Diurnal cycling of rhizosphere bacterial communities is associated with shifts in carbon metabolism.

BACKGROUND: The circadian clock regulates plant metabolic functions and is an important component in plant health and productivity. Rhizosphere bacteria play critical roles in plant growth, health, and development and are shaped primarily by soil communities. Using Illumina next-generation sequencing and high-resolution mass spectrometry, we characterized bacterial communities of wild-type (Col-0) Arabidopsis thaliana and an acyclic line (OX34) ectopically expressing the circadian clock-associated cca1 transcription factor, relative to a soil control, to determine how cycling dynamics affected the microbial community. Microbial communities associated with Brachypodium distachyon (BD21) were also evaluated. RESULTS: Significantly different bacterial community structures (P = 0.031) were observed in the rhizosphere of wild-type plants between light and dark cycle samples. Furthermore, 13% of the community showed cycling, with abundances of several families, including Burkholderiaceae, Rhodospirillaceae, Planctomycetaceae, and Gaiellaceae, exhibiting fluctuation in abundances relative to the light cycle. However, limited-to-no cycling was observed in the acyclic CCAox34 line or in soil controls. Significant cycling was also observed, to a lesser extent, in Brachypodium. Functional gene inference revealed that genes involved in carbohydrate metabolism were likely more abundant in near-dawn, dark samples. Additionally, the composition of organic matter in the rhizosphere showed a significant variation between dark and light cycles. CONCLUSIONS: The results of this study suggest that the rhizosphere bacterial community is regulated, to some extent, by the circadian clock and is likely influenced by, and exerts influences, on plant metabolism and productivity. The timing of bacterial cycling in relation to that of Arabidopsis further suggests that diurnal dynamics influence plant-microbe carbon metabolism and exchange. Equally important, our results suggest that previous studies done without relevance to time of day may need to be reevaluated with regard to the impact of diurnal cycles on the rhizosphere microbial community.

Localized micronutrient patches induce lateral root foraging and chemotropism in Nicotiana attenuata.

Nutrients are distributed unevenly in the soil. Phenotypic plasticity in root growth and proliferation may enable plants to cope with this variation and effectively forage for essential nutrients. However, how micronutrients shape root architecture of plants in their natural environments is poorly understood. We used a combination of field and laboratory-based assays to determine the capacity of Nicotiana attenuata to direct root growth towards localized nutrient patches in its native environment. Plants growing in nature displayed a particular root phenotype consisting of a single primary root and a few long, shallow lateral roots. Analysis of bulk soil surrounding the lateral roots revealed a strong positive correlation between lateral root placement and micronutrient gradients, including copper, iron and zinc. In laboratory assays, the application of localized micronutrient salts close to lateral root tips led to roots bending in the direction of copper and iron. This form of chemotropism was absent in ethylene and jasmonic acid deficient lines, suggesting that it is controlled in part by these two hormones. This work demonstrates that directed root growth underlies foraging behavior, and suggests that chemotropism and micronutrient-guided root placement are important factors that shape root architecture in nature.

Species-specific regulation of herbivory-induced defoliation tolerance is associated with jasmonate inducibility.

Induced changes in root carbohydrate pools are commonly assumed to determine plant defoliation tolerance to herbivores. However, the regulation and species specificity of these two traits are not well understood. We determined herbivory-induced changes in root carbohydrates and defoliation tolerance in seven different solanaceous plant species and correlated the induced changes in root carbohydrates and defoliation tolerance with jasmonate inducibility. Across species, we observed strong species-specific variation for all measured traits. Closer inspection revealed that the different species fell into two distinct groups: Species with a strong induced jasmonic acid (JA) burst suffered from a reduction in root carbohydrate pools and reduced defoliation tolerance, while species with a weak induced JA burst maintained root carbohydrate pools and tolerated defoliation. Induced JA levels predicted carbohydrate and regrowth responses better than jasmonoyl-L-isoleucine (JA-Ile) levels. Our study shows that induced JA signaling, root carbohydrate responses, and defoliation tolerance are closely linked, but highly species specific, even among closely related species. We propose that defoliation tolerance may evolve rapidly via changes in the plant's defense signaling network.

Auxin Is Rapidly Induced by Herbivore Attack and Regulates a Subset of Systemic, Jasmonate-Dependent Defenses.

