Petunia Mutant Research Articles

Cell layer-specific expression of the homeotic MADS-box transcription factor PhDEF contributes to modular petal morphogenesis in petunia.

Floral homeotic MADS-box transcription factors ensure the correct morphogenesis of floral organs, which are organized in different cell layers deriving from distinct meristematic layers. How cells from these distinct layers acquire their respective identities and coordinate their growth to ensure normal floral organ morphogenesis is unresolved. Here, we studied petunia (Petunia × hybrida) petals that form a limb and tube through congenital fusion. We identified petunia mutants (periclinal chimeras) expressing the B-class MADS-box gene DEFICIENS in the petal epidermis or in the petal mesophyll, called wico and star, respectively. Strikingly, wico flowers form a strongly reduced tube while their limbs are almost normal, while star flowers form a normal tube but greatly reduced and unpigmented limbs, showing that petunia petal morphogenesis is highly modular. These mutants highlight the layer-specific roles of PhDEF during petal development. We explored the link between PhDEF and petal pigmentation, a well-characterized limb epidermal trait. The anthocyanin biosynthesis pathway was strongly downregulated in star petals, including its major regulator ANTHOCYANIN2 (AN2). We established that PhDEF directly binds to the AN2 terminator in vitro and in vivo, suggesting that PhDEF might regulate AN2 expression and therefore petal epidermis pigmentation. Altogether, we show that cell layer-specific homeotic activity in petunia petals differently impacts tube and limb development, revealing the relative importance of the different cell layers in the modular architecture of petunia petals.

Physiological changes besides the enhancement of pigmentation in Petunia hybrida caused by overexpression of PhAN2, an R2R3-MYB transcription factor.

Ectopic expression of PhAN2 in vegetative tissue can improve regeneration and adventitious rooting but inhibit axillary bud outgrowth of petunia, while overexpression specifically in flowers could shorten longevity. Anthocyanin 2 has been only treated as a critical positive regulation factor of anthocyanin biosynthesis in petunia flowers. To determine if this gene had other functions in plant growth, we overexpressed this gene in an an2 mutant petunia cultivar driven by promoters with different strengths or tissue specificity. Various physiological processes of transformants in different growth stages and environments were analyzed. Besides the expected pigmentation improvement in different tissues, the results also showed that ectopic expression of AN2 could improve the regeneration skill but inhibit the axillary bud germination of in vitro plants. Moreover, the rooting ability of shoot tips of transformants was significantly improved, while some transgenic lines' flower longevity was shortened. Gene expression analysis showed that the transcripts level of AN2, partner genes anthocyanin 1 (AN1), anthocyanin 11 (AN11), and target gene dihydroflavonol 4-reductase (DFR) was altered in the different transgenic lines. In addition, ethylene biosynthesis-related genes 1-aminocyclopropane-1-carboxylic acid synthase (ACS1) and ACC oxidase (ACO1) were upregulated in rooting and flower senescence processes but at different time points. Overall, our data demonstrate that the critical role of this AN2 gene in plant growth physiology may extend beyond that of a single activator of anthocyanin biosynthesis.

Petunia PLEIOTROPIC DRUG RESISTANCE 1 Is a Strigolactone Short-Distance Transporter with Long-Distance Outcomes.

Phytohormones of the strigolactone (SL) family have been characterized as negative regulators of lateral bud outgrowth and triggers of symbioses between plants and mycorrhizal fungi. SLs and their precursors are synthesized in root tips as well as along shoot and root vasculature; they either move shoot-wards and regulate plant architecture or are exuded from roots into the soil to establish mycorrhizal symbiosis. Owing to the difficulty in quantification of SL in shoot tissues because of low abundance, it is not yet clear how SL distribution in plants is regulated at short- and long-distances from SL biosynthetic and target tissues. To address this question, we grafted wild-type scions and rootstocks from different petunia mutants for SL biosynthesis/transport and investigated SL activity by quantifying lateral bud outgrowth in the main shoot. Based on these results, we show that (i) the previously reported petunia SL transporter PLEIOTROPIC DRUG RESISTANCE 1 (PDR1) directly accounts for short-distance SL transport and (ii) long-distance transport of SLs seems to be partially and not directly dependent on PDR1. These data suggest that the root-to-shoot transport of SLs occurs either via the vasculature bundle through transporters other than PDR1 or involves SL precursors that are not substrates of PDR1.

