A NOVEL ROOT TRAIT TO IMPROVE MAIZE DROUGHT TOLERANCE AND CARBON SEQUESTRATION: LIGNIFIED OUTER CORTICAL PARENCHYMA CELLS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1025251
• Grant No.: 2021-67013-33723
• Cumulative Award Amt.: $500,000.00
• Proposal No.: 2020-03632
• Project Start Date: Jan 15, 2021
• Project End Date: Jan 14, 2025
• Grant Year: 2021
• 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
We have recently discovered that genotypic variation for root cortical anatomy in maize is associated with substantial variation in plant performance in dry, hard soils. Drought is a primary limitation to global crop production. The development of maize cultivars with enhanced drought tolerance and carbon sequestration through the construction of roots with more recalcitrant compounds is an important goal for global agriculture. As we show in this proposal, maize genotypes show substantial variation for MCS (Multiseriate Cortical Sclerenchyma) which is associated with greater root lignin content, better penetration of hard soil, and better plant growth under drought. The overall objective of this project is to evaluate and develop this trait as a tool to improve drought resistance and carbon sequestration in maize and in other crops. Specifically, we will: 1) confirm the physiological utility of the MCS phenotype to improve plant water status and growth under drought in field and greenhouse environments, 2) identify genes underlying natural variation in MCS and develop stocks that will support mechanistic assessment of its regulation and utility, and 3) evaluate the utility of MCS for improved carbon sequestration from the atmosphere into deep soil domains. Defining and understanding traits enhancing drought resistance is of considerable importance for keeping U.S. agriculture competitive while ending world hunger.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
203	1510	1020	100%

Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
1510 - Corn;

Field Of Science
1020 - Physiology;

Keywords

anatomy

carbon sequestration

cortex

drought

gwas

lignin

maize

parenchyma

root

Goals / Objectives
Objectives: We have recently discovered that genetic variation for outer cortical cell traits in maize is associated with substantial variation in plant performance in dry, hard soils. Small outer cortical sclerenchyma cells with thick cell walls (MCS, multiseriate cortical sclerenchyma) display variation in root growth and water acquisition under drought. The overall objective of this project is to evaluate and develop this trait as a tool to improve drought resistance and carbon sequestration in maize, with potential application to other crops. Specifically, we will:1. Confirm the physiological utility of MCS to improve plant water status and growth under drought in field and greenhouse environments.2. Identify genetic loci underlying natural variation in MCS and develop stocks that will support mechanistic assessment of its regulation and utility. 3. Evaluate the utility of MCS for improved carbon sequestration from the atmosphere into deep soil domains.

