Evidence that intragenic recombination contributes to allelic diversity of the S-RNase gene at the self-incompatibility (S) locus in Petunia inflata.
For Solanaceae type self-incompatibility, discrimination between self and nonself pollen by the pistil is controlled by the highly polymorphic S-RNase gene. To date, the mechanism generating the allelic diversity of this gene is largely unknown. Natural populations offer a good opportunity to address this question because they likely contain different alleles that share recent common progenitors. We identified 19 S haplotypes from a natural population of Petunia inflata in Argentina, used reverse transcriptase-polymerase chain reaction to obtain cDNAs for 15 alleles of the S-RNase gene, and sequenced all the cDNAs. Phylogenetic studies revealed that five of these alleles and two previously identified alleles form a major clade, and that the 5' region of S(19) allele was derived from an ancestor allele closely related to S(2), whereas its 3' region was derived from an ancestor allele closely related to S(8). A similar evolutionary relationship was found among S(3), S(12), and S(15) alleles. These findings suggest that intragenic recombination contributed to the generation of the allelic diversity of the S-RNase gene. Two additional findings emerged from the sequence comparisons. First, the nucleotide sequence of the S(1) allele identified in this work is completely identical to that of the previously identified S(1) allele of a different origin. Second, in the two hypervariable regions HVa and HVb, thought to be involved in determining S allele specificity, S(6) and S(9) alleles differ only by four nucleotides, all in HVb, resulting in two amino acid differences. The implications of these findings are discussed. (+info)
Petunia Ap2-like genes and their role in flower and seed development.
We have isolated three Apetala2 (Ap2)-like genes from petunia and studied their expression patterns by in situ hybridization. PhAp2A has a high sequence similarity to the A function gene Ap2 from Arabidopsis and a similar expression pattern during flower development, suggesting that they are cognate orthologs. PhAp2B and PhAp2C encode for AP2-like proteins that belong to a different subgroup of the AP2 family of transcription factors and exhibit divergent, nearly complementary expression patterns during flower development compared with PhAp2A. In contrast, all three PhAp2 genes are strongly expressed in endosperm. The phenotype of the petunia A-type mutant blind cannot be attributed to mutations in the petunia Ap2 homologs identified in this study, and reverse genetics strategies applied to identify phap2a mutants indicate that PhAp2A might not be essential for normal perianth development in petunia. Nevertheless, we show that PhAp2A is capable of restoring the homeotic transformations observed in flowers and seed of the ap2-1 mutant of Arabidopsis. Although the interspecific complementation proves that PhAp2A encodes a genuine Ap2 ortholog from petunia, additional factors may be involved in the control of perianth identity in this species. (+info)
Silencing of the tapetum-specific zinc finger gene TAZ1 causes premature degeneration of tapetum and pollen abortion in petunia.
TAZ1 (TAPETUM DEVELOPMENT ZINC FINGER PROTEIN1; renamed from PEThy; ZPT3-2) cDNA was first isolated as an anther-specific cDNA from petunia. Here, we report a functional characterization that includes analysis of spatial and temporal expression profiles and examination of anther phenotypes in TAZ1-silenced plants. TAZ1 showed a biphasic expression pattern. In the premeiotic phase, TAZ1 transcripts were found to accumulate in all cell types of the anther except the tapetum and gametophytic tissues, whereas the postmeiotic phase of anther development was characterized by expression exclusively in the tapetum. Silencing of TAZ1 by cosuppression resulted in aberrant development and precocious degeneration of the tapetum, followed by extensive microspore abortion that started soon after their release from pollen tetrads. A few pollen grains that survived showed reduced flavonol accumulation, defects in pollen wall formation, and poor germination rates. This study demonstrates an essential role for TAZ1 in the postmeiotic phase of tapetum development. (+info)
CYP92B1, A cytochrome P450, expressed in petunia flower buds, that catalyzes monooxidation of long-chain fatty acids.
In higher plants, long-chain fatty acid hydroperoxides are intermediates in the synthesis of a diverse group of bioactive compounds. We used the reverse trascriptase-polymerase chain reaction to isolate a gene responsible for the oxidization of fatty acids from Petunia hybrida. A P450 cDNA not isolated earlier, CYP92B1, contained an open reading frame predicted to encode a polypeptide consisting of 510 amino acid residues. The transcript of the cyp92B1 gene was expressed at a high level in the early stage of flower development. CYP92B1 cDNA was expressed in a yeast, Saccharomyces cerevisiae, and recombinant yeast microsomes containing CYP92B1, a hemoprotein, metabolized lauric acid, linoleic acid, and linolenic acid. (+info)
Proteomics of light-harvesting proteins in different plant species. Analysis and comparison by liquid chromatography-electrospray ionization mass spectrometry. Photosystem I.
