Arbuscular mycorrhizal fungi influence the intraspecific competitive ability of plants under field and glasshouse conditions.

MAIN CONCLUSION: Nicotiana attenuata's capacity to interact with arbuscular mycorrhizal fungi influences its intraspecific competitive ability under field and glasshouse conditions, but not its overall community productivity. Arbuscular mycorrhizal (AM) fungi can alter the nutrient status and growth of plants, and they can also affect plant-plant, plant-herbivore, and plant-pathogen interactions. These AM effects are rarely studied in populations under natural conditions due to the limitation of non-mycorrhizal controls. Here we used a genetic approach, establishing field and glasshouse communities of AM-harboring Nicotiana attenuata empty vector (EV) plants and isogenic plants silenced in calcium- and calmodulin-dependent protein kinase expression (irCCaMK), and unable to establish AM symbioses. Performance and growth were quantified in communities of the same (monocultures) or different genotypes (mixed cultures) and both field and glasshouse experiments returned similar responses. In mixed cultures, AM-harboring EV plants attained greater stalk lengths, shoot and root biomasses, clearly out-competing the AM fungal-deficient irCCaMK plants, while in monocultures, both genotypes grew similarly. Competitive ability was also reflected in reproductive traits: EV plants in mixed cultures outperformed irCCaMK plants. When grown in monocultures, the two genotypes did not differ in reproductive performance, though total leaf N and P contents were significantly lower independent of the community type. Plant productivity in terms of growth and seed production at the community level did not differ, while leaf nutrient content of phosphorus and nitrogen depended on the community type. We infer that AM symbioses drastically increase N. attenuata's competitive ability in mixed communities resulting in increased fitness for the individuals harboring AM without a net gain for the community.

ZEITLUPE facilitates the rhythmic movements of Nicotiana attenuata flowers.

Circadian organ movements are ubiquitous in plants. These rhythmic outputs are thought to be regulated by the circadian clock and auxin signalling, but the underlying mechanisms have not been clarified. Flowers of Nicotiana attenuata change their orientation during the daytime through a 140° arc to balance the need for pollinators and the protection of their reproductive organs. This rhythmic trait is under the control of the circadian clock and results from bending and re-straightening movements of the pedicel, stems that connect flowers to the inflorescence. Using an explant system that allowed pedicel growth and curvature responses to be characterized with high spatial and temporal resolution, we demonstrated that this movement is organ autonomous and mediated by auxin. Changes in the growth curvature of the pedicel are accompanied by an auxin gradient and dorsiventral asymmetry in auxin-dependent transcriptional responses; application of auxin transport inhibitors influenced the normal movements of this organ. Silencing the expression of the circadian clock component ZEITLUPE (ZTL) arrested changes in the growth curvature of the pedicel and altered auxin signalling and responses. IAA19-like, an Aux/IAA transcriptional repressor that is circadian regulated and differentially expressed between opposite tissues of the pedicel, and therefore possibly involved in the regulation of changes in organ curvature, physically interacted with ZTL. Together, these results are consistent with a direct link between the circadian clock and the auxin signalling pathway in the regulation of this rhythmic floral movement.

The Clock Gene TOC1 in Shoots, Not Roots, Determines Fitness of Nicotiana attenuata under Drought.

The highly conserved core circadian clock component TIMING OF CAB EXPRESSION1 (TOC1) contextualizes environmental stress responses in plants, for example by gating abscisic acid signaling and suppressing thermoresponsive growth. Selective interaction of TOC1 with PHYTOCHROME B under far-red-enriched light suggests a connection between circadian gating of light responses and sensitivity to ABA, an important regulator of growth and stress responses, including under drought. However, the fitness consequences of TOC1 function, particularly in the root, are poorly understood. Here, we used the desert annual, Nicotiana attenuata, to investigate the function of TOC1 in shoots and roots for maintaining fitness under drought, in both field and glasshouse experiments. Despite marked decreases in leaf water loss, TOC1-deficient lines failed to maintain fitness in response to drought stress as measured by total seed capsule production. Restoring TOC1 transcript levels in shoots via micrografting was sufficient to restore wild-type drought responses under field conditions. Microarrays identified a coexpression module in leaves strongly linking red and far-red light signaling to drought responses in a TOC1-dependent manner, but experiments with phytochrome-deficient lines revealed that the effects of TOC1 deficiency under drought cannot be attributed to changes in red/far-red light perception alone. Taken together, these results elucidate the sophisticated, tissue-dependent role of the circadian clock in maintaining fitness in the face of long-term abiotic stresses such as drought.

