Source: PENNSYLVANIA STATE UNIVERSITY submitted to NRP
OPTIMIZING ROOT METAXYLEM PHENOTYPES TO IMPROVE DROUGHT TOLERANCE IN MAIZE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1012027
Grant No.
2017-67013-26192
Cumulative Award Amt.
$470,000.00
Proposal No.
2016-10511
Multistate No.
(N/A)
Project Start Date
Mar 15, 2017
Project End Date
Mar 14, 2023
Grant Year
2017
Program Code
[A1152]- Physiology of Agricultural Plants
Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
408 Old Main
UNIVERSITY PARK,PA 16802-1505
Performing Department
Plant Science
Non Technical Summary
This project will evaluate the potential to improve the drought tolerance of corn by selecting plants with smaller but more numerous xylem vessels, which are the cellular structures in roots that carry water from the soil to the leaves. Corn is the most important crop in the USA and globally. Drought stress is the most important limitation for corn production in the USA and globally, and drought stress is expected to become more severe in coming decades as a result of global climate change. We have observed that corn lines with a large number of small vessels perform better under drought. In this project we will confirm and extend these results, and also identify genes that are responsible for this natural variation.Defining and understanding traits improving the drought tolerance of crops is needed to breed crop varieties with better yield, better drought tolerance, and less reliance on irrigation. Insufficient information exists regarding the value of xylem vessels in crop breeding. Should the importance of xylem vessels for drought tolerance be demonstrated, an entirely new tool will be available for targeted crop improvement. This project will provide validated breeding targets, germplasm sources, molecular markers, and candidate genes that could be used directly in maize breeding. Our close collaboration with maize geneticists will ensure that any useful results will be readily applied in the maize community. While this project focuses on maize, xylem vessels may also be useful breeding targets in wheat, rice, legumes, and other crops. This project therefore addresses novel scientific issues that are of demonstrable relevance to human welfare.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2031510102070%
2011510108030%
Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1510 - Corn;