Plant responses to herbivore attack are regulated by phytohormonal networks. To date, the role of the auxin indole-3-acetic acid (IAA) in this context is not well understood. We quantified and manipulated the spatiotemporal patterns of IAA accumulation in herbivore-attacked Nicotiana attenuata plants to unravel its role in the regulation of plant secondary metabolism. We found that IAA is strongly, rapidly, and specifically induced by herbivore attack. IAA is elicited by herbivore oral secretions and fatty acid conjugate elicitors and is accompanied by a rapid transcriptional increase of auxin biosynthetic YUCCA-like genes. IAA accumulation starts 30 to 60 s after local induction and peaks within 5 min after induction, thereby preceding the jasmonate (JA) burst. IAA accumulation does not require JA signaling and spreads rapidly from the wound site to systemic tissues. Complementation and transport inhibition experiments reveal that IAA is required for the herbivore-specific, JA-dependent accumulation of anthocyanins and phenolamides in the stems. In contrast, IAA does not affect the accumulation of nicotine or 7-hydroxygeranyllinalool diterpene glycosides in the same tissue. Taken together, our results uncover IAA as a rapid and specific signal that regulates a subset of systemic, JA-dependent secondary metabolites in herbivore-attacked plants.

A Nicotiana attenuata cell wall invertase inhibitor (NaCWII) reduces growth and increases secondary metabolite biosynthesis in herbivore-attacked plants.

Plant invertases are sucrolytic enzymes that are essential for the regulation of carbohydrate metabolism and source-sink relationships. While their activity has been well documented during abiotic and biotic stresses, the role of proteinaceous invertase inhibitors in regulating these changes is unknown. Here, we identify a putative Nicotiana attenuata cell wall invertase inhibitor (NaCWII) which is strongly up-regulated in a jasmonate (JA)-dependent manner following simulated attack by the specialist herbivore Manduca sexta. To understand the role of NaCWII in planta, we silenced its expression by RNA interference and measured changes in primary and secondary metabolism and plant growth following simulated herbivory. NaCWII-silenced plants displayed a stronger depletion of carbohydrates and a reduced capacity to increase secondary metabolite pools relative to their empty vector control counterparts. This coincided with the attenuation of herbivore-induced CWI inhibition and growth suppression characteristic of wild-type plants. Together our findings suggest that NaCWII may act as a regulatory switch located downstream of JA accumulation which fine-tunes the plant's balance between growth and defense metabolism under herbivore attack. Although carbohydrates are not typically viewed as key factors in plant growth and defense, our study shows that interfering with their catabolism strongly influences plant responses to herbivory.

Plant vascular architecture determines the pattern of herbivore-induced systemic responses in Arabidopsis thaliana.

The induction of systemic responses in plants is associated with the connectivity between damaged and undamaged leaves, as determined by vascular architecture. Despite the widespread appreciation for studying variation in induced plant defense, few studies have characterized spatial variability of induction in the model species, Arabidopsis thaliana. Here we show that plant architecture generates fine scale spatial variation in the systemic induction of invertase and phenolic compounds. We examined whether the arrangement of leaves along the stem (phyllotaxy) produces predictable spatial patterns of cell-wall bound and soluble invertase activities, and downstream phenolic accumulation following feeding by the dietary specialist herbivore, Pieris rapae and the generalist, Spodoptera exigua. Responses were measured in leaves within and outside of the damaged orthostichy (leaves sharing direct vascular connections), and compared to those from plants where source-sink transport was disrupted by source leaf removal and by an insertional mutation in a sucrose transporter gene (suc2-1). Following herbivore damage to a single, middle-aged leaf, induction of cell-wall and soluble invertase was most pronounced in young and old leaves within the damaged orthostichy. The pattern of accumulation of phenolics was also predicted by these vascular connections and was, in part, dependent on the presence of source leaves and intact sucrose transporter function. Induction also occurred in leaves outside of the damaged orthostichy, suggesting that mechanisms may exist to overcome vascular constraints in this system. Our results demonstrate that systemic responses vary widely according to orthostichy, are often herbivore-specific, and partially rely on transport between source and sink leaves. We also provide evidence that patterns of induction are more integrated in A. thaliana than previously described. This work highlights the importance of plant vascular architecture in determining patterns of systemic induction, which is likely to be ecologically important to insect herbivores and plant pathogens.

Jasmonate-dependent depletion of soluble sugars compromises plant resistance to Manduca sexta.

Jasmonates regulate plant secondary metabolism and herbivore resistance. How they influence primary metabolites and how this may affect herbivore growth and performance are not well understood. We profiled sugars and starch of jasmonate biosynthesis-deficient and jasmonate-insensitive Nicotiana attenuata plants and manipulated leaf carbohydrates through genetic engineering and in vitro complementation to assess how jasmonate-dependent sugar accumulation affects the growth of Manduca sexta caterpillars. We found that jasmonates reduce the constitutive and herbivore-induced concentration of glucose and fructose in the leaves across different developmental stages. Diurnal, jasmonate-dependent inhibition of invertase activity was identified as a likely mechanism for this phenomenon. Contrary to our expectation, both in planta and in vitro approaches showed that the lower sugar concentrations led to increased M. sexta growth. As a consequence, jasmonate-dependent depletion of sugars rendered N. attenuata plants more susceptible to M. sexta attack. In conclusion, jasmonates are important regulators of leaf carbohydrate accumulation and this determines herbivore growth. Jasmonate-dependent resistance is reduced rather than enhanced through the suppression of glucose and fructose concentrations, which may contribute to the evolution of divergent resistance strategies of plants in nature.

