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At least 235 records · Page 13

RNAi and genome editing of sugarcane: Progress and prospects

SUMMARY Sugarcane, which provides 80% of global table sugar and 40% of biofuel, presents unique breeding challenges due to its highly polyploid, heterozygous, and frequently aneuploid genome. Significant progress has been made in developing genetic resources, including the recently completed reference genome of the sugarcane cultivar R570 and pan‐genomic resources from sorghum, a closely related diploid species. Biotechnological approaches including RNA interference (RNAi), overexpression of transgenes, and gene editing technologies offer promising avenues for accelerating sugarcane improvement. These methods have successfully targeted genes involved in important traits such as sucrose accumulation, lignin biosynthesis, biomass oil accumulation, and stress response. One of the main transformation methods—biolistic gene transfer or Agrobacterium ‐mediated transformation—coupled with efficient tissue culture protocols, is typically used for implementing these biotechnology approaches. Emerging technologies show promise for overcoming current limitations. The use of morphogenic genes can help address genotype constraints and improve transformation efficiency. Tissue culture‐free technologies, such as spray‐induced gene silencing, virus‐induced gene silencing, or virus‐induced gene editing, offer potential for accelerating functional genomics studies. Additionally, novel approaches including base and prime editing, orthogonal synthetic transcription factors, and synthetic directed evolution present opportunities for enhancing sugarcane traits. These advances collectively aim to improve sugarcane's efficiency as a crop for both sugar and biofuel production. This review aims to discuss the progress made in sugarcane methodologies, with a focus on RNAi and gene editing approaches, how RNAi can be used to inform functional gene targets, and future improvements and applications.

Brant, Eleanor [Agronomy Department, Plant Molecul↗

Attaining the promise of plant gene editing at scale

Crop improvement relies heavily on genetic variation that arises spontaneously through mutation. Modern breeding methods are very adept at combining this genetic variation in ways that achieve remarkable improvements in plant performance. Novel traits have also been created through mutation breeding and transgenesis. The advent of gene editing, however, marks a turning point: With gene editing, synthetic variation will increasingly supplement and, in some cases, supplant the genetic variation that occurs naturally. We are still in the very early stages of realizing the opportunity provided by plant gene editing. At present, typically only one or a few genes are targeted for mutation at a time, and most mutations result in loss of gene function. New technological developments, however, promise to make it possible to perform gene editing at scale. RNA virus vectors, for example, can deliver gene-editing reagents to the germ line through infection and create hundreds to thousands of diverse mutations in the progeny of infected plants. With developmental regulators, edited somatic cells can be induced to form meristems that yield seed-producing shoots, thereby increasing throughput and shrinking timescales for creating edited plants. As these approaches are refined and others developed, they will allow for accelerated breeding, the domestication of orphan crops and the reengineering of metabolism in a more directed manner than has ever previously been possible.

59 BASIC BIOLOGICAL SCIENCES↗

TGCM: (T)rait, (G)ene, and (C)rop Growth (M)odel Directed Targeted Gene Characterization in Sorghum (Final Technical Report)

Understanding which genes control important crop traits could help scientists develop better bioenergy and food crops more efficiently. However, plant genomes contain tens of thousands of genes, and testing each one individually is expensive and time-consuming. This project developed computational tools to predict which genes are most likely to matter, allowing researchers to focus their efforts where they will have the greatest impact. This project developed and validated integrated approaches combining machine learning, quantitative genetics, and crop growth modeling to improve the efficiency of functional gene characterization in sorghum (Sorghum bicolor), a critical bioenergy and food security crop. The research addressed a fundamental challenge in plant biology: the majority of genes in plant genomes lack experimentally validated functions, making it difficult to prioritize which genes to study using resource-intensive reverse genetics approaches.

60 APPLIED LIFE SCIENCES↗

Eco-evolutionary optimality as a means to improve vegetation and land-surface models

Global vegetation and land-surface models embody interdisciplinary scientific understanding of the behaviour of plants and ecosystems, and are indispensable to project the impacts of environmental change on vegetation and the interactions between vegetation and climate. Furthermore, systematic errors and persistently large differences among carbon and water cycle projections by different models highlight the limitations of current process formulations. In this review, focusing on core plant functions in the terrestrial carbon and water cycles, we show how unifying hypotheses derived from eco-evolutionary optimality (EEO) principles can provide novel, parameter-sparse representations of plant and vegetation processes. We present case studies that demonstrate how EEO generates parsimonious representations of core, leaf-level processes that are individually testable and supported by evidence. EEO approaches to photosynthesis and primary production, dark respiration and stomatal behaviour are ripe for implementation in global models. EEO approaches to other important traits, including the leaf economics spectrum and applications of EEO at the community level are active research areas. Independently tested modules emerging from EEO studies could profitably be integrated into modelling frameworks that account for the multiple time scales on which plants and plant communities adjust to environmental change.

