Quantum Dots: A New Technique to Assess Mycorrhizal Contributions to Plant Nitrogen Across a Fire-Altered Landscape

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1 Mission Kearney Foundation of Soil Science: Understanding and Managing Soil-Ecosystem Functions Across Spatial and Temporal Scales Progress Report: , 1/1/ /31/2007 Quantum Dots: A New Technique to Assess Mycorrhizal Contributions to Plant Nitrogen Across a Fire-Altered Landscape Kathleen Treseder 1,2 *, Matthew Whiteside 1, and David LeBauer 2 The overarching objective of our research is to develop a better understanding of the contributions of mycorrhizal fungi (root symbionts) to the nitrogen (N) nutrition of plants in human-impacted ecosystems of Southern California. Toward this goal, we have (1) developed a novel nanotechnological technique to measure the uptake of organic N by plants and fungi, (2) established a size gradient of fragments of coastal sage scrub in which to apply this technique, and (3) conducted a global synthesis of plant responses to N additions to place our results within a larger scale. We focused on habitat fragmentation because it is one of the most striking aspects of global change operating in Southern California. About 90% of coastal sage scrub habitat has been lost due to urban development and agriculture. Nitrogen uptake is an important issue in restoration and conservation of remaining coastal sage scrub fragments, because the growth of many plant species is N limited. Objective 1: Development of a nanotechnological technique to measure the uptake of organic N by plants and fungi. Uptake of organic nutrients by fungi and plants is difficult to study in natural systems. As a proof-of-method, we assessed quantum dots (fluorescent nanoscale semiconductors) as a new tool to observe uptake and translocation of organic N by fungi and plants. Approach: We conjugated quantum dots to the amino groups of glycine, arginine, and chitosan, and incubated them with Penicillium fungi (a saprotroph), and annual bluegrass (Poa annua) inoculated with arbuscular mycorrhizal fungi. As experimental controls, we incubated fungi and bluegrass samples with substrate-free quantum dots as well as unbound quantum dot substrate mixtures. We also field-tested quantum dots by applying them to soils. In this case, minirhizotron cameras were used to image the movement of quantum dots belowground, and UV flashlights were used to visualize quantum dot uptake in plant shoots. Results: Penicillium fungi, annual bluegrass, and arbuscular mycorrhizal fungi all showed uptake and translocation of quantum dot-labeled organic N, but no uptake of quantum dot controls (fig. 1). Quantum dots in soil-based systems could be tracked belowground in minirhizotron images, and could be viewed aboveground in plant shoots with the naked eye (fig. 2). Discussion: We are working with a promising tool to quantify organic N uptake by plants and their mycorrhizal fungi. Our ability to use minirhizotrons and handheld UV flashlights to nondestructively assess quantum dot movement in soil systems indicates that we should be able to apply this technique to our fragmented sites. This experiment is one of the first to demonstrate direct uptake of organic N by arbuscular mycorrhizal fungi. A manuscript describing this work has been submitted to Ecology. Departments of 1 Ecology and Evolutionary Biology and 2 Earth System Science, University of California, Irvine *Principal Investigator