Значение вторичных метаболитов в формировании устойчивости к мучнистой росе деревьев 16-летних культур дуба черешчатого

Значение вторичных метаболитов в формировании устойчивости к мучнистой росе деревьев 16-летних культур дуба черешчатого

Evolution of tonoplast P-ATPase transporters involved in vacuolar acidification.

Petunia mutants (Petunia hybrida) with blue flowers defined a novel vacuolar proton pump consisting of two interacting P-ATPases, PH1 and PH5, that hyper-acidify the vacuoles of petal cells. PH5 is similar to plasma membrane H(+) P3A -ATPase, whereas PH1 is the only known eukaryoticP3B -ATPase. As there were no indications that this tonoplast pump is widespread in plants, we investigated the distribution and evolution of PH1 and PH5. We combined database mining and phylogenetic and synteny analyses of PH1- and PH5-like proteins from all kingdoms with functional analyses (mutant complementation and intracellular localization) of homologs from diverse angiosperms. We identified functional PH1 and PH5 homologs in divergent angiosperms. PH5 homologs evolved from plasma membrane P3A -ATPases, acquiring an N-terminal tonoplast-sorting sequence and new cellular function before angiosperms appeared. PH1 is widespread among seed plants and related proteins are found in some groups of bacteria and fungi and in one moss, but is absent in most algae, suggesting that its evolution involved several cases of gene loss and possibly horizontal transfer events. The distribution of PH1 and PH5 in the plant kingdom suggests that vacuolar acidification by P-ATPases appeared in gymnosperms before flowers. This implies that, next to flower color determination, vacuolar hyper-acidification is required for yet unknown processes.

FUNCTIONAL CHARACTERIZATION OF DENDROBIUM AND ONCIDIUM DFR IN PETUNIA HYBRIDA MODEL

ISHS XXVII International Horticultural Congress - IHC2006: International Symposium on Plant Biotechnology: From Bench to Commercialization FUNCTIONAL CHARACTERIZATION OF DENDROBIUM AND ONCIDIUM DFR IN PETUNIA HYBRIDA MODEL

A petunia mutant affected in intracellular accommodation and morphogenesis of arbuscular mycorrhizal fungi

The regulation of the arbuscular mycorrhizal (AM) symbiosis is largely under the control of a genetic programme of the plant host. This programme includes a common symbiosis signalling pathway that is shared with the root nodule symbiosis. Whereas this common pathway has been investigated in detail, little is known about the mycorrhiza-specific regulatory steps upstream and downstream of the common pathway. To get further insight in the regulation of the AM symbiosis, a transposon-mutagenized population of Petunia hybrida was screened for mutants with defects in AM development. Here, we describe a petunia mutant, penetration and arbuscule morphogenesis1 (pam1), which is characterized by a strong decrease in colonization by three different AM fungi. Penetrating hyphae are frequently aborted in epidermal cells. Occasionally the fungus can progress to the cortex, but fails to develop arbuscules. The resulting hyphal colonization of the cortex in mutant plants does not support symbiotic acquisition of phosphate and copper by the plant. Expression analysis of three petunia orthologues of the common SYM genes LjPOLLUX, LjSYMRK and MtDMI3 indicates that pam1 is not mutated in these genes. We conclude that the PAM1 gene may play a specific role in intracellular accommodation and morphogenesis of the fungal endosymbiont.

Shoot meristem maintenance is controlled by a GRAS-gene mediated signal from differentiating cells.

Plant shoot development depends on the perpetuation of a group of undifferentiated cells in the shoot apical meristem (SAM). In the Petunia mutant hairy meristem (ham), shoot meristems differentiate postembryonically as continuations of the subtending stem. HAM encodes a putative transcription factor of the GRAS family, which acts non-cell-autonomously from L3-derived tissue of lateral organ primordia and stem provasculature. HAM acts in parallel with TERMINATOR (PhWUSCHEL) and is required for continued cellular response to TERMINATOR and SHOOTMERISTEMLESS (PhSTM). This reveals a novel mechanism by which signals from differentiating tissues extrinsically control stem cell fate in the shoot apex.