59 BASIC BIOLOGICAL SCIENCES↗

Toward a coordinated understanding of hydro‐biogeochemical root functions in tropical forests for application in vegetation models

Summary Tropical forest root characteristics and resource acquisition strategies are underrepresented in vegetation and global models, hampering the prediction of forest–climate feedbacks for these carbon‐rich ecosystems. Lowland tropical forests often have globally unique combinations of high taxonomic and functional biodiversity, rainfall seasonality, and strongly weathered infertile soils, giving rise to distinct patterns in root traits and functions compared with higher latitude ecosystems. We provide a roadmap for integrating recent advances in our understanding of tropical forest belowground function into vegetation models, focusing on water and nutrient acquisition. We offer comparisons of recent advances in empirical and model understanding of root characteristics that represent important functional processes in tropical forests. We focus on: (1) fine‐root strategies for soil resource exploration, (2) coupling and trade‐offs in fine‐root water vs nutrient acquisition, and (3) aboveground–belowground linkages in plant resource acquisition and use. We suggest avenues for representing these extremely diverse plant communities in computationally manageable and ecologically meaningful groups in models for linked aboveground–belowground hydro‐nutrient functions. Tropical forests are undergoing warming, shifting rainfall regimes, and exacerbation of soil nutrient scarcity caused by elevated atmospheric CO 2 . The accurate model representation of tropical forest functions is crucial for understanding the interactions of this biome with the climate.

Plant Sciences↗

Tradeoffs between phenotypic plasticity and local adaptation influence the ecophysiology of the moss, Sphagnum magellanicum

Bryophytes are a diverse plant group and are functionally different from vascular plants. Yet, their peculiarities are rarely considered in the theoretical frameworks for plants. Currently, we lack information about the magnitude and the importance of intraspecific variability in the ecophysiology of bryophytes and how these might translate to local adaptation—a prerequisite for adaptive evolution. Capitalizing on two ecologically distinct (hummock and hollow) phenotypes of Sphagnum magellanicum, we explored the magnitude and pattern of intraspecific variability in this species and asked whether the environmental-mediated changes in shoot and physiological traits are due to phenotypic plasticity or local adaptation. Size, pigmentation, and habitat type that distinguished the phenotypes in the field did not influence the trait responses under a transplant and factorial experiment. In addition, the magnitude and pattern of trait variability (e.g., branch, stem and capitulum mass) changed with the treatments, which suggest that trait responses were due largely to phenotypic plasticity. The trait responses also suggest that the ecophysiological needs for mosses to grow in clumps, where they maintain a uniform growth may have an overriding effect over the potential for a fixed adaptive response to environmental heterogeneity, which would constrain local adaptation. In this paper, we conclude that extending the trait-based framework to mosses or making comparisons between mosses and vascular plants under any theoretical framework would only be meaningful to the extent that growth form and dispersal strategies are considered.

59 BASIC BIOLOGICAL SCIENCES↗

Root angle is controlled by EGT1 in cereal crops employing an antigravitropic mechanism

Root angle in crops represents a key trait for efficient capture of soil resources. Root angle is determined by competing gravitropic versus antigravitropic offset (AGO) mechanisms. Here we report a root angle regulatory gene termed ENHANCED GRAVITROPISM1 ( EGT1 ) that encodes a putative AGO component, whose loss-of-function enhances root gravitropism. Mutations in barley and wheat EGT1 genes confer a striking root phenotype, where every root class adopts a steeper growth angle. EGT1 encodes an F-box and Tubby domain-containing protein that is highly conserved across plant species. Haplotype analysis found that natural allelic variation at the barley EGT1 locus impacts root angle. Gravitropic assays indicated that Hvegt1 roots bend more rapidly than wild-type. Transcript profiling revealed Hvegt1 roots deregulate reactive oxygen species (ROS) homeostasis and cell wall-loosening enzymes and cofactors. ROS imaging shows that Hvegt1 root basal meristem and elongation zone tissues have reduced levels. Atomic force microscopy measurements detected elongating Hvegt1 root cortical cell walls are significantly less stiff than wild-type. In situ analysis identified HvEGT1 is expressed in elongating cortical and stele tissues, which are distinct from known root gravitropic perception and response tissues in the columella and epidermis, respectively. We propose that EGT1 controls root angle by regulating cell wall stiffness in elongating root cortical tissue, counteracting the gravitropic machinery’s known ability to bend the root via its outermost tissues. We conclude that root angle is controlled by EGT1 in cereal crops employing an antigravitropic mechanism.