Project Methods
Activity 1. Confirm the physiological utility of the MCS phenotype for improved drought resistance in greenhouse mesocosms and in the fieldMesocosm studieswill 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 MCS, in addition to transgenic 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. If our hypotheses are correct, genotypes with MCS will have greater water acquisition during drought, leading to better water status and better growth under water stress than genotypes with the non-MCS phenotype. Genotypes with MCS enable the plant to penetrate dry, hard soils to access water in deep soil domains compared to non-MCS genotypes. In addition, genotypes with MCS that have a reduced hydraulic conductivity that will enable soil water banking and the conservation of soil water throughout the growth season. Since the utility of MCS has not been rigorously tested in crops, it is important to determine if it entails tradeoffs for other plant functions.Field workwill enable us to determine if MCS improves plant growth and grain yield under water stress in the field, considering varying drought regimes (sporadic vs. terminal), varying soil types, belowground competition among neighboring plants, effects of soil drying on soil hardness, high soil surface temperatures, and other biotic and abiotic stresses associated with drought. These factors could be important for the utility of MCS but are not captured in mesocosm studies in the greenhouse. This is important since MCS phenotypes may have interactions withe.g. radial water and nutrient transport or soil penetration, which should be understood before this trait can be deployed in crop breeding programs.Activity 2: Discover genes underlying natural variation in MCS phenotypesGoal 1) Phenotypic analysis:This project will focus on the WiDiv association panel (primarily as described in). This panel is a diverse set of inbred lines that have been selected to flower in the northern Midwest, but that contains a wide diversity of maize germplasm including representation of tropical alleles included as germplasm enhancement of maize (GEM) lines (https://usda-gem.public.iastate.edu/).Goal 2) Candidate gene discovery:Genetic resources for genome-wide association mapping (GWAS) using the WiDiv association panel have been previously developed and published. Genotype-by-sequencing (GBS) data has been collected on all lines using theApeKIprocedure. GWAS analysis is conducted using the Q-K model implemented in GAPIT and FARMCPU. Significance determination is determined either genome-wide using Bonferroni or false discovery rate (FDR) or based on a restricted set of candidate genes identified by pathway or transcriptional network analysis. For multiple reasons, the most significant SNP is not always in the functional gene but may be in a gene model adjacent to the causal gene. For this reason, multiple levels of assessment are used to sift through regional candidates (e.g. +/- five annotated gene models). A microarray-based maize gene-atlas provided to the community via MaizeGDB - has been developed 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 but are not due to factors in LD with SNPs in our set. A goal of this project is to discover at least four candidate genes (we can handle a maximum of 6) that are highly likely to contribute to variation to MCS, and thoroughly assess them in our corroboration pipeline.Goal 3) Candidate gene corroboration and stock development for mechanistic studies:Corroboration of genes will be done via transgenic plants and, if available, transposon insertion lines. The approach to transgenic analysis 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.Activity 3: Evaluate the utility of MCS for improved carbon sequestration in soilThis activity will utilize both growth chamber experimentation and field site assessments in PA and WI to determine the utility of MCS for increased deep soil carbon sequestration. Growth chamber experiments will allow the manipulation of specific environmental factors to quantify their respective roles in the decomposition of plant material. Field sites will allow for evaluation of decompositionin situunder natural field conditions with natural microbial, protozoans, micro-arthropods and earthworm communities. Existing lines contrasting for MCS will be grown in soil mesocosms (described above) for 40 days. Fresh roots of different ages and root whorls will be collected, gently washed, surface sterilized, dried, and weighed. Dry root material from all whorls collected from distal, mid, and apical sections of each root will be weighed and placed into decomposition bags (5 x 5 cm polyester, 50 µm mesh). The bags will be heat sealed to prevent root loss. Bags will be subjected to the pre-determined treatments in the growth chamber and field settings (see below).In the growth chamber, mesh bags will be buried in an Alfisol or Entisol, representing soils in major crop production areas in the United States. After 3, 6, and 9 months, the mesh bags will be extracted, gently washed of soil, dried, and weighed. Decomposition rates will be measured as the change in dry root biomass between the initial and final weights of the root material. In each collected mesh bag, we will profile the total microbial (bacterial and fungal) communities and their respective biomasses. These will allow us to calculate differences in microbiome composition and structure across treatments (i.e. beta-diversity) and the distribution on microbial taxa, community richness, and diversity in each sample (i.e. alfa-diversity).The field work will enable us to quantify decomposition rates in the presence of more realistic biotic factors. Roots of genotypes contrasting in MCS of different ages and originating from different whorls will be studied. Field soil coringwill enable the collection of roots located in different soil depths in the field. Roots will be washed of soil, surface sterilized, dried, weighed, subsampled for C:N ratio measurements and lignin content, and placed in decomposition bags. Roots sealed in polyester mesh bags will be buried at 10 cm depth in field soil in an Entisol soil in WI and an Alfisol in PA.Soil water content will be tracked.At collection, roots will be gently washed of soil, dried, and weighed to measure decomposition rates and subsampled to determine C:N ratios and lignin content.