Field Of Science
1080 - Genetics; 1020 - Physiology;
Goals / Objectives
Objectives: We have recently discovered that genetic variation for root metaxylem vessel phenotypes in maize is associated with substantial variation in hydraulic health and improved resistance to water deficit stress. The overall objective of this project is to evaluate and develop this trait as a tool to improve drought resistance in maize and other crops. Specifically, we will:Confirm the physiological utility of the 'many-small' versus 'few-large' metaxylem vessel phenotypes to improve plant water status under drought;Discover genes underlying natural variation in metaxylem phenotypes, especially number and size of metaxylem vessels;Evaluate the utility of the 'many-small' versus 'few-large' metaxylem phenotypes to improve growth and yield under managed drought in two field environments.
Project Methods
Activity 1. Confirm the physiological utility of the 'many small' versus 'few large' metaxylem vessel phenotypes for improved drought resistance in greenhouse mesocosmsThe goal of this objective is to evaluate the physiological costs and benefits of the many small (MS) vs few large (FL) metaxylem vessel phenotypes for water acquisition from drying soil. Mesocosm studies will permit evaluation of this hypothesis by allowing detailed monitoring of plant water status, leaf gas exchange, root and shoot growth, and water acquisition over time. Existing inbred lines contrasting for MXV, in addition to transgenics and transposon lines (from activity 2) will be grown in soil mesocosms maintained at field capacity or subjected to progressive water stress by irrigating at a fraction of the non-stressed treatment to achieve a 30-60 % reduction in shoot growth. Soil water content from representative mesocosms will be tracked with a multiplexed TDR-100 system (Campbell Scientific) placed at various depths in the soil logged by a CR21X (Campbell Scientific). Leaf stomatal conductance and CO2 assimilation will be monitored with a LI 6400 (LI-COR Biosciences). In a typical study plants will be destructively harvested at 14, 28, and 42 days after planting for analysis of tissue water status, biomass, root growth and architecture, leaf area, and root anatomy. The mesocosms have plastic liners that permit the soil column to be removed intact. These columns will be subdivided to determine root length and soil water content with depth. Root length, diameter, and branching will be measured by image analysis (WinRhizo Pro, Regent Instruments). Root anatomical phenotypes will be quantified by analysis (RootScan2) of images gathered from Laser Ablation Tomography. Leaf water status will be measured as relative water content and leaf water potential with a pressure gauge (PMS model 1000). Root hydraulic conductance will be measured in 10 cm samples using a modified pressure apparatus. Activity 2: Discover genes underlying natural variation in metaxylem phenotypes Goal 1: Phenotypic analysisAnalysis of constitutive trait expression under non-stress conditions: 500 lines from the WiDiv have been grown at ARBC in 2013- 2015. All samples were analyzed using laser ablation tomography and RootScan2.Analysis of plasticity for trait expression under stress: The WiDiv was grown in 2016 and will be grown again in 2017 at ARBC in two replications under both non-stress and water stress conditions. MXV phenotypes will be evaluated under each regime, and plasticity will be determined as the difference in values under stress relative to the non-stress control.Goal 2: Candidate gene discoveryGenotype-by-sequencing (GBS) data has been collected on all lines using the ApeKI procedure. We have re-sequenced 32 targeted lines, and also contributed sequence to the HapMap2 analysis [43]. We have completed an RNA-seq analysis of this panel using RNA from whole-seedlings including roots. This analysis yielded over 3 million SNPs from which we filtered a set of ca. 200,000 that have an allele frequency greater than 5% and have minimal missing data. GWAS analysis is conducted using the Q-K model implemented in GAPIT. Significance determination is determined either genome-wide using Bonferroni or FDR, or based on a restricted set of candidate genes identified by pathway or transcriptional network analysis. We have developed a microarray-based maize gene atlas that includes RNA-seq analysis of 24 unique dissected root tissues. The gene atlas will allow us to identify promising candidates based on expression on relevant tissues. Expression levels of candidate genes in the WiDiv set is also used as covariate for association analysis as a way to capture causal expression differences that associate with our phenotype.Goal 3: Candidate gene corroboration and stock development for mechanistic studiesCorroboration of genes will be via transgenic plants and, if available, transposon insertion lines. The approach to transgenic analysis (knockdown by RNAi or artificial miRNA; constitutive or targeted overexpression; and/or expression of alternative alleles) will be determined based on the predicted expression alteration that will best recapitulate and extend phenotypic variation due to the predicted allelic contrast of the candidate gene. Transgenes will be introduced into HiII and backcrossed into B73. Transposon lines will be searched for in the publicly available uniform Mu and Ac/Ds sequence tagged resources.Activity 3. Evaluate the utility of metaxylem phenotypes for drought resistance in the fieldThis activity will utilize two contrasting water stress environments: 1) automated field rainout shelters in PA and 2) the Apache Root Biology Center (ARBC) in AZ. Existing maize lines and genetic stocks generated in activity 2 contrasting for MXV phenotypes will be planted with and without progressive water stress. Plant growth, plant and soil water status, and root development will be monitored through time. Some plants will be excavated each week to determine the progression of nodal root and crown development as it relates to vegetative and reproductive development. Plant water status will be measured as midday stomatal conductance (LI-6400), leaf relative water content, predawn water potential (PMS 1000), and the rate of sap flow (Dynagage sap flow sensors). Soil water content with depth will be monitored with Dynamax PR2 multi depth soil moisture probes (10, 20, 30, 40, 60, and 100 cm), and TDR probes (TDR100, Campbell Scientific) installed at various depths (15, 25, and 40 cm). Destructive harvests and soil coring (Giddings) during anthesis will be used to measure root length density with depth (WinRhizo), gravimetric soil water content, and soil water potential (Dew Point meter), leaf area, plant biomass partitioning, and anatomical analysis (LAT/Rootscan2). Fully bordered subplots will be left in the field for determination of yield components. Depth of water extraction will be estimated from the natural abundance of H218O in shoot tissue, calibrated against H218O stratification with soil depth. At flowering, soil samples will be collected at several depths and at the same time plant stems will be collected close to where soil was sampled. Cryogenic vacuum distillation will be used to extract soil water and plant stem water. The water samples will be analyzed by mass spectrometry.

Progress 03/15/17 to 03/14/23

Outputs
Target Audience:Crop breeders, crop scientists,policy makers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided PhD training for Stephanie Kelin How have the results been disseminated to communities of interest?Lynch presented results at 50 invited talks at universities and research conferences. The Lynch lab web site about root biology gets about 70k visitors per year. What do you plan to do during the next reporting period to accomplish the goals?Project is complete