59 BASIC BIOLOGICAL SCIENCES↗

State of Wildfires

Fire is an essential component of ecosystems and acts as key driver of biogeochemical cycling with impacts on vegetation structure and composition, soil conditions, and climate feedbacks. Fire behavior and effects vary based on the types of fuel, fuel dryness, and frequency of ignition. Due to changing climate and land use patterns, fire danger is increasing in many regions globally, and fires are having increasingly devastating impacts on human health, infrastructure, and ecosystem services. Recurrent fires help to determine the distribution of trees and grasses, and overall fuel load, which inform the behavior of future fires. Process-based models can be used to capture multi-scale impacts at the forest stand-level up to the landscape level, and across minutes to centuries, but must capture variation in fire behavior and intensity as a function of the fuel and climate, from high intensity forest crown fires within boreal regions to low intensity rapid grass fires of the tropics. Fire model development demonstrates our ability to capture large scale fire influenced biogeography and vegetation distribution at the earth system scale through vegetation traits and fire feedbacks. At the individual stand scale the importance of interactions and feedbacks between above and below ground process is essential to capturing the vegetation dynamics across boreal forest systems. Improving the mechanics of including plant physiology and specifically live fuel moisture content is the next step to advancing the capability of process based models to inform fire research. Advances in the testing and creation of a mechanistic live fuel moisture model demonstrate the foundation for future live fuel dynamics research that can be informed by field and remote sensing information. Improved remote sensing, technological and modeling capabilities support a more comprehensive and cohesive fire response that will be better equipped to overcome current barriers and anticipate the new reality of fires in a warming world.

wildfires↗

Changing Interactions Between Trace Gas Fluxes, Belowground Chemistry, and Plant Traits Across an Arctic Thermokarst Landscape

Arctic permafrost soils are increasingly subject to thermokarst that is, abrupt ground subsidence caused by thaw. Wetlands can form within these depressions, leading to changes in organic matter decomposition and gas fluxes (CO 2 , CH 4 , N 2 O, NH 3 ). Thermokarst wetlands tend to be dominated by graminoids, while surrounding upland tussock tundra tends to be dominated by mixed communities of shrubs and graminoids. Here, to investigate how thermokarst alters the land-atmosphere exchange of C and N gases in Arctic tundra, we analyzed soil, porewater, above- and belowground biomass, and measured gas fluxes across dominant plant functional types (PFTs) within a lowland thermokarst wetland and adjacent upland tussock tundra. Both locations were overall sinks of CO 2 , sources of CH 4 , and sources of both N 2 O and NH 3 . We found that thermokarst wetlands emitted enough CH 4 to generate a positive radiative forcing in CO 2 equivalents (+1.2 μmol m −2 s −1 CO 2 -eq), counteracting the high CO 2 uptake. In contrast, the upland tussock tundra had a net negative radiative forcing (−1.2 μmol m −2 s −1 CO 2 -eq). Differences in gas flux and soil chemistry between upland and lowland are primarily driven by flooded conditions present in thermokarst wetland. Additionally, root biomass from graminoids across both lowlands and uplands significantly correlated with CH 4 fluxes, supporting previous observations of plant-mediated transport of CH 4 . Graminoid cover was correlated with increases in low molecular weight dissolved organic carbon, possibly associated with root exudates that fuel methanogenesis. Forb cover in the upland tussock tundra was significantly correlated with nine soil chemical variables, indicating that forbs may influence local soil chemistry or conversely, that soil chemistry controls where forbs grow. Overall, our findings indicate the variability in gas fluxes in the upland tussock tundra is partially controlled by PFT cover, while thermokarst wetlands emit enough CH 4 to counteract CO 2 uptake, with implications for carbon budget changes in Arctic systems.

