4.1.1 Morphometric analysis of flower organs
Gene expression profiles in reproductive tissues showed spatial and temporal variability (Mascarenhas 1989; Honys and Twell 2004). To optimize for comparative gene expression analyses between reduced (sex) and unreduced (apomeiosis) pollen at a single developmental stage, the morphological development of antherheads was staged depending on the flower bud length. The identification of an allometric relationship between the flower bud size and the gametophyte stage of anthers would set up a fast and non-destructive method to screen for appropiate anther developmental stages for RNA isolation and subsequent transcriptome comparisons.
Analyses of early flower development from about 780 flower buds in six diploid Boechera genotypes showed a linear relationship between bud length (i.e. from the receptaculum to the tip of the outermost sepal) and flower organ size (e.g. stage S4 to S12 antherheads: R2apo=0.87, F=1273.50, p<0.001; R2sex=0.95, F=4237.23, p<0.001; Fig. 5). Only minor variations among genotypes, but no differences between sex and apomixis were detected (general linear model for regression line slopes (b) of stage S4 to S12 antherheads: H0: bapo=bsex; R2=0.95, F(44, 8) = 0.68, p=0.804; Fig. 5). Bud stages S1 to S3, which most likely can be assigned to Arabidopsis flower developmental stages 1 to 5 (sensu Smyth et al. (1990)) were excluded from flower organ analysis because the majority of flower organs are at primordial stages or are prior to emergence.
In a second step we examined the correlation of antherhead length with male gametogenesis stages according to their flower bud correlates. Different from other tissues, such as the tapetum, microsporogenous tissues undergo several systematic histodifferentiation steps, and was thus reported as appropiate “marker” for anther development (Scott et al., 1991). We decided to focus on four major histodifferentiation steps of male gametophyte development which are characterized by their pre-meiotic, meiotic and post-meiotic appearance (Fig. 6, Table 1). Despite some marker-specific variations in the correlation quality, flower bud length predicts the gametophytic stage of anthers with relative high accuracy (N=455; Sp: 100%, PMC: 100%, Me: 52.27% and Msp: 58.24%). Thereby, the sporocyte (Sp) stage is prevalently correlated with flower buds of 0.2 to 0.3 mm length (antherhead length: <100µm), flower buds between0.3 and 0.9 mm length (~100 to ~430µm) exhibit a high correlation with pollen mother cells (PMCs), and microspores (Msp) are highly enriched in flower buds greater
than 1.1 mm (>610µm). Meiotically-active gametophytes (Me) are predominant in flower buds with 0.9 to 1.1 mm length (Sex: ~550 to 610 and 760µm; Apo: ~430 to 610µm).
Figure 5. Correlation of flower organs and flower bud length for sexual and apomictic Boechera
genotypes.
Error bars show the standard deviation (SD) of single flower organ sizes for each flower bud stage (see also Fig. 6 and Table 1). Total number of examined flower buds per genotypes given in parentheses.
Figure 6. Light microscope analysis of Boechera antherhead development and corresponding
gametophyte stages during pollen formation.
(A) Flower buds and appropriate antherheads of different developmental stages S3 to S12 in a sexual genotype. (B-E) Light microscopy images of semi-thin sections of resin-embedded antherheads after histological staining displaying the development of the sporocyte into a mature microspore, used for cytological validation of major gametophyte stages (B – Sp; C - PMC; D - Me; E – Msp, Table 1). E, endodermis; P, parietal cells; Sp, sporocyte; V, vascular region; C, connective; T, tapetum; PMC, pollen mother cell; En, endothecium; MC, meiotic cell; Me, meiosis; Msp, microspores.
Table 1. Developmental markers of Boechera pollen formation corresponding to gametophyte
and flower stages.
4.1.2 Heterochronic development of male gametophytes in sexual and apomictic Boechera
Divergent apomictic taxa (e.g. Panicum, Tripsacum) share an accelerated development of their apomeiotic ovules relative to their sexual counterparts (Savidan 2007), an observation which is mirrored by temporal shifts (heterochrony) between both reproductive modes on the transcriptome level (Sharbel et al., 2010). Such a desynchronization of the flower development in apomicts is speculated to result from the temporal alteration of the sexual pathway without disrupting it (Grimanelli 2003). Under this viewpoint we tested, whether microgametogenesis and anther elongation growth are synchronized in the same way between sexual and apomictic Boechera flower buds. Therefore, the data set was analysed per genotype and the comparison was focussed on anthers of flower bud stage S8 to S12. Statistical analysis exhibited, that in apomictic genotypes antherhead length predicts all gametophytic stages of anther development, whereas in all sexual genotypes meiotic and microspore stages strongly overlapped in antherhead size class, which together point to heterochronic development between sexual and apomictic antherheads (one-way ANOVA with Tukey-HSD post hoc test, between *p<10-2 and ***p<10-9, Fig. 7A). Similar to the observations in ovule
tissues, the onset of meiosis in apomictic antherheads appears to be accelerated compared to sexual anthers (Fig. 7B and C).
Figure 7. Correlation of antherhead length with different gametophyte stages among sexual
and apomictic Boechera.
(A) Horizontal bars above boxplots demonstrate significant comparisons between gametophytic stages within each genotype (*p<0.05; **p<0.01; ***p<0.001, n.s. = not significant), as conducted for a 95% binomial proportion confidence interval with a one-way ANOVA including Tukey-HSD post hoc test (Supplemental Table 5). Despite observations of antherhead-related gametophyte stages for flower bud stages S3 to S12, the correlation analysis focuses only on S8 to S12 (refer to Figure 1 and 2). Graphs display frequencies of microspores, meiotic and pollen mother cells (PMC) relative to antherhead length bins in (B) sexual and (C) apomictic Boechera. The threshold for statistical significance of a stage- specific PMC enrichment according to a two-tailed Fisher’s exact test was ***p<0.001 (white boxes).
4.1.3 PMC stage suitable for comparative gene expression analyses
Several criteria were proposed for selection of apomixis candidate genes, such as the specific expression of the candidate at the onset of meiosis and the ability of producing unreduced gametes by circumvention of meiosis checkpoints, which otherwise would override the abnormal behaviour (Grimanelli et al., 2001). In addition, comparative gene expression profiling of sexual and apomictic ovule transcriptomes demonstrated a spike in gene expression change at the megaspore mother cell stage (Sharbel et al.,
2010). Thus, the observation of altered male meiocyte fate at the diplotene stage of prophase I in facultative and obligate apomicts (see 4.2.1, Fig. 8), pointing to deregulation of genes at premeiotic stages, together with the high correlation between antherhead length and gametophyte stage, led to the selection of antherheads with a length of 400±30 µm (S8) for collection of total RNA. Anthers at bud stage S8 are significantly enriched for PMCs being close to meiosis in both sexual and apomictic genotypes (one-tailed Fisher’s exact test, ***p<0.001, Fig. 7B and C, Supplemental Table 5).