Impacts
What was accomplished under these goals? Metaxylem perforation plate phenotypes as regulators of drought tolerance in maize We assess how the structure and frequency of perforation plates along the length of metaxylem affect water use strategies in maize throughin silicomodeling, controlled environment studies, and field trials. Significant variation for the prominence and frequency of perforation plates along the length of metaxylem vessels was observed across a diversity of maize accessions. Measurements of perforation plates had high heritability and phenotypes were consistent across leaves, brace roots, and subterranean roots. Fluid velocity models suggested that the observed variation for perforation plates in maize has strong effects on water transport through xylem conduits, which was supported byin situmeasures of water transport across root segments. Maize genotypes with less pronounced perforation plates long metaxylem vessels had greater vulnerability to cavitation events under water stress and displayed lower rates of transpiration under water-stressed conditions. Reduced water use in these genotypes appeared to contribute to a strategy of conserving soil moisture for sustained water capture at later stages of terminal drought stress, as evidenced by their improved plant water status and larger yields at later stages of terminal drought. Root metaxylem and architecture phenotypes integrate to regulate water use under drought stress At the genus and species level, variationin root anatomy and architecture may interact to affectstrategies of drought avoidance. To investigate this idea,root anatomy and architecture of the drought-sensitive common bean (Phaseolus vulgaris)and drought-adapted tepary bean (Phaseolus acutifolius) were analyzed in relation to water use under terminal drought. Intraspecific variation for metaxylem anatomy and axial conductance was found in the roots of both species. Genotypes with high-conductance root metaxylem phenotypes acquired and transpired more water per unit leaf area, shoot mass, and root mass than genotypes with low-conductance metaxylem phenotypes. Interspecific variation in root architecture and root depth was observed whereP. acutifoliushas a deeper distribution of root length thanP. vulgaris. In the deeper-rootedP. acutifolius,genotypes with high root conductance were better able to exploit deep soil water than genotypes with low root axial conductance. Contrastingly, in the shallower-rootedP. vulgaris, genotypes with low root axial conductance had improved water status through conservation of soil moisture for sustained water capture later in the season. These results indicate that metaxylem morphology interacts with root system depth to determine a strategy of drought avoidanceandillustrate synergism among architectural and anatomical phenotypes for root function. Root Secondary Growth: An Unexplored Component of Soil Resource Acquisition Despite recent progress in elucidating the molecular basis of secondary growth (cambial growth), the functional implications of this developmental process remain poorly understood. Targeted studies exploring how abiotic and biotic factors affect this process, as well as the relevance of secondary growth to fitness of annual dicotyledonous crop species under stress are almost entirely absent from the literature. Specifically, the physiological role of secondary growth in roots has been completely neglected yet entails a unique array of implications for plant performance that are distinct from secondary growth in shoot tissue. Since roots are directly responsible for soil resource capture, understanding of the fitness landscape of root phenotypes is important in both basic and applied plant biology. Interactions between root secondary growth, edaphic conditions, and soil resource acquisition may have significant effects on plant fitness. Our intention here is not to provide a comprehensive review of a sparse and disparate literature, but rather to highlight knowledge gaps, propose hypotheses, and identify opportunities for novel and agriculturally relevant research pertaining to secondary growth of roots. This viewpoint 1) summarizes evidence from our own studies and other published work, 2) proposes hypotheses regarding the fitness landscape of secondary growth of roots in annual dicotyledonous species for abiotic and biotic stress, and 3) highlights the importance of directing research efforts to this topic within an agricultural context.Secondary growth of the roots of annual dicots has functional significance with regards to soil resource acquisition and transport, interactions with soil organisms, and carbon sequestration. Research on these topics would contribute significantly toward understanding the agronomic value of secondary growth of roots for crop improvement. Integrated maize root phenotypes for drought tolerance In order to test the hypothesis that multiple integrated root phenotypes would co-optimize drought tolerance, we phenotyped the root anatomy and architecture of 400 mature maize genotypes under well-watered and water-stressed conditions in the field. We found substantial variation in all 23 root phenes measured. A phenotypic bulked segregant analysis revealed that bulks representing the best and worst performers in the field displayed distinct root phenotypes. In contrast to the worst bulk, the root phenotype of the best bulk under drought consisted of greater cortical aerenchyma formation, more numerous and narrower metaxylem vessels, and thicker nodal roots. Partition against medians (PAM) clustering revealed several clusters of unique root phenotypes related to plant performance under water stress. Clusters associated with improved drought tolerance consisted of phene states that likely enable greater soil exploration by reallocating internal resources to greater root construction (increased aerenchyma content, larger cortical cells, fewer cortical cell files), restrict uptake of water to conserve soil moisture (reduced hydraulic conductance, narrow metaxylem vessels), and improve penetrability of hardening, drying soils (thick roots with a larger proportion of stele, smaller distal cortical cells). We propose that the most drought tolerant integrated phenotypes merit consideration as breeding ideotypes. Shared genetic architecture underlying root metaxylem phenotypes under drought stress in cereals Root metaxylem are phenotypically diverse structures whose function is related to their anatomy, particularly under drought stress. Much research has dissected the genetic machinery underlying metaxylem phenotypes in dicots, but monocots are relatively unexplored. In maize (Zea mays), a robust pipeline integrated a GWAS of root metaxylem phenes under well-watered and water stress conditions with a gene co-expression network to identify candidate genes most likely to impact metaxylem phenotypes. We identified several promising candidate genes in 14 gene co-expression modules inferred to be functionally relevant to xylem development. We also identified five gene candidates that co-localized in multiple root metaxylem phenes in both well-watered and water stress conditions. Using a rice GWAS conducted in parallel, we detected overlapping genetic architecture influencing root metaxylem phenotypes by identifying eight pairs of syntenic candidate genes significantly associated with metaxylem phenes. There is evidence that the genes of these syntenic pairs may be involved in biosynthetic processes related to the cell wall, hormone signaling, oxidative stress responses, and drought responses. Our study demonstrates a powerful new strategy for identifying promising gene candidates and suggests several gene candidates that may enhance our understanding of vascular development and responses to drought in cereals.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Sidhu, JS, I Ajmera, S Arya, JP Lynch. 2023. RootSlice: functional structural modeling of root anatomy. Plant, Cell, and Environment 46:1671-1690 https://doi.org/10.1111/pce.14552
