Sporophytic control of pollen meiotic progression is mediated by tapetum expression of ABORTED MICROSPORES

Abstract

Pollen development is dependent on the tapetum, a sporophytic anther cell layer surrounding the microspores that functions in pollen wall formation but is also essential for meiosis-associated development. There is clear evidence of crosstalk and co-regulation between the tapetum and microspores, but how this is achieved is currently not characterized. ABORTED MICROSPORES (AMS), a tapetum transcription factor, is important for pollen wall formation, but also has an undefined role in early pollen development. We conducted a detailed investigation of chromosome behaviour, cytokinesis, radial microtubule array (RMA) organization, and callose formation in the ams mutant. Early meiosis initiates normally in ams, shows delayed progression after the pachytene stage, and then fails during late meiosis, with disorganized RMA, defective cytokinesis, abnormal callose formation, and microspore degeneration, alongside abnormal tapetum development. Here, we show that selected meiosis-associated genes are directly repressed by AMS, and that AMS is essential for late meiosis progression. Our findings indicate that AMS has a dual function in tapetum-meiocyte crosstalk by playing an important regulatory role during late meiosis, in addition to its previously characterized role in pollen wall formation. AMS is critical for RMA organization, callose deposition, and therefore cytokinesis, and is involved in the crosstalk between the gametophyte and sporophytic tissues, which enables synchronous development of tapetum and microspores.

Keywords: ABORTED MICROSPORES; AMS; anther; callose; cytokinesis; male sterile; meiosis; pollen development; radial microtubule array; tapetum.

Fig. 1.

ams is male sterile, with abnormal tapetum and tetrads. TEM anther sections of wild-type (WT) Col-0 (A, C, E, G, I) and ams mutant (B, D, F, H, J). (A, B) TEM sections of pollen mother cells (PMC) pre-meiosis and tapetum in WT (A) and ams (B), showing normal tapetum (T) and PMC, middle layer (ML), endodermis (En), and epidermis (Ep). Meiosis progresses normally in WT (C, E), whereas irregularities occur in the ams tapetum with pre-vacuolation (v) mutant tapetal cells (D, F). Connections between tapetum cells and PMC are seen in WT (C, E, arrows), whilst abnormal/compressed connections are seen in the ams mutant (D, F, arrows). WT (G) showing callose cell wall deposition on developing meiocytes with cytokinesis occurring to form tetrads, while ams (H) has abnormal callose accumulation and separation. Normal tetrads are seen in WT (I) stage (vacuoles: v), whereas abnormal tetrads and highly vacuolated (v) tapetum cells are observed in ams (J). Scale bars: 5 μm (C, D, G, H) and 10 μm (the rest).

Fig. 2.

Male sterile mutants have normal early meiosis development. DAPI staining of wild-type (Col) and male sterile mutants ams, dyt1, tdf1, ms188, and ms1 meiocytes during male meiosis. The presence of five bivalents is clear at metaphase I and metaphase II in all lines, with correct separation occurring in metaphase II. During telophase II there is the balanced formation of four sets of five chromosomes with the correct formation of tetrads in all lines observed. Scale bars: 10 μm.

Fig. 3.

Chromosome pairing, synapsis and nuclear envelope formation appear normal in ams. (A) Localization of the synaptonemal complex protein ZYP1 (red) and the axis-associated protein ASY1 (green) at zygotene stage in WT and ams. The distribution of both proteins appears normal indicating that chromosome pairing and synapsis occur in the absence of AMS. Chromosomes are counterstained with DAPI (blue). Scale bars: 5 μm. (B) Nucleoporin localization in WT and ams pollen mother cells. Similar nuclear envelope expression patterns of AtSUN2 in wild-type and ams at telophase II with AtSUN2 (green) antibody, counterstained using DAPI (blue). Strongest SUN2 signal was observed in ams. Scale bars: 5 μm.

Fig. 4.