FLOOZY of petunia is a flavin mono-oxygenase-like protein required for the specification of leaf and flower architecture.

The mechanisms that determine the relative positions of floral organs, and thereby their numbers, is a poorly understood aspect of flower development. We isolated a petunia mutant, floozy (fzy), in which the formation of floral organ primordia in the outermost three floral whorls and one of the two bracts at the base of the flower is blocked at an early stage. In addition, fzy mutants fail to generate secondary veins in leaves and bracts and display a decreased apical dominance in the inflorescence. FZY encodes an enzyme with homology to flavin mono-oxygenases and appears to be the ortholog of YUCCA genes of Arabidopsis. FZY is expressed in young leafs and bracts and in developing flowers. In young floral meristems FZY is expressed in the center of the meristem dome and, later, expression becomes localized on the flanks of the initiating petal and stamen primordia and at several sites in maturing anthers and carpels. These findings indicate that FZY is involved in synthesizing a signaling compound that is required for floral organ initiation and specification of the vascularization pattern in leaves. Although fzy mutants contain normal auxin levels, ectopic expression of FZY results in excessive auxin accumulation, suggesting that the signaling compound is auxin.

Flavonoid gene expression and UV photoprotection in transgenic and mutant Petunia leaves

The effects of UVB radiation on plant growth rate, gene expression and flavonoid content in wild-type, and in transgenic and mutant F3′H deficient Petunia lines have been studied for the first time. In wild-type Petunia, UVB induced an increase in total levels of flavonols and this was due to an up-regulation of several genes in the phenylpropanoid pathway. Furthermore, UVB induced a higher rate of production of dihydroxylated flavonols than mono-hydroxylated equivalents. Thus, the ratio of quercetin ( ortho-dihydroxylated) to kaempferol (monohydroxylated) increased. In the F3H deficient mutant line, increasing UVB resulted in up-regulation of all of the basic flavonoid biosynthetic genes. Total flavonoids increased to levels significantly higher than in control plants, and the predominant flavonoid was kaempferol. The leaves of these plants grew at a significantly slower rate than comparably treated wild-type plants under ambient or enhanced UVB radiation. This suggests that the predominance of quercetin in the wild-type confers a protective advantage that is not matched in the mutant, even with higher overall flavonoid levels. In contrast, the antisense F3H construct produced an unexpected down-regulation of C4H, CHS and CHI transcription. This resulted in lower total flavonoid production in these plants. The growth rate of these plants was not impaired in UVB to a statistically significant extent, and the Q:K ratio did not change with increasing UVB radiation. This investigation has established a likely correlation between the effect of UVB on plant growth rate, the level of activity of the F3′H gene, and the proposed photoprotection afforded by an increased Q:K ratio.

White anther: A petunia mutant that abolishes pollen flavonol accumulation, induces male sterility, and is complemented by a chalcone synthase transgene

A mutation in an inbred line of petunia (Petunia hybrida) produces a reduction in the deep-purple corolla pigmentation and changes the anther color from yellow to white. In addition, the mutant, designated white anther (wha), is functionally male sterile. The inability of pollen from wha plants to germinate in vitro provides a physiological basis for the lack of seed set observed in self-crosses of the mutant. Biochemical complementation with nanomolar amounts of kaempferol, a flavonol aglycone, confirms that the inability of the wha pollen to germinate is due to a lack of this essential compound. Transgenic complementation with a functional ChsA (Chalcone synthase A) cDNA suggests that the genetic lesion responsible for the wha phenotype is in Chs, the gene for the first enzyme in the flavonol biosynthesis pathway. The genetic background of the parental line, as well as the pollen phenotype, allowed us to deduce that the wha mutation is in ChsA. To our knowledge, wha is the first induced, nontransgenic Chs mutant described in petunia, and analysis of the mutation confirms earlier molecular and genetic observations that only two Chs genes (A and J) are expressed in reproductive tissues and that they are differentially regulated in corolla and anther.

Genetic control of branching pattern and floral identity during Petunia inflorescence development.

