The goal of this project is to determine the physiological and genetic basis underlying the potential production of more nutrient dense crops in farming systems. This will enable farmers interested in organic or conventional practices to integrate soil fertility systems that favor more sustainable production. It will allow plant breeders to develop crop cultivars suited for organic production.
Previously, we demonstrated that tomato fruit grown under an organic fertilizer regime had elevated phytonutrient content compared to tomato fruit grown under a conventional fertilizer regime. Using a comprehensive transcriptome analysis, we tested the following hypotheses: 1.) Growth under organic fertilizer regime will result in differential expression of the tomato genome and 2.) Genes and pathways associated with phytonutrients that were observed to be significantly higher under organic fertilizer regime will demonstrate higher expression. Both hypotheses tested true, indicating an adjustment of the plants’ genomic activity in response to a different nitrogen regime. We identified genes and associated pathways –among them, lycopene, ascorbate, soluble solids, and salvage pathways –which are expressed at higher levels under organic conditions (Sharpe et al., 2019 https://www.biorxiv.org/content/10.1101/755769v1).
The next logical step is to investigate the genomic response of roots after exposure to organic vs conventional fertilizer, to provide further understanding of how organic and conventional nitrogen are metabolized. We propose to test the following hypotheses: 1.) Different fertilizer regimes will elicit differential expression of nitrogen metabolism genes, and 2.) A different root/soil microbiome will be fostered under the two nitrogen conditions. This research includes a time course root/microbiome-targeted transcriptome analysis, focusing on genes and pathways associated with nitrogen metabolism and on the microbial symbionts activity favored under the different fertilizer conditions. Gene-based knowledge generated will facilitate identification of genotypes that utilize organic fertilizer more efficiently.
Short-term: We aim to increase knowledge of the relationship between phytonutrient content and underlying gene expression changes in plants when grown under organic or conventional soil fertility management systems in a model crop plant (tomato). We clarify here that promoting organic over conventional is not the goal of this study; rather, we hope to provide a basis for understanding of gene expression and metabolic differences, which may in turn result in nutritional differences, between the organic and conventional treatments. We aim to develop a model for comprehensive analysis in the field of crop production. We seek to use our study results to apply for grant funding in the USDA’s AFRI program in order to conduct a more comprehensive study, which will include field research.
Intermediate-term: Knowledge generated from the project, particularly with regards to organic vs. conventional nitrogen uptake and utilization as well as corresponding phytonutrient profiles in the different fertilizer regimes, will assist organic and sustainable producers to develop management plans for soil fertility. Understanding of gene function and of markers associated with genes expressed differentially under organic or conventional conditions will help plant breeders in the crossing and selection of more efficient crop cultivars that optimize both nutritional quality and yields of crops grown using more sustainable farming practices.
Long-term: Increase consumption of foods with improved nutritional quality, thereby contributing to the health of American children and adults and reducing health care costs. Implement more sustainable farming practices, particularly related to soil fertility and pest management, will enhance the quality of U.S. agro ecosystems. Contribute to the long-term economic viability of American farmers.
Summary of ongoing work:
Plants were grown as per the experimental design. Soil and root samples were collected and frozen in liquid nitrogen. Currently, RNA extractions from soil samples are being performed. Samples will be checked for quality and submitted for total RNAseq as proposed.
The materials and methods used were as described in a recent publication from the lab by Sharpe et al., 2019 https://www.biorxiv.org/content/10.1101/755769v1.full
Tomato seeds (Solanum lycopersicum L.) ‘Oregon Spring’ sourced from Johnny’s Selected Seeds, Winslow, ME, were sown in LC1 Professional Growing Mix (Sun Grow Horticulture, Bellevue, WA). Glasshouse temperatures were maintained at 21.1/18.3°C (day/night) with a 14 h day, supplemented with high-pressure sodium lamps and 10h night photoperiod. Three-week emergent seedlings were fertilized with a BioLink All Purpose Fertilizer 5-5-5 (Westbridge Agricultural Products, Vista, CA) solution at a concentration of 4 mL/L tap water representing the organic nutrient treatment (ORG). The seedlings grown under conventional fertilizer (CONV) received a Peters 20-10-20 solution (1.02 g/L tap water). Both treatments received one liter per week of their respective nutrient treatment.