Progress 01/15/21 to 01/14/25

Outputs
Target Audience:The target audiences for this project include: 1) Fellow researchers and students, who seek to understand how crops plants adapt to drought and soil infertility, and how crop plants can be harnessed to improve biosequestration of atmospheric carbon dioxide. 2) The crop breeding community, in both public and private sectors, who are interested in the identification and characterization of crop traits that improve drought tolerance, reduce the requirement for intensive nitrogen fertilization, and improve biosequestration of atmospheric carbon dioxide, all of which are significant economic and agronomic motivations for crop improvement. 3) The research funding and donor communities, in both public and private sectors, in both domestic and international arenas, who seek to prioritize research investments to those avenues most likely to deliver social benefits in a reasonable time frame. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project supported the postdoctoral training of Cody DePew, Gordon Custer, and undergraduate assistants at Penn State. This project has also supported the training of the PhD student Courtney Tharp (Plant Biology Program, Penn State). How have the results been disseminated to communities of interest?In addition to refereed journal articles, Dini-Andreote, Schneider, and Lynch made numerous presentations to scientific colleagues throughout the span of this project. The Lynch lab website about root biology receives approximately 70k visitors per year. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We have conducted multiple field studies to determine the fitness landscape of MCS under drought and suboptimal N availability in maize. We found that MCS is associated with drought adaptation in maize as part of a cluster of root phenotypes including large root diameter with a large proportion of root cross-sectional area occupied by the stele (Klein et al, Plant Physiology 2020, 183:1011-1025). In wheat, we found that MCS is associated with drought adaptation when it is combined with high expression of root cortical senescence (manuscript in preparation). We analyzed post-decomposition characteristics from MCS and non-MCS root phenotypes of Maize to determine the effect of MCS on root persistency in soil as a potential carbon sequestration mechanism. Interestingly, we found MCS lines to degrade slightly faster than non-MCS in root litter bags overwintered in field soil. The analysis of microbiome composition of these litter bags showed statistically significant differences in bacterial communities associated with the decomposition of MCS and non-MCS roots. Anatomical imaging of commonly available model maize lines indicated that MCS was not present in lines available for transgenic manipulation. Homologous genes from MCS GWAS analysis were identified in Arabidopsis to assess lignin phenotypes more rapidly and broadly than in Maize. We have acquired these lines and are in the process of verifying their genetic background. We used the structural-functional plant/soil model OpenSimRoot v2 to evaluate the importance of MCS in maize domestication as an adaptation to changing soil mechanical impedance regimes. Our results reveal that increasing Holocene atmospheric CO2 concentrations permitted the appearance of reduced NRN and MCS between 12000 to 8000 years before present (yBP), promoting deeper root systems. The advent of irrigation by 6000 yBP switched nitrogen distribution from topsoil to subsoil domains, a change that increased the utility of reduced NRN and MCS. Our results suggest that root phenotypes that enhance plant performance under nitrogen stress were important for maize adaptation to changing agricultural practices in Tehuacan Valley. Our results support the hypothesis that anthropogenic modifications to the soil environment shaped the root phenotypes of modern maize. MCS is part of a suite of responses of roots to hard soil mediated by ethylene. Another is that roots stop elongating when they encounter hard soil. Simulations of ancient soils indicate that this response is useful in native soil but became less useful during crop domestication with the advent of irrigation and cultivation in Neolithic agriculture. We hypothesized that maize root phenotypes with greater plasticity (meaning reduced elongation in response to mechanical impedance, i.e. a 'stop signal') have fitness advantages over phenotypes with reduced plasticity (i.e. unimpeded root elongation) in native soils, by reallocating root foraging to softer, presumably wetter, soil domains, and that the value of the stop signal reduced with soil cultivation and crop domestication. We used OpenSimRoot to simulate native and cultivated soils and evaluated maize root phenotypes with varying axial and lateral root penetration ability in water, nitrogen, and impedance regimes associated with Neolithic agriculture. The stop signal was advantageous in native soils but was less beneficial in cultivated irrigated soils. Reduced root foraging in hard, dry topsoil enabled root growth in deeper domains where water is available, resulting in an improved balance of resource expenditure and acquisition. The importance of the stop signal is evident in modern high-input agroecosystems in which drought is a limiting factor. These results support the hypotheses that the reduction of lateral root growth by mechanical impedance is adaptive in native soil, but became less adaptive with soil cultivation and irrigation associated with Neolithic agriculture. The importance of MCS as a root phenotype pertains to the fact that it enhances the penetration of hard soils by physically reinforcing the root