality and impaired pollen release. The results obtained indicate that NEC1 might be involved in the development of stomium cells, which are ruptured during the normal process of anther dehiscence to release mature pollen. Southern analysis revealed the presence of a highly homologous NEC1-like gene, named NEC2, in the P. hybrida genome. The presence of NEC2 was confirmed by segregation analysis and sequencing of genomic clones. The implications of these results and possible reasons why no visually obvious phenotype in nectaries could be produced by co-suppression or transposon insertion mutagenesis are discussed.