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Plant volatile emissions can recruit predators of herbivores for indirect defense and attract pollinators to aid in pollination. Although volatiles involved in defense and pollinator attraction are primarily emitted from leaves and flowers, respectively, they will co‐evolve if their underlying genetic basis is intrinsically linked, due either to pleiotropy or to genetic linkage. However, direct evidence of co‐evolving defense and floral traits is scarce. We characterized intraspecific variation of herbivory‐induced plant volatiles (HIPVs), the key components of indirect defense against herbivores, and floral volatiles in wild tobacco Nicotiana attenuata. We found that variation of (E)‐β‐ocimene and (E)‐α‐bergamotene contributed to the correlated changes in HIPVs and floral volatiles among N. attenuata natural accessions. Intraspecific variations of (E)‐β‐ocimene and (E)‐α‐bergamotene emissions resulted from allelic variation of two genetically co‐localized terpene synthase genes, NaTPS25 and NaTPS38, respectively. Analyzing haplotypes of NaTPS25 and NaTPS38 revealed that allelic variations of NaTPS25 and NaTPS38 resulted in correlated changes of (E)‐β‐ocimene and (E)‐α‐bergamotene emission in HIPVs and floral volatiles in N. attenuata. Together, these results provide evidence that pleiotropy and genetic linkage result in correlated changes in defenses and floral signals in natural populations, and the evolution of plant volatiles is probably under diffuse selection.

The circadian clock contextualizes plant responses to environmental signals. Plants use temporal information to respond to herbivory, but many of the functional roles of circadian clock components in these responses, and their contribution to fitness, remain unknown. We investigate the role of the central clock regulator TIMING OF CAB EXPRESSION 1 (TOC1) in Nicotiana attenuata's defense responses to the specialist herbivore Manduca sexta under both field and glasshouse conditions. We utilize 15 N pulse-labeling to quantify nitrogen incorporation into pools of three defense compounds: caffeoylputrescine (CP), dicaffeoyl spermidine (DCS) and nicotine. Nitrogen incorporation was decreased in CP and DCS and increased in nicotine pools in irTOC1 plants compared to empty vector (EV) under control conditions, but these differences were abolished after simulated herbivory. Differences between EV and irTOC1 plants in nicotine, but not phenolamide production, were abolished by treatment with the ethylene agonist 1-methylcyclopropene. Using micrografting, TOC1's effect on nicotine was isolated to the root and did not affect the fitness of heterografts under field conditions. These results suggest that the circadian clock contributes to plant fitness by balancing production of metabolically expensive nitrogen-rich defense compounds and mediating the allocation of resources between vegetative biomass and reproduction.

SummaryPlant volatile emissions can recruit predators of herbivores for indirect defence and attract pollinators to aid in pollination. Although volatiles involved in defence and pollinator attraction are primarily emitted from leaves and flowers, respectively, they will co-evolve if their underlying genetic basis is intrinsically linked, either due to pleiotropy or genetic linkage. However, direct evidence of co-evolving defence and floral traits is scarce.We characterized intra-specific variation of herbivory-induced plant volatiles (HIPVs), the key components of indirect defence against herbivores, and floral volatiles in the wild tobacco Nicotiana attenuata.We found that variation of (E)-β-ocimene and (E)-α-bergamotene contributed to the correlated changes in HIPVs and floral volatiles among N. attenuata natural accessions. Intra-specific variations of (E)-β-ocimene and (E)-α-bergamotene emissions resulted from allelic variation of two genetically co-localized terpene synthase genes, NaTPS25 and NaTPS38 respectively.Analyzing haplotypes of NaTPS25 and NaTPS38 revealed that allelic variations of NaTPS25 and NaTPS38 resulted in correlated changes of (E)-β-ocimene and (E)-α-bergamotene emission in HIPVs and floral volatiles in N. attenuata.Together, these results provide evidence that pleiotropy and genetic linkage result in correlated changes in defences and floral signals in natural populations, and the evolution of plant volatiles is likely under diffuse selection.

Plant trait diversity is known to influence population yield, but the scale at which this happens remains unknown: divergent individuals might change yields of immediate neighbors (neighbor scale) or of plants across a population (population scale). We use Nicotiana attenuata plants silenced in mitogen-activated protein kinase 4 (irMPK4) – with low water-use efficiency (WUE) – to study the scale at which water-use traits alter intraspecific population yields. In the field and glasshouse, we observed overyielding in populations with low percentages of irMPK4 plants, unrelated to water-use phenotypes. Paired-plant experiments excluded the occurrence of overyielding effects at the neighbor scale. Experimentally altering field arbuscular mycorrhizal fungal associations by silencing the Sym-pathway gene NaCCaMK did not affect reproductive overyielding, implicating an effect independent of belowground AMF interactions. Additionally, micro-grafting experiments revealed dependence on shoot-expressed MPK4 for N. attenuata to vary its yield per neighbor presence. We find that variation in a single gene, MPK4, is responsible for population overyielding through a mechanism, independent of irMPK4’s WUE phenotype, at the aboveground, population scale., eLife digest Whether on farmland or in a forest, plants do not grow in isolation. Plants compete with their neighbors over limited space and resources, and individual plants respond to this competition in different ways by changing how much they grow and how they use resources. The efficiency with which crop plants use water, for example, is one trait that is dramatically influenced by neighboring plants and is of increasing concern given the warming climate. Understanding the effects of interactions between individual plants in a population as a whole is complicated, especially in natural plant communities where neighbors are often from different species. For this reason, McGale et al. took a different approach and looked at neighbors that were all from the same species and differed only in the activity of a single gene. The species in question was coyote tobacco, a plant that is native to western North America. McGale et al. used genetic engineering to silence a gene called MPK4, which was known from previous studies to have the effect of reducing water-use efficiency. Some of these ‘water-inefficient’ plants were then grown in mixed populations with plants that had normal levels of MPK4. In experiments conducted both in a glasshouse and at a field station in the Utah desert, McGale et al. found that populations with a low percentage of the MPK4-silenced plants were actually more productive than ‘monocultures’ that were all one type or the other. Further analysis showed that the increase in productivity did not depend on the different soil nutrient or water use of the different populations, or even the density of the plants in the populations. Pairs of plants grown in single pots essentially ruled out any interactions between immediate neighbors being responsible for the increased productivity, suggesting that that effect must instead emerge at the level of the population. Perhaps unexpectedly, McGale et al. also found that the MPK4-silenced plants and control plants did not actually differ in how they used water when grown in the field (previous studies had all been conducted in glasshouses), indicating that this trait also could not explain the observed population-level effect. Finally, experiments that involved grafting the shoots of one plant onto the roots of another suggested that the effect most likely comes from the aboveground parts of the plant. Ecologists have previously noted that more diverse populations typically have higher productivity. This new finding that a small percentage of slightly different plants in an otherwise uniform population can increase overall productivity will likely to be of special interest to researchers looking to boost the efficiency of agricultural ecosystems. Also, since MPK4 is highly conserved, and thus likely to be found in many plant species, this could be an interesting trait with which to study the interactions of natural plant communities.

