Single gene mutation in a plant MYB transcription factor causes a major shift in pollinator preference.

Understanding the molecular basis of reproductive isolation and speciation is a key goal of evolutionary genetics. In the South American genus Petunia, the R2R3-MYB transcription factor MYB-FL regulates the biosynthesis of UV-absorbing flavonol pigments, a major determinant of pollinator preference. MYB-FL is highly expressed in the hawkmoth-pollinated P. axillaris, but independent losses of its activity in sister taxa P. secreta and P. exserta led to UV-reflective flowers and associated pollinator shifts in each lineage (bees and hummingbirds, respectively). We created a myb-fl CRISPR mutant in P. axillaris and studied the effect of this single gene on innate pollinator preference. The mutation strongly reduced the expression of the two key flavonol-related biosynthetic genes but only affected the expression of few other genes. The mutant flowers were UV reflective as expected but additionally contained low levels of visible anthocyanin pigments. Hawkmoths strongly preferred the wild-type P. axillaris over the myb-fl mutant, whereas both social and solitary bee preference depended on the level of visible color of the mutants. MYB-FL, with its specific expression pattern, small number of target genes, and key position at the nexus of flavonol and anthocyanin biosynthetic pathways, provides a striking example of evolution by single mutations of large phenotypic effect.

MYB-FL controls gain and loss of floral UV absorbance, a key trait affecting pollinator preference and reproductive isolation.

Adaptations to new pollinators involve multiple floral traits, each requiring coordinated changes in multiple genes. Despite this genetic complexity, shifts in pollination syndromes have happened frequently during angiosperm evolution. Here we study the genetic basis of floral UV absorbance, a key trait for attracting nocturnal pollinators. In Petunia, mutations in a single gene, MYB-FL, explain two transitions in UV absorbance. A gain of UV absorbance in the transition from bee to moth pollination was determined by a cis-regulatory mutation, whereas a frameshift mutation caused subsequent loss of UV absorbance during the transition from moth to hummingbird pollination. The functional differences in MYB-FL provide insight into the process of speciation and clarify phylogenetic relationships between nascent species.

Hawkmoth pollinators decrease seed set of a low-nectar Petunia axillaris line through reduced probing time.

Although deception of floral pollinators is well known among orchids, the majority of animal-pollinated plants secure pollination by nectar rewards. The costs and benefits of nectar production remain poorly understood. Here, we developed a crossing design to introgress a low-nectar-volume locus of Petunia integrifolia into the genetic background of P. axillaris. The resulting introgression line resembled P. axillaris but produced only one-third of the nectar volume. When exposed simultaneously to low-nectar and wild-type P. axillaris plants, hawkmoth pollinators reduced their probing duration on low-nectar plants but otherwise did not show any signs of discrimination against these plants. However, reduced probing duration resulted in reduced seed production in the low-nectar plants despite their higher reproductive potential as evidenced by hand pollination. In line with this interpretation, we found a positive correlation between probing duration and seed set, and hawkmoth pollination of low-nectar plants that were manually supplemented with nectar to parental levels yielded seed sets similar to hand pollination. Thus, a simple self-serving pollinator behavior--the adjustment of probing time in response to nectar volume--may select against reducing nectar and protect many plant-pollinator mutualisms against a drift toward parasitism.

Pollinator choice in Petunia depends on two major genetic Loci for floral scent production.

BACKGROUND: Differences in floral traits, such as petal color, scent, morphology, or nectar quality and quantity, can lead to specific interactions with pollinators and may thereby cause reproductive isolation. Petunia provides an attractive model system to study the role of floral characters in reproductive isolation and speciation. The night-active hawkmoth pollinator Manduca sexta relies on olfactory cues provided by Petunia axillaris. In contrast, Petunia exserta, which displays a typical hummingbird pollination syndrome, is devoid of scent. The two species can easily be crossed in the laboratory, which makes it possible to study the genetic basis of the evolution of scent production and the importance of scent for pollinator behavior. RESULTS: In an F2 population derived from an interspecific cross between P. axillaris and P. exserta, we identified two quantitative trait loci (QTL) that define the difference between the two species' ability to produce benzenoid volatiles. One of these loci was identified as the MYB transcription factor ODORANT1. Reciprocal introgressions of scent QTL were used for choice experiments under controlled conditions. These experiments demonstrated that the hawkmoth M. sexta prefers scented plants and that scent determines choice at a short distance. When exposed to conflicting cues of color versus scent, the insects display no preference, indicating that color and scent are equivalent cues. CONCLUSION: Our results show that scent is an important flower trait that defines plant-pollinator interactions at the level of individual plants. The genetic basis underlying such a major phenotypic difference appears to be relatively simple and may enable rapid loss or gain of scent through hybridization.

Speciation genes in the genus Petunia.

A major innovation in angiosperms is the recruitment of animal pollinators as a means to enhance the efficiency and specificity of pollen transfer. The implementation of this reproductive strategy involved the rapid and presumably coordinated evolution of multiple floral traits. A major question concerns the molecular identity of the genetic polymorphisms that specify the phenotypic differences between distinct pollination syndromes. Here, we report on our work with Petunia, an attractive model system for quantitative plant genetics and genomics. From interspecific crosses, we obtained F2 plants that differed in the length of the floral tube or the size of the limb. We used these plants to study the behaviour of the hawkmoth pollinator, Manduca sexta. Plants with larger limbs were preferentially visited, consistent with the notion that flower size affects visibility under low light conditions. The moths also displayed an innate preference for shorter tubes. However, in those cases that flowers with long tubes were chosen, the animals fed for equal time. Thus, the perception of tube length may help the moths, early on, to avoid those plants that are more difficult to handle.

