Unknown,Transcriptomics,Genomics,Proteomics

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Unraveling the KNOTTED1 regulatory network in maize meristems

ABSTRACT: KNOTTED1(KN1)-like homeobox (KNOX) transcription factors function in plant meristems, self-renewing structures consisting of stem cells and their immediate daughters. Despite their importance for plant development, the genomic network targeted by KNOX proteins is poorly understood. Using ChIP-seq, we defined the KN1 cistrome in maize inflorescences and found that KN1 binds to several thousand loci. To understand how these binding occupancies correlate with changes in transcriptional regulation, we performed RNA-seq on immature ears and tassels, and compared expression profiles between normal and loss-of-function kn1 plants, in addition to immature leaves from normal and gain-of-function Kn1 plants. We found that 643 of the KN1 targets were modulated in one or multiple tissues, with a strong enrichment for transcription factors (including other homeobox genes) and genes participating in several hormonal pathways, most significantly auxin, implicating KN1 at the crossroads of plant hormone signaling. The loss-of-function kn1 phenotype is reminiscent of auxin mutants and kn1 mis-expression in leaves correlates with increased auxin signaling. Our results demonstrate that KN1 plays a key role in orchestrating the upper levels of a hierarchical gene regulatory network that impacts plant meristem identity and function. For loss-of-function mutant, kn1-e1 homozygote were compared to heterozygote or wild-type siblings (ears, tassels and shoot apical meristem). For gain-of-function mutant, Kn1-N homozygote were compared to wild-type siblings.Kn1-N heterozygote were compared to wild-type siblings. For each genotype and tissue, 2 biological replicates were sequenced, except for tassels (3 biological replicates). The whole data set thus includes a total of 20 samples/libraries.

ORGANISM(S): Zea mays

SUBMITTER: Maria Mejia Guerra

PROVIDER: E-GEOD-38487 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Unraveling the KNOTTED1 regulatory network in maize meristems.

Bolduc Nathalie N Yilmaz Alper A Mejia-Guerra Maria Katherine MK Morohashi Kengo K O'Connor Devin D Grotewold Erich E Hake Sarah S

Genes & development 20120801 15

KNOTTED1 (KN1)-like homeobox (KNOX) transcription factors function in plant meristems, self-renewing structures consisting of stem cells and their immediate daughters. We defined the KN1 cistrome in maize inflorescences and found that KN1 binds to several thousand loci, including 643 genes that are modulated in one or multiple tissues. These KN1 direct targets are strongly enriched for transcription factors (including other homeobox genes) and genes participating in hormonal pathways, most signi ...[more]

PMID: 22855831

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Project description:In this study we used the maize (Zea mays) inflorescence to investigate gene networks that modulate determinacy, specifically the decision to allow branch growth. We characterized developmental transitions by associating spatiotemporal expression profiles with morphological changes resulting from genetic perturbations that disrupt steps in a pathway controlling branching. These are the RNA-seq datasets used in this study. We profiled changes in gene expression during normal maize ear and tassel development and in developing maize ear primordia upon genetic perturbation of the RAMOSA branching pathway. For the wild-type ear and tassel developmental series, greenhouse-grown B73 inbred plants were used. 10mm ears were collected and sectioned as follows from tip to base along the developmental gradient: tip 1mm sampled (tip; Inflorescence Meristem/Spikelet Pair Meristem), next 1mm discarded, next 1mm sampled (mid; Spikelet Meristem), next 2mm discarded, next 2 mm sampled (base; Floral Meristem), and immediately frozen in liquid nitrogen. Sections from ~30 sampled ears were pooled for each of 2 biological replicates to represent tip, mid, and base stages. Tassels were hand-dissected, measured, separated by stage: 1-2mm (stg1), 3-4mm (stg2), and 5-7mm (stg3), and immediately frozen in liquid N. For each stage, ~20-30 tassels were pooled for each of 2 biological replicates. For ramosa mutant series, segregating families (1:1) of ra1-R, ra2-R, and ra3-fea1 mutant alleles, all introgressed at least 6 times into the B73 inbred background, were grown at CSHL Uplands Farm. Field-grown plants were genotyped and collected 6-7 weeks after germination (V7-V8 stage). First and second ear primordia were immediately hand-dissected, measured, and frozen in liquid nitrogen. For ra1, ra2 and ra3 mutants and wild-type controls, ears were pooled into two size classes: 1) 1mm class included a range of 0.7-1.5mm sized ears and nine ears were pooled for each of 2 biological replicates; 2) 2mm class included a range of 1.8-2.5mm sized ears and six ears were pooled for each of three biological replicates. Wild-type samples were proportional mixtures of heterozygote siblings segregating in ra1, ra2, and ra3 populations. Variability factors (e.g. ear size within class, ear rank on the plant, and time of collection) were distributed evenly across pooled samples.

Project description:Small RNAs (21-24 nt) are pivotal regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in diverse eukaryotes, including most if not all plants. MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are the two major types, both of which have a demonstrated and important role in plant development, stress responses and pathogen resistance. In this work, we used a deep sequencing approach (Sequencing-By-Synthesis, or SBS) to develop sequence resources of small RNAs from different maize tissues (including leaves, ears and tassels) collected from wild-type plants of the B73 variety. The high depth of the resulting datasets enabled us to examine in detail critical small RNA features as size distribution, tissue-specific regulation and sequence conservation between different organs in this species. We also developed database resources and a dedicated website (http://smallrna.udel.edu/) with computational tools for allowing other users to identify new miRNAs or siRNAs involved in specific regulatory pathways, verify the degree of conservation of these sequences in other plant species and map small RNAs on genes or larger regions of the maize genome under study. Small RNA libraries were derived from leaves, ears and tassels of maize variety B73 (wild-type). Plants were grown in a flood irrigated plot at the University of Arizona (Tucson, AZ, USA) in 2007 and organs were pooled from several plants for each library. Young leaves were collected from 6-weeks-old seedlings. Post-meiotic immature ears were harvested from 10- and 11-week old plants while pre-meiotic tassels were collected from 8-week old plants. Total RNA was isolated using the Plant RNA Purification Reagent (Invitrogen) and submitted to Illumina (Hayward, CA, http://www.illumina.com) for small RNA library construction using approaches described in (Lu et al., 2007) with minor modifications. The small RNA libraries were sequenced with the Sequencing-By-Synthesis (SBS) technology by Illumina. PERL scripts were designed to remove the adapter sequences and determine the abundance of each distinct small RNA. We thank Lyudmila Sidorenko and Vicki Chandler for providing the plant material and Kan Nobuta for assistance with the computational methods.

Dataset Information

Unraveling the KNOTTED1 regulatory network in maize meristems

Publications

Unraveling the KNOTTED1 regulatory network in maize meristems.

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