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Siangliw JL, B Thunnom, MA Natividad, MR Quintana, D Chebotarov, KL McNally, JP Lynch, KM Brown, A Henry. 2022. Response of Southeast Asian rice root architecture and anatomy phenotypes to drought stress. Frontiers in Plant Science, 13 , https://doi.org/10.3389/fpls.2022.1008954
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: 3. Massas ASF, JD Burridge, HM Schneider, D Debouck, KM Brown, JP Lynch. 2022. Comparative phenomics of root architecture and anatomy in Phaseolus species. Crop Science https://doi.org/10.1002/csc2.20838
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Fonta JE, P Vejchasarn, A Henry, JP Lynch, KM Brown. 2022. Many paths to one goal: Identifying integrated rice root phenotypes for diverse drought environments. Frontiers in Plant Science doi: 10.3389/fpls.2022.959629
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Fonta JE, J Giri, P Vejchasarn, JP Lynch, KM Brown. 2022. Spatiotemporal responses to drought in rice roots. Plant Soil https://doi.org/10.1007/s11104-022-05527-w
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Lynch JP. The importance of edaphic stress interactions for plant function: important yet poorly understood drivers of plant production in future climates. Field Crops Research Volume 283, 1 July 2022, 108547 https://doi.org/10.1016/j.fcr.2022.108547
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Clement C, HM Schneider, DB Dresb�ll, JP Lynch, K Thorup-Kristensen. 2022. Root and xylem anatomy varies with root length, root order, soil depth, and environment in intermediate wheatgrass Thinopyrum intermedium and Alfalfa Medicago sativa: Implications for water uptake. Annals of Botany 130(3):367-382. doi: 10.1093/aob/mcac058.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Lopez Valdivia Ivan Alden Perkins, Hannah Schneider, Miguel Vallebueno-Estrada, James Burridge, Eduardo Gonz�lez-Orozco, Aurora Montufar, Rafael Montiel, Jonathan Lynch, Jean-Philippe Vielle-Calzada 2022. Gradual domestication of root traits in the earliest maize from Tehuacan PNAS Vol. 119 No. 17 e2110245119
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Galindo-Casta�eda T, JP Lynch, J Six, M Hartmann. 2022. Improving soil resource uptake by plants through capitalizing synergies between root architecture and anatomy and root-associated microorganisms. Frontiers in Plant Science,Volume 13 - 2022 | https://doi.org/10.3389/fpls.2022.827369
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Strock CF, HM Schneider, JP Lynch. 2022. Anatomics: high-throughput phenotyping of plant anatomy. TIPS https://doi.org/10.1016/j.tplants.2022.02.009
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Guang-Jun Wu, Xucun Jia, Christopher Strock, Shu-Ting Dong, Ji-Wang Zhang, Bin Zhao, Jonathan P. Lynch, Peng Liu. 2022. Root anatomical phenotypes related to growth under low nitrogen availability in maize (Zea mays L.) hybrids. Plant Soil 10.1007/s11104-022-05331-6
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Lynch JP, SJ Mooney, CF Strock, HM Schneider. 2022. Future roots for future soils. Plant, Cell & Environment 45:620-636, https://doi.org/10.1111/pce.14213
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Ajmera I, Henry A, Radanielson AM, Klein SP, Ianevski A, Bennett MJ, Band LR, JP Lynch. 2022. Integrated root phenotypes for low nitrogen tolerance in rice. Plant, Cell & Environment 45: 805-822. http://dx.doi.org/10.1111/pce.14284
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Lynch JP, CF. Strock, HM Schneider, JS Sidhu, I Ajmera, T Galindo-Casta�eda, SP Klein, MT Hanlon. 2021. Root anatomy and soil resource capture. Plant and Soil (466),21-63 10.1007/s11104-021-05010-y
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Strock, CF, JD Burridge, MD Niemiec, KM Brown, JP Lynch. 2020. Root metaxylem phenotypes interact with root architecture to regulate water use under drought stress. Plant, Cell Environment 1-19https://doi.org/10.1111/pce.13875
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Strock CF, JP Lynch. 2020. Root secondary growth: An unexplored component of soil resource acquisition. Annals of Botany 126:205218, doi: 10.1093/aob/mcaa068
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Schneider HM, JP Lynch. 2020. Should root plasticity be a crop breeding target? Frontiers in Plant Science 11: 546. doi: 10.3389/fpls.2020.00546
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Klein SP, HM Schneider, AC Perkins, KM Brown, JP Lynch. 2020. Multiple integrated root phenotypes are associated with improved drought tolerance. Plant Physiology 83, 10111025, www.plantphysiol.org/cgi/doi/10.1104/pp.20.00211
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Schneider HM, Klein SP, Hanlon MT, Brown KM, Kaeppler SM, Lynch JP. Genetic control of root anatomical plasticity in maize. 2020. The Plant Genome 13 https://doi.org/10.1002/tpg2.20003
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Lynch JP. 2019 Root phenotypes for improved nutrient capture: an underexploited opportunity for global agriculture. New Phytologist 223: 548564 doi: 10.1111/nph.15738
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Galindo-Casta�eda T, KM Brown, G Kuldau, G Roth, N Wenner, S Ray, JP Lynch. 2019. Root cortical anatomy is associated with differential pathogenic and symbiotic fungal colonization in maize. Plant, Cell, and Environment 42:2999- 3014.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Yang JT, HM Schneider, KM Brown, JP Lynch. 2019. Genotypic variation and nitrogen stress effects on root anatomy in maize are node-specific. Journal of Experimental Botany 70 53115325.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Hall B, A Lanba, JP Lynch. 2019. Three-dimensional analysis of biological systems via a novel laser ablation technique. J. Laser Appl. 31, 022602.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Strock CF, LM De la Riva, JP Lynch. 2018. Reduction in root secondary growth as a strategy for phosphorus acquisition. Plant Physiology, 176:691-703. DOI:https://doi.org/10.1104/pp.17.01583.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Klein SP, JE Reeger, SM Kaeppler, KM Brown, JP Lynch. 2020. Shared genetic architecture underlying root metaxylem phenotypes under drought stress in cereals. BioRxiv https://doi.org/10.1101/2020.11.02.365247
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Schneider HM, CF Strock, MT Hanlon, DJ Vanhees, AC Perkins, IB Ajmera, JS Sidhu, SJ Mooney KM Brown, JP Lynch. 2021. Multiseriate cortical sclerenchyma enhance root penetration in compacted soils. PNAS Vol. 118(6) e2012087118 https://doi.org/10.1073/pnas.2012087118