A main determinant of inflorescence architecture is the site where floral meristems are initiated. We show that in wild-type Petunia bifurcation of the inflorescence meristem yields two meristems of approximately equal size. One terminates into a floral meristem and the other maintains its inflorescence identity. By random transposon mutagenesis we have generated two mutants in which the architecture of the inflorescence is altered. In the extra petals- (exp) mutant the inflorescence terminates with the formation of a single terminal flower. Phenotypic analysis showed that exp is required for the bifurcation of inflorescence meristems. In contrast, the aberrant leaf and flower- (alf) mutant is affected in the specification of floral meristem identity while the branching pattern of the inflorescence remains unaltered. A weak alf allele was identified that, after bifurcation of the inflorescence meristem, yields a 'floral' meristem with partial inflorescence characteristics. By analysing independent transposon dTph1 insertion alleles we show that the alf locus encodes the Petunia FLORICAULA/LEAFY homolog. In situ hybridisation shows that alf is expressed in the floral meristem and also in the vegetative meristem. Differences and similarities between these Petunia mutants and mutations affecting inflorescence architecture in other species will be discussed.

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.

Calmodulin binding to glutamate decarboxylase is required for regulation of glutamate and GABA metabolism and normal development in plants.

Glutamate decarboxylase (GAD) catalyzes the decarboxylation of glutamate to CO2 and gamma-aminobutyrate (GABA). GAD is ubiquitous in prokaryotes and eukaryotes, but only plant GAD has been shown to bind calmodulin (CaM). Here, we assess the role of the GAD CaM-binding domain in vivo. Transgenic tobacco plants expressing a mutant petunia GAD lacking the CaM-binding domain (GADdeltaC plants) exhibit severe morphological abnormalities, such as short stems, in which cortex parenchyma cells fail to elongate, associated with extremely high GABA and low glutamate levels. The morphology of transgenic plants expressing the full-length GAD (GAD plants) is indistinguishable from that of wild-type (WT) plants. In WT and GAD plant extracts, GAD activity is inhibited by EGTA and by the CaM antagonist trifluoperazine, and is associated with a CaM-containing protein complex of approximately 500 kDa. In contrast, GADdeltaC plants lack normal GAD complexes, and GAD activity in their extracts is not affected by EGTA and trifluoperazine. We conclude that CaM binding to GAD is essential for the regulation of GABA and glutamate metabolism, and that regulation of GAD activity is necessary for normal plant development. This study is the first to demonstrate an in vivo function for CaM binding to a target protein in plants.

Transgene inactivation in Petunia hybrida is influenced by the properties of the foreign gene.

Petunia mutant RL01 was transformed with maize A1 and gerbera gdfr cDNAs, which both encode dihydroflavonol-4-reductase (DFR) activity. The same Agrobacterium vector and the same version of the CaMV 35S promoter were used in both experiments. Transformation with the cDNAs resulted in production of pelargonidin pigments in the transformants. However, the A1 and gdfr transformants showed clearly different phenotypes. The flowers of the primary A1 transformants were pale and showed variability in pigmentation during their growth, while the flowers of the gdfr transformants showed intense and highly stable coloration. The color difference in the primary transformants was reflected in the expression levels of the transgenes as well as in the levels of anthocyanin pigment. As previously reported by others, the instability in pigmentation in the A1 transformants was more often detected in clones with multiple copies of the transgene and was associated with methylation of the 35S promoter and of the transgene cDNA itself. In the gdfr transformants, the most intense pigmentation was observed in plants with multiple transgenes in their genome. Only rarely was partial methylation of the 35S promoter detected, while the gdfr cDNA always remained in an unmethylated state. We conclude that the properties of the transgene itself strongly influence the inactivation process. The dicotyledonous gdfr cDNA with a lower GC content and fewer possible methylation sites is more 'compatible' the genomic organization of petunia and this prevents it being recognized as a foreign gene and hence silenced by methylation.

Targeted gene inactivation in petunia by PCR-based selection of transposon insertion mutants.

Establishment of loss-of-function phenotypes is often a key step in determining the biological function of a gene. We describe a procedure to obtain mutant petunia plants in which a specific gene with known sequence is inactivated by the transposable element dTph1. Leaves are collected from batches of 1000 plants with highly active dTph1 elements, pooled according to a three-dimensional matrix, and screened by PCR using a transposon- and a gene-specific primer. In this way individual plants with a dTph1 insertion can be identified by analysis of about 30 PCRs. We found insertion alleles for various genes at a frequency of about 1 in 1000 plants. The plant population can be preserved by selfing all the plants, so that it can be screened for insertions in many genes over a prolonged period.

