ORCID IDs: 0000-0002-6182-800X (J.H.); 0000-0001-6612-3570 (S.V.); 0000-0002-9564-8146 (K.M.M.).With the emergence of massively parallel sequencing, genomewide expression data production has reached an unprecedented level. This abundance of data has greatly facilitated maize research, but may not be amenable to traditional analysis techniques that were optimized for other data types. Using publicly available data, a gene coexpression network (GCN) can be constructed and used for gene function prediction, candidate gene selection, and improving understanding of regulatory pathways. Several GCN studies have been done in maize (Zea mays), mostly using microarray datasets. To build an optimal GCN from plant materials RNA-Seq data, parameters for expression data normalization and network inference were evaluated. A comprehensive evaluation of these two parameters and a ranked aggregation strategy on network performance, using libraries from 1266 maize samples, were conducted. Three normalization methods and 10 inference methods, including six correlation and four mutual information methods, were tested. The three normalization methods had very similar performance. For network inference, correlation methods performed better than mutual information methods at some genes. Increasing sample size also had a positive effect on GCN. Aggregating single networks together resulted in improved performance compared to single networks.Maize (Zea mays) is the most widely produced crop in United States, and U.S. agriculture accounted for 36% of world maize production in 2015 (USDA, 2016). Maize has also been in the center of genetics research for more than 100 years, including McClintock's pioneering work with transposable elements (reviewed by McClintock, 1983;Fedoroff, 2012). Due to recent technological advances in nucleic acid sequencing and the availability of the maize genome sequence (Schnable et al., 2009), maize genomics research has been greatly expedited.RNA-sequencing (RNA-Seq) has become the favored technique for detecting genomewide expression patterns. RNA-Seq has some advantages over microarray analysis of gene expression, including single base-pair resolution, detection of novel transcripts, and the ability to analyze transcript abundance without existing genome information (reviewed by Wang et al., 2009;Han et al., 2015;Conesa et al., 2016). RNA-Seq data provides information about single nucleotide polymorphisms, which facilitates genomewide association studies (Fu et al., 2013;Li et al., 2013a;Lonsdale et al., 2013;Fadista et al., 2014). Because of its widespread adaptability, greater than 5000 Illumina platform Maize RNA-Seq libraries (Fig. 1A) are available in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database (Leinonen et al., 2010), adding to the body of data that can be used to study the maize genome.The maize genome is large and heterogeneous, and the genome annotation is still far from complete (Cigan et al., 2005;Ficklin and Feltus, 2011). Although recent work has...

42 43 44 45 Acknowledgments 46 3 We are grateful to Nathan Springer for providing the SeqCap DNA methylation data. 47 This work was funded by the National Science Foundation funds to K.M.M (CMB-48 035919) and start-up funds from the University of Washington Bothell School of STEM 49 to T.F.M. P.A.C. was supported by a grant from NSF (IOS-1802848). 50 51 Significance statement 52 MOP1-dependent gene expression changes in response to ABA were identified as 53 having synergistic and combinatorial direct and indirect effects. Overlapping regulatory 54 networks were uncovered, reinforcing the idea that epigenetic regulation is crucial to 55 plant response and adaptation to abiotic stress. 56 57 58 4 ABSTRACT 59 60 Plants are subjected to extreme environmental conditions and must adapt rapidly. The 61 phytohormone abscisic acid (ABA) accumulates during abiotic stress, signaling 62 transcriptional changes that trigger physiological responses. Epigenetic modifications 63 often facilitate transcription, particularly at genes exhibiting temporal, tissue-specific and 64 environmentally-induced expression. In maize (Zea mays), MEDIATOR OF 65 PARAMUTATION 1 (MOP1) is required for progression of an RNA-dependent 66 epigenetic pathway that regulates transcriptional silencing of loci genomewide. MOP1 67 function has been previously correlated with genomic regions adjoining particular types 68 of transposable elements and genic regions, suggesting that this regulatory pathway 69 functions to maintain distinct transcriptional activities within genomic spaces, and that 70 loss of MOP1 may modify the responsiveness of some loci to other regulatory 71 pathways. As critical regulators of gene expression, MOP1 and ABA pathways each 72 regulate specific genes. To determine whether loss of MOP1 impacts ABA-responsive 73 gene expression in maize, mop1-1 and Mop1 homozygous seedlings were subjected to 74 exogenous ABA and RNA-sequencing. A total of 3,242 differentially expressed genes 75 (DEGs) were identified in four pairwise comparisons. Overall, ABA-induced changes in 76 gene expression were enhanced in mop1-1 homozygous plants. The highest number of 77 DEGs were identified in ABA-induced mop1-1 mutants, including many transcription 78 factors; this suggests combinatorial regulatory scenarios including direct and indirect 79 transcriptional responses to genetic disruption (mop1-1) and/or stimulus-induction of a 80 hierarchical, cascading network of responsive genes. Additionally, a modest increase in 81 5 CHH methylation at putative MOP1-RdDM loci in response to ABA was observed in 82 some genotypes, suggesting that epigenetic variation might influence environmentally-83 induced transcriptional responses in maize. 84 85 INTRODUCTION 86 87As sessile organisms, plants must adapt rapidly to fluctuating and often extreme abiotic 88 stress conditions that negatively impact crop productivity and yield, such as water 89 deprivation/drought, high salinity, nutrient deficiency and extreme temperatures. In 90 addition to its role in plant develop...

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Plants respond to abiotic stress stimuli, such as water deprivation, through a hierarchical cascade that includes detection and signaling to mediate transcriptional and physiological changes. The phytohormone abscisic acid (ABA) is well-characterized for its regulatory role in these processes in response to specific environmental cues. ABA-mediated changes in gene expression have been demonstrated to be temporally-dependent, however, the genome-wide timing of these responses are not well-characterized in the agronomically important crop plant Zea mays (maize). ABA-mediated responses are synergistic with other regulatory mechanisms, including the plant-specific RNA-directed DNA methylation (RdDM) epigenetic pathway. Our prior work demonstrated that after relatively long-term ABA induction (8 h), maize plants homozygous for the mop1-1 mutation, defective in a component of the RdDM pathway, exhibit enhanced transcriptional sensitivity to the phytohormone. At this time-point, many hierarchically positioned transcription factors are differentially expressed resulting in primary (direct) and secondary (indirect) transcriptional outcomes. To identify more immediate and direct MOP1-dependent responses to ABA, we conducted a transcriptomic analysis using mop1-1 mutant and wild type plants treated with ABA for 1 h. One h of ABA treatment was sufficient to induce unique categories of differentially expressed genes (DEGs) in mop1-1. A comparative analysis between the two time-points revealed that distinct epigenetically-regulated changes in gene expression occur within the early stages of ABA induction, and that these changes are predicted to influence less immediate, indirect transcriptional responses. Homology with MOP1-dependent siRNAs and a gene regulatory network (GRN) were used to identify putative immediate and indirect targets, respectively. By manipulating two key regulatory networks in a temporal dependent manner, we identified genes and biological processes regulated by RdDM and ABA-mediated stress responses. Consistent with mis-regulation of gene expression, mop1-1 homozygous plants are compromised in their ability to recover from water deprivation. Collectively, these results indicate transcriptionally and physiologically relevant roles for MOP1-mediated regulation of gene expression of plant responses to environmental stress.