Progress 03/15/21 to 03/14/22

Outputs
Target Audience:Plant Scientists, crop breeders Changes/Problems:With collaborators at the Wisconsin Crop Improvement Center, we are developing CRISPR/Cas9-edited mutants with targeted deletions within our gene candidates. Due to Covid restrictions over the previous year, development of these mutant lines has taken longer than anticipated, so we request additional time to fulfill this goal. What opportunities for training and professional development has the project provided?This project is supporting the PhD training of Stephanie Klein and undergraduate assistants at Penn State. This project is supporting the Post-doctoral training of Vai Lor at UW-Madison. How have the results been disseminated to communities of interest?Lynch presented results at 10 invited talks at universities and research conferences. The Lynch lab web site about root biology gets about 70k visitors per year. What do you plan to do during the next reporting period to accomplish the goals?With collaborators at the Wisconsin Crop Improvement Center, we are developing CRISPR/Cas9-edited mutants with targeted deletions within our gene candidates. Due to Covid restrictions over the previous year, development of these mutant lines has taken longer than anticipated, so we request additional time to fulfill this goal. For our physiology work, we have continued analyzing data collected from multiple field locations and conducted studies in silico to complement our field findings. Results are forthcoming. We also are preparing two manuscripts: one centered on our genetics and genome-wide association work and a second discussing physiological trends associated with drought stress. The latter is nearer completion, and we anticipate submitted for review by June 2022. The genetics study is contingent on the mutant maize lines, though we anticipate submitting for publication by fall 2022.

