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.     

A specialist root herbivore exploits defensive metabolites to locate nutritious tissues

The most valuable organs of plants are often particularly rich in essential elements, but also very well defended. This creates a dilemma for herbivores that need to maximise energy intake while minimising intoxication. We investigated how the specialist root herbivore Diabrotica virgifera solves this conundrum when feeding on wild and cultivated maize plants. We found that crown roots of maize seedlings were vital for plant development and, in accordance, were rich in nutritious primary metabolites and contained higher amounts of the insecticidal 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and the phenolic compound chlorogenic acid. The generalist herbivores Diabrotica balteata and Spodoptera littoralis were deterred from feeding on crown roots, whereas the specialist D. virgifera preferred and grew best on these tissues. Using a 1,4-benzoxazin-3-one-deficient maize mutant, we found that D. virgifera is resistant to DIMBOA and other 1,4-benzoxazin-3-ones and that it even hijacks these compounds to optimally forage for nutritious roots.     

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.     

Jasmonic acid induces rapid changes in carbon transport and partitioning in Populus

Here, we tested whether rapid changes in carbohydrate transport and partitioning to storage organs would be induced by jasmonic acid (JA), a plant-produced signal of herbivore attack known to induce resistance. Carbon-11, introduced as (11)CO(2), was used to track real-time carbohydrate transport and partitioning nondestructively in Populus species before and 12 h after application of JA to a single leaf. Jasmonic acid resulted in more rapid [(11)C]-photosynthate export from both local and systemic leaves, as well as greater partitioning of [(11)C]-photosynthate to the stem and roots. In Populus tremuloides, following JA treatment, leaf starch decreased, but there was no change in photosynthetic rates or leaf soluble sugar concentration, indicating that recent photosynthate was diverted from starch accumulation in the leaf to other plant organs. Increasing the supply of photosynthate to roots and stems may shield resources from folivorous predators, and may also facilitate both storage and nutrient uptake, and ultimately lead to greater tolerance, either by enhancing regrowth capacity or by replacing nutrients consumed by herbivores.     

Dynamic Precision Phenotyping Reveals Mechanism of Crop Tolerance to Root Herbivory

