Radiocarbon Flux Measurements Provide Insight into Why a Pyroligneous Acid Product Stimulates Plant Growth.

Agriculture in the 21st century faces many formidable challenges with the growing global population. Increasing demands on the planet's natural resources already tax existing agricultural practices. Today, many farmers are using biochemical treatments to improve their yields. Commercialized organic biostimulants exist in the form of pyroligneous acid generated by burning agricultural waste products. Recently, we examined the mechanisms through which a commercial pyroligneous acid product, Coriphol™, manufactured by Corigin Solutions, Inc., stimulates plant growth. During the 2023 growing season, outdoor studies were conducted in soybean to examine the effects of different Coriphol™ treatment concentrations on plant growth. Plant height, number of leaves, and leaf size were positively impacted in a dose-dependent manner with 2 gallon/acre soil treatments being optimal. At harvest, this level of treatment boosted crop yield by 40%. To gain an understanding of why Coriphol™ improves plant fitness, follow-up laboratory-based studies were conducted using radiocarbon flux analysis. Here, radioactive 11CO2 was administered to live plants and comparisons were made between untreated soybean plants and plants treated at an equivalent Coriphol™ dose of 2 gallons/acre. Leaf metabolites were analyzed using radio-high-performance liquid chromatography for [11C]-chlorophyll (Chl) a and b components, as well as [11C]-ß-carotene (ß-Car) where fractional yields were used to calculate metabolic rates of synthesis. Altogether, Coriphol™ treatment boosted rates of Chl a, Chl b, and ß-Car biosynthesis 3-fold, 2.6-fold, and 4.7-fold, respectively, and also increased their metabolic turnover 2.2-fold, 2.1-fold, and 3.9-fold, respectively. Also, the Chl a/b ratio increased from 3.1 to 3.4 with treatment. Altogether, these effects contributed to a 13.8% increase in leaf carbon capture.

Radiocarbon Flux Measurements Reveal Mechanistic Insight into Heat-Stress Induction of Nicotine Biosynthesis in Nicotiana attenuata.

The effect of high-temperature (HT) stress on nicotine biosynthesis in Nicotiana attenuata was examined. Nicotine content was measured in mature leaves, young sink leaves, and in roots from well-watered plants grown at 25 °C as controls and from plants exposed to 38 °C and 43 °C temperatures applied for 24, 48, 72, and 96 h duration. At 38 °C, all leaf nicotine levels were significantly less than control plants for up to 72 h exposure but rose sharply thereafter to levels significantly greater than controls with 96 h exposure. In contrast, plants exposed to 43 °C never exhibited a reduction in leaf nicotine content and showed an increase in content with just 48 h exposure. Using radioactive 11CO2 and 13NO3-, we found that HT stress reduced both CO2 fixation and nitrate uptake. Furthermore, radiocarbon flux analysis revealed that 'new' carbon partitioning (as 11C) into the 11C-radiolabeled amino acid (AA) pool was significantly reduced with HT stress as were yields of [11C]-aspartic acid, an important AA in nicotine biosynthesis, and its beta-amido counterpart [11C]-asparagine. In contrast, [12C]-aspartic acid levels appeared unaffected at 38 °C but were elevated at 43 °C relative to controls. [12C]-Asparagine levels were noted to be elevated at both stress temperatures. Since HT reductions in carbon input and nitrogen uptake were noted to impede de novo AA biosynthesis, protein degradation at HT was examined as a source of AAs. Here, leaf total soluble protein (TSP) content was reduced 39% with long exposures to both stress temperatures. However, Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) which was 41% TSP appeared unaffected. Altogether, these results support the theory that plant proteins other than Rubisco degrade at elevated temperatures freeing up essential AAs in support of nicotine biosynthesis.

Studies Using Mutant Strains of Azospirillum brasilense Reveal That Atmospheric Nitrogen Fixation and Auxin Production Are Light Dependent Processes.