Impacts
What was accomplished under these goals? We identified several integrated root phenotypes, or combinations of single root phenes, that are associated with drought tolerance. Integrated root phenotypes associated with improved drought tolerance consisted of root characteristics that enabled greater soil exploration by reallocating internal resources to greater root construction (increased aerenchyma content, larger cortical cells, fewer cortical cell files), restricted uptake of water to conserve soil moisture (reduced hydraulic conductance, narrow metaxylem vessels), and greater penetrability of hardening, drying soils (thick roots with a larger proportion of stele, smaller distal cortical cells). These findings were published in Plant Physiology (Klein et al. 2020). We have conducted experiments to determine the optimal root hydraulic architecture, or combination of root traits most relevant to water uptake and transport, for drought tolerance. In a series of field and greenhouse experiments, we have found evidence that the optimal root hydraulic architecture may change with worsening drought. Under mild drought, a root hydraulic architecture that enables slower extraction of deep water stores may be advantageous (i.e. many and small root metaxylem vessels, few nodal roots, steep-angled roots). However, under severe drought, a root hydraulic architecture that facilitates rapid extraction of surface water may be advantageous (i.e. few and large root metaxylem vessels, few nodal roots, shallow angled roots). We are validating these findings in silico using SimRoot. We have identified several candidate genes potentially underlying the natural variation in root metaxylem phenes and will validate the function of these genes using gene-edited loss-of-function and overexpression maize mutants. Three gene candidates have been advanced into this pipeline, which have been shown to be highly expressed in either of two root tissues where root metaxylem develop: the root stele and the meristematic/elongation zone 3 days after sowing. The resulting transgenic maize lines will be functionally evaluated in the greenhouse. Summary of progress in 2021: We have made substantial progress in the last year. For the genome-wide association study, we have selected two gene candidates to advance through the mutant development pipeline. With collaborators at the Wisconsin Crop Improvement Center, we are developing CRISPR/Cas9-edited mutants with targeted deletions within our gene candidates. Due to Covid restrictions over the previous year, development of these mutant lines has taken longer than anticipated, so we request additional time to fulfill this goal. For our physiology work, we have continued analyzing data collected from multiple field locations and conducted studies in silico to complement our field findings. Results are forthcoming. We also are preparing two manuscripts: one centered on our genetics and genome-wide association work and a second discussing physiological trends associated with drought stress. The latter is nearer completion, and we anticipate submitted for review by June 2022. The genetics study is contingent on the mutant maize lines, though we anticipate submitting for publication by fall 2022. Taken as a whole, our results to date indicate that several root traits acting in concert are associated with improved drought tolerance in corn. This is important to the nation since corn is a principal Amercian crop that is the mainstay of agricultural economies and rural livelihoods in many states, and since drought is the single greatest risk to corn production, and this risk is expected to grow in years ahead because of the effects of climate change on weather patterns. Identification of root traits improving drought tolerance and their genetic control provides tools to crop breeders to develop more resilient, productive corn varieties for US production. Improved drought tolerance is also useful for many developing nations in Africa and Latin America where corn is a staple crop. Improving food secuity and farm income in these regions benefits the US by increasing economic development and political stability, and in Latin America, by reducing economic pressures driving illegal immigration to the US. Root traits we identify in corn may also be useful in other cereal crops that are important in the US, especially sorghum.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Schneider HM, CF Strock, MT Hanlon, DJ Vanhees, AC Perkins, IB Ajmera, JS Sidhu, SJ Mooney KM Brown, JP Lynch. 2021. Multiseriate cortical sclerenchyma enhance root penetration in compacted soils. PNAS Vol. 118(6) e2012087118 https://doi.org/10.1073/pnas.2012087118
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Schneider HM, JT Yang, KM Brown, JP Lynch. 2021. Nodal root diameter and node number interact to influence nitrogen stress tolerance in maize (Zea mays L.). Plant Direct http://doi.org/10.1002/pld3.310
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Lynch JP, CF. Strock, HM Schneider, JS Sidhu, I Ajmera, T Galindo-Casta�eda, SP Klein, MT Hanlon. 2021. Root Anatomy and Soil Resource Capture PLSO (466),21-63 https://doi.org/10.1007/s11104-021-05010-y
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Lynch JP 2021. Root Biology in the 21st century: challenges and opportunities. Viewpoint. Annals of Botany, 128:i-ii. DOI: 10.1093/aob/mcab062
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Lynch JP Harnessing root architecture to address global challenges. 2021. The Plant Journal, http://doi.org/10.1111/tpj.15560
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Sch�fer ED, MR Owen, JA Postma, C Kuppe, CK Black, JP Lynch 2021. Simulating Crop Root Systems Using OpenSimRoot Plant Systems Biology, 293-323 https://link.springer.com/protocol/10.1007/978-1-0716-1816-5_15


Progress 03/15/20 to 03/14/21

Outputs
Target Audience:Plant scientists, crop breeders Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project is supporting the PhD training of Stephanie Klein and undergraduate assistants at Penn State. This project is supporting the Post-doctoral training of Vai Lor at UW-Madison. How have the results been disseminated to communities of interest?Lynch presented results at 10invited talks at universities and research conferences. The Lynch lab web site about root biology gets about 70k visitors per year. What do you plan to do during the next reporting period to accomplish the goals?We have identified several promising gene targets from Genome Wide Association Analysis (GWAS) for genes that appear to regulating metxylem phenotypes in maize. In 2021 we plan to develop transgenic lines in which three of thesegenes have been knocked out or overexpressed in collaboration with Shawn Kaeppler at the Wisconsin Crop Innovation Center. We will then measure the metaxylem phenotypes of these lines to confirm their role in regulatingmetaxylem phenotypes.

