My desire to return back to the world of soil brought me to my second post-doc with Laura Meredith and Malak Tfaily where I have been studying the impact of drought on carbon cycling by plants and soil microbes as part of the water and life dynamics (WALD) campaign at the tropical rainforest at Biosphere 2. This large-scale 66-day drought experiment allowed me to be part of an international research group, each focusing on different aspects of the rainforest, including water, air, plants, and soil. My research focus is on how drought impacts carbon cycling and metabolic processes of microbes from the roots-rhizosphere-bulk soil continuum using multi-omics techniques.
Soil Microbial Carbon Cycling
To examine the effect of drought on soil microbial carbon cycling, we will be combining multiple ‘omics datasets including metabolomics, volatilomics, metagenomics, and metatranscriptomics from samples spanning the duration of pre-drought and drought conditions to recreate what metabolic pathways are active and identify VOC and metabolite-cycling genes. Are specific research questions is: How does drought affect soil microbial activity and carbon allocation towards energy vs. biosynthesis? And, what impact does this have on microbial contribution to atmospheric gas exchange (CO2 and VOC)? To answer these, we will approach from two angles: 1) Bulk-level by examining soil microbial activity through metagenomics, metatranscriptomics, and metabolomics, and 2) Stable isotope labeling where we will track how carbon from position-specific 13C-pyruvate is allocated into CO2, primary metabolites, and volatile organic compounds (VOCs).
Plant root metabolic pathways and carbon allocation
To investigate drought-response strategies for individual plant species, we focused on Clitoria, a canopy tree, and Piper and Hibiscus, understory plants and hypothesized that the roots from the three plant species will have different active metabolic pathways and carbon allocation strategies during drought due to their different ecological roles (trees vs. plants, understory vs. canopy). To address this hypothesis, we performed the following: 1) position-specific (C1 or C2) 13C-pyruvate labelling to observe carbon-allocation strategies, 2) 1H NMR (nuclear magnetic resonance) spectroscopy to measure all primary and 13C-labelled metabolites in roots, 3) MALDI (Matrix-assisted laser desorption/ionization) to observe distribution of metabolites within the root tissue , and 4) nanoSIMS (nanoscale secondary ion mass spectroscopy) to observe distribution of 13C label within root tissue both at ambient and drought conditions on washed (rhizosphere removed) and unwashed (rhizosphere remained intact) roots.
Our results showed that the three plant species responded differently to drought. NMR showed Piper and Hibiscus metabolic profiles are most impacted by drought, while Clitoria’s are most impacted by whether the roots were washed or unwashed. MALDI data showed that in response to drought both Piper and Hibiscus accumulated sugars in their roots, however, Piper also accumulated lignin, while Hibiscus accumulated tannins and fatty acids. This pattern corresponded to changes observed in 13C labeled metabolites where Piper showed a slowing down of acetate production while Hibiscus maintained acetate and ethanol production via glycolysis, which could have proceeded fatty acid production. Meanwhile, MALDI showed that Clitoria accumulated more sugars in its unwashed vs washed roots, as compared to Piper which showed no difference in the washed vs. unwashed roots. Furthermore, NanoSIMS images showed that Clitoria unwashed roots had clustered regions of 13C enrichment as compared to the washed roots which showed more uniform distribution. Overall, Piper and Hibiscus both respond strongly to drought, but in slightly different ways, and, while Clitoria does not respond strongly to drought in its root metabolome, there is a response to the presence of rhizosphere microbes. Clitoria is a legume, therefore, these differences between washed and unwashed roots could indicate its strong symbiotic interactions with rhizosphere microbes (Honeker et al., submitted to Environmental Science & Technology).
Linnea Honeker 