Performing Department
(N/A)
Non Technical Summary
The storage root is a prime example of a type of specialized organ that can accumulate and store theproducts of photosynthesis allowing plant survival under abiotic stress episodes. When phosphorusavailability is limited, plants use a multifaceted set of strategies to increase the availability of phosphorus in the soil. In sweetpotato, the favorable or lack thereof of storage root development undersuboptimal phosphorus availability is not understood and provides an opportunity for research as a novelstorage root model organism to further our understanding of storage root formation under stress.Recently, we have observed sweetpotato genotypes displaying variable responses to suboptimal phosphorusavailability. The overall objective is to elucidate the mechanisms and the genetic architectureunderlying the reduced secondary root growth of developing roots in sweetpotato. The objectives of this research are to1) measure the extent of reduced secondary root growth and anatomical components that stimulatedecreases in root radial thickening under phosphorus stress, 2) assess the physiological mechanism of rootnon-structural carbohydrate allocation under phosphorus stress, and 3) Identify quantitative trait loci,candidate genes, and gene pathways underlying the natural phenotypic variation in root secondaryroot growth and carbohydrate status.It's crucial to define and understand mechanisms that enhance nutrient stress tolerance to keep American agriculture competitive, end world hunger, and adapt to climate change, which is partof NIFA's grand challenges. Understanding root secondary growth under edaphic nutrient stress could be a game-changer for improving the nutrient stress tolerance of root and tuber crops. Although this project focuses on sweetpotatoes, the findings could be applied to other crops such as cassava, potatoes, sugar beets, and carrots. Therefore, this mechanism may have broad utility in agriculture. By addressing novel scientific issues relevant to human welfare, this project sheds light on the importance of root formation under nutrient stress and offers a new perspective on how to improve crop productivity. The data presented in this project is the first demonstration of the physiological value of these mechanisms under reduced phosphorus availability.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The central goal aims to understand the anatomical, physiological, and genetic factors underlying P-stress-induced reprogramming of root architecture. This offers a unique opportunity in understudied root crops where improved crop adaptation to P-stress often results in a penalty on the most important agronomic trait, i.e., storage root yield.Objective 1:Measure the extent of reduced secondary root growth and anatomical components that stimulate decreases in root radial thickening (root girth) under reduced P availability.Objective 2:Assess the physiological mechanism of root non-structural carbohydrate (NSC) flux (i.e., status; accumulation and remobilization) under reduced P availability both in greenhouse and field scenarios.Objective 3:Identify quantitative trait loci (QTL), candidate genes, and gene pathways underlying the natural phenotypic variation in root secondary root growth and carbohydrate status storage.
Project Methods
Studies will be conducted in soil mesocosms, in aeroponics growth systems, and in a low phosphorus field siteat PSU. Greenhouse mesocosms and aeroponics structures are used to reliably generate realistic P stress conditions for sweetpotatoes. The Low Phosphorus Field 300 was designed for studies of plant root adaptations for low soil P availability and consists of a 3-acre research facility containing six blocks of high and low phosphorus (P) split-plots in Hagerstown silt loam soil. The low P plots do not receive P fertilizer and have available soil P levels between 7-10 ppm. High P plots are fertilized to maintain high P levels. Root samples collected in each environment will be used for anatomical phenotyping. Multiple cross-sectional images of each sample will be captured with LAT.Anatomical features will be measured in RootScan2, image analysis software. Mesocosm and aeroponics studieswill permit the evaluation of this hypothesis by allowing detailed monitoring of the effects of P stress and control conditions on root and shoot growth over time. The TB and DP populations will be grown in soil mesocosms and aeroponics and maintained at control or P deficit concentrations by irrigating with established P fertilizers regimes.Physiological growth parameters such as leaf stomatal conductance and CO2 assimilation will be monitored with a LI 6800 (LI-COR Biosciences). For this study, plants will be destructively harvested 30 days after planting for analysis of root growth and architecture, root anatomy, biomass, leaf area, and P content. Specifically, mesocosms have plastic liners that permit the soil column to be removed intact. Root length, diameter, and branching will be measured by image analysis (RhizoVision Explorer; open-source software for root image analysis). Root anatomical traits (e.g., TCSA, stele area, aerenchyma, and metaxylem components, among others) will be quantified by analysis (RootScan2) of images gathered from LAT. Leaf P content will be measured spectrophotometrically after ashing leaf tissue at 500°C for 16 h. Structural