cortex. We developed a new analytical model to understand the tensile stress-strain behavior of a single root axis, which is the first to incorporate root anatomical features, to reduce the existing uncertainty in predictions. Stele and cortex biomechanical properties are substantially different, affecting the tensile behavior of plant roots. Accounting for these anatomical traits increased the accuracy of predicting root biomechanical properties from tensile tests. Root anatomy is an important determinant of root metabolic costs, soil exploration, and soil resource capture. Root anatomy varies substantially within and among plant species. As a tool to better understand the biological relevance of root anatomy, we developed RootSlice, a multicellular functional-structural model of root anatomy. RootSlice can capture phenotypically accurate root anatomy in three dimensions of different root classes and developmental zones of both monocotyledonous and dicotyledonous species. MCS interacts with other root anatomical phenotypes to determine plant fitness. We discovered that one of these is cell size. Cortical cell size is an important phene determining root metabolic cost, but the underlying physiological mechanism is unclear. We used in silico and empirical approaches to show that vacuolar occupancy in cortical parenchyma cells regulates root metabolic cost. We also show that vacuolar occupancy is associated with cortical cell diameter and cell length, phenes that are under distinct genetic control and hold the potential for improving crop yields under edaphic stress. We discovered the width of root cell walls as another anatomical trait of interest. Increased root cortical parenchyma wall width (CPW) can improve tolerance to drought stress in maize by reducing the metabolic costs of soil exploration. Significant variation for CPW was observed in maize germplasm. Under water stress in the field, increased CPW is correlated with better water acquisition and doubled yield. We identified candidate genes underlying CPW. We propose CPW as a new trait that has utility under edaphic stress meriting further investigation. To examine the utility of MCS in carbon sequestration, we started to look at the differential composition of rhizosphere bacterial communities of 8 maize lines with or without MCS in natural drought conditions (Fort Collins, CO). For these samples, total rhizosphere DNA was extracted and subjected to target sequencing of the bacterial 16S rRNA gene. These data revealed a statistically marginal difference between rhizosphere bacterial communities in MCS and non-MCS maize lines. These differences align with some of the variations detected in the tea bag root decomposition experiment. In brief, this experiment consisted of fragmenting root materials from these maize lines into <1cm pieces, transferring these root fragments into mesh bags, followed by incubation in soil. The treatments also included variations in soil moisture regime and soil nitrogen content. The results revealed a strong significant variation in bacterial communities between MCS and non-MCS lines, and to a minor extend across abiotic treatment variables (moisture and nitrogen content). Only marginal differences were observed in biomass degradation over the period of root material incubation in soil (3-9 months). Collectively, these results point to a complex interaction between root-associated bacterial communities and bacterial degraders of root tissues that are dynamically modulated by plant root phenes and abiotic soil properties.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Sidhu JS, I Lopez-Valdivia, CF Strock, HM Schneider, JP Lynch. 2024. Cortical parenchyma wall width (CPW) regulates root metabolic cost and maize performance under suboptimal water availability Journal of Experimental Botany, Volume 75, Issue 18, 27 September 2024, Pages 57505767, https://doi.org/10.1093/jxb/erae191
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Klein SP, SM Kaeppler, KM Brown, JP Lynch. 2024. Integrating GWAS with a gene co-expression network best prioritizes candidate genes associated with root metaxylem phenes in maize. The Plant Genome 17 https://doi.org/10.1002/tpg2.20489
• Type: Peer Reviewed Journal Articles Status: Awaiting Publication Year Published: 2024 Citation: Du, Pengzhen; Jonathan Lynch; Zhengli Sun; Feng-Min Li 2024. Does root cortical burden and root axial water transport capacity affect crop yield under stress? A meta-analysis. Plant and Soil In press 8-1-24
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Rangarajan H, JP Lynch. 2024. Did crop domestication change the fitness landscape of root response to soil mechanical impedance? An in-silico analysis. Annals of Botany, mcae201, https://doi.org/10.1093/aob/mcae201
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Lynch JP, T Galindo-Casta�eda, HM Schneider, JS Sidhu, H Rangarajan, LM York. 2023. Root Phenotypes for Improved Nitrogen Capture. Plant and Soil 10.1007/s11104-023-06301-2
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Galindo-Casta�eda T, Martin Hartmann, JP Lynch. 2024. Location: root architecture structures rhizosphere microbial associations. JXB 75:594604, https://doi.org/10.1093/jxb/erad421
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Meijer GJ, JP Lynch, JG Chimungu, KW Loades. 2024. Root anatomy and biomechanical properties: Are plant roots individual elements or the sum of their parts? PLSO https://doi.org/10.1007/s11104-024-06507-y
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Sidhu JS, KM Brown, JP Lynch. 2024. Cortical cell size regulates root metabolic cost The Plant Journal 118(5):1343-1357. doi: 10.1111/tpj.16672. Epub 2024 Feb 10