As the use of microbial inoculants in agriculture rises, it becomes important to understand how the environment may influence microbial ability to promote plant growth. This work examines whether there are light dependencies in the biological functions of Azospirillum brasilense, a commercialized prolific grass-root colonizer. Though classically defined as non-phototrophic, A. brasilense possesses photoreceptors that could perceive light conducted through its host's roots. Here, we examined the light dependency of atmospheric biological nitrogen fixation (BNF) and auxin biosynthesis along with supporting processes including ATP biosynthesis, and iron and manganese uptake. Functional mutants of A. brasilense were studied in light and dark environments: HM053 (high BNF and auxin production), ipdC (capable of BNF, deficient in auxin production), and FP10 (capable of auxin production, deficient in BNF). HM053 exhibited the highest rate of nitrogenase activity with the greatest light dependency comparing iterations in light and dark environments. The ipdC mutant showed similar behavior with relatively lower nitrogenase activity observed, while FP10 did not show a light dependency. Auxin biosynthesis showed strong light dependencies in HM053 and FP10 strains, but not for ipdC. Ferrous iron is involved in BNF, and a light dependency was observed for microbial 59Fe2+ uptake in HM053 and ipdC, but not FP10. Surprisingly, a light dependency for 52Mn2+ uptake was only observed in ipdC. Finally, ATP biosynthesis was sensitive to light across all three mutants favoring blue light over red light compared to darkness with observed ATP levels in descending order for HM053 > ipdC > FP10.

Plants Utilize Suberin Biopolymers as a Vector for Transmitting Visible Light through Their Roots.

Plants conduct light from their aboveground tissues belowground to their root system. This phenomenon may influence root growth and perhaps serve to stimulate natural biological functions of the microorganisms associating with them. Here we show that light transmission in maize roots largely occurs within the endodermis, a region rich in suberin polyester biopolymers. Using cork as a natural resource rich in suberin polymers, we extracted, depolymerized, and examined light transmission in the visible and infrared regions. Suberin co-monomers dissolved in toluene showed no evidence of enhanced light transmission over that of the pure solvent in the visible light region and reduced light transmission in the infrared region. However, when these co-monomers were catalytically repolymerized using Bi(OTf)3, light transmission through suspended polymers significantly increased 1.3-fold in the visible light region over that in pure toluene, but was reduced in the infrared region.

Treatments with Liquid Smoke and Certain Chemical Constituents Prevalent in Smoke Reduce Phloem Vascular Sectoriality in the Sunflower with Improvement to Growth.

Many higher plants possess a physiological organization that is based upon the carbon economy of their parts. While photosynthates are partitioned according to the relative strength of the plant's sink tissues, in many species there is also a very close relationship between partitioning, phyllotaxy and vascular connectivity giving rise to sectorial patterns of allocation. Here, we examined the influence of smoke and certain chemical constituents prevalent in smoke including, catechol, resorcinol and hydroquinone on phloem vascular sectoriality in common sunflower (Helianthis annuus L.), as a model plant for sectoriality. By administering radioactive carbon-11 to a single source leaf as 11CO2, 11C-photosynthate allocation patterns were examined using autoradiography. A 1:200 aqueous dilution of liquid smoke treated soil caused 2.6-fold and 2.5-fold reductions in phloem sectoriality in sink leaves and roots, respectively. Treatment with catechol (1,2-d ihydroxybenzene) or resorcinol (1,3-dihydroxybenzene), polyphenolic constituents that are prevalent in smoke, caused similar reductions in phloem sectoriality in the same targeted sink tissues. However, treatment with hydroquinone (1,4-dihydroxybenzene) had no effect. Finally, the longer-term effects of smoke exposure on plant growth and performance were examined using outdoor potted plants grown over the 2022 season. Plants exposed to liquid smoke treatments of the soil on a weekly basis had larger thicker leaves possessing 35% greater lignin content than untreated control plants. They also had thicker stems although the lignin content was the same as controls. Additionally, plants exposed to treatment produced twice the number of flowers with no difference in their disk floret diameters as untreated controls. Altogether, loss of phloem sectoriality from exposure to liquid smoke in the sunflower model benefited plant performance.

Azospirillum brasilense Bacteria Promotes Mn2+ Uptake in Maize with Benefits to Leaf Photosynthesis.

Azospirillum brasilense is a prolific grass-root colonizing bacteria well-known for its ability to promote plant growth in several cereal crops. Here we show that one of the mechanisms of action in boosting plant performance is through increased assimilation of the micronutrient manganese by the host. Using radioactive 52Mn2+ (t½ 5.59 d), we examined the uptake kinetics of this micronutrient in young maize plants, comparing the performance of three functional mutants of A. brasilense, including HM053, a high auxin-producing and high N2-fixing strain; ipdC, a strain with a reduced auxin biosynthesis capacity; and FP10, a strain deficient in N2-fixation that still produces auxin. HM053 had the greatest effect on host 52Mn2+ uptake, with a significant increase seen in shoot radioactivity relative to non-inoculated controls. LA-ICP-MS analysis of root sections revealed higher manganese distributions in the endodermis of HM053-inoculated plants and overall higher manganese concentrations in leaves. Finally, increased leaf manganese concentration stimulated photosynthesis as determined by measuring leaf fixation of radioactive 11CO2 with commensurate increases in chlorophyll concentration.