Land-atmosphere feedback↗

GWAS identifies candidate genes controlling adventitious rooting in Populus trichocarpa

Adventitious rooting (AR) is critical to the propagation, breeding, and genetic engineering of trees. The capacity for plants to undergo this process is highly heritable and of a polygenic nature; however, the basis of its genetic variation is largely uncharacterized. To identify genetic regulators of AR, we performed a genome-wide association study (GWAS) using 1148 genotypes of Populus trichocarpa. GWASs are often limited by the abilities of researchers to collect precise phenotype data on a high-throughput scale; to help overcome this limitation, we developed a computer vision system to measure an array of traits related to adventitious root development in poplar, including temporal measures of lateral and basal root length and area. GWAS was performed using multiple methods and significance thresholds to handle non-normal phenotype statistics and to gain statistical power. These analyses yielded a total of 277 unique associations, suggesting that genes that control rooting include regulators of hormone signaling, cell division and structure, reactive oxygen species signaling, and other processes with known roles in root development. Numerous genes with uncharacterized functions and/or cryptic roles were also identified. These candidates provide targets for functional analysis, including physiological and epistatic analyses, to better characterize the complex polygenic regulation of AR.

59 BASIC BIOLOGICAL SCIENCES↗

Retrospective analysis of biochemical limitations to photosynthesis in 49 species: C 4 crops appear still adapted to pre‐industrial atmospheric [ CO 2 ]

Abstract Leaf CO 2 uptake ( A ) in C 4 photosynthesis is limited by the maximum apparent rate of PEPc carboxylation ( V pmax ) at low intercellular [CO 2 ] ( c i ) with a sharp transition to a c i ‐saturated rate ( V max ) due to co‐limitation by ribulose‐1:5‐bisphosphate carboxylase/oxygenase (Rubisco) and regeneration of PEP. The response of A to c i has been widely used to determine these two parameters. V max and V pmax depend on different enzymes but draw on a shared pool of leaf resources, such that resource distribution is optimized, and A maximized, when V max and V pmax are co‐limiting. We collected published A / c i curves in 49 C 4 species and assessed variation in photosynthetic traits between phylogenetic groups, and as a function of atmospheric [CO 2 ]. The balance of V max ‐ V pmax varied among evolutionary lineages and C 4 subtypes. Operating A was strongly V max ‐limited, such that re‐allocation of resources from V pmax towards V max was predicted to improve A by 12% in C 4 crops. This would not require additional inputs but rather altered partitioning of existing leaf nutrients, resulting in increased water and nutrient‐use efficiency. Optimal partitioning was achieved only in plants grown at pre‐industrial atmospheric [CO 2 ], suggesting C 4 crops have not adjusted to the rapid increase in atmospheric [CO 2 ] of the past few decades.

Pignon, Charles P.↗

Root Pulling Force Across Drought in Maize Reveals Genotype by Environment Interactions and Candidate Genes

High-throughput, field-based characterization of root systems for hundreds of genotypes in thousands of plots is necessary for breeding and identifying loci underlying variation in root traits and their plasticity. We designed a large-scale sampling of root pulling force, the vertical force required to extract the root system from the soil, in a maize diversity panel under differing irrigation levels for two growing seasons. We then characterized the root system architecture of the extracted root crowns. We found consistent patterns of phenotypic plasticity for root pulling force for a subset of genotypes under differential irrigation, suggesting that root plasticity is predictable. Using genome-wide association analysis, we identified 54 SNPs as statistically significant for six independent root pulling force measurements across two irrigation levels and four developmental timepoints. For every significant GWAS SNP for any trait in any treatment and timepoint we conducted post hoc tests for genotype-by-environment interaction, using a mixed model ANOVA. We found that 8 of the 54 SNPs showed significant GxE. Candidate genes underlying variation in root pulling force included those involved in nutrient transport. Although they are often treated separately, variation in the ability of plant roots to sense and respond to variation in environmental resources including water and nutrients may be linked by the genes and pathways underlying this variation. While functional validation of the identified genes is needed, our results expand the current knowledge of root phenotypic plasticity at the whole plant and gene levels, and further elucidate the complex genetic architecture of maize root systems.

Woods, Patrick↗

Distinctiveness of genes contributing to growth of Pseudomonas syringae in diverse host plant species

A variety of traits are necessary for bacterial colonization of the interior of plant hosts, including well-studied virulence effectors as well as other phenotypes contributing to bacterial growth and survival within the apoplast. High-throughput methods such as transposon sequencing (TnSeq) are powerful tools to identify such genes in bacterial pathogens. However, there is little information as to the distinctiveness of traits required for bacterial colonization of different hosts. Here, we utilize randomly barcoded TnSeq (RB-TnSeq) to identify the genes that contribute to the ability of Pseudomonas syringae strain B728a to grow within common bean (Phaseolus vulgaris), lima bean (Phaseolus lunatus), and pepper (Capsicum annuum); species representing two different plant families. The magnitude of contribution of most genes to apoplastic fitness in each of the plant hosts was similar. However, 50 genes significantly differed in their fitness contributions to growth within these species. These genes encoded proteins in various functional categories including polysaccharide synthesis and transport, amino acid metabolism and transport, cofactor metabolism, and phytotoxin synthesis and transport. Six genes that encoded unannotated, hypothetical proteins also contributed differentially to growth in these hosts. The genetic repertoire of a relatively promiscuous pathogen such as P. syringae may thus be shaped, at least in part, by the conditional contribution of some fitness determinants.