Conditional male fertility in maize

This study compares conditional male fertility (CMF) in maize and petunia. CMF is a reversible defect in pollen germination or tube growth; pollen is nonfunctional in self-crosses but fully functional in outcrosses or when supplied with specific flavonol aglycones at pollination. CMF occurs in maize and petunia mutants that lack chalcone synthase (CHS) activity and therefore do not synthesize flavonols. In maize CMF seedlings and developing male florets, CHS transcripts accumulate to high levels, yet western blot analysis using an anti-CHS antiserum does not detect any CHS protein. This is in contrast to CMF petunia, where no CHS RNA is detected (Vogt et al. 1994). While CMF petunia pollen requires flavonols to germinate, CMF maize pollen germinates and grows both in vivo and in vitro without the addition of flavonols. However, pollen tubes abort after 12 h of growth which explains the lack of seed set in self crosses (Mo et al. 1992). Pollen tubes of CMF maize have an unusual morphology in vivo, with heavy callose deposits throughout the tube and tips that burst within the silk. Normal tube morphology and seed set are restored by adding flavonols to the silks at pollination. As previously shown with petunia, fecundity (seed set) may be enhanced in maize by adding quercetin and kaempferol at pollination.

Cloning and expression of cytochrome P450 genes controlling flower colour.

Blue and violet flowers generally contain derivatives of delphinidin; red and pink flowers generally contain derivatives of cyanidin or pelargonidin. Differences in hydroxylation patterns of these three major classes of anthocyanidins are controlled by the cytochrome P450 enzymes flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase. Here we report on the isolation of complementary DNA clones of two different flavonoid 3',5'-hydroxylase genes that are expressed in petunia flowers. Restriction-fragment length polymorphism mapping and complementation of mutant petunia lines showed that the flavonoid 3',5'-hydroxylase genes correspond to the genetic loci Hf1 and Hf2.

Ectopic expression of pMADS3 in transgenic petunia phenocopies the petunia blind mutant.

We cloned a MADS-box gene, pMADS3, from Petunia hybrida, which shows high sequence homology to the Arabidopsis AGAMOUS and Antirrhinum PLENA. pMADS3 is expressed exclusively in stamens and carpels of wild-type petunia plants. In the petunia mutant blind, which shows homeotic conversions of corolla limbs into antheroid structures with pollen grains and small parts of sepals into carpelloid tissue, pMADS3 is expressed in all floral organs as well as in leaves. Ectopic expression of pMADS3 in transgenic petunia leads to phenocopies of the blind mutant, i.e., the formation of antheroid structures on limbs and carpelloid tissue on sepals. Transgenic tobacco plants that overexpress pMADS3 exhibit an even more severe phenotype, with the sepals forming a carpel-like structure encasing the interior floral organs. Our results identify BLIND as a negative regulator of pMADS3, which specifies stamens and carpels during petunia flower development.

Functional analysis of petunia floral homeotic MADS box gene pMADS1.

The petunia mutant green petal (gp, line PLV) shows a homeotic effect in one floral whorl, that is, the conversion of petal to sepal. We demonstrate that this mutant contains a chromosomal deletion, including the petunia MADS box gene pMADS1. Second whorl petal development in this null mutant can be restored with a CaMV 35S-pMADS1 transgene, demonstrating the essential role of pMADS1 in this process. Because gp (PLV) shows only a minor effect on stamen development, the homeotic effects of pMADS1 are different from those of B-type genes in Antirrhinum and Arabidopsis. Two other MADS box genes, pMADS2 and fbp1 (Angenent et al. 1992), require pMADS1 to maintain expression in the second whorl. However, in the absence of pMADS1 these two genes continue to be expressed in the third whorl. The functions assigned to pMADS1 are further supported by experiments in which we phenocopy gp by cosuppression of pMADS1 gene expression. The flowers, obtained through cosuppression and phenotype restoration, display different degrees of sepal to petal conversion. Analysis of these flowers indicate that pMADS1 controls growth under the zone of petal and stamen initiation, which causes the corolla tube and stamen filaments to emerge as a congenitally fused structure.