Flower movement balances pollinator needs and pollen protection.

Flower signaling and orientation are key characteristics that determine a flower's pollinator guild. However, many flowers actively move during their daily cycle, changing both their detectability and accessibility to pollinators. The flowers of the wild tobacco Nicotiana attenuata orientate their corolla upward at sunset and downward after sunrise. Here, we investigated the effect of different flower orientations on a major pollinator of N. attenuata, the hawkmoth Manduca sexta. We found that although flower orientation influenced the flight altitude of the moth in respect to the flower, it did not alter the moth's final flower choice. These behavioral observations were consistent with the finding that orientation did not systematically change the spatial distribution of floral volatiles, which are major attractants for the moths. Moreover, hawkmoths invested the same amount of time into probing flowers at different orientations, even though they were only able to feed and gather pollen from horizontally and upward-oriented flowers, but not from downward-facing flowers. The orientation of the flower was hence crucial for a successful interaction between N. attenuata and its hawkmoth pollinator. Additionally, we also investigated potential adverse effects of exposing flowers at different orientations to natural daylight levels, finding that anther temperature of upward-oriented flowers was more than 7°C higher than for downward-oriented flowers. This increase in temperature likely caused the significantly reduced germination success that was observed for pollen grains from upward-oriented flowers in comparison to those of downward and horizontally oriented flowers. These results highlight the importance of flower reorientation to balance pollen protection and a successful interaction of the plant with its insect pollinators by maintaining the association between flower volatiles and flower accessibility to the pollinator.

Fitness consequences of a clock pollinator filter in Nicotiana attenuata flowers in nature.

Nicotiana attenuata flowers, diurnally open, emit scents and move vertically to interact with nocturnal hawkmoth and day-active hummingbird pollinators. To examine the fitness consequences of these floral rhythms, we conducted pollination trials in the plant's native habitat with phase-shifted flowers of plants silenced in circadian clock genes. The results revealed that some pollination benefits observed under glasshouse conditions were not reproduced under natural field conditions. Floral arrhythmicity increased pollination success by hummingbirds, while reducing those by hawkmoths in the field. Thus, floral circadian rhythms may influence a plant's fitness by filtering pollinators leading to altered seed set from outcrossed pollen.

Circadian clock component, LHY, tells a plant when to respond photosynthetically to light in nature.

The circadian clock is known to increase plant growth and fitness, and is thought to prepare plants for photosynthesis at dawn and dusk; whether this happens in nature was unknown. We transformed the native tobacco, Nicotiana attenuata to silence two core clock components, NaLHY (irLHY) and NaTOC1 (irTOC1). We characterized growth and light- and dark-adapted photosynthetic rates (Ac ) throughout a 24 h day in empty vector-transformed (EV), irLHY, and irTOC1 plants in the field, and in NaPhyA- and NaPhyB1-silenced plants in the glasshouse. The growth rates of irLHY plants were lower than those of EV plants in the field. While irLHY plants reduced Ac earlier at dusk, no differences between irLHY and EV plants were observed at dawn in the field. irLHY, but not EV plants, responded to light in the night by rapidly increasing Ac . Under controlled conditions, EV plants rapidly increased Ac in the day compared to dark-adapted plants at night; irLHY plants lost these time-dependent responses. The role of NaLHY in gating photosynthesis is independent of the light-dependent reactions and red light perceived by NaPhyA, but not NaPhyB1. In summary, the circadian clock allows plants not to respond photosynthetically to light at night by anticipating and gating red light-mediated in native tobacco.