The light-harvesting proteins (Lhca) of photosystem I (PSI) from four monocot and five dicot species were extracted from plant material, separated by reversed-phase high-performance liquid chromatography (HPLC) and subsequently identified on the basis of their intact molecular masses upon on-line hyphenation with electrospray ionization mass spectrometry. Although their migration behavior in gel electrophoresis was very similar, the elution times among the four antenna types in reversed-phase-HPLC differed significantly, even more than those observed for the light-harvesting proteins of photosystem II. Identification of proteins is based on the good agreement between the measured intact molecular masses and the values calculated on the basis of their nucleotide-derived amino acid sequences, which makes the intact molecular masses applicable as intact mass tags. These values match excellently for Arabidopsis, most probably because of the availability of high-quality DNA sequence data. In all species examined, the four antennae eluted in the same order, namely Lhca1 > Lhca3 > Lhca4 > Lhca2. These characteristic patterns enabled an unequivocal assignment of the proteins in preparations from different species. Interestingly, in all species examined, Lhca1 and Lhca2 were present in two or three isoforms. A fifth antenna protein, corresponding to the Lhca6 gene, was found in tomato (Lycopersicon esculentum). However PSI showed a lower heterogeneity than photosystem II. In most plant species, Lhca2 and Lhca4 proteins are the most abundant PSI antenna proteins. The HPLC method used in this study was found to be highly reproducible, and the chromatograms may serve as a highly confident fingerprint for comparison within a single and among different species for future studies of the PSI antenna. (+info)
cDNA cloning, heterologous expressions, and functional characterization of malonyl-coenzyme a:anthocyanidin 3-o-glucoside-6"-o-malonyltransferase from dahlia flowers.
In the flowers of important ornamental Compositae plants, anthocyanins generally carry malonyl group(s) at their 3-glucosyl moiety. In this study, for the first time to our knowledge, we have identified a cDNA coding for this 3-glucoside-specific malonyltransferase for anthocyanins, i.e. malonyl-coenzyme A:anthocyanidin 3-O-glucoside-6"-O-malonyltransferase, from dahlia (Dahlia variabilis) flowers. We isolated a full-length cDNA (Dv3MaT) on the basis of amino acid sequences specifically conserved among anthocyanin acyltransferases of the versatile plant acyltransferase family. Dv3MaT coded for a protein of 460 amino acids. Quantitative real-time PCR analyses of Dv3MaT showed that the transcript was present in accordance with the distribution of 3MaT activities and the anthocyanin accumulation pattern in the dahlia plant. The Dv3MaT cDNA was expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity and characterized. The recombinant Dv3MaT catalyzed the regiospecific transfer of the malonyl group from malonyl-coenzyme A (K(m), 18.8 microM) to pelargonidin 3-O-glucoside (K(m), 46.7 microM) to produce pelargonidin 3-O-6"-O-malonylglucoside with a k(cat) value of 7.3 s(-1). The other enzymatic profiles of the recombinant Dv3MaT were closely related to those of native anthocyanin malonyltransferase activity in the extracts of dahlia flowers. Dv3MaT cDNA was introduced into petunia (Petunia hybrida) plants whose red floral color is exclusively provided by cyanidin 3-O-glucoside and 3,5-O-diglucoside. Thirteen transgenic lines of petunia were found to produce malonylated products of these anthocyanins (11-63 mol % of total anthocyanins in the flower). The spectral stability of cyanidin 3-O-6"-O-malonylglucoside at the pHs of intracellular milieus of flowers was significantly higher than that of cyanidin 3-O-glucoside. Moreover, 6"-O-malonylation of cyanidin 3-O-glucoside effectively prevented the anthocyanin from attack of beta-glucosidase. These results suggest that malonylation should serve as a strategy for pigment stabilization in the flowers. (+info)
Isolation and properties of floral defensins from ornamental tobacco and petunia.
The flowers of the solanaceous plants ornamental tobacco (Nicotiana alata) and petunia (Petunia hybrida) produce high levels of defensins during the early stages of development. In contrast to the well-described seed defensins, these floral defensins are produced as precursors with C-terminal prodomains of 27 to 33 amino acids in addition to a typical secretion signal peptide and central defensin domain of 47 or 49 amino acids. Defensins isolated from N. alata and petunia flowers lack the C-terminal domain, suggesting that it is removed during or after transit through the secretory pathway. Immunogold electron microscopy has been used to demonstrate that the N. alata defensin is deposited in the vacuole. In addition to the eight canonical cysteine residues that define the plant defensin family, the two petunia defensins have an extra pair of cysteines that form a fifth disulfide bond and hence define a new subclass of this family of proteins. Expression of the N. alata defensin NaD1 is predominantly flower specific and is most active during the early stages of flower development. NaD1 transcripts accumulate in the outermost cell layers of petals, sepals, anthers, and styles, consistent with a role in protection of the reproductive organs against potential pathogens. The floral defensins inhibit the growth of Botrytis cinerea and Fusarium oxysporum in vitro, providing further support for a role in protection of floral tissues against pathogen invasion. (+info)
The MADS box gene FBP2 is required for SEPALLATA function in petunia.
The ABC model, which was accepted for almost a decade as a paradigm for flower development in angiosperms, has been subjected recently to a significant modification with the introduction of the new class of E-function genes. This function is required for the proper action of the B- and C-class homeotic proteins and is provided in Arabidopsis by the SEPALLATA1/2/3 MADS box transcription factors. A triple mutant in these partially redundant genes displays homeotic conversion of petals, stamens, and carpels into sepaloid organs and loss of determinacy in the center of the flower. A similar phenotype was obtained by cosuppression of the MADS box gene FBP2 in petunia. Here, we provide evidence that this phenotype is caused by the downregulation of both FBP2 and the paralog FBP5. Functional complementation of the sepallata mutant by FBP2 and our finding that the FBP2 protein forms multimeric complexes with other floral homeotic MADS box proteins indicate that FBP2 represents the same E function as SEP3 in Arabidopsis. (+info)