ARR17 controls dioecy in Populus by repressing B-class MADS-box gene expression

Abstract

The number of dioecious species for which the genetic basis of sex determination has been resolved is rapidly increasing. Nevertheless, the molecular mechanisms downstream of the sex determinants remain largely elusive. Here, by RNA-sequencing early-flowering isogenic aspen (Populus tremula) lines differing exclusively for the sex switch gene ARR17, we show that a narrowly defined genetic network controls differential development of female and male flowers. Although ARR17 encodes a type-A response regulator supposedly involved in cytokinin (CK) hormone signalling, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated arr17 knockout only affected the expression of a strikingly small number of genes, indicating a specific role in the regulation of floral development rather than a generic function in hormone signalling. Notably, the UNUSUAL FLORAL ORGANS (UFO) gene, encoding an F-box protein acting as a transcriptional cofactor with LEAFY (LFY) to activate B-class MADS-box gene expression, and the B-class gene PISTILLATA (PI), necessary for male floral organ development, were strongly de-repressed in the arr17 CRISPR mutants. Our data highlight a CK-independent role of the poplar response regulator ARR17 and further emphasize the minimal differences between female and male individuals. This article is part of the theme issue 'Sex determination and sex chromosome evolution in land plants'.

Keywords: cytokinin; dioecy; flower development; poplar; single gene sex determination.

Figure 1.

Transcriptome variation and ARR17 expression in female and male flower buds of early-flowering aspen lines. (a) Principal component analysis (PCA) of transcriptome variation of early-flowering female (magenta) and male arr17 CRISPR (blue) aspen (P. tremula) lines. Different symbols indicate different sampling days (i.e. days 5, 10, 15 and 20 after the start of flower induction). The first principal component PC1 explains 71% and the second principal component PC2 11% of the total variance. (b) ARR17 expression occurs in a narrow temporal window during poplar flower development. Average relative expression (n = 3) of ARR17 expression determined via qRT-PCR over a developmental time course in the same two genotypes shown in (a). ARR17 expression peaks at day 20. Error bars indicate the standard error of the mean (SEM).

Figure 2.

PISTILLATA (PI) and UNUSUAL FLORAL ORGANS (UFO) are strongly upregulated in arr17 CRISPR mutants on day 20. Volcano plot showing 29 549 expressed genes. Significantly differentially expressed genes (female versus arr17 CRISPR lines) at day 20 (p_adj < 0.05 and abs(log₂FC > 1.5)) are depicted in red. PI and UFO are highlighted by filled symbols. The dashed grey line indicates the p-value significance threshold.

Figure 3.

Potential downstream pathway of the sex switch ARR17. For the development of male flower organs (i.e. stamens), the B-class MADS-box genes PISTILLATA (PI) and APETALA 3 (AP3) are essential as they form a heterodimer (dashed square) with C-class and E-class genes AGAMOUS (AG) and SEPALLATA (SEP), respectively. These genes are regulated transcriptionally by genes such as LEAFY (LFY) and the cofactor UNUSUAL FLORAL ORGANS (UFO) (as part of the SCF complex), which potentially represses a factor (depicted as ‘X') via degradation [50] that would repress B-class gene expression. (Online version in colour.)