2 Objective 2: Establishment of a size gradient of fragments of coastal sage scrub. We hypothesized that habitat fragmentation alters mycorrhizal communities by excluding plant species that harbor ectomycorrhizal fungi, because ectomycorrhizal host plants tend to require greater undisturbed area to establish. Ectomycorrhizal fungi are thought to possess a greater uptake capacity for organic N than do arbuscular mycorrhizal fungi. As a result, ecosystem-level exploitation of organic N by plants should decline, followed by a reduction in standing plant biomass and net primary productivity (NPP). Approach: We selected six habitat fragments with areas that ranged from 23 to 5,414 m 2. These fragments are located in Newport Back Bay, Orange County. They are surrounded locally by foot and horse trails and regionally by urban development. In the past year, we have installed minirhizotron tubes and have collected baseline data on mycorrhizal community structure, plant canopy height, and plant diversity. Results: We have found that plant diversity declines with decreasing habitat area (fig. 3). This decline is accompanied by a reduction in maximum canopy height in the smaller fragments. Likewise, ectomycorrhizal fungi tend to represent a smaller portion of the mycorrhizal community as fragment size diminishes, although this pattern is only marginally significant. The shift in mycorrhizal fungi may occur because ectomycorrhizal host plants are less abundant in the smaller fragments. Discussion: Our results to date support our hypotheses that a reduction in habitat size alters plant communities, with consequences for mycorrhizal community structure and standing plant biomass. We will continue to conduct these assessments throughout the next year, as well as quantify NPP. In addition, we are now ready to apply quantum dots in the sites to assess organic N uptake by mycorrhizal fungi and plant roots. Note that the focus on habitat fragmentation is a revision of our original proposal to study fire recovery; we were forced to change our plans owing to a large fire in 2007 that burned most of our fire chronosequence sites. Objective 3: A global synthesis of plant responses to N additions. We performed a metaanalysis of 126 N addition experiments to evaluate N limitation of NPP in terrestrial ecosystems. We tested the hypothesis that N limitation is widespread among biomes and influenced by geography and climate. Approach: We used the response ratio (RANPP fertilized /ANPP control ) of plant growth in fertilized to control plots. Results: We found that most ecosystems are N limited with an average 29% growth response to N (i.e., R=1.29). R was significant within temperate forests (R=1.19), tropical forest (R=1.60), temperate grasslands (R=1.53), tropical grasslands (R=1.26), wetlands (R=1.16), and tundra (R=1.35), but not deserts. Eight tropical forest studies had been conducted on very young volcanic soils in Hawaii, and this subgroup was strongly N-limited (R=2.13). The degree of N- limitation in the remainder of the tropical forest studies (R=1.20) was comparable to that of temperate forests. Moreover, forest R did not vary with latitude. Grassland response increased with latitude, but was independent of temperature and precipitation. 2

3 Discussion. These results suggest that the global N and C cycles interact strongly, and that geography can mediate ecosystem response to N within certain biome types. This work is in press in Ecology. Figure 1. Superimposed (white light and fluorescence) confocal laser scans of Penicillium solitum uptake of QD-ON conjugates. (A) At 2 hours of incubation with QD-glycine, fungal hyphae showed evidence of uptake. (B) At 6 hours, less labeled glycine was seen in solution and more within the hyphae. (C) After 24 hours, very little labeled glycine was seen outside of the hyphae. (D) After 24 hours, QD-arginine fluorescence appeared in the cytoplasm but not in vesicles (arrows). (E) Uptake of QD labeled chitosan after 5 hours of incubation. (F) QD control (quantum dots unbound to glycine) after 24 hours of incubation showed no signs of uptake. Scale bar is 10 µm. 3

4 Figure 2. Fluorescence of QD-glycine in a plant and fungi using field imaging techniques. (A-D) Unmagnified digital images of a Poa annua individual incubated with orange excitation QDglycine. (A) White light view (B) UV view (C) Root detail (D) Blade detail. Orange fluorescence indicates presence of QDs. Scale bar is (A, B) 1 cm, (C) 5 mm, (D) 500 µm. (E-G) Minirhizotron images of fungal hyphae uptake of QD-glycine (E) White light image pre-qd injection (F) UV image pre-qd injection (G) 2 hours after injection of QD-glycine, UV minirhizotron images showed hyphal uptake (arrow) of the labeled glycine. Hypha is ~2mm long. 4

5 8 6 P < r2 = Plant species richness (#/fragment) P = r 2 = 0.74 Maximum canopy ht (cm) root colonizatio Ratio of ECM:A P = 0.06 r 2 = Fragment area (m 2 ) Figure 3. Plant diversity, canopy height, and mycorrhizal community structure in a size gradient of coastal sage scrub fragments in Newport Back Bay. 5

6 This research was funded by the Kearney Foundation of Soil Science: Understanding and Managing Soil-Ecosystem Functions Across Spatial and Temporal Scales, Mission ( The Kearney Foundation is an endowed research program created to encourage and support research in the fields of soil, plant nutrition, and water science within the Division of Agriculture and Natural Resources of the University of California. 6