Examining effects of rhizobacteria in relieving abiotic crop stresses using carbon-11 radiotracing.

In agriculture, plant growth promoting bacteria (PGPB) are increasingly used for reducing environmental stress-related crop losses through mutualistic actions of these microorganisms, activating physiological and biochemical responses, building tolerances within their hosts. Here we report the use of radioactive carbon-11 (t½ 20.4 min) to examine the metabolic and physiological responses of Zea mays to Azospirillum brasilense (HM053) inoculation while plants were subjected to salinity and low nitrogen stresses. Host metabolism of "new" carbon resources (as 11 C) and physiology including [11 C]-photosynthate translocation were measured in response to imposed growth conditions. Salinity stress caused shortened, dense root growth with a 6-fold increase in foliar [11 C]-raffinose, a potent osmolyte. ICP-MS analyses revealed increased foliar Na+ levels at the expense of K+ . HM053 inoculation relieved these effects, reinstating normal root growth, lowering [11 C]-raffinose levels while increasing [11 C]-sucrose and its translocation to the roots. Na+ levels remained elevated with inoculation, but K+ levels were boosted slightly. Low nitrogen stress yielded longer roots possessing high levels of anthocyanins. Metabolic analysis revealed significant shifts in "new" carbon partitioning into the amino acid pool under low nitrogen stress, with significant increases in foliar [11 C]-glutamate, [11 C]-aspartate, and [11 C]-asparagine, a noted osmoprotectant. 11 CO2 fixation and [11 C]-photosynthate translocation also decreased, limiting carbon supply to roots. However, starch levels in roots were reduced under nitrogen limitation, suggesting that carbon repartitioning could be a compensatory action to support root growth. Finally, inoculation with HM053 re-instated normal root growth, reduced anthocyanin, boosted root starch, and returned 11 C-allocation levels back to those of unstressed plants.

Carbon-11 Radiotracing Reveals Physiological and Metabolic Responses of Maize Grown under Different Regimes of Boron Treatment.

In agriculture, boron is known to play a critical role in healthy plant growth. To dissect the role of boron in maize metabolism, radioactive carbon-11 (t½ 20.4 min) was used to examine the physiological and metabolic responses of 3-week-old B73 maize plants to different levels of boron spanning 0 mM, 0.05 mM, and 0.5 mM boric acid (BA) treatments. Growth behavior, of both shoots and roots, was recorded and correlated to plant physiological responses. 11CO2 fixation, leaf export of [11C]-photosynthates, and their rate of transport increased systematically with increasing BA concentrations, while the fraction of [11C]-photosynthates delivered to the roots under 0 mM and 0.5 mM BA treatments was lower than under 0.05 mM BA treatment, likely due to changes in root growth. Additionally, solid-phase extraction coupled with gamma counting, radio-fluorescence thin layer chromatography, and radio-fluorescence high-performance liquid chromatography techniques applied to tissue extracts provided insight into the effects of BA treatment on 'new' carbon (as 11C) metabolism. Most notable was the strong influence reducing boron levels had on raising 11C partitioning into glutamine, aspartic acid, and asparagine. Altogether, the growth of maize under different regimes of boron affected 11CO2 fixation, its metabolism and allocation belowground, and altered root growth. Finally, inductively coupled plasma mass spectrometry provided insight into the effects of BA treatment on plant uptake of other essential nutrients. Here, levels of boron and zinc systematically increased in foliar tissues with increasing BA concentration. However, levels of magnesium, potassium, calcium, manganese, and iron remained unaffected by treatment. The rise in foliar zinc levels with increased BA concentration may contribute to improved 11CO2 fixation under these conditions.

Examining the Effects of the Nitrogen Environment on Growth and N2-Fixation of Endophytic Herbaspirillum seropedicae in Maize Seedlings by Applying 11C Radiotracing.

