BonnMu: A Sequence-Indexed Resource of Transposon-Induced Maize Mutations for Functional Genomics Studies.

Sequence-indexed insertional libraries in maize (Zea mays) are fundamental resources for functional genetics studies. Here, we constructed a Mutator (Mu) insertional library in the B73 inbred background designated BonnMu A total of 1,152 Mu-tagged F2-families were sequenced using the Mu-seq approach. We detected 225,936 genomic Mu insertion sites and 41,086 high quality germinal Mu insertions covering 16,392 of the annotated maize genes (37% of the B73v4 genome). On average, each F2-family of the BonnMu libraries captured 37 germinal Mu insertions in genes of the Filtered Gene Set (FGS). All BonnMu insertions and phenotypic seedling photographs of Mu-tagged F2-families can be accessed via MaizeGDB.org Downstream examination of 137,410 somatic and germinal insertion sites revealed that 50% of the tagged genes have a single hotspot, targeted by Mu By comparing our BonnMu (B73) data to the UniformMu (W22) library, we identified conserved insertion hotspots between different genetic backgrounds. Finally, the vast majority of BonnMu and UniformMu transposons was inserted near the transcription start site of genes. Remarkably, 75% of all BonnMu insertions were in closer proximity to the transcription start site (distance: 542 bp) than to the start codon (distance: 704 bp), which corresponds to open chromatin, especially in the 5' region of genes. Our European sequence-indexed library of Mu insertions provides an important resource for functional genetics studies of maize.

Stability of Single-Parent Gene Expression Complementation in Maize Hybrids upon Water Deficit Stress.

Heterosis is the superior performance of F1 hybrids compared with their homozygous, genetically distinct parents. In this study, we monitored the transcriptomic divergence of the maize (Zea mays) inbred lines B73 and Mo17 and their reciprocal F1 hybrid progeny in primary roots under control and water deficit conditions simulated by polyethylene glycol treatment. Single-parent expression (SPE) of genes is an extreme instance of gene expression complementation, in which genes are active in only one of two parents but are expressed in both reciprocal hybrids. In this study, 1,997 genes only expressed in B73 and 2,024 genes only expressed in Mo17 displayed SPE complementation under control and water deficit conditions. As a consequence, the number of active genes in hybrids exceeded the number of active genes in the parental inbred lines significantly independent of treatment. SPE patterns were substantially more stable to expression changes by water deficit treatment than other genotype-specific expression profiles. While, on average, 75% of all SPE patterns were not altered in response to polyethylene glycol treatment, only 17% of the remaining genotype-specific expression patterns were not changed by water deficit. Nonsyntenic genes that lack syntenic orthologs in other grass species, and thus evolved late in the grass lineage, were significantly overrepresented among SPE genes. Hence, the significant overrepresentation of nonsyntenic genes among SPE patterns and their stability under water limitation might suggest a function of these genes during the early developmental manifestation of heterosis under fluctuating environmental conditions in hybrid progeny of the inbred lines B73 and Mo17.

Non-syntenic genes drive RTCS-dependent regulation of the embryo transcriptome during formation of seminal root primordia in maize (Zea mays L.).

Seminal roots of maize are pivotal for early seedling establishment. The maize mutant rootless concerning crown and seminal roots (rtcs) is defective in seminal root initiation during embryogenesis. In this study, the transcriptomes of wild-type and rtcs embryos were analyzed by RNA-Seq based on histological results at three stages of seminal root primordia formation. Hierarchical clustering highlighted that samples of each genotype grouped together along development. Determination of their gene activity status revealed hundreds of genes specifically transcribed in wild-type or rtcs embryos, while K-mean clustering revealed changes in gene expression dynamics between wild-type and rtcs during embryo development. Pairwise comparisons of rtcs and wild-type embryo transcriptomes identified 131 transcription factors among 3526 differentially expressed genes [false discovery rate (FDR) <5% and |log2Fc|≥1]. Among those, functional annotation highlighted genes involved in cell cycle control and phytohormone action, particularly auxin signaling. Moreover, in silico promoter analyses identified putative RTCS target genes associated with transcription factor action and hormone metabolism and signaling. Significantly, non-syntenic genes that emerged after the separation of maize and sorghum were over-represented among genes displaying RTCS-dependent expression during seminal root primordia formation. This might suggest that these non-syntenic genes came under the transcriptional control of the syntenic gene rtcs during seminal root evolution. Taken together, this study provides first insights into the molecular framework underlying seminal root initiation in maize and provides a starting point for further investigations of the molecular networks underlying RTCS-dependent seminal root initiation.

Complexity and specificity of the maize (Zea mays L.) root hair transcriptome.

Root hairs are tubular extensions of epidermis cells. Transcriptome profiling demonstrated that the single cell-type root hair transcriptome was less complex than the transcriptome of multiple cell-type primary roots without root hairs. In total, 831 genes were exclusively and 5585 genes were preferentially expressed in root hairs [false discovery rate (FDR) ≤1%]. Among those, the most significantly enriched Gene Ontology (GO) functional terms were related to energy metabolism, highlighting the high energy demand for the development and function of root hairs. Subsequently, the maize homologs for 138 Arabidopsis genes known to be involved in root hair development were identified and their phylogenetic relationship and expression in root hairs were determined. This study indicated that the genetic regulation of root hair development in Arabidopsis and maize is controlled by common genes, but also shows differences which need to be dissected in future genetic experiments. Finally, a maize root view of the eFP browser was implemented including the root hair transcriptome of the present study and several previously published maize root transcriptome data sets. The eFP browser provides color-coded expression levels for these root types and tissues for any gene of interest, thus providing a novel resource to study gene expression and function in maize roots.