Piriformospora indica and Sebacina vermifera increase growth performance at the expense of herbivore resistance in Nicotiana attenuata.

A Sebacinales species was recovered from a clone library made from a pooled rhizosphere sample of Nicotiana attenuata plants from 14 native populations. Axenic cultures of the related species, Piriformospora indica and Sebacina vermifera, were used to examine their effects on plant performance. Inoculation of N. attenuata seeds with either fungus species stimulated seed germination and increased growth and stalk elongation. S. vermifera inoculated plants flowered earlier, produced more flowers and matured more seed capsules than did non-inoculated plants. Jasmonate treatment during rosette-stage growth, which slows growth and elicits herbivore resistance traits, erased differences in vegetative, but not reproductive performance resulting from S. vermifera inoculation. Total nitrogen and phosphorous contents did not differ between inoculated and control plants, suggesting that the performance benefits of fungal inoculation did not result from improvements in nutritional status. Since the expression of trypsin proteinase inhibitors (TPI), defensive proteins which confer resistance to attack from Manduca sexta larvae, incur significant growth and fitness costs for the plant, we examined the effect of S. vermifera inoculation on herbivore resistance and TPI activity. After 10 days of feeding on S. vermifera-inoculated plants, larval mass was 46% higher and TPI activity was 48% lower than that on non-inoculated plants. These results suggest that Sebacina spp. may interfere with defense signaling and allow plants to increase growth rates at the expense of herbivore resistance mediated by TPIs.

The composition and timing of flower odour emission by wild Petunia axillaris coincide with the antennal perception and nocturnal activity of the pollinator Manduca sexta.

In the genus Petunia, distinct pollination syndromes may have evolved in association with bee-visitation (P. integrifolia spp.) or hawk moth-visitation (P. axillaris spp). We investigated the extent of congruence between floral fragrance and olfactory perception of the hawk moth Manduca sexta. Hawk moth pollinated P. axillaris releases high levels of several compounds compared to the bee-pollinated P. integrifolia that releases benzaldehyde almost exclusively. The three dominating compounds in P. axillaris were benzaldehyde, benzyl alcohol and methyl benzoate. In P. axillaris, benzenoids showed a circadian rhythm with an emission peak at night, which was absent from P. integrifolia. These characters were highly conserved among different P. axillaris subspecies and P. axillaris accessions, with some differences in fragrance composition. Electroantennogram (EAG) recordings using flower-blends of different wild Petunia species on female M. sexta antennae showed that P. axillaris odours elicited stronger responses than P. integrifolia odours. EAG responses were highest to the three dominating compounds in the P. axillaris flower odours. Further, EAG responses to odour-samples collected from P. axillaris flowers confirmed that odours collected at night evoked stronger responses from M. sexta than odours collected during the day. These results show that timing of odour emissions by P. axillaris is in tune with nocturnal hawk moth activity and that flower-volatile composition is adapted to the antennal perception of these pollinators.

The orf13 T-DNA gene of Agrobacterium rhizogenes confers meristematic competence to differentiated cells.

Plant infections by the soil bacterium Agrobacterium rhizogenes result in neoplastic disease with the formation of hairy roots at the site of infection. Expression of a set of oncogenes residing on the stably integrated T-DNA is responsible for the disease symptoms. Besides the rol (root locus) genes, which are essential for the formation of hairy roots, the open reading frame orf13 mediates cytokinin-like effects, suggesting an interaction with hormone signaling pathways. Here we show that ORF13 induced ectopic expression of KNOX (KNOTTED1-like homeobox) class transcription factors, as well as of several genes involved in cell cycle control in tomato (Lycopersicon esculentum). ORF13 has a retinoblastoma (RB)-binding motif and interacted with maize (Zea mays) RB in vitro, whereas ORF13, bearing a point mutation in the RB-binding motif (ORF13*), did not. Increased cell divisions in the vegetative shoot apical meristem and accelerated formation of leaf primordia were observed in plants expressing orf13, whereas the expression of orf13* had no influence on cell division rates in the shoot apical meristem, suggesting a role of RB in the regulation of the cell cycle in meristematic tissues. On the other hand, ectopic expression of LeT6 was not dependent on a functional RB-binding motif. Hormone homeostasis was only altered in explants of leaves, whereas in the root no effects were observed. We suggest that ORF13 confers meristematic competence to cells infected by A. rhizogenes by inducing the expression of KNOX genes and promotes the transition of infected cells from the G1 to the S phase by binding to RB.

The ethanolic fermentation pathway supports respiration and lipid biosynthesis in tobacco pollen.

Rapid pollen tube growth requires a high rate of sugar metabolism to meet energetic and biosynthetic demands. Previous work on pollen sugar metabolism showed that tobacco pollen carry out efficient ethanolic fermentation concomitantly with a high rate of respiration (Bucher et al., 1995). Here we show that the products of fermentation, acetaldehyde and ethanol, are further metabolised in a pathway that bypasses mitochondrial PDH. The enzymes involved in this pathway are pyruvate decarboxylase, aldehyde dehydrogenase and acetyl-CoA synthetase. Radiolabelling experiments show that during tobacco pollen tube growth label of 14C-ethanol is incorporated into CO2 as well as into lipids and other higher molecular weight compounds. A role for the glyoxylate cycle appears unlikely since activity of malate synthase, a key enzyme of the glyoxylate cycle, could not be detected.