Progress 01/15/23 to 01/14/24

Outputs
Target Audience:Scientists, crop breeders, policy makers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project is supporting the postdoctoral training of Cody DePew, Gordon Custer, and undergraduate assistants at Penn State. This project has also supported the training of the PhD student Courtney Tharp (Plant Biology Program, Penn State). How have the results been disseminated to communities of interest?Dini-Andreote presented data on topics related to this project at 1 international conference and 3 seminar talks internal to Penn State. Schneider presented data related to this project at 4 conferences (2 presentations and 2 posters) and during 3 international seminar talks. Lynch presented 6 seminars this year. The Lynch lab website about root biology receives approximately 70k visitors per year. What do you plan to do during the next reporting period to accomplish the goals?In 2024 we will: Confirm that MCS is related to drought tolerance in wheat in concert with root cortical senescence. Validate role of ethylene sensitivity in MCS formation, root depth, and drought tolerance. Publish several papers related to the findings of these studies.

Impacts
What was accomplished under these goals? We have conducted multiple field studies to determine the fitness landscape of MCS under drought and suboptimal N availability in maize. We found that MCS is associated with drought adaptation in maize as part of a cluster of root phenotypes including large root diameter with a large proportion of root cross sectional area occupied by the stele (Klein et al, Plant Physiology 2020, 183:1011-1025). In wheat, we found that MCS is associated with drought adaptation when it is combined with high expression of root cortical senescence (in preparation). We analyzed post-decomposition characteristics from MCS and non-MCS phenotypes of Maize to determine the effect of MCS on root durability as a potential carbon sequestration source. MCS lines degraded more quickly than non-MCS in root litter bags overwintered in field soil. Analysis of microbiome composition showed statistically significant differences in microbe communities between MCS and non-MCS roots. Anatomical imaging of commonly available model maize lines indicated that MCS was not present in lines available for transgenic manipulation. Homologous genes from MCS GWAS analysis were identified in Arabidopsis to assess lignin phenotypes more rapidly and broadly than in Maize. We have acquired these lines and are in the process of verifying their genetic background. We used the structural-functional plant/soil model OpenSimRoot v2 to evaluate the importance of MCS in maize domestication as an adaptation to changing soil mechanical impedance regimes. We reconstructed the root phenotypes of maize and teosinte, as well as the soil and atmospheric environments of the Tehuacan Valley - an important site of maize domestication - over the last 18,000 years using a combination of ancient DNA, paleobotany, and functional-structural modeling. Our results reveal that increasing Holocene atmospheric CO2 concentrations permitted the appearance of reduced NRN and MCS between 12000 to 8000 years before present (yBP), promoting deeper root systems. The advent of irrigation by 6000 yBP switched nitrogen distribution from topsoil to subsoil domains, a change which increased the utility of reduced NRN and MCS. Comparison of allelic frequencies among ancient samples ranging from 5500 to 500 yBP suggest that increased SRN may have appeared around 3500 yBP, coinciding with a period of increased human population, agricultural intensification, and soil degradation. Our results suggest that root phenotypes that enhance plant performance under nitrogen stress were important for maize adaptation to changing agricultural practices in Tehuacan Valley. Our results support the hypothesis that anthropogenic modifications to the soil environment shaped the root phenotypes of modern maize (Lopez-Valdivia 2024, PhD dissertation, The Pennsylvania State University, manuscript in preparation). To examine the utility of MCS in carbon sequestration, we started to look at the differential composition of rhizosphere bacterial communities of 8 maize lines with or without MCS in natural drought conditions (Fort Collins, CO). For these samples, total DNA was extracted and subjected to target gene sequencing. These data may reveal the extent to which this localized root trait affects the recruitment of bacterial taxa with potential implications for plant performance, and, later on, affecting root decomposition dynamics. Besides, we established a tea bag root decomposition experiment in pots. For that, we processed root tissue from plants grown during the 2021 field season, dried this tissue, and cut the tissue into <1cm pieces. These samples were sealed in mesh bags in pots of field soil of varying water content (including moisture regime) and soil nitrogen content. We sampled these pots at two distinct time points (i.e., 3 and 9 months after incubation). Surprisingly, the analysis of bacterial communities in these bags at the initial time point has already indicated a strong treatment effect. This suggests that MCS and non-MCS roots are colonized by distinct bacterial decomposers and that these communities and the decomposition rate are affected by soil moisture and N content. As for the second time-point, samples are currently being processed to include the analysis of bacterial and fungal communities, in line with microscopic assessments of root structure decomposition.