The highly conserved core circadian clock component TIMING OF CAB EXPRESSION 1 (TOC1) contextualizes environmental stress responses in plants, for example by gating abscisic acid (ABA) signaling and suppressing thermoresponsive growth. Selective interaction of TOC1 with PHYTOCHROME B (PHYB) 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 (irTOC1) 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 co-expression 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.

Functional variation is known to influence population yield, but the scale at which this happens is still unknown. Relevant signals might only reach immediate neighbors of a phenotypically diverse plant (neighbor-scale) or conversely may distribute across the population (population-scale). We useNicotiana attenuatasilenced in mitogen-activated protein kinase 4 (irMPK4), plants with low water-use efficiency (WUE), to study the scale at which water-use traits alter intraspecific population yields. In the field and glasshouse, populations with low percentages of irMPK4 plants planted among isogenic control plants produced maximum overall growth and yield. Through paired-plant and local-plant-configuration analyses, we determined that this occurred at the population scale. However, we find that this effect was not due to irMPK4’s WUE phenotype. With micro-grafting, we additionally show thatMPK4-deficiency may mediate the response at the population-scale: shoot-expressedMPK4is required forN. attenuatato change yield in response to a neighbor.

All plants need a nutrient called phosphorus to grow and thrive. Phosphorus is found in soil, but the supply is limited so plants often struggle to acquire enough of it. To overcome this problem, many plants form friendly relationships (or symbioses) with certain fungi in the soil known as arbuscular mycorrhizal fungi. The fungi colonize plant roots and supply phosphorus and other nutrients in return for sugars and various molecules. Although many crop plants – including barley and potatoes – are able to form these symbioses, farmers commonly apply fertilizers containing phosphate and other nutrients to their fields to increase the amount of food they produce. Breeding new crop varieties that are better at forming symbioses with the fungi could reduce the need for fertilizers. However, the methods currently available to study these relationships are laborious and time-consuming, typically requiring samples of plant roots to be examined in a laboratory. Wang, Schafer et al. used an approach called metabolomics to search for molecules in coyote tobacco plants that indicate the plants have formed symbioses with arbuscular mycorrhizal fungi. The experiments found that a group of molecules called blumenols accumulate in the roots and also in the shoots and leaves of plants with these symbioses, but not in the tobacco plants that were not able to associate with the fungi. Experiments in several other plant species including tomato, potato and barley produced similar findings, suggesting that the blumenols may be a useful and potentially universal indicator of symbioses between many different plants and fungi. Measuring the levels of blumenols in plant shoots and leaves is much quicker and easier than current methods of identifying fungal symbioses in plant root samples. Therefore, blumenols may be a useful tool for plant breeders who would like to screen large numbers of plants for these symbioses, and breed crops that negotiate better interactions with the beneficial fungi.

Plants are the primary producers in most terrestrial ecosystems and have complex defense systems to protect their produce. Defense-deficient, high-yielding agricultural monocultures attract abundant nonhuman consumers, but are alternatively defended through pesticide application and genetic engineering to produce insecticidal proteins such as Cry1Ac (Bacillus thuringiensis). These approaches alter the balance between yield protection and maximization but have been poorly contextualized to known yield-defense trade-offs in wild plants. The native plant Nicotiana attenuata was used to compare yield benefits of plants transformed to be defenseless to those with a full suite of naturally evolved defenses, or additionally transformed to ectopically produce Cry1Ac. An insecticide treatment allowed us to examine yield under different herbivore loads in N. attenuata's native habitat. Cry1Ac, herbivore damage, and growth parameters were monitored throughout the season. Biomass and reproductive correlates were measured at season end. Non-Cry1Ac-targeted herbivores dominated on noninsecticide-treated plants, and increased the yield drag of Cry1Ac-producing plants in comparison with endogenously defended or undefended plants. Insecticide-sprayed Cry1Ac-producing plants lagged less in stalk height, shoot biomass, and flower production. In direct comparison with the endogenous defenses of a native plant, Cry1Ac production did not provide yield benefits for plants under observed herbivore loads in a field study.

Summary 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.