Impacts
What was accomplished under these goals? We identified several integrated root phenotypes, or combinations of single root phenes, that are associated with drought tolerance. Integrated root phenotypes associated with improved drought tolerance consisted of root characteristics that enabled greater soil exploration by reallocating internal resources to greater root construction (increased aerenchyma content, larger cortical cells, fewer cortical cell files), restricted uptake of water to conserve soil moisture (reduced hydraulic conductance, narrow metaxylem vessels), and greater penetrability of hardening, drying soils (thick roots with a larger proportion of stele, smaller distal cortical cells). These findings were published in Plant Physiology (Klein et al. 2020). We have conducted experiments to determine the optimal root hydraulic architecture, or combination of root traits most relevant to water uptake and transport, for drought tolerance. In a series of field and greenhouse experiments, we have found evidence that the optimal root hydraulic architecture may change with worsening drought. Under mild drought, a root hydraulic architecture that enables slower extraction of deep water stores may be advantageous (i.e. many and small root metaxylem vessels, few nodal roots, steep-angled roots). However, under severe drought, a root hydraulic architecture that facilitates rapid extraction of surface water may be advantageous (i.e. few and large root metaxylem vessels, few nodal roots, shallow-angled roots). We are validating these findingsin silicousing SimRoot. We have identified several candidate genes potentially underlying the natural variation in root metaxylem phenes and will validate the function of these genes using gene-edited loss-of-function and overexpression maize mutants. Three gene candidates have been advanced into this pipeline, which have been shown to be highly expressed in either of two root tissues where root metaxylem develop: the root stele and the meristematic/elongation zone 3 days after sowing. The resulting transgenic maize lines will be functionally evaluated in the greenhouse.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Schneider HM, Klein SP, Hanlon MT, Brown KM, Kaeppler SM, Lynch JP. Genetic control of root anatomical plasticity in maize (2020) The Plant Genome 13 https://doi.org/10.1002/tpg2.20003
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Klein SP, HM Schneider, AC Perkins, KM Brown, JP Lynch. Multiple integrated root phenotypes are associated with improved drought tolerance. Plant Physiology 83, 10111025, www.plantphysiol.org/cgi/doi/10.1104/pp.20.00211
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Schneider HM, JP Lynch. Should root plasticity be a crop breeding target? Frontiers in Plant Science 11: 546. doi:�10.3389/fpls.2020.00546
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Strock CF, JP Lynch Root secondary growth: An unexplored component of soil resource acquisition Annals of Botany 126:205218, doi: 10.1093/aob/mcaa068
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Strock, CF, JD Burridge, MD Niemiec, KM Brown, JP Lynch. 2021. Root metaxylem phenotypes interact with root architecture to regulate water use under drought stress. PCE 44:49-67 https://doi.org/10.1111/pce.13875
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Levin KA, MR Tucker, CF Strock, JP Lynch, DE Mather. 2021 Three-dimensional imaging reveals that positions of cyst nematode feeding sites relative to xylem vessels differ between susceptible and resistant wheat. Plant Cell Reports, 40:393-403 https://doi.org/10.1007/s00299-020-02641-w
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Klein SP, JE Reeger, SM Kaeppler, KM Brown, JP Lynch. Shared genetic architecture underlying root metaxylem phenotypes under drought stress in cereals. BioRxiv https://www.biorxiv.org/content/10.1101/2020.11.02.365247v2
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Schneider HM, JT Yang, KM Brown, JP Lynch. 2021. Nodal root diameter and node number interact to influence nitrogen stress tolerance in maize (Zea mays L.). Plant Direct accepted Jan 25 2021 (BioRxiv doi:�https://doi.org/10.1101/2020.12.23.424185)


Progress 03/15/19 to 03/14/20

Outputs
Target Audience:Plant scientists, crop breeders Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project is supporting the PhD training of Stephanie Klein and undergraduate assistants at Penn State. This project is supporting the Post-doctoral training of Vai Lor at UW-Madison. How have the results been disseminated to communities of interest?Lynch presented results at 12 invited talks at universities and research conferences. Klein presented results at 1 invited talk and 2poster presentations at research conferences. The Lynch lab web site about root biology gets about 70k visitors per year. What do you plan to do during the next reporting period to accomplish the goals?We are working on following up on gene discovery activities and further dissecting the physiological differences associated with the contrasting metaxylem phenotypes in the field and greenhouse.

Impacts
What was accomplished under these goals? We identified several integrated root phenotypes, or combinations of single phenes, that are associated with drought tolerance.Clusters associated with improved drought tolerance consisted of phene states that enabled greater soil exploration by reallocating internal resources to greater root construction (increased aerenchyma content, larger cortical cells, fewer cortical cell files), restricted uptake of water to conserve soil moisture (reduced hydraulic conductance, narrow metaxylem vessels), and greater penetrability of hardening, drying soils (thick roots with a larger proportion of stele, smaller distal cortical cells). We have found evidence that variation in MXV size and number is associated with functional tradeoffs under drought. Maize lines with "small" MXV were more likely to be more drought resistant than lines with "large" vessels. MXV phenes are plastic and most often become smaller and less numerous under drought stress, which has implications for water use and availability. We have identified several candidate genes related to variation in metaxylem phenes and will validate the function of these genes using transgenic maize lines. Two of the priority candidate genes have been successfully cloned and sequenced. The sequencing results indicate that the primary gene transcript expressed in the root system is different from the primary transcript on MaizeGDB. This information will be used to generate transgenic plants overexpressing the primary transcripts of candidate genes. We are also targeting the candidate genes with CRISPR/Cas9 to generate loss-of-function mutants. We have generated the desired CRISPR/Cas9 vectors and will be initiating the transformation of maize. The resulting transgenic maize lines will be functionally evaluated in the greenhouse.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Lynch JP. 2019 Root phenotypes for improved nutrient capture: an underexploited opportunity for global agriculture. New Phytologist 223: 548564 doi: 10.1111/nph.15738
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Yang JT,�HM Schneider, KM Brown, JP Lynch.�2019. Genotypic variation and nitrogen stress effects on root anatomy in maize are node-specific. Journal of Experimental Botany �70: 53115325
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Galindo-Casta�eda T, KM Brown, G Kuldau, G Roth, N Wenner, S Ray, JP Lynch. 2019. Root cortical anatomy is associated with differential pathogenic and symbiotic fungal colonization in maize. Plant, Cell, and Environment 42:2999-3014.
  • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Schneider HM, SP Klein, MT Hanlon, KM Brown, SM Kaeppler, JP Lynch. Genetic control of root anatomical plasticity in maize. The Plant Genome accepted Nov 1 2019


