Prezygotic mate selection is only partially correlated with the expression of NaS-like RNases and affects offspring phenotypes.

Nicotiana attenuata styles preferentially select pollen from among accessions with corresponding expression patterns of NaS-like-RNases (SLRs), and the postpollination ethylene burst (PPEB) is an accurate predictor of seed siring success. However, the ecological consequences of mate selection, its effect on the progeny, and the role of SLRs in the control of ethylene signaling remain unknown. We explored the link between the magnitude of the ethylene burst and expression of the SLRs in a set of recombinant inbred lines (RILs), dissected the genetic underpinnings of mate selection through genome-wide association study (GWAS), and examined its outcome for phenotypes in the next generation. We found that high levels of PPEB are associated with the absence of SLR2 in most of the tested RILs. We identified candidate genes potentially involved in the control of mate selection and showed that pollination of maternal genotypes with their favored pollen donors produces offspring with longer roots. When the maternal genotypes are only able to select against nonfavored pollen donors, the selection for such positive traits is abolished. We conclude that plants' ability of mate choice contributes to measurable changes in progeny phenotypes and is thus likely a target of selection.

Exploring the future of GM technology in sustainable local food systems in Colombia.

The security of Earth's food systems is challenged by shifting regional climates. While agricultural processes are disrupted by climate change, they also play a large role in contributing to destabilizing greenhouse gases. Finding new strategies to increase yields while decreasing agricultural environmental impacts is essential. Tropical agriculture is particularly susceptible to climate change: local, smallholder farming, which provides a majority of the food supply, is high risk and has limited adaptation capacity. Rapid, inexpensive, intuitive solutions are needed, like the implementation of genetically modified (GM) crops. In the Latin American tropics, high awareness and acceptance of GM technologies, opportunities to test GM crops as part of local agricultural educations, and their known economic benefits, support their use. However, this is not all that is needed for the future of GM technologies in these areas: GM implementation must also consider environmental and social sustainability, which can be unique to a locality. Primarily from the perspective of its educators, the potential of a rural Colombian university in driving GM implementation is explored, including the role of this type of university in producing agricultural engineers who can innovate with GM to meet regionally-dependent environmental and cultural needs that could increase their sustainability.

Integrating plant and fungal quantitative genetics to improve the ecological and agricultural applications of mycorrhizal symbioses.

Finding and targeting genes that quantitatively contribute to agricultural and ecological processes progresses food production and conservation efforts. Typically, quantitative genetic approaches link variants in a single organism's genome with a trait of interest. Recently, genome-to-genome mapping has found genome variants interacting between species to produce the result of a multiorganism (including multikingdom) interaction. These were plant and bacterial pathogen genome interactions; plant-fungal coquantitative genetics have not yet been applied. Plant-mycorrhizae symbioses exist across most biomes, for a majority of land plants, including crop plants, and manipulate many traits from single organisms to ecosystems for which knowing the genetic basis would be useful. The availability of Rhizophagus irregularis mycorrhizal isolates, with genomic information, makes dual-genome methods with beneficial mutualists accessible and imminent.

TOC1 in Nicotiana attenuata regulates efficient allocation of nitrogen to defense metabolites under herbivory stress.

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.

Allelic differences of clustered terpene synthases contribute to correlated intraspecific variation of floral and herbivory-induced volatiles in a wild tobacco.

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)-ß-ocimene and (E)-α-bergamotene contributed to the correlated changes in HIPVs and floral volatiles among N. attenuata natural accessions. Intraspecific variations of (E)-ß-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)-ß-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.

Determining the scale at which variation in a single gene changes population yields.

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.

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 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.

Blumenols as shoot markers of root symbiosis with arbuscular mycorrhizal fungi.

High-through-put (HTP) screening for functional arbuscular mycorrhizal fungi (AMF)-associations is challenging because roots must be excavated and colonization evaluated by transcript analysis or microscopy. Here we show that specific leaf-metabolites provide broadly applicable accurate proxies of these associations, suitable for HTP-screens. With a combination of untargeted and targeted metabolomics, we show that shoot accumulations of hydroxy- and carboxyblumenol C-glucosides mirror root AMF-colonization in Nicotiana attenuata plants. Genetic/pharmacologic manipulations indicate that these AMF-indicative foliar blumenols are synthesized and transported from roots to shoots. These blumenol-derived foliar markers, found in many di- and monocotyledonous crop and model plants (Solanum lycopersicum, Solanum tuberosum, Hordeum vulgare, Triticum aestivum, Medicago truncatula and Brachypodium distachyon), are not restricted to particular plant-AMF interactions, and are shown to be applicable for field-based QTL mapping of AMF-related genes.

The Active Jasmonate JA-Ile Regulates a Specific Subset of Plant Jasmonate-Mediated Resistance to Herbivores in Nature.

The jasmonate hormones are essential regulators of plant defense against herbivores and include several dozen derivatives of the oxylipin jasmonic acid (JA). Among these, the conjugate jasmonoyl isoleucine (JA-Ile) has been shown to interact directly with the jasmonate co-receptor complex to regulate responses to jasmonate signaling. However, functional studies indicate that some aspects of jasmonate-mediated defense are not regulated by JA-Ile. Thus, it is not clear whether JA-Ile is best characterized as the master jasmonate regulator of defense, or if it regulates more specific aspects. We investigated possible functions of JA-Ile in anti-herbivore resistance of the wild tobacco Nicotiana attenuata, a model system for plant-herbivore interactions. We first analyzed the soluble and volatile secondary metabolomes of irJAR4xirJAR6, asLOX3, and WT plants, as well as an RNAi line targeting the jasmonate co-receptor CORONATINE INSENSITIVE 1 (irCOI1), following a standardized herbivory treatment. irJAR4xirJAR6 were the most similar to WT plants, having a ca. 60% overlap in differentially regulated metabolites with either asLOX3 or irCOI1. In contrast, while at least 25 volatiles differed between irCOI1 or asLOX3 and WT plants, there were few or no differences in herbivore-induced volatile emission between irJAR4xirJAR6 and WT plants, in glasshouse- or field-collected samples. We then measured the susceptibility of jasmonate-deficient vs. JA-Ile-deficient plants in nature, in comparison to wild-type (WT) controls, and found that JA-Ile-deficient plants (irJAR4xirJAR6) are much better defended even than a mildly jasmonate-deficient line (asLOX3). The differences among lines could be attributed to differences in damage from specific herbivores, which appeared to prefer either one or the other jasmonate-deficient phenotype. We further investigated the elicitation of one herbivore-induced volatile known to be jasmonate-regulated and to mediate resistance to herbivores: (E)-α-bergamotene. We found that JA was a more potent elicitor of (E)-α-bergamotene emission than was JA-Ile, and when treated with JA, irJAR4xirJAR6 plants emitted 20- to 40-fold as much (E)-α-bergamotene than WT. We conclude that JA-Ile regulates specific aspects of herbivore resistance in N. attenuata. This specificity may allow plants flexibility in their responses to herbivores and in managing trade-offs between resistance, vs. growth and reproduction, over the course of ontogeny.

Cry1Ac production is costly for native plants attacked by non-Cry1Ac-targeted herbivores in the field.

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.

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.