Colleen Doherty

A major challenge for humankind is to feed the increasing human population in a sustainable manner. According to UN������������������s development programme extreme hunger and malnutrition is a major barrier to development in many countries: 795 million people are estimated to be chronically undernourished as of 2014, often as a direct consequence of environmental degradation, drought and loss of biodiversity. The Sustainable Development Goals (SDGs) aim to end hunger and malnutrition by 2030. Improved agricultural productivity is a critical part of achieving the SDG goal 2, Zero Hunger. Currently more than one third of crop yields are lost due to abiotic and biotic stress factors, such as drought, salinity, pests and disease. To minimize this yield gap and to simultaneously reduce the environmental impact of current agricultural practices, future crop production needs to be achieved on sub-optimal soils with reduced input of fertilizers and pesticides (���������������more with less������������������). These challenges have increased the awareness of the importance of the plant microbiome for improved agricultural practices. Plants are colonized by an astounding number of microorganisms that can have profound effects on seed germination, seedling vigour, plant growth and development, nutrition, diseases and productivity. Thus, the plants can be viewed as holobionts that benefits from its microbiome in terms of specific functions and traits. In return, plants transfer a substantial part of their photosynthetically fixed carbon directly into symbionts and into their immediate surroundings thereby supporting the microbial community and influencing its composition and activities. For the vast majority of plant-associated microorganisms, however, there is little knowledge of their specific impact on crop growth and crop resilience and the mechanisms underlying microbiome-plant interactions. Hence, a critical step in developing new microbiome-assisted approaches to quantitatively and predictably improve crop resilience management strategies is deciphering the hyperdiverse plant microbiome. In particular, we need to identify keystone microorganisms and mechanisms involved in plant growth promotion and protection against biotic and abiotic stresses. To that end, systems-based analyses combined with deep-learning and modelling are essential to decode the taxonomic diversity and functional potential of plant microbiomes. The overall aim of this multidisciplinary research program is to develop a scalable system-based strategy to harness the functional potential of plant microbiomes for improving crop resilience. More specifically, we will focus on experimental analyses and modelling of the phyllosphere microbiome of wheat (Triticum aestivum), one of the most important cereal food crops worldwide. The phyllosphere microbiome is defined here as the collective microbial communities inhabiting both the leaf surface as well as the internal leaf tissue. We will zoom in on the microbiome of flag leaves of wheat, as the flag leaf is a major determinant (up to 45%) of wheat yield. To do this, we combine renowned academic expertise in microbiology, chemistry, DNA and RNA sequencing, bioinformatics, machine-learning and modelling with company support in plant breeding and agronomy to deliver novel approaches and technologies.

Several carbon capture mechanisms have emerged in plant systems that provide unique advantages to plants depending on their environment. For example, while most plants use a C3 photosynthesis mechanism, C4 and CAM carbon capture mechanisms can increase water use efficiency or temperature tolerance. These advantages have been well-characterized in the atmospheric CO2 levels on Earth, but in enclosed human habitats such as those needed for long-term space flight, CO2 levels far exceed that of Earth���s atmosphere. Altered CO2 levels affect nutritional content and water use efficiency, but this research has used CO2 levels below that on enclosed human habitats. This proposed work would examine how high CO2 levels affect the plant physiology and nutritional content of edible microgreens that use different photosynthetic mechanisms: C3, C4, and CAM. We will monitor physiological characteristics and the nutritional profile across different CO2 levels for these microgreen species with C3, C4, and CAM photosynthesis. The combined effects of altered CO2 levels and other spaceflight relevant stresses such as water availability will be examined to understand if these different photosynthetic mechanisms can provide advantages to enhance plant productivity in space environments. These results would provide important baseline information on plant nutrition and performance that is needed for planning long-term space missions and thus would address the following objectives of the solicitation and NASA program goals: Decadal Survey- Priority 3: A systematic suite of plant biology experiments to elucidate mechanisms by which plants respond and adapt to spaceflight, and to facilitate their eventual use in Bioregenerative Life Support Systems; PB-1 How does gravity affect plant growth, development & metabolism (e.g. photosynthesis, reproduction, lignin formation, plant defense mechanisms) and PB-3 How can horticultural approaches for sustained production of edible crops in space be both improved and implemented (especially as related to water and nutrient provision in the root zone)?

The purpose of this project is to develop a handheld Mueller matrix polarimeter that can be deployed to measure leaves in transmission. Leaves from different corn varieties will be quantified using both this handheld unit and our laboratory unit (an imaging Mueller Matrix polarimeter). These data will be compared to ground truth from e.g., enzymatic, colorimetric, 1D-NMR, and Mass-spectrometry based analyses, to correlate polarimetry measurements to metabolic concentration. Additionally, we will investigate polarization in reflection using a hyperspectral imaging polarimeter to quantify polarization������������������s ability to correct for bidirectional reflectance effects from canopy level measurements.