Tissue-Specific Emission of (E)-α-Bergamotene Helps Resolve the Dilemma When Pollinators Are Also Herbivores.

More than 87% of flowering plant species are animal-pollinated [1] and produce floral scents and other signals to attract pollinators. These floral cues may however also attract antagonistic visitors, including herbivores [2]. The dilemma is exacerbated when adult insects pollinate the same plant that their larvae consume. It remains largely unclear how plants maximize their fitness under these circumstances. Here we show that in the night-flowering wild tobacco Nicotiana attenuata, the emission of a sesquiterpene, (E)-α-bergamotene, in flowers increases adult Manduca sexta moth-mediated pollination success, while the same compound in leaves is known to mediate indirect defense against M. sexta larvae [3, 4]. Forward and reverse genetic analyses demonstrated that both herbivory-induced and floral (E)-α-bergamotene are regulated by the expression of a monoterpene-synthase-derived sesquiterpene synthase (NaTPS38). The expression pattern of NaTPS38 also accounts for variation in (E)-α-bergamotene emission among natural accessions. These results highlight that differential expression of a single gene that results in tissue-specific emission of one compound contributes to resolving the dilemma for plants when their pollinators are also herbivores. Furthermore, this study provides genetic evidence that pollinators and herbivores interactively shape the evolution of floral signals and plant defense.

Fitness consequences of altering floral circadian oscillations for Nicotiana attenuata.

Ecological interactions between flowers and pollinators are all about timing. Flower opening/closing and scent emissions are largely synchronized with pollinator activity, and a circadian clock regulates these rhythms. However, whether the circadian clock increases a plant's reproductive success by regulating these floral rhythms remains untested. Flowers of Nicotiana attenuata, a wild tobacco, diurnally and rhythmically open, emit scent and move vertically through a 140° arc to interact with nocturnal hawkmoths. We tethered flowers to evaluate the importance of flower positions for Manduca sexta-mediated pollinations; flower position dramatically influenced pollination. We examined the pollination success of phase-shifted flowers, silenced in circadian clock genes, NaZTL, NaLHY, and NaTOC1, by RNAi. Circadian rhythms in N. attenuata flowers are responsible for altered seed set from outcrossed pollen.

Hawkmoths evaluate scenting flowers with the tip of their proboscis.

Pollination by insects is essential to many ecosystems. Previously, we have shown that floral scent is important to mediate pollen transfer between plants (Kessler et al., 2015). Yet, the mechanisms by which pollinators evaluate volatiles of single flowers remained unclear. Here, Nicotiana attenuata plants, in which floral volatiles have been genetically silenced and its hawkmoth pollinator, Manduca sexta, were used in semi-natural tent and wind-tunnel assays to explore the function of floral scent. We found that floral scent functions to increase the fitness of individual flowers not only by increasing detectability but also by enhancing the pollinator's foraging efforts. Combining proboscis choice tests with neurophysiological, anatomical and molecular analyses we show that this effect is governed by newly discovered olfactory neurons on the tip of the moth's proboscis. With the tip of their tongue, pollinators assess the advertisement of individual flowers, an ability essential for maintaining this important ecosystem service.

Silencing Nicotiana attenuata LHY and ZTL alters circadian rhythms in flowers.

The rhythmic opening/closing and volatile emissions of flowers are known to attract pollinators at specific times. That these rhythms are maintained under constant light or dark conditions suggests a circadian clock involvement. Although a forward and reverse genetic approach has led to the identification of core circadian clock components in Arabidopsis thaliana, the involvement of these clock components in floral rhythms has remained untested, probably because of the weak diurnal rhythms in A. thaliana flowers. Here, we addressed the role of these core clock components in the flowers of the wild tobacco Nicotiana attenuata, whose flowers open at night, emit benzyl acetone (BA) scents and move vertically through a 140° arc. We first measured N. attenuata floral rhythms under constant light conditions. The results suggest that the circadian clock controls flower opening, BA emission and pedicel movement, but not flower closing. We generated transgenic N. attenuata lines silenced in the homologous genes of Arabidopsis LATE ELONGATED HYPOCOTYL (LHY) and ZEITLUPE (ZTL), which are known to be core clock components. Silencing NaLHY and NaZTL strongly altered floral rhythms in different ways, indicating that conserved clock components in N. attenuata coordinate these floral rhythms.