Leaf-herbivore attack reduces carbon reserves and regrowth from the roots via jasmonate and auxin signaling.

Herbivore attack leads to resource conflicts between plant defensive strategies. Photoassimilates are required for defensive compounds and carbon storage below ground and may therefore be depleted or enriched in the roots of herbivore-defoliated plants. The potential role of belowground tissues as mediators of induced tolerance-defense trade-offs is unknown. We evaluated signaling and carbohydrate dynamics in the roots of Nicotiana attenuata following Manduca sexta attack. Experimental and natural genetic variability was exploited to link the observed metabolite patterns to plant tolerance and resistance. Leaf-herbivore attack decreased sugar and starch concentrations in the roots and reduced regrowth from the rootstock and flower production in the glasshouse and the field. Leaf-derived jasmonates were identified as major regulators of this root-mediated resource-based trade-off: lower jasmonate levels were associated with decreased defense, increased carbohydrate levels and improved regrowth from the rootstock. Application and transport inhibition experiments, in combination with silencing of the sucrose non-fermenting (SNF) -related kinase GAL83, indicated that auxins may act as additional signals that regulate regrowth patterns. In conclusion, our study shows that the ability to mobilize defenses has a hidden resource-based cost below ground that constrains defoliation tolerance. Jasmonate- and auxin-dependent mechanisms may lead to divergent defensive plant strategies against herbivores in nature.

Flexible resource allocation during plant defense responses.

Plants are organisms composed of modules connected by xylem and phloem transport streams. Attack by both insects and pathogens elicits sometimes rapid defense responses in the attacked module. We have also known for some time that proteins are often reallocated away from pathogen-infected tissues, while the same infection sites may draw carbohydrates to them. This has been interpreted as a tug of war in which the plant withdraws critical resources to block microbial growth while the microbes attempt to acquire more resources. Sink-source regulated transport among modules of critical resources, particularly carbon and nitrogen, is also altered in response to attack. Insects and jasmonate can increase local sink strength, drawing carbohydrates that support defense production. Shortly after attack, carbohydrates may also be drawn to the root. The rate and direction of movement of photosynthate or signals in phloem in response to attack is subject to constraints that include branching, degree of connection among tissues, distance between sources and sinks, proximity, strength, and number of competing sinks, and phloem loading/unloading regulators. Movement of materials (e.g., amino acids, signals) to or from attack sites in xylem is less well understood but is partly driven by transpiration. The root is an influential sink and may regulate sink-source interactions and transport above and below ground as well as between the plant and the rhizosphere and nearby, connected plants. Research on resource translocation in response to pathogens or herbivores has focused on biochemical mechanisms; whole-plant research is needed to determine which, if any, of these plant behaviors actually influence plant fitness.

Temporal changes in allocation and partitioning of new carbon as (11)C elicited by simulated herbivory suggest that roots shape aboveground responses in Arabidopsis.

Using the short-lived isotope (11)C (t(1/2) = 20.4 min) as (11)CO(2), we captured temporal changes in whole-plant carbon movement and partitioning of recently fixed carbon into primary and secondary metabolites in a time course (2, 6, and 24 h) following simulated herbivory with the well-known defense elicitor methyl jasmonate (MeJA) to young leaves of Arabidopsis (Arabidopsis thaliana). Both (11)CO(2) fixation and (11)C-photosynthate export from the labeled source leaf increased rapidly (2 h) following MeJA treatment relative to controls, with preferential allocation of radiolabeled resources belowground. At the same time, (11)C-photosynthate remaining in the aboveground sink tissues showed preferential allocation to MeJA-treated, young leaves, where it was incorporated into (11)C-cinnamic acid. By 24 h, resource allocation toward roots returned to control levels, while allocation to the young leaves increased. This corresponded to an increase in invertase activity and the accumulation of phenolic compounds, particularly anthocyanins, in young leaves. Induction of phenolics was suppressed in sucrose transporter mutant plants (suc2-1), indicating that this phenomenon may be controlled, in part, by phloem loading at source leaves. However, when plant roots were chilled to 5°C to disrupt carbon flow between above- and belowground tissues, source leaves failed to allocate resources belowground or toward damaged leaves following wounding and MeJA treatment to young leaves, suggesting that roots may play an integral role in controlling how plants respond defensively aboveground.

