OPTIMIZING ROOT METAXYLEM PHENOTYPES TO IMPROVE DROUGHT TOLERANCE IN MAIZE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: EXTENDED
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1012027
• Grant No.: 2017-67013-26192
• Project No.: PENW-2016-10511
• Proposal No.: 2016-10511
• Program Code: A1152
• Project Start Date: Mar 15, 2017
• Project End Date: Mar 14, 2023
• Grant Year: 2017

Project Director
Lynch, J. P.

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
203	1510	1020	70%
201	1510	1080	30%

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;

Keywords

drought

maize

phenomics

root anatomy

water relations

xylem

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 03/15/21 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