Co-PI Nielsen will be responsible for overseeing all experimental design and computational components of this project. This includes QTL analyses, RNA-Seq data QC and processing, eQTL analyses, cross-species eQTL analyses, and network analysis. Nielsen will mentor the Ph.D. student selected for this project; this student will be selected from students in the NCSU Bioinformatics Graduate program. Under Nielsen������������������s supervision, the student will perform most of the analyses required for this project. Nielsen will also co-mentor the students involved with the summer internship program with St. Augustine������������������s. Dr. Doherty will serve as Co-I for the plant stress response, molecular, and biochemical analysis and validation of candidate regulatory interactions. Specifically, Dr. Doherty will oversee in: Aim 1: The phenotyping of the plant-response traits, monitoring general growth parameters and physiology related sensitivity due to WNT and nematode presence. Aim 2: Extraction of RNA, preparation and sequencing of the libraries for the RNA samples. QC will be performed on all samples prior to sequencing. Dr. Doherty will oversee library construction in her lab and will coordinate getting the samples sequenced. Aim 4: In this objective Dr. Doherty will oversee the validation of the predicted mechanisms. Again, the Doherty lab will be responsible for extracting RNA and preparing libraries from the for RNA-seq or generating constructs and IP-ing for ChIP-Seq. Once prepared and checked for quality, Dr. Doherty will coordinate sequencing of the libraries, will retrieve and store the data and will process the resulting data and evaluate the success of the predictions and the targets identified. The Doherty lab will assist with evaluating the success of the approach and interpreting the candidate targets.

Identification and testing of promoters for crops. Using our motif identification method Identify cis-regulatory regions and test their functionality.

Despite their abundance in US soils, REEs are dispersed and challenging to extract. The economic and environmental costs of extraction combined with the low resale value of REEs has resulted in decreased US production of REEs. The lack of US companies with the resources and interest in continuing to harvest REE generates a national security risk as the availability of these essential components is under foreign control. Current methods of REE extraction require the use of aqueous chemical treatments. New advances include the use of bacterial filters to capture REE. While these have contributed to reducing the cost of extraction, these approaches still require significant capital investment and a large amount of water to excavate and recapture the REE. Thus, these are approaches that can only be economically employed in areas with large deposits of REEs. The successful completion of this proposed research will result in an economical plant bio-mining system to extract REE from soil and waste sources. The low costs and minimal footprint of the plant bio-miners will provide an efficient and scalable approach that can be deployed in areas ranging from small fields and consumer waste areas to large mines and reclamation areas. The low upfront costs will encourage the use of plant bio-miners thus reducing the scarcity of REEs and eliminating our dependence on foreign REE sources. The design and engineering of the plant bio-mining system will provide tools to understand how plants regulate uptake and distribute REEs and calcium, which REEs mimic. Small doses of REE can enhance tolerance to abiotic stress. The development of this system to accumulate REEs in plants will help us design REE-inspired treatments to mimic these positive effects on abiotic stress responses and enhance tolerance to drought and heat stress.

The circadian clock, an internal timekeeping mechanism, enables organisms to temporally organize their molecular and biochemical activities so that they are optimally timed with environmental conditions. Many environmental responses are gated by the circadian clock. Responses to environmental stimuli vary depending on the time of day or season that it is perceived by the organism. We examined microarray and RNA-Seq data sets available in GeneLab and found that components of the circadian clock are often identified as differentially expressed in response to gravity in multiple studies in Arabidopsis. This finding led us to investigate the potential for gating of gravitropic responses such as root bending. We observed that the time of day the stimulus is provided affects the magnitude of the gravitropic response. Further, this response is altered in a circadian clock mutant. Based on these findings we hypothesize that, like other environmental stimuli, the response to gravity is gated by the circadian clock. We propose to investigate if there is an integrated relationship between the circadian clock and gravity. First, we propose to determine if the time of day and year can impact the observed effects of microgravity indicating that these factors should be considered in all microgravity research. Secondly we will examine if microgravity affects circadian-regulated activities and the timing of all aspects of physiology and development.

Borlaug Fellowship Program for Sri Lanka fellow to spend 12 weeks at NC State conducting research to identify novel mechanisms, gene candidates, and superior alleles for saline tolerance by RNA-seq analysis of Sri Lankan rice germplasm.

Plants are essential for life on earth and for long duration spaceflight and colonization. However, the spaceflight environment is not ideal for plant growth and understanding how plants sense and respond to this environment is critical for enabling plant growth in space. The objective of this study is to identify mechanisms regulating plant responses to spaceflight and microgravity. GeneLab contains several datasets of molecular responses of Arabidopsis grown in spaceflight or onboard the International Space Station. However, there is little overlap among these datasets suggesting that microgravity-specific responses may be masked by the differences in experimental conditions. We evaluated distinct gene sets and identified common cis-regulatory elements. We propose to investigate these top candidates and reveal overarching regulatory pathways. We will carry out Yeast 1Hybrid library screening to identify their target transcription factors. We will also generate Arabidopsis plants with either reduced (knockdown) or increased (over expression) expression of these factors and study their response to simulated microgravity and other stresses. This approach is more inclusive than the overlap of specific gene responses across experiments and is particularly beneficial for identifying patterns in limited data sets. The results from this study will provide valuable information on the potential primary effectors governing plant responses to spaceflight and microgravity.

This project aims to develop cellular sorting technologies and methods for microbiome constituents. The goal will be to sort microbes based on their detected rare earth element concentrations.