Abstract

Beyond Soil: Nitrogen Cycling and Plant Uptake in Weathered Bedrock Rhizospheres of Deeply Rooted Californian Forests

By

Kelsey Lee Crutchfield-Peters

Doctor of Philosophy in Integrative Biology

University of California, Berkeley

Professor Todd E. Dawson, Chair

Across diverse ecosystems worldwide, plant roots and their associated microbiome (rhizospheres) are known to extend beyond soil horizons and into weathered bedrock where they drive water and carbon cycling meters below the base of soil. Little is known however about how deep rhizospheres drive nutrient cycling in weathered bedrock horizons. In my dissertation research, I combine biogeochemical, plant ecophysiological and genetic techniques to explore how nitrogen (N)–the most limiting nutrient to plant growth in terrestrial ecosystems–is cycled throughout a deep rooting profile. My research is based at the Eel River Critical Zone Observatory in northern CA, where more than a decade of research has demonstrated the importance of the weathered bedrock in forest and watershed function. I build upon this work to understand the role of the weathered bedrock vadose (unsaturated) zone in deep N cycling and explore how dominant plant species acquire and cycle nitrogen in soil versus weathered bedrock. In chapter one, I investigate N cycling dynamics in the weathered bedrock vadose zone (WBVZ), asking: How do the chemical forms and concentrations of biologically available dissolved N change year-round? And what is the source and fate of this dissolved N? I take advantage of a novel field instrument called the Vadose Zone Monitoring System (VMS) to sample water every 1.5 meters throughout a WBVZ, from the base of soil down to groundwater (~16 m depth). Alongside my colleagues, I have sampled water approximately every two weeks for more than 2 years for Total N, Total Carbon (TIC/TOC), inorganic N (ammonium, NH4+, and nitrate, NO3-) as well as CO2 and O2 gasses across the entire VMS depth profile. From these data I demonstrate that (1) biologically available N is on the same order of magnitude as temperate forest soils and is seasonally-cycled in the WBVZ, (2) the dominant form of that N is organic N and it is strongly linked to dissolved organic carbon (DOC) dynamics, and (3) decreases in N concentration in the WBVZ is both dependent on incoming precipitation and linked to periods of high forest-level metabolic activity, suggesting plant use of N from the WBVZ. Additionally, ?13C-DOC values from the VMS suggest that dissolved organic matter in the vadose zone is derived from fresh plant sources and not lithologically derived from parent material at the site. I conclude that deep N cycles are driven by rhizospheres meters below the base of soil and represent a major gap in our current understanding of deeply rooted ecosystems. In chapter two, I build upon my findings in chapter 1 and explore the uptake of plant-available N in Douglas fir (Pseudotsuga menziesii), a dominant tree species at the Eel River CZO that can extend dense root networks into fractured rock horizons. Using an isotopic labeling experiment, I address the following questions: How do soil-grown versus rock-grown fine roots differ in their N uptake capacity? And what sources of nitrogen are preferred between soil and rock grown roots? I dug seven 1 m deep pits at the Eel River CZO and sampled terminal fine root tips from mature P. menziesii trees for a dual 15N and 13C tracer experiment. I incubated fine roots from either soil or weathered bedrock fractures in one of three labeled nutrient solutions (15NH4Cl, K15NO3- or 15N-glycine) at 10, 40 or 100 micromolar concentrations. After incubation, I calculate N uptake capacity for soil and rock grown roots for each treatment based on the degree of 15N isotopic enrichment. For the glycine treatments, I also estimated 13C uptake to assess in-tact amino acid uptake. I found that rock-grown roots have a similar N uptake capacity to soil-grown roots, but that rock grown roots have slightly lower uptake rates. I also found that both soil- and rock-grown roots prefer NH4+, which is the dominant form of inorganic N available at our site. Second to NH4+, all roots took up glycine at higher rates than NO3-––which had consistently low uptake across all concentrations. However, there was no significant uptake of 13C in any of the glycine treatments, indicating that the ectomycorrhizal symbionts which associate with P. menziesii rapidly decarboxylated the glycine to acquire the N. Ultimately, I found that despite drastic differences in the soil and saprolite environments, the mycorrhizal fine roots of P. menziesii are equally capable of acquiring N. Furthermore, they are primed to take advantage of the most available forms of N at the site: both inorganic-N in the form of NH4+ as well as organic N through the action of rapid depolymerization of more complex organic molecules. These findings support the potential of deep roots in the WBVZ to take advantage of the two most common forms of N that are dynamically cycled: NH4+ and organic N. In my third and final chapter I use RNA-seq to explore differential gene expression in P. menziesii fine roots sampled from soil and weathered rock, asking: How does differential gene expression change broadly in soil versus bedrock environments? And, more specifically, how does expression of N uptake genes (e.g., Ammonium transporter (AMT) and nitrate transporter (NRT) gene families) in P. menziesii fine roots differ between roots grown in these two environments? To address these questions, I collected mycorrhizal fine root tips of P. menziesii from soil and saprolite horizons from 6 trees in summer 2020 and 7 trees from summer 2021 and extracted total plant RNA for Illumina 150 PE Sequencing. From this work I identify the P. menziesii ortholog of the MADs-box gene, DAL20 as the singular gene that is consistently upregulated in rock-grown roots between summers 2020 and 2021. In other conifers, DAL20 is exclusively expressed in root tissue and its close homolog in Arabidopsis thaliana increases root apical meristem size and results in increased primary root growth when upregulated. I found that expansion genes, involved in loosening the cell wall to allow for cell expansion, were also upregulated in rock environments. While there were no major differences in N uptake genes (AMT or NRT families) between soil and saprolite environments, there were consistent annual patterns in expression in AMT and NRT expression regardless of year or substrate (e.g., soil or rock). There was one AMT gene that is consistently highly expressed above all others, and it is the 4th most highly expressed of the 23,397 genes analyzed for differential gene expression. In other study systems, patterns like this in AMT expression were linked to transfer of N from mycorrhizal symbionts. Together, these patterns suggest a rock environment ‘syndrome’ in genes that enhance root growth and flexibility and point toward a unique gene possibly involved in N transfer to the plant from the mycorrhizal symbiont.