Novel application of 2-[(18)F]fluoro-2-deoxy-D-glucose to study plant defenses.

INTRODUCTION: Since its first use in humans in 1976, 2-[¹8F]fluoro-2-deoxy-d-glucose (¹8FDG) continues to serve as a tracer to measure tissue glucose metabolism in medical imaging. Here we demonstrate a novel use for this tracer to study glycoside biosynthesis in plants as a measure of plant response to defense induction. METHODS: Coupling autoradiography with radio high-performance liquid chromatography analysis of tissue extracts, we examined the combined effects of leaf wounding and treatment using the potent plant defense hormone, methyl jasmonate (MeJA), to measure tracer distribution and tracer use in secondary defense chemistry in Arabidopsis thaliana. We hypothesized that competing sinks like roots and reproductive tissues, as well as vascular architecture, would impact the induction of phenolic defenses of the plant that make use of glucose in glycoside formation by altering distribution and metabolic utilization of ¹8FDG. RESULTS: Our studies showed that leaf orthostichy defined the major route of ¹8FDG transport in both vegetative and reproductive plants when a single petiole was cut as the entry point for tracer introduction. However, when nonorthostichous leaves were damaged and treated with MeJA, ¹8FDG was transported in its intact form to these leaves 3 h later, where it was incorporated into phenolic glycosides. CONCLUSIONS: Our work demonstrates a new use for ¹8FDG in plant science with insights into carbohydrate allocation that contradict conclusions of previous studies showing transport of resources away from damaged sites.

11C-imaging: methyl jasmonate moves in both phloem and xylem, promotes transport of jasmonate, and of photoassimilate even after proton transport is decoupled.

The long-distance transport and actions of the phytohormone methyl jasmonate (MeJA) were investigated by using the short-lived positron-emitting isotope 11C to label both MeJA and photoassimilate, and compare their transport properties in the same tobacco plants (Nicotiana tabacum L.). There was strong evidence that MeJA moves in both phloem and xylem pathways, because MeJA was exported from the labeled region of a mature leaf in the direction of phloem flow, but it also moved into other parts of the same leaf and other mature leaves against the direction of phloem flow. This suggests that MeJA enters the phloem and moves in sieve tube sap along with photoassimilate, but that vigorous exchange between phloem and xylem allows movement in xylem to regions which are sources of photoassimilate. This exchange may be enhanced by the volatility of MeJA, which moved readily between non-orthostichous vascular pathways, unlike reports for jasmonic acid (which is not volatile). The phloem loading of MeJA was found to be inhibited by parachloromercuribenzenesulfonic acid (PCMBS) (a thiol reagent known to inhibit membrane transporters), and by protonophores carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and 2,4-dinitrophenol (DNP) suggesting proton co-transport. MeJA was found to promote both its own transport and that of recent photoassimilate within 60 min. Furthermore, we found that MeJA can counter the inhibitory effect of the uncoupling agent, CCCP, on sugar transport, suggesting that MeJA affects the plasma membrane proton gradient. We also found that MeJA's action may extend to the sucrose transporter, since MeJA countered the inhibitory effects of the sulfhydryl reagent, PCMBS, on the transport of photoassimilate.

Stimulating natural defenses in poplar clones (OP-367) increases plant metabolism of carbon tetrachloride.

Groundwater contamination by carbon tetrachloride (CCl4) presents a health risk as a potential carcinogen and pollutant that is capable of depleting the ozone layer. Although use of poplar trees in a phytoremediation capacity has proven to be cost effective for cleaning contaminated sites, minimizing leaf emission of volatile contaminants remains a pressing issue. We hypothesized that recently fixed carbon plays a key role in CCl4 metabolism in planta yielding nonvolatile trichloroacetic acid (TCA) and that the extent of this metabolism can be altered by heightening plant defenses. Labeling intact leaves with (11)CO2 (t 1/2 20.4 m) can test this hypothesis, because the extremely short half-life of the tracer reflects only those processes involving recently fixed carbon. Using radio-HPLC analysis, we observed [(11)C]TCA from leaf extract from poplar clones (OP-367) whose roots were exposed to a saturated solution of CCl4 (520 ppm). Autoradiography of [(11)C]photosynthate showed increased leaf export and partitioning to the apex within 24 h of CCl4 exposure, suggesting that changes in plant metabolism and partitioning of recently fixed carbon occur rapidly. Additionally, leaf CCl4 emissions were highest in the morning, when carbon pools are low, suggesting a link between contaminant metabolism and leaf carbon utilization. Further, treatment with methyljasmonate, a plant hormone implicated in defense signal transduction, reduced leaf CCl4 emissions two-fold due to the increased formation of TCA.