Herbaspirillum seropedicae, as an endophyte and prolific root colonizer of numerous cereal crops, occupies an important ecological niche in agriculture because of its ability to promote plant growth and potentially improve crop yield. More importantly, there exists the untapped potential to harness its ability, as a diazotroph, to fix atmospheric N2 as an alternative nitrogen resource to synthetic fertilizers. While mechanisms for plant growth promotion remain controversial, especially in cereal crops, one irrefutable fact is these microorganisms rely heavily on plant-borne carbon as their main energy source in support of their own growth and biological functions. Biological nitrogen fixation (BNF), a microbial function that is reliant on nitrogenase enzyme activity, is extremely sensitive to the localized nitrogen environment of the microorganism. However, whether internal root colonization can serve to shield the microorganisms and de-sensitize nitrogenase activity to changes in the soil nitrogen status remains unanswered. We used RAM10, a GFP-reporting strain of H. seropedicae, and administered radioactive 11CO2 tracer to intact 3-week-old maize leaves and followed 11C-photosynthates to sites within intact roots where actively fluorescing microbial colonies assimilated the tracer. We examined the influence of administering either 1 mM or 10 mM nitrate during plant growth on microbial demands for plant-borne 11C. Nitrogenase activity was also examined under the same growth conditions using the acetylene reduction assay. We found that plant growth under low nitrate resulted in higher nitrogenase activity as well as higher microbial demands for plant-borne carbon than plant growth under high nitrate. However, carbon availability was significantly diminished under low nitrate growth due to reduced host CO2 fixation and reduced allocation of carbon resources to the roots. This response of the host caused significant inhibition of microbial growth. In summary, internal root colonization did little to shield these endophytic microorganisms from the nitrogen environment.

Plant-Growth-Promoting Bacteria Can Impact Zinc Uptake in Zea mays: An Examination of the Mechanisms of Action Using Functional Mutants of Azospirillum brasilense.

Among the PGPB, the genus Azospirillum-with an emphasis on A. brasilense-is likely the most studied microorganism for mitigation of plant stress. Here, we report the investigation of functional mutants HM053, ipdC and FP10 of A. brasilense to understand how the biological functions of these microorganisms can affect host Zn uptake. HM053 is a Nif+ constitutively expressed strain that hyper-fixes N2 and produces high levels of the plant's relevant hormone auxin. FP10 is a Nif- strain deficient in N2-fixation. ipdC is a strain that is deficient in auxin production but fixes N2. Zn uptake was measured in laboratory-based studies of 3-week-old plants using radioactive 65Zn2+ (t½ 244 days). Principal Component Analysis was applied to draw out correlations between microbial functions and host 65Zn2+ accumulation. Additionally, statistical correlations were made to our prior data on plant uptake of radioactive 59Fe3+ and 59Fe2+. These correlations showed that low microbial auxin-producing capacity resulted in the greatest accumulation of 65Zn. Just the opposite effect was noted for 59Fe where high microbial auxin-producing capacity resulted in the greatest accumulation of that tracer.

From the Outside in: An Overview of Positron Imaging of Plant and Soil Processes.

Positron-emitting nuclides have long been used as imaging agents in medical science to spatially trace processes non-invasively, allowing for real-time molecular imaging using low tracer concentrations. This ability to non-destructively visualize processes in real time also makes positron imaging uniquely suitable for probing various processes in plants and porous environmental media, such as soils and sediments. Here, we provide an overview of historical and current applications of positron imaging in environmental research. We highlight plant physiological research, where positron imaging has been used extensively to image dynamics of macronutrients, signalling molecules, trace elements, and contaminant metals under various conditions and perturbations. We describe how positron imaging is used in porous soils and sediments to visualize transport, flow, and microbial metabolic processes. We also address the interface between positron imaging and other imaging approaches, and present accompanying chemical analysis of labelled compounds for reviewed topics, highlighting the bridge between positron imaging and complementary techniques across scales. Finally, we discuss possible future applications of positron imaging and its potential as a nexus of interdisciplinary biogeochemical research.

Defense Priming in Nicotiana tabacum Accelerates and Amplifies 'New' C/N Fluxes in Key Amino Acid Biosynthetic Pathways.

