The male sterile 8 mutation of maize disrupts the temporal progression of the transcriptome and results in the mis-regulation of metabolic functions

Abstract

SUMMARYMaize anther ontogeny is complex, with the expression of more than 30 000 genes over 4 days of cell proliferation, cell fate acquisition and the start of meiosis. Although many male-sterile mutants disrupt these key steps, few have been investigated in detail. The terminal phenotypes of Zea mays (maize) male sterile 8 (ms8) are small anthers exhibiting meiotic failure. Here, we document much earlier defects: ms8 epidermal cells are normal in number but fail to elongate, and there are fewer, larger tapetal cells that retain, rather than secrete, their contents. ms8 meiocytes separate early, have extra space between them, occupied by excess callose, and the meiotic dyads abort. Thousands of transcriptome changes occur in ms8, including ectopic activation of genes not expressed in fertile siblings, failure to express some genes, differential expression compared with fertile siblings and about 40% of the differentially expressed transcripts appear precociously. There is a high correlation between mRNA accumulation assessed by microarray hybridization and quantitative real-time reverse transcriptase polymerase chain reaction. Sixty-three differentially expressed proteins were identified after two-dimensional gel electrophoresis followed by liquid chromatography tandem mass spectroscopy, including those involved in metabolism, plasmodesmatal remodeling and cell division. The majority of these were not identified by differential RNA expression, demonstrating the importance of proteomics in defining developmental mutants.

Maize csmd1 exhibits pre-meiotic somatic and post-meiotic microspore and somatic defects but sustains anther growth

“…Chromosome behavior in meiocytes and microspores was observed by hematoxyliniron-aceto-carmine staining (Chang and Neuffer 1989). Propidium iodide (CalBiochem, now supplied by Merck http://www.merck-chemicals.com) staining with Triton treatment, which resulted in a nuclear stain for confocal imaging (Leica TCS SP5, Leica Microsystem), was performed as described previously (Kelliher and Walbot 2011;Wang et al 2010); cell and organ dimension measurements were calculated with VOLOCITY software (PerkinElmer, http://www.perkinelmer.com). For meiocytes and microspores, anther locule wall and anther locule dimension comparisons, four biological replicates were used from fertile and csmd1 plants.…”

“…Laser Dissecting Microscope (Wang et al 2009). Aniline blue (Sigma-Aldrich, http://www.sigmaaldrich.com) staining and callose fluorimetric quantitative assay were done as described previously (Kohle et al 1985;Wang et al 2010). Four biological replicates were used from fertile and csmd1 plants for callose fluorimetric quantitative assay.…”

“…Sample dissection and bright-field microscopy followed a published protocol (Wang et al 2010). Anthers from the upper floret were pooled from several spikelets after careful developmental staging.…”

“…In maize, both somatic cell and PMC differentiation abnormalities prior to meiosis cause failure of PMC differentiation or meiotic arrest in prophase I in the mutants analyzed thus far. Examples include msca1 in which anther cells differentiate as leaf cell types (Chaubal et al 2003), mac1 which lacks a tapetum and middle layer but has too many PMCs (Sheridan et al 1996), ms23 and ms32 (Chaubal et al 2000) both of which have a double layer ''pre-tapetal'' cell type that fails to support the PMCs, and ms8, in which there are epidermal and tapetal defects (Wang et al 2010). In all of these cases of pre-meiotic arrest, anthers growth ceases at about 2 mm, the length typical of the early meiotic stage in fertile anthers.…”

“…Given the high cost of producing hybrid corn by detasseling, however, reintroduction of male sterility, if properly managed, could be warranted. Maize also has notable advantages for investigation of male reproductive development, principally the early carpel abortion and large cohorts of anthers maturing in near synchrony on the tassel (Bedinger and Fowler 2009;Wang et al 2010). Analysis of male-sterile mutants that disrupt normal somatic and haploid cell differentiation should help pinpoint mechanisms underlying the normal progression of developmental events, and could be used as the future foundation for reliable hybrid seed production.…”