Publications

• Type: Journal Articles Status: Published Year Published: 2024 Citation: Meijer GJ, JP Lynch, JG Chimungu, KW Loades. 2024. Root anatomy and biomechanical properties: Are plant roots individual elements or the sum of their parts? PLSO in press
• Type: Journal Articles Status: Published Year Published: 2024 Citation: Lopez-Valdivia I, H Rangarajan, M Vallebueno-Estrada, JP Lynch. 2024. Exploring yield stability and the fitness landscape of maize landrace root phenotypes in silico. BioRxiv 9-24

Progress 01/15/22 to 01/14/23

Outputs
Target Audience:Scientists, graduate students and crop breeders Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project is supporting the postdoctoral training of Cody DePew, Gordon Custer, and undergraduate assistants at Penn State. How have the results been disseminated to communities of interest?Dini-Andreote presented data on topics related to this project at 2 international conferences. Schneider presented data related to this project at several seminar talks. Lynch presented 6 seminars this year. The Lynch lab website about root biology receives approximately 70k visitors per year. What do you plan to do during the next reporting period to accomplish the goals?In 2023 we will: Decomposition analysis. Assess effect of MCS on decomposition resistance/carbon sequestration. Determine microbiome community surrounding decomposing MCS and non-MCS roots from samples collected during 2022. Conduct elemental analysis to determine C:N ratio and redox status of MCS roots before and after decomposition, and its effects on microbiome communities. Conduct genetic analysis and anatomical screening for Arabidopsis homologs involved in MCS development. Perform further analysis of anatomical features from MCS roots grown in various stress conditions in the field. Publish several papers related to the findings of these studies.

Impacts
What was accomplished under these goals? We are analyzing data gathered from field studies to determine the fitness landscape of MCS under drought and suboptimal N availability in maize. We are analyzing post-decomposition characteristics from MCS and non-MCS phenotypes of Maize to determine the effect of MCS on root durability as a potential carbon sequestration source. Laser ablation tomography imaging shows the extent of decomposition. Analysis of microbiome composition shows statistically significant differences in microbe communities between MCS and non-MCS roots. Anatomical imaging of commonly available model maize lines indicated that MCS was not present in lines available for transgenic manipulation. Homologous genes from MCS GWAS analysis were identified in Arabidopsis to assess lignin phenotypes more rapidly and broadly than in Maize. We have acquired these lines and are in the process of verifying their genetic background. We are using the structural-functional plant/soil model OpenSimRoot v2 to evaluate the importance of MCS in maize domestication as an adaptation to changing soil mechanical impedance regimes. We have also started assessing the change in ethylene sensitivity in maize lines. Ethylene signaling is important for MCS development. We have collected root samples from a wheat diversity panel under drought from Mexico to determine if MCS formation is associated with drought tolerance. These have been imaged and are now being analyzed.