In the struggle to survive herbivory by leaf-feeding insects, plants employ multiple strategies to defend themselves. One mechanism by which plants increase resistance is by intensifying their responsiveness in the production of certain defense agents to create a rapid response. Known as defense priming, this action can accelerate and amplify responses of metabolic pathways, providing plants with long-lasting resistance, especially when faced with waves of attack. In the work presented, short-lived radiotracers of carbon administered as 11CO2 and nitrogen administered as 13NH3 were applied in Nicotiana tabacum, to examine the temporal changes in 'new' C/N utilization in the biosynthesis of key amino acids (AAs). Responses were induced by using topical application of the defense hormone jasmonic acid (JA). After a single treatment, metabolic partitioning of recently fixed carbon (designated 'new' carbon and reflected as 11C) increased through the shikimate pathway, giving rise to tyrosine, phenylalanine and tryptophan. Amplification in 'new' carbon fluxes preceded changes in the endogenous (12C) pools of these AAs. Testing after serial JA treatments revealed that fluxes of 'new' carbon were accelerated, amplified and sustained over time at this higher rate, suggesting a priming effect. Similar results were observed with recently assimilated nitrogen (designated 'new' nitrogen reflected as 13N) with its partitioning into serine, glycine and glutamine, which play important roles supporting the shikimate pathway and downstream secondary metabolism. Finally, X-ray fluorescence imaging revealed that levels of the element Mn, an important co-factor for enzyme regulation in the shikimate pathway, increased within JA treated tissues, suggesting a link between plant metal ion regulation and C/N metabolic priming.

Quantifying Plant-Borne Carbon Assimilation by Root-Associating Bacteria.

Herbaspirillum seropedicae is a rhizobacteria that occupies a specialized ecological niche in agriculture. As an endophyte and prolific grass root colonizer it has the potential to promote plant growth, enhancing crop yield in many cereal crops. While the mechanisms for plant growth promotion are controversial, the one irrefutable fact is these microorganisms rely heavily on plant-borne carbon as their main energy source in support of their biological functions. Unfortunately, the tools and technology enabling researchers to trace carbon exchange between plants and the microorganisms associating with them has been limiting. Here, we demonstrate that radioactive 11CO2 administered to intact maize leaves with translocation of 11C-photosynthates to roots can provide a 'traceable' source of carbon whose assimilation by microbial organisms can be quantified with enormous sensitivity. Fluorescence root imaging of RAM10, a green fluorescent protein (GFP) reporting strain of H. seropedicae, was used to identify regions of high microbial colonization. Microbes were mechanically removed from these regions via sonication in saline solution and extracts were subjected to fluorescence measurement and gamma counting to correlate carbon-11 atoms with numbers of colony forming units. The method has potential to translate to other microorganisms provided they possess an optical reporting trait.

Synthesis of l-[4-11 C]Asparagine by Ring-Opening Nucleophilic 11 C-Cyanation Reaction of a Chiral Cyclic Sulfamidate Precursor.

The development of a convenient and rapid method to synthesize radiolabeled, enantiomerically pure amino acids (AAs) as potential positron emission tomography (PET) imaging agents for mapping various biochemical transformations in living organisms remains a challenge. This is especially true for the synthesis of carbon-11-labeled AAs given the short half-life of carbon-11 (11 C, t1/2 =20.4 min). A facile synthetic pathway to prepare enantiomerically pure 11 C-labeled l-asparagine was developed using a partially protected serine as a starting material with a four-step transformation providing a chiral five-membered cyclic sulfamidate as the radiolabeling precursor. Its structure and absolute configuration were confirmed by X-ray crystallography. Utilizing a [11 C]cyanide nucleophilic ring opening reaction followed by selective acidic hydrolysis and deprotection, enantiomerically pure l-[4-11 C]asparagine was synthesized. Further optimization of reaction parameters, including base, metal ion source, solvent, acid component, reaction temperature and reaction time, a reliable two-step method for synthesizing l-[4-11 C]asparagine was presented: within a 45±3 min (n=5, from end-of-bombardment), the desired enantiomerically pure product was synthesized with the initial nucleophilic cyanation yield of 69±4 % (n=5) and overall two-step radiochemical yield of 53±2 % (n=5) based on starting [11 C]HCN, and with radiochemical purity of 96±2 % (n=5).

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.

In vivo quantitative imaging of photoassimilate transport dynamics and allocation in large plants using a commercial positron emission tomography (PET) scanner.