Progress 03/15/18 to 03/14/19

Outputs
Target Audience:Our primary target audiences are plant biologists and crop breeders. Changes/Problems:With the help of new collaborators at Colorado State, we added a field study at the agricultural research farm outside Ft. Collins, CO. This field study was conducted from May to October in 2018 and was a replication of our field experiment in the rainout shelters in Rock Springs, PA. What opportunities for training and professional development has the project provided?This project is supporting the PhD training of Stephanie Klein and undergraduate assistants at Penn State. This project is supporting the Post-doctoral training of Vai Lor at UW-Madison. How have the results been disseminated to communities of interest?Lynch presented results at5 invited talks at universities and research conferences. Klein presented results at 1 invited talk and 3 poster presentations at research conferences. The Lynch lab web site about root biology gets about 30k visitors per year. What do you plan to do during the next reporting period to accomplish the goals?We are working on following up on gene discovery activities and further dissecting the physiological differences associated with the contrasting metaxylem phenotypes in the field and greenhouse.

Impacts
What was accomplished under these goals? We have made substantial progress in all objectives. We have found evidence that variation in MXV size and number is associated with functional tradeoffs. Maize lines with "small" MXV were more likely to be more drought resistant than lines with "large" vessels. MXV phenes are plastic and most often become smaller and less numerous under drought stress. We have also discovered a related yet distinct root phene, MXV length, which exhibits substantial variation and is also plastic in response to drought. A preliminary study relating MXV length to drought tolerance suggested that maize lines with "longer" vessels were more drought tolerant, though additional studies are needed. We have identified several candidate genes related to variation in metaxylem phenes and will validate the function of these genes using transgenic maize lines Two of the priority candidate genes has been successfully cloned and sequenced. The sequencing result indicate that the primary gene transcript expressed in the root system is different from the primary transcript on MaizeGDB. This information will be used to generate transgenic plants overexpressing the primary transcripts of candidate genes. We are also targeting the candidate genes with CRISPR/Cas9 to generate loos-of-function mutants. We have generated the desired CRISPR/Cas9 vectors and will be initiating the transformation of maize. The resulting transgenic maize lines will be functionally evaluated in the greenhouse.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Lynch JP 2018. Rightsizing root phenotypes for drought resistance. J Experimental Botany, Vol. 69, No. 13 pp. 3279⿿3292, 2018


Progress 03/15/17 to 03/14/18

Outputs
Target Audience:Our primary target audiences are plant biologists and crop breeders. Changes/Problems:The renovations to the rainout shelters in Rock Springs, PA, were significantly delayed. Because the renovations were not complete by the start of the field season, we were unable to conduct field experiments during this year. We adapted several of the PA field trials into a greenhouse studies, though we plan to conduct the field trials as originally conceived during the upcoming field season. What opportunities for training and professional development has the project provided?This project is supporting the PhD training of Stephanie Klein and undergraduate assistants at Penn State. How have the results been disseminated to communities of interest?Lynch presented results at 15 invited talks at universities and research conferences. The Lynch lab web site about root biology gets about 30k visitors per year. What do you plan to do during the next reporting period to accomplish the goals?Follow up on gene discovery activities, better understand the contrasting phenotypes in maize root crowns in the field.

Impacts
What was accomplished under these goals? We have made substantial progress in all objectives. We have found evidence that variation in MXV size and number is associated with functional tradeoffs. Maize lines with "small" MXV were more likely to be more drought tolerant than lines with "large" vessels. MXV phenes are plastic and most often become smaller and less numerous under drought stress. We have also discovered a related yet distinct root phene, MXV length, which exhibits substantial variation and is also plastic in response to drought. A preliminary study relating MXV length to drought tolerance suggested that maize lines with "longer" vessels were more drought tolerant, though additional studies are needed. We have identified several candidate genes related to variation in metaxylem phenes that we are now confirming by generating transgenic maize lines. These maize lines will be functionally validated in the greenhouse.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Rightsizing root phenotypes for drought resistance. J Exp Botany, in press