Identification and characterization of circadian clock genes in a native tobacco, Nicotiana attenuata.

BACKGROUND: A plant's endogenous clock (circadian clock) entrains physiological processes to light/dark and temperature cycles. Forward and reverse genetic approaches in Arabidopsis have revealed the mechanisms of the circadian clock and its components in the genome. Similar approaches have been used to characterize conserved clock elements in several plant species. A wild tobacco, Nicotiana attenuata has been studied extensively to understand responses to biotic or abiotic stress in the glasshouse and also in their native habitat. During two decades of field experiment, we observed several diurnal rhythmic traits of N. attenuata in nature. To expand our knowledge of circadian clock function into the entrainment of traits important for ecological processes, we here report three core clock components in N. attenuata. RESULTS: Protein similarity and transcript accumulation allowed us to isolate orthologous genes of the core circadian clock components, LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION 1/PSEUDO-RESPONSE REGULATOR 1 (TOC1/PRR1), and ZEITLUPE (ZTL). Transcript accumulation of NaLHY peaked at dawn and NaTOC1 peaked at dusk in plants grown under long day conditions. Ectopic expression of NaLHY and NaZTL in Arabidopsis resulted in elongated hypocotyl and late-flowering phenotypes. Protein interactions between NaTOC1 and NaZTL were confirmed by yeast two-hybrid assays. Finally, when NaTOC1 was silenced in N. attenuata, late-flowering phenotypes under long day conditions were clearly observed. CONCLUSIONS: We identified three core circadian clock genes in N. attenuata and demonstrated the functional and biochemical conservation of NaLHY, NaTOC1, and NaZTL.

Tissue specific diurnal rhythms of metabolites and their regulation during herbivore attack in a native tobacco, Nicotiana attenuata.

Ecological performance is all about timing and the endogenous clock that allows the entrainment of rhythms and anticipation of fitness-determining events is being rapidly characterized. How plants anticipate daily abiotic stresses, such as cold in early mornings and drought at noon, as well as biotic stresses, such as the timing of pathogen infections, is being explored, but little is known about the clock's role in regulating responses to insect herbivores and mutualists, whose behaviors are known to be strongly diurnally regulated and whose attack is known to reconfigure plant metabolomes. We developed a liquid chromatography-mass spectrometry procedure and analyzed its output with model-based peak picking algorithms to identify metabolites with diurnal accumulation patterns in sink/source leaves and roots in an unbiased manner. The response of metabolites with strong diurnal patterns to simulated attack from the specialist herbivore, Manduca sexta larvae was analyzed and annotated with in-house and public databases. Roots and leaves had largely different rhythms and only 10 ions of 182 oscillating ions in leaves and 179 oscillating ions in roots were rhythmic in both tissues: root metabolites mainly peaked at dusk or night, while leaf metabolites peaked during the day. Many oscillating metabolites showed tissue-specific regulation by simulated herbivory of which systemic responses in unattacked tissues were particularly pronounced. Diurnal and herbivory-elicited accumulation patterns of disaccharide, phenylalanine, tyrosine, lyciumoside I, coumaroyl tyramine, 12-oxophytodienoic acid and jasmonic acid and those of their related biosynthetic transcripts were examined in detail. We conclude that oscillating metabolites of N. attenuata accumulate in a highly tissue-specific manner and the patterns reveal pronounced diurnal rhythms in the generalized and specialized metabolism that mediates the plant's responses to herbivores and mutualists. We propose that diurnal regulation will prove to an important element in orchestrating a plant's responses to herbivore attack.