59 BASIC BIOLOGICAL SCIENCES↗

Canopy height and climate dryness parsimoniously explain spatial variation of unstressed stomatal conductance

The spatio-temporal variation of stomatal conductance directly regulates photosynthesis, water partitioning, and biosphere-atmosphere interactions. While many studies have focused on stomatal response to stresses, the spatial variation of unstressed stomatal conductance remains poorly determined, and is usually characterized in land surface models (LSMs) simply based on plant functional type (PFT). Here, we derived unstressed stomatal conductance at the ecosystem-scale using observations from 115 global FLUXNET sites. When aggregated by PFTs, the across-PFT pattern was highly consistent with the parameterizations of LSMs. However, PFTs alone captured only 17% of the variation in unstressed stomatal conductance across sites. Within the same PFT, unstressed stomatal conductance was negatively related to climate dryness and canopy height, which explained 45% of the total spatial variation. Our results highlight the importance of plant environment interactions in shaping stomatal traits. The trait-environment relationship established here provides an empirical approach for improved parameterizations of stomatal conductance in LSMs.

Yanlan Liu↗

Genetic and Functional Diversity Help Explain Pathogenic, Weakly Pathogenic, and Commensal Lifestyles in the Genus Xanthomonas

The genus Xanthomonas has been primarily studied for pathogenic interactions with plants. However, besides host and tissue-specific pathogenic strains, this genus also comprises nonpathogenic strains isolated from a broad range of hosts, sometimes in association with pathogenic strains, and other environments, including rainwater. Based on their incapacity or limited capacity to cause symptoms on the host of isolation, nonpathogenic xanthomonads can be further characterized as commensal and weakly pathogenic. This study aimed to understand the diversity and evolution of nonpathogenic xanthomonads compared to their pathogenic counterparts based on their cooccurrence and phylogenetic relationship and to identify genomic traits that form the basis of a life history framework that groups xanthomonads by ecological strategies. We sequenced genomes of 83 strains spanning the genus phylogeny and identified eight novel species, indicating unexplored diversity. While some nonpathogenic species have experienced a recent loss of a type III secretion system, specifically the hrp2 cluster, we observed an apparent lack of association of the hrp2 cluster with lifestyles of diverse species. We performed association analysis on a large data set of 337 Xanthomonas strains to explain how xanthomonads may have established association with the plants across the continuum of lifestyles from commensals to weak pathogens to pathogens. Presence of distinct transcriptional regulators, distinct nutrient utilization and assimilation genes, transcriptional regulators, and chemotaxis genes may explain lifestyle-specific adaptations of xanthomonads.

59 BASIC BIOLOGICAL SCIENCES↗

Severe and mild drought cause distinct phylogenetically linked shifts in the blue grama (Bouteloua gracilis) rhizobiome

Plants rely on a diverse rhizobiome to regulate nutrient acquisition and plant health. With increasing severity and frequency of droughts worldwide due to climate change, untangling the relationships between plants and their rhizobiomes is vital to maintaining agricultural productivity and protecting ecosystem diversity. While some plant physiological responses to drought are generally conserved, patterns of root exudation (release of small metabolites shown to influence microbes) and the consequential effects on the plant rhizobiome can differ widely across plant species under drought. To address this knowledge gap, we conducted a greenhouse study using blue grama ( Bouteloua gracilis ), a drought-tolerant C4 grass native to shortgrass prairie across North American plains, as a model organism to study the effect of increasing drought severity (ambient, mild drought, severe drought) on root exudation and the rhizobiome. Our previous results demonstrated physiological effects of increasing drought severity including an increase in belowground carbon allocation through root exudation and shifts in root exudate composition concurrent with the gradient of drought severity. This work is focused on the rhizobiome community structure using targeted sequencing and found that mild and severe drought resulted in unique shifts in the bacterial + archaeal and fungal communities relative to ambient, non-droughted controls. Specifically, using the change in relative abundance between ambient and drought conditions for each ZOTU as a surrogate for population-scale drought tolerance (e.g., as a response trait), we found that rhizobiome response to drought was non-randomly distributed across the phylogenies of both communities, suggesting that Planctomycetota , Thermoproteota (formerly Thaumarchaeota ), and the Glomeromycota were the primary clades driving these changes. Correlation analyses indicated weak correlations between droughted community composition and a select few root exudate compounds previously implicated in plant drought responses including pyruvic acid, D-glucose, and myoinositol. This study demonstrates the variable impacts of drought severity on the composition of the blue grama rhizobiome and provides a platform for hypothesis generation for targeted functional studies of specific taxa involved in plant-microbe drought responses.