Spindle formation morphology in wild-type (WT, Col 0) and male sterile meiocytes. The spindle was detected by immunostaining with anti-α-tubulin antibody (green) and chromosomes were counterstained with DAPI (blue). Tubulin localization is similar in WT Col-0 and ams at prophase I (A, B), with normal spindle morphology during metaphase I in Col-0 (C) and ams (D). Radial microtubule arrays (RMA) however are disorganized in ams (F) compared with WT (E); this is also observed in the upstream male sterile mutants dyt1 (G) and tdf1 (H), but not the downstream mutant ms188 (I). Nucleus positioning within the tetrad is also unbalanced forming a ‘triad’ like shape in dyt1, tdf1, and ams (G–I). Scale bars: 5 μm.

Fig. 5.

Callose staining of male meiocytes of tapetum defective mutants. Callose staining of wild-type (WT) Col-0 (A) and ams (B) tetrads during meiosis initially showed similar callose production, suggesting normal initiation of callose biosynthesis. Callose deposition, forming thick walls surrounding the tetrads, was subsequently seen in WT (C), whereas this was abnormal and disorganized in ams tetrads (D), dyt1 (E), and tdf1 mutants (F), and the later stage tapetum mutants, ms188 (G) and ms1 (H), showed normal callose deposition and thick, ordered callose layers surrounding the tetrads. Scale bars: 10 µm.

Fig. 6.

Meiotic progression over time in ams compared with wild-type. Detection of EdU labelling in pollen mother cells across a time course of sampled cells (2, 8, 18, 28, 32, and 42 h). White, EdU Alexa Fluor 488. (A) Wild-type (WT) Col-0 showing normal meiotic progression, with tetrads observed after 32 h. (B) ams showed delayed progression through meiosis after pachytene stage, with occasional tetrads observed only after 42 h. Scale bar: 10 μm.

Fig. 7.

AMS binds directly to the promoters of selected meiotic-associated genes to regulate their expression. (A) ChIP–qPCR analysis of the enrichment of AMS regulatory targets compared with WBC27 positive control. Predicted E-boxes in the promoter region represented by dark vertical lines and promoter regions analysed by ChIP–qPCR and EMSA represented by dotted line and arrows. (B) Fold enrichment represents the fold change in +Ab (antibody) compared with −Ab samples, normalized to WBC27 fold change. qPCR data were gathered from three biological and two technical replicates. *Significant changes based on Student’s t-test, P<0.05. Error bars represent SD. (C) EMSA using digoxigenin-labelled probes without AMS protein or unlabelled probes (lane 1); lanes 2–4 show AMS protein and DIG-labelled probe with increasing amount of competitor DNA (10× and 100×, respectively). Gel retardation indicates the binding of the AMS to promoters of the target genes. (D) Relative expression values based on qRT-PCR analysis measured in whole inflorescence in wild-type (Col-0), ams, and AMSprom:AMS-GR-YFP in WT Col-0 background, showing up-regulation in ams mutant and down-regulation in the AMSprom:AMS-GR-YFP line 24 h after AMS induction by DEX. Expression was normalized to wild-type for ams and to AMS-GR-YFP without DEX for AMS-GR-YFP 24 h after DEX. *Significant changes based on Student’s t-test, P<0.05. Error bars represent SE.

Fig. 8.

Proposed regulatory network for AMS. Tapetum regulatory pathway based on network published in Ferguson et al. (2017). AMS is directly regulated by DYT1 through TDF1, and itself directly regulates MS188, which regulates MS1. AMS has a published role in sporopollenin wall formation alongside MS188. We propose that it is the loss of AMS in the early tapetum mutants that cause the phenotypes observed in ams, tdf1, and dyt1 mutants, as ms188 and ms1 develop normally. We have shown a novel role for AMS in the correct regulation of RMA localization (green lines in meiocytes) during telophase II, for correct cytokinesis, callose wall deposition (yellow material in meiocyte) to produce a functional tetrad. AMS is also important for fully functional tapetum cells that have a high energy requirement during meiosis (darker background colour represents this, and black circles indicate lipidic tapetosomes and elaioplasts) to provide for the developing pollen. We have shown that AMS appears to directly negatively regulate at least four meiosis-associated genes, possibly through interaction in the tapetum, and propose that AMS has a role in the final stages of meiosis through their regulation. n, nucleus; v, vacuole. Arrows: regulation; lines ending with a line: repression; lines ending with circle: protein interactions; dashed lines indicate a minor role in regulation of network (as predicted by modelling; Ferguson et al. 2017); red lines indicate a major role in regulation.