Six-weeks-old plants of similar size and vigor from each treatment were transferred to individual 24-liter pots. Potting media for the ORG nutrient treatment consisted of a mix of LC1 Professional Growing Mix (Sun Grow Horticulture, Bellevue, WA), Whitney Farms Compost (Scotts, Marysville, OH), and sifted soil in a ratio by volume of 15:5:1. The CONV plants were potted in 100 percent LC1 Professional Growing Mix. Upon transplantation, individual plants were fertilized with 150 mL of applicable nutrient solution once per week. Twelve plants in each nutrient treatment were selected at week seven and arranged in a randomized complete block design consisting of six blocks. Plants were provided 1 liter of water every other day and the dosage of weekly nutrient treatments was increased to 500 mL beginning at week eight. Lateral shoots below the first flower cluster were removed as they appeared. Starting in week 11, the CONV nutrient solution concentration was increased from 1.02 g/L of Peters 20-10-20 to 1.25 g/L. Beginning in week 12, nutrient treatments were augmented with micronutrients. The ORG treatment was amended with BioLink Micronutrient Fertilizer (Westbridge Agricultural Products, Vista, CA) at 3.9 mL/L tap water and the CONV treatment was augmented with calcium phosphate monobasic monohydrate at 166 mg/L tap water. Concentrations of macronutrients were equivalent in both treatments, with total nitrogen, total phosphorus and total potassium concentrations of 260 ppm, 99 ppm and 235/220 ppm (CONV/ORG respectively) based on laboratory analyses (Analytical Science Laboratory, University of Idaho, Moscow, ID). Nitrogen forms varied greatly between CONV and ORG treatments with nitrate constituting the dominant form in CONV, and organic forms (i.e. amino acids and proteins) predominating the ORG nutrient solution. In week 13, the air temperature in the glasshouse was increased to 23.3/20°C (day/night) and the nutrient dosage was increased to 750 mL per plant every other day. In week 14 the nutrient dosage was increased to 1 liter every other day.
Plant tissue and Soil samples: Plants and soil were sampled throughout the experiment during the predetermined time points mentioned in the time course analysis section below and all tissue and soil sample collections were made in triplicate. Plant root samples were collected, carefully washed, and weighed while a sub-sample of the roots was frozen in liquid nitrogen. The soil samples were divided into two portions: the bulk soil in the ground and the rhizosphere soil that was in direct contact with the plant roots.
Time Course Analysis: A time-course collection of soil samples was implemented to sequence the changes in the metatranscriptome. Soil and tissue samples were collected throughout the experiment and are being utilized for RNA extraction and purification. Time points (TP) for soil and tissue collection are as follows: TP-0, right before transplanting, TP-1, pre-flowering stage around 30 Days after Planting (DAP), TP-2, flowering stage around 70 DAP, TP-3.1, fruiting stage of immature green fruit around 90-100 DAP, TP-3.2, ripening stage of Breaker fruits around 110 DAP, and TP-4, mature red fruit stage around 125 DAP.
Soil RNA Extraction
- Prior to nucleic acid extraction all the solutions were prepared in diethyl polycarbonate (DEPC) treated water. All the glassware and plasticware were autoclaved prior to use.
- Hexadecyltrimethylammonium bromide (CTAB) extraction buffer – mix equal volumes of 10% (wt/vol) CTAB in 0.7 M NaCl with 240 mM potassium phosphate buffer (pH 8.0).
- RNA precipitation solution – dissolve 30% (wt/vol) polyethylene glycol 8000 in 1.6 M NaCl solution.
- 5 g (wet soil) was weighed and transferred to 2 mL screw cap centrifuge tubes (ThermoFisher Scientific, Waltham, MA, USA) containing 0.5 g (0.5 uM size) glass beads (RPI research products, Mt Prospect, IL, USA).
- For RNA extraction, 0.5 mL of CTAB extraction buffer and 0.5 mL phenol-chloroform-isoamyl alcohol (25:24:1) (pH 7.15) was added to the soil samples.
- Samples were then lysed for 3 min using Qiagen TissueLyser-II (Hilden, Germany) with machine speed set at 30 1/s. The samples were then centrifuged at 14,000 rpm at 4oC for 10 min.
- The aqueous phase was then transferred to fresh 1.5 mL Eppendorf tube and equal volume of chloroform-isoamyl alcohol (24:1) was added.
- Samples were centrifuged at 14,000 rpm at 4oC for 10 min to remove the residual phenol.
- The aqueous layer was transferred to a fresh 1.5 mL Eppendorf tube.
- For RNA precipitation, 2 volumes of RNA precipitation solution was added to the extracted aqueous layer and incubated at room temperature for 2 h.
- Samples were then centrifuged at 14,000 rpm at 4oC for 10 min and supernatant was gently discarded.
- The pellet was then washed twice in ice cold 100% ethanol and air dried prior to resuspension in 50 µL DEPC treated water.
- The extracted RNA was treated with DNase using Ambion Turbo DNA-free kit (Invitrogen, Carlsbad, CA, USA), for downstream applications using manufacturers guidelines.
Currently there are no results to report. RNA is being isolated from the soil and root samples collected as part of the experiment. Most of the efforts have been focused on obtaining high quality RNA from soil samples. For this various RNAse inhibitors have been evaluated. Use of Aurine Trichloroacetic acid provides the best results.
Educational & Outreach Activities
Thus far, we have engaged in discussion with visitors to lab regarding the anticipated education and outreach activities. As we are still waiting on greenhouse space to begin the project (which will become available in November), we have been limited as to the extent of activities that can be conducted at this time.