BACKGROUND: Although important aspects of whole-plant carbon allocation in crop plants (e.g., to grain) occur late in development when the plants are large, techniques to study carbon transport and allocation processes have not been adapted for large plants. Positron emission tomography (PET), developed for dynamic imaging in medicine, has been applied in plant studies to measure the transport and allocation patterns of carbohydrates, nutrients, and phytohormones labeled with positron-emitting radioisotopes. However, the cost of PET and its limitation to smaller plants has restricted its use in plant biology. Here we describe the adaptation and optimization of a commercial clinical PET scanner to measure transport dynamics and allocation patterns of (11)C-photoassimilates in large crops. RESULTS: Based on measurements of a phantom, we optimized instrument settings, including use of 3-D mode and attenuation correction to maximize the accuracy of measurements. To demonstrate the utility of PET, we measured (11)C-photoassimilate transport and allocation in Sorghum bicolor, an important staple crop, at vegetative and reproductive stages (40 and 70 days after planting; DAP). The (11)C-photoassimilate transport speed did not change over the two developmental stages. However, within a stem, transport speeds were reduced across nodes, likely due to higher (11)C-photoassimilate unloading in the nodes. Photosynthesis in leaves and the amount of (11)C that was exported to the rest of the plant decreased as plants matured. In young plants, exported (11)C was allocated mostly (88 %) to the roots and stem, but in flowering plants (70 DAP) the majority of the exported (11)C (64 %) was allocated to the apex. CONCLUSIONS: Our results show that commercial PET scanners can be used reliably to measure whole-plant C-allocation in large plants nondestructively including, importantly, allocation to roots in soil. This capability revealed extreme changes in carbon allocation in sorghum plants, as they advanced to maturity. Further, our results suggest that nodes may be important control points for photoassimilate distribution in crops of the family Poaceae. Quantifying real-time carbon allocation and photoassimilate transport dynamics, as demonstrated here, will be important for functional genomic studies to unravel the mechanisms controlling phloem transport in large crop plants, which will provide crucial insights for improving yields.

Robust biological nitrogen fixation in a model grass-bacterial association.

Nitrogen-fixing rhizobacteria can promote plant growth; however, it is controversial whether biological nitrogen fixation (BNF) from associative interaction contributes to growth promotion. The roots of Setaria viridis, a model C4 grass, were effectively colonized by bacterial inoculants resulting in a significant enhancement of growth. Nitrogen-13 tracer studies provided direct evidence for tracer uptake by the host plant and incorporation into protein. Indeed, plants showed robust growth under nitrogen-limiting conditions when inoculated with an ammonium-excreting strain of Azospirillum brasilense. (11)C-labeling experiments showed that patterns in central carbon metabolism and resource allocation exhibited by nitrogen-starved plants were largely reversed by bacterial inoculation, such that they resembled plants grown under nitrogen-sufficient conditions. Adoption of S. viridis as a model should promote research into the mechanisms of associative nitrogen fixation with the ultimate goal of greater adoption of BNF for sustainable crop production.

Radiosynthesis of 3-indolyl[1-¹¹C]acetic acid for phyto-PET-imaging: an improved production procedure and formulation method.

An improved production procedure and formulation method for the carbon-11 radiolabeled phytohormone, 3-indolyl-[l-(11)C]acetic acid ([(11)C]IAA), was developed by modifying selected original reaction parameters. This updated procedure both doubled the yield (from 25.9±6.7% (n=12) to 61.0±0.3% (n=10)) and increased the concentration (0.2-0.4 GBq/0.15-0.3 mL), enabling us to provide the radiotracer [(11)C]IAA suitable for in vivo phyto-PET-imaging studies. The specific activity was improved by more than a factor of three (26.7±5.6 GBq/µmol to 82.5±36.1 GBq/µmol). The total synthesis time for both production and formulation was 81.8±3.0 min (n=10). In addition, a streamlined semi-remote controlled production system, containing five processing modules, was designed and built for routine [(11)C]IAA production. This integrated system facilitated routine high radiation level production of [(11)C]IAA while minimizing radiation exposure to the production chemists.

Induced carbon reallocation and compensatory growth as root herbivore tolerance mechanisms.

Upon attack by leaf herbivores, many plants reallocate photoassimilates below ground. However, little is known about how plants respond when the roots themselves come under attack. We investigated induced resource allocation in maize plants that are infested by the larvae Western corn rootworm Diabrotica virgifera virgifera. Using radioactive (11) CO(2), we demonstrate that root-attacked maize plants allocate more new (11) C carbon from source leaves to stems, but not to roots. Reduced meristematic activity and reduced invertase activity in attacked maize root systems are identified as possible drivers of this shoot reallocation response. The increased allocation of photoassimilates to stems is shown to be associated with a marked thickening of these tissues and increased growth of stem-borne crown roots. A strong quantitative correlation between stem thickness and root regrowth across different watering levels suggests that retaining photoassimilates in the shoots may help root-attacked plants to compensate for the loss of belowground tissues. Taken together, our results indicate that induced tolerance may be an important strategy of plants to withstand belowground attack. Furthermore, root herbivore-induced carbon reallocation needs to be taken into account when studying plant-mediated interactions between herbivores.

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.