The western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte) is a major pest of maize (Zea mays) that is well adapted to most crop management strategies. Breeding for tolerance is a promising alternative to combat WCR but is currently constrained by a lack of physiological understanding and phenotyping tools. We developed dynamic precision phenotyping approaches using ¹¹C with positron emission tomography, root autoradiography, and radiometabolite flux analysis to understand maize tolerance to WCR. Our results reveal that WCR attack induces specific patterns of lateral root growth that are associated with a shift in auxin biosynthesis from indole-3-pyruvic acid to indole-3-acetonitrile. WCR attack also increases transport of newly synthesized amino acids to the roots, including the accumulation of Gln. Finally, the regrowth zones of WCR-attacked roots show an increase in Gln turnover, which strongly correlates with the induction of indole-3-acetonitrile-dependent auxin biosynthesis. In summary, our findings identify local changes in the auxin biosynthesis flux network as a promising marker for induced WCR tolerance.The western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte; Supplemental Fig. S1) is a voracious pest of maize (Zea mays). Larvae hatch in the soil during late spring and immediately begin feeding on the crop’s root system. Over time, active feeding can result in substantial root damage with significant loss of water and/or nutrient uptake, thus weakening plants (Flint-Garcia et al., 2009). Plants also become highly susceptible to lodging when major damage is inflicted upon the anchoring root system. Taken together, these effects can result in significant corn yield losses and management costs totaling between $650 million to $1 billion in the U.S. annually (Flint-Garcia et al., 2009; Gray et al., 2009).History reveals the enormous resilience and adaptability of this pest and just how quickly it can evolve to overcome management strategies. For example, resistance to application of chemical pesticides, including cyclodienes (benzene hexachloride, aldrin) and organophosphates (methyl parathion), was seen over just a 10-year period of their use in Nebraska’s cornfields during the 1950s and 1990s, respectively (Ball and Weekman, 1963; Meinke et al., 1998). Alternate management practices, including rotation of corn with other crops on a seasonal basis, was generally considered the best choice for management since 1909 (Levine et al., 2002). In east/central Illinois, 95% to 98% of cropland had adopted a management strategy using only soybean as the rotation crop. Unfortunately, the enthusiastic adoption of this strategy over a broad area combined with the efficacy of the technique created a strong selection that favored a less common D. v. virgifera phenotype with reduced egg laying fidelity to cornfields. Over time, natural selection afforded a strong reproductive advantage to females laying their eggs in soybean fields. Since the late 1990s, a strain of the western corn rootworm with resistance to crop rotation can be found in parts of Illinois, Indiana, and parts of bordering states (Gray et al., 2009; Levine et al., 2002).More recently, D. v. virgifera resistance to deployed genetically modified organisms has been reported. First introduced into the market to target this pest back in 2003, genetically altered Bt-maize expressing one or more proteins from the soil bacteria Bacillus thuringiensis provided enhanced plant defenses to larval feeding. When a vulnerable insect ate the Bt-containing plant, the protein became activated in its gut, forming a toxin that paralyzed the digestive system and caused it to stop feeding. Unfortunately, resistance began to show within three generations of selection (Meihls et al., 2008).An alternative strategy to reduce the negative impact of D. v. virgifera attack without triggering counter adaptations in the pest is plant tolerance, which relies on a plant’s capacity to maintain growth and yield even in the presence of substantial damage. While D. v. virgifera-tolerant maize germplasms exhibiting slight to moderate tolerances to D. v. virgifera have been reported (Flint-Garcia et al., 2009), more effective lines are needed. Unfortunately, we know very little about the underlying mechanisms for crop tolerance. Over the years, one resounding message has been that the physiological processes affected by herbivory should be better characterized before breeding tools can be leveraged in a rational way to generate improved varieties that maintain high yields under herbivore pressure (Riedell, 1990). Rational decision making in the breeding selection process requires rigorous phenotyping; however, present phenotyping tools tell us little about the plasticity of root systems, especially when it comes to understanding mechanisms for crop tolerance to attack belowground. It was recently suggested that the timing for allocation of newly fixed carbon resources as soluble sugars between leaves, stalks, and root systems, and their coordination with mobilization of other resources including amino acids, may play significant roles in determining the ability of maize plants to survive an attack by D. v. virgifera (Orians et al., 2011; Robert et al., 2014).In this work, our systematic evaluation of the physiological, metabolic, and genetic basis for root regrowth as a tolerance trait sheds new light on the regulation of the growth hormone auxin (indole-3-acetic acid [IAA]) and its role in this process. Radioactive decay of ¹¹C (β⁺ emitter, t_1/2 = 20.4 min), dynamic whole-plant positron emission tomography, root autoradiography, and radiometabolite flux analyses allowed us to map the transport, allocation and metabolism of carbon and nitrogen resources against genetic and radiolabeled biochemical markers including [¹¹C]IAA, [¹¹C]indole, [¹¹C]indole-3-acetonitrile ([¹¹C]IAN), [¹¹C]indole-3-acetamide ([¹¹C]IAM), and l-[5-¹¹C]Gln (Supplemental Fig. S2). Taken together, these tools enabled us for the first time, to our knowledge, to rigorously map out the auxin biosynthesis flux network at regional tissue levels and in turn provide new insights on auxin regulation and its coordination with the availability of a key amino acid, l-Gln. The developed phenotyping tools can now be employed for the rapid identification and selection of D. v. virgifera-tolerant maize germplasm.     

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.     

Expression of Group B Protective Surface Protein (BPS) by Invasive and Colonizing Isolates of Group B Streptococci

Group B protective surface protein (BPS) is expressed on the cell surface of some group B streptococcal (GBS) (Streptococcus agalactiae) strains and adds to the identification by capsular polysaccharide (CPS), and c or R proteins. We investigated the prevalence of BPS among GBS clinical isolates (303 invasive, 4122 colonizing) collected over 11 years in four American cities. Hot HCl cell extracts were tested by immunoprecipitation in agarose with rabbit antisera to BPS; the alpha (α) and beta (β) components of c protein; R1, R3, and R4 species of R protein; and CPS serotypes Ia–VIII. BPS was found in 155 isolates (seven invasive, 148 colonizing). Of these, 87 were Ia, 37 II, 20 V; none were III. BPS was expressed usually with another protein: a species of R by 87 or a component of c by 39. The predominant CPS/protein profiles with BPS were Ia/R1,BPS and II/c(α + β),BPS. Thus, along with CPS serotype and other surface proteins, BPS can be a valuable marker for precise strain characterization of unique GBS clinical isolates with complex surface protein profiles.     

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.