Transcriptomic and anatomical complexity of primary, seminal, and crown roots highlight root type-specific functional diversity in maize (Zea mays L.).

Maize develops a complex root system composed of embryonic and post-embryonic roots. Spatio-temporal differences in the formation of these root types imply specific functions during maize development. A comparative transcriptomic study of embryonic primary and seminal, and post-embryonic crown roots of the maize inbred line B73 by RNA sequencing along with anatomical studies were conducted early in development. Seminal roots displayed unique anatomical features, whereas the organization of primary and crown roots was similar. For instance, seminal roots displayed fewer cortical cell files and their stele contained more meta-xylem vessels. Global expression profiling revealed diverse patterns of gene activity across all root types and highlighted the unique transcriptome of seminal roots. While functions in cell remodeling and cell wall formation were prominent in primary and crown roots, stress-related genes and transcriptional regulators were over-represented in seminal roots, suggesting functional specialization of the different root types. Dynamic expression of lignin biosynthesis genes and histochemical staining suggested diversification of cell wall lignification among the three root types. Our findings highlight a cost-efficient anatomical structure and a unique expression profile of seminal roots of the maize inbred line B73 different from primary and crown roots.

Extensive tissue-specific transcriptomic plasticity in maize primary roots upon water deficit.

Water deficit is the most important environmental constraint severely limiting global crop growth and productivity. This study investigated early transcriptome changes in maize (Zea mays L.) primary root tissues in response to moderate water deficit conditions by RNA-Sequencing. Differential gene expression analyses revealed a high degree of plasticity of the water deficit response. The activity status of genes (active/inactive) was determined by a Bayesian hierarchical model. In total, 70% of expressed genes were constitutively active in all tissues. In contrast, <3% (50 genes) of water deficit-responsive genes (1915) were consistently regulated in all tissues, while >75% (1501 genes) were specifically regulated in a single root tissue. Water deficit-responsive genes were most numerous in the cortex of the mature root zone and in the elongation zone. The most prominent functional categories among differentially expressed genes in all tissues were 'transcriptional regulation' and 'hormone metabolism', indicating global reprogramming of cellular metabolism as an adaptation to water deficit. Additionally, the most significant transcriptomic changes in the root tip were associated with cell wall reorganization, leading to continued root growth despite water deficit conditions. This study provides insight into tissue-specific water deficit responses and will be a resource for future genetic analyses and breeding strategies to develop more drought-tolerant maize cultivars.

Transcriptomic complexity in young maize primary roots in response to low water potentials.

BACKGROUND: Widespread and more frequently occurring drought conditions are a consequence of global warming and increase the demand for tolerant crop varieties to feed the growing world population. A better understanding of the molecular mechanisms underlying the water deficit response of crops will enable targeted breeding strategies to develop robust cultivars. RESULTS: In the present study, the transcriptional response of maize (Zea mays L.) primary roots to low water potentials was monitored by RNA sequencing (RNA-Seq) experiments. After 6 h and 24 h of mild (-0.2 MPa) and severe (-0.8 MPa) water deficit conditions, the primary root transcriptomes of seedlings grown under water deficit and control conditions were compared. The number of responsive genes was dependent on and increased with intensification of water deficit treatment. After short-term mild and severe water deficit 249 and 3,000 genes were differentially expressed, respectively. After a 24 h treatment the number of affected genes increased to 7,267 and 12,838 for mild and severe water deficit, respectively, including more than 80% of the short-term responsive genes. About half of the differentially expressed genes were up-regulated and maximal fold-changes increased with treatment intensity to more than 300-fold. A consensus set of 53 genes was differentially regulated independently of the nature of deficit treatment. Characterization revealed an overrepresentation of the Gene Ontology (GO) categories "oxidoreductase activity" and "heme binding" among regulated genes connecting the water deficit response to ROS metabolism. CONCLUSION: This study gives a comprehensive insight in water deficit responsive genes in young maize primary roots and provides a set of candidate genes that merit further genetic analyses in the future.

The Aux/IAA gene rum1 involved in seminal and lateral root formation controls vascular patterning in maize (Zea mays L.) primary roots.

The maize (Zea mays L.) Aux/IAA protein RUM1 (ROOTLESS WITH UNDETECTABLE MERISTEMS 1) controls seminal and lateral root initiation. To identify RUM1-dependent gene expression patterns, RNA-Seq of the differentiation zone of primary roots of rum1 mutants and the wild type was performed in four biological replicates. In total, 2 801 high-confidence maize genes displayed differential gene expression with Fc ≥2 and FDR ≤1%. The auxin signalling-related genes rum1, like-auxin1 (lax1), lax2, (nam ataf cuc 1 nac1), the plethora genes plt1 (plethora 1), bbm1 (baby boom 1), and hscf1 (heat shock complementing factor 1) and the auxin response factors arf8 and arf37 were down-regulated in the mutant rum1. All of these genes except nac1 were auxin-inducible. The maize arf8 and arf37 genes are orthologues of Arabidopsis MP/ARF5 (MONOPTEROS/ARF5), which controls the differentiation of vascular cells. Histological analyses of mutant rum1 roots revealed defects in xylem organization and the differentiation of pith cells around the xylem. Moreover, histochemical staining of enlarged pith cells surrounding late metaxylem elements demonstrated that their thickened cell walls displayed excessive lignin deposition. In line with this phenotype, rum1-dependent mis-expression of several lignin biosynthesis genes was observed. In summary, RNA-Seq of RUM1-dependent gene expression in maize primary roots, in combination with histological and histochemical analyses, revealed the specific regulation of auxin signal transduction components by RUM1 and novel functions of RUM1 in vascular development.