Goemann, Hannah M.↗

NGEE Arctic Plant Traits: Fine Roots, Kougarok Road Mile Marker 64, Seward Peninsula, Alaska, 2016

Soil cores were collected from twelve vegetation biomass plots at the Kougarok hillslope in late July of 2016. The sampled plots were located across six ecotypes present at this site (n=2 replicates per ecotype). Soil cores were separated into depth intervals in the field and frozen for transport to Oak Ridge National Laboratory. Data package contains one *.csv and one *.pdf.Laboratory sample processing of these soils generated depth-specific data on soil properties as well as fine root biomass, length, %C, %N, delta13C, and delta15N. Only live fine roots were analyzed. Fine roots were not separated by species or plant functional type.The Next-Generation Ecosystem Experiments: Arctic (NGEE Arctic), a research effort to reduce uncertainty in Earth System Models by developing a predictive understanding of carbon-rich Arctic ecosystems and feedbacks to climate. NGEE Arctic was supported by the Department of Energy’s Office of Biological and Environmental Research.The NGEE Arctic project had two field research sites: 1) located within the Arctic polygonal tundra coastal region on the Barrow Environmental Observatory (BEO) and the North Slope near Utqiagvik (Barrow), Alaska and 2) multiple areas on the discontinuous permafrost region of the Seward Peninsula north of Nome, Alaska.Through observations, experiments, and synthesis with existing datasets, NGEE Arctic provided an enhanced knowledge base for multi-scale modeling and contributed to improved process representation at global pan-Arctic scales within the Department of Energy’s Earth system Model (the Energy Exascale Earth System Model, or E3SM), and specifically within the E3SM Land Model component (ELM).

54 ENVIRONMENTAL SCIENCES↗

Time‐series multi‐omics analysis of micronutrient stress in Sorghum bicolor reveals iron and zinc crosstalk and regulatory network conservation

Micronutrient stress impacts growth, biomass production, and grain yield in crops. Multi-omics studies are valuable resources in identifying genes for functional studies and trait improvement, such as accumulation of Fe or Zn under deficient or excess conditions for bioenergy or grain agriculture. We conducted transcriptomics and ionomics analyses on Sorghum bicolor BTx623, grown under Fe and Zn limited and excess conditions over a 21-day period. To identify early and late transcriptional response in roots and leaves, 180 RNAseq libraries were sequenced for differential expression and co-expression network analyses. Fe and Zn accumulation was measured using ICP-MS at each time point, and a fluorometer was used to estimate chlorophyll content in leaves. Among the four treatments, Fe limitation and Zn excess resulted in the largest phenotypic effects and transcriptional response in roots and leaves. Several of the reduction (Strategy I) and chelation (Strategy II) strategy genes that improve bioavailability of Fe and Zn in plant roots often used by non-grass and grass species, respectively, were differentially expressed. Gene regulatory network (GRN) analysis of roots revealed enrichment of genes from Fe limiting and Zn excess which strongly connect to homologues of SbFIT, SbPYE, and SbBTS as hub genes. The GRN for leaf responses showed homologues of SbPYE and SbBTS as hubs connecting genes for chloroplast biosynthesis, Fe-S cluster assembly, photosynthesis, and ROS scavenging. Expression analyses suggest sorghum uses Strategy II genes for Fe and Zn uptake, as expected, but can also utilize Strategy I genes, which may be advantageous in variable moisture environments. We found strong overlap between Fe and Zn responsive GRNs, indicative of micronutrient crosstalk. We also found conservation of root and leaf GRNs, and known homologous genes suggest strong constraints on homeostasis networks in plants. These data will provide a resource for functional genetics to enhance micronutrient transport in sorghum, and opportunities to conduct further comparative GRN analysis across diverse crops species.

59 BASIC BIOLOGICAL SCIENCES↗