carbohydrates (i.e., lignin) will be measured using the Acetyl Bromide Soluble (ABSL) assay, used for total lignin quantification in plant biomass. We will use multivariate analysis to identify multiple root phenotypes that may be associated with the performance of sweetpotato under reduced P availability.This activity will utilize two contrasting P stress environments: the study will be conducted at the Russell E. Larson Agricultural Research Farm at Rock Springs, Pennsylvania (40.709746°N, 77.956965°W). A split-plot design will be used with two P levels: two 0.05-ha low-P fields (10 ppm mean available P by Mehlich-3 [ICP]) split into two, 0.025-ha blocks each and two 0.05-ha high-P fields (38 ppm mean available P) split into two, 0.025-ha blocks each. Each sweetpotato entry will be randomized within each block. All plots will be fertilized according to each treatment, with soil nutrient levels adjusted to meet sweetpotato requirements as determined by soil tests at the beginning of each growing season. Each sweetpotato entry will have five replications within each P treatment. Average maximum/minimum temperatures for this site are 27°C/17°C, average total rainfall is approximately 18.5 cm, and average light/dark photoperiod is about 14.5/9.5 h. Midday photosynthetic active radiation is approximately 1,500 to 2,000 μmol photons m-2 s-2. Soil is a Hagerstown silt loam (fine, mixed, semi-active, mesic Typic Hapludalf). Plant growth, plant physiological status, and root development will be monitored through time. A subset of plants will be excavated at 30 DAP to determine the initiation and progression of ARs-to-SRs under both treatments. Destructive harvests and soil coring at 120 DAP will be used to measure root length density with depth (RhizoVision Explorer), leaf area, plant biomass partitioning, and anatomical analysis (LAT/Rootscan2). Fully bordered subplots will be left in the field for determination of yield components.The TB and DP populations will be grown in soil micro-mesocosms (described above) for 30 days and field-harvested storage roots will be harvested at 120 DAP. Fresh ARs and SRs will be collected, gently washed, surface sterilized, dried, and weighed. Main fractions of glucose, fructose, sucrose, and starch from excised roots (ARs and SRs) will be determined by an enzyme-coupled reaction assay based on hexokinase and glucose-6-phosphate dehydrogenase. Phosphoglucose-isomerase and invertase will be used to convert fructose and sucrose stoichiometrically to glucose for subsequent assays. Root extracts will be evaporated to dryness at 24°C and assayed directly along with sucrose, fructose, and glucose calibration standards. Root starch will be assayed in insoluble debris following the three extractions of sugars with 80% methanol. Water will be added, and starch hydrated by heating to 121°C for 10 min in an autoclave. Starch will be completely hydrolyzed to glucose with amyloglucosidase, and aliquots will be assayed for glucose as described above.Root respiration rates will be determined immediately after washing for two 10-cm segments taken from representative ARs at the 10 cm nearest to the stem node (proximal), 10 cm at the middle of the root axis (medial), and 10 cm from the growing tip back (distal).Root respiration rates will be measured using a Li-Cor 6800 gas-exchange system with a modified respiration chamber. All measurements will be performed under ambient greenhouse conditions, with the sealed chamber being submerged in a water bath kept at 28°C and baseline sample chamber and reference chamber CO2 concentration of 400 μmol mol-1.Phenotyping of constitutive trait expression under stress and non-stress conditions: individuals from both populations will be grown (augmented block design with replicated checks) under reduced P availability and high P conditions. A preliminary analysis of the data is described above. All samples will be imaged using LAT and phenotyped using RootScan2 as described above.NGS-based genotyping: Using a novel and patented quantitative reduced representation sequencing (qRRS) approach, we have generated a high-density dosage-based SNP data for both populations. The ngsComposer software was used for demultiplexing and quality filtering of the NGS reads. The qRRS approach allows for uniform low-cost deep sequencing across loci and samples, thus a stringent read depth threshold of 45x at each locus/sample rather than average read depth across samples. Variant calling was performed with GATK as implemented within the GBSapp pipeline, which applies multiple QC and best practices. The variant calls for the TB and DP populations include 25,120 and 155,398 high-fidelity markers, respectively.Linkage, QTL and Genome-Wide Association analysis:Genome-wide association (GWA) analysis will be performed using GWASpoly as previously described in our previous studies as shown in our work on the genetic control of continuous storage root formation and bulking in sweetpotato. For the GWAS approach, we will model various gene actions ie. additive, diploidized additive, triplex dominant, duplex dominant, and simplex dominant. The GWAS model will control for population structure using the realized kinship matrix based on the Van Raden method and specifying the ploidy as 6 (i.e., hexaploid). FDR tests, Bonferroni correction, and permutations tests will be applied to control for the false positive rate.