Global transcriptome analysis of two ameiotic1 alleles in maize anthers: defining steps in meiotic entry and progression through prophase I

Abstract

Background: Developmental cues to start meiosis occur late in plants. Ameiotic1 (Am1) encodes a plant-specific nuclear protein (AM1) required for meiotic entry and progression through early prophase I. Pollen mother cells (PMCs) remain mitotic in most am1 mutants including am1-489, while am1-praI permits meiotic entry but PMCs arrest at the leptotene/zygotene (L/Z) transition, defining the roles of AM1 protein in two distinct steps of meiosis. To gain more insights into the roles of AM1 in the transcriptional pre-meiotic and meiotic programs, we report here an in depth analysis of gene expression alterations in carefully staged anthers at 1 mm (meiotic entry) and 1.5 mm (L/Z) caused by each of these am1 alleles.

Results: 1.0 mm and 1.5 mm anthers of am1-489 and am1-praI were profiled in comparison to fertile siblings on Agilent® 4 × 44 K microarrays. Both am1-489 and am1-praI anthers are cytologically normal at 1.0 mm and show moderate transcriptome alterations. At the 1.5-mm stage both mutants are aberrant cytologically, and show more drastic transcriptome changes. There are substantially more absolute On/Off and twice as many differentially expressed genes (sterile versus fertile) in am1-489 than in am1-praI. At 1.5 mm a total of 4,418 genes are up- or down-regulated in either am1-489 or am1-praI anthers. These are predominantly stage-specific transcripts. Many putative meiosis-related genes were found among them including a small subset of allele-specific, mis-regulated genes specific to the PMCs. Nearly 60% of transcriptome changes in the set of transcripts mis-regulated in both mutants (N = 530) are enriched in PMCs, and only 1% are enriched in the tapetal cell transcriptome. All array data reported herein will be deposited and accessible at MaizeGDB http://www.maizegdb.org/.

Conclusions: Our analysis of anther transcriptome modulations by two distinct am1 alleles, am1-489 and am1-praI, redefines the role of AM1 as a modulator of expression of a subset of meiotic genes, important for meiotic progression and provided stage-specific insights into the genetic networks associated with meiotic entry and early prophase I progression.

Figure 1

Maize tassel and anther development. Fertile maize anthers contain four locules. At ~0.1 mm there are about 50 cells in each locule. The line drawing of a transverse section (a) shows that the L2-derived (L2-d) cells are distinguishable from the L1-derived epidermis (ep) within a locule. At 0.3 mm (b) the L2-d has developed into endothecium (en), secondary parietal (2p) and archesporial cell types (ar). At 1.0 mm (c) middle layer (ml) and tapetum (tp) are apparent after a periclinal division of the 2p layer cells. Apart from the general morphology of no anther exertion, the mature tassel on the male-sterile am1-praI plant (e) is highly similar to a fertile sibling tassel (d) with the same number of tassel branches. In contrast, the am1-489 tassels (g) have fewer side branches (5-6) than fertile siblings (f). The individual reproductive units on the tassel are spikelets (sp). There are two florets in each spikelet, and each floret contains 3 anthers (h). The adaxial, upper florets used in the array experiment are developmentally 1 day ahead of the abaxial, lower florets. Anthers on a male sterile am1-praI plant (i) can reach 4-5 mm in length before they shrivel and degenerate; am1-489 anthers show growth arrest at 2-2.5 mm (data not shown). Based on cytological evidence and published results, anther sizes and the developmental timeline showing days after locular primordium initiation are diagrammed (j).

Figure 2

Cytological staging of pollen mother cells (PMCs). (a) Acetocarmine-stained chromosomes in PMCs in anthers of various sizes. PMCs in both fertile and mutant anthers at 1.0 mm are mostly at interphase. PMCs in 1.5 mm fertile anthers are at the leptotene or zygotene stage of meiotic prophase I. PMCs in both 1.5 mm and 2.0 mm am1-489 anthers show unsynchronized, mitotic characteristics with 20 univalents; am1-praI central cells start meiosis successfully when anthers reach ~2.0 mm but arrest at the L/Z transition. (b) Developmental timing of meiotic stages at the corresponding anther lengths in fertile anthers.

Figure 3

Differential expression between mutant and fertile anthers. Venn diagrams of up- or down-regulated genes in am1 alleles compared to the common fertile dataset. Number of transcripts unique to a stage or allele or expressed in common: (a) between two stages; (b) between alleles; and (c) comparison of the two mutant alleles at 1.5 mm, showing the number of differentially expressed transcripts unique to each allele and the common set (including the oppositely regulated with duplicates removed).

Figure 4

Gene ontology annotation. Percentage of each gene ontology category of differentially expressed transcripts in mutant anthers compared to fertile (excluding unknowns) found in: 489_1.0: 1.0 mm am1-489; 489_1.5: 1.5 mm am1-489; pra_1.0: 1.0 mm am1-praI; pra_1.5: 1.5 mm am1-praI; 489+pra_1.0: both am1-489 and am1-praI at 1.0 mm; 489+pra_1.5: both am1-489 and am1-praI at 1.5 mm. Categories are in alphabetical order, starting from the bottom of the list.

Figure 5

Heat map of 297 PMC-enriched, differentially expressed genes in mutant (S) versus fertile (F) anthers.

Figure 6

Heat map of 37 PMC-enriched genes that clustered with the Am1 gene expression profile as in Figure 5.

Figure 7

Heat map of 45 PMC-enriched genes that clustered with the Skp1B and Rad54 expression profiles as in Figure 5.