miR167 limits anther growth to potentiate anther dehiscence

In flowering plants, anther dehiscence and pollen release are essential for sexual reproduction. Anthers dehisce after cell wall degradation weakens stomium cell junctions in each anther locule, and desiccation creates mechanical forces that open the locules. Either effect or both together may break stomium cell junctions. The microRNA miR167 negatively regulates ARF6 and ARF8, which encode Auxin Response transcription