Publications

Progress 01/15/21 to 01/14/22

Outputs
Target Audience:Researchers, crop breeders, graduate students, postdoctoral scholars Changes/Problems:Because of COVID issues we were delayed in contracting our postdoctoral scholar Dr. Depew by 10 months. What opportunities for training and professional development has the project provided?This project is supporting the postdoctoral training of US citizens Cody DePew, Gordon Custer, and undergraduate assistants at Penn State. How have the results been disseminated to communities of interest?Dini-Andreote presented data on topics related to this project at 2 international conferences. Schneider presented data related to this project at 4 conferences (2 presentations and 2 posters) and during 3 international seminar talks. Lynch presented 6 seminars this year. The Lynch lab website about root biology receives approximately 70k visitors per year. What do you plan to do during the next reporting period to accomplish the goals?Additional analysis of the 2021 field data is being performed to understand the effect of MCS on drought and nitrogen stress tolerance. Field experiments for drought (rain-out shelters) and nitrogen stress will be repeated and confirmed in PA (PSU Rock Springs Facility) during the 2022 field season in maize MCS lines. These experiments will include more detailed analysis of water usage to complement data obtained from wheat in the greenhouse. We also plan to obtain anatomical samples from an elite wheat diversity panel at the International Maize and Wheat Improvement Center (CIMMYT) in Texcoco, Mexico in March 2022. We have identified several gene candidates by genome-wide association mapping (GWAS) that may be relevant in MCS formation and regulation. One strong candidate gene was an F-box protein, a family of regulatory proteins that may be involved in development and regulation of traits such as MCS. Working with WCIC, a maize knock-out line is being generated to test this hypothesis. We will then test this mutant's ability to produce root MCS. At 3 and 6 months, samples of decomposing MCS and non-MCS root material will be collected, dried, and weighed to measure the effect of MCS on decomposition. We will also identify the microbiome associated with decomposing roots in various conditions.

Impacts
What was accomplished under these goals? Preliminary field studies of MCS in maize during drought and nitrogen stress were expanded to better understand the implications of root MCS under these conditions. These larger experiments compared 6 to 8 maize lines with or without MCS in natural drought conditions (Fort Collins, CO), artificially-imposed drought by rain-out shelters in the field (State College, PA), and fields with depleted nitrogen availability (Rock Springs, PA). We have collected and are processing samples from the 2021 field season to examine shoot biomass, maize yield, and rooting depth in these conditions. Previous experiments compared only 3 lines each of MCS and non-MCS lines. By more than doubling sample size, these data may reveal subtle effects not observed in previous studies, such as MCS plasticity under nitrogen stress. Unlike many traits where drought tolerance is correlated with increased rooting depth, roots with MCS do not appear larger or deeper in these experiments. We have determined that roots with MCS have a reduced hydraulic conductivity, which may lead to water-banking and thus drought resistance in the field. We are currently growing wheat lines with and without MCS in the greenhouse while accurately measuring soil water content daily to test this hypothesis. These experiments are performed in the greenhouse in pots filled with field soil and compacted to replicate field conditions. These pots include a top layer of tilled, organic-rich soil, and a lower layer of harder, clay-rich soil. Detailed analysis of daily water usage is being measured to determine if water-banking by MCS contributes to drought tolerance. To examine the utility of MCS in carbon sequestration, we will determine the composition of microbial communities in the rhizosphere of dead and living roots. We have collected rhizosphere samples to analyze the microbiome of MCS and non-MCS maize lines grown in drought conditions. We have also collected root tissue from plants grown during the 2021 field season, dried this tissue, and cut the tissue into <1cm pieces. These samples are sealed in mesh bags in pots of field soil of varying water content and pH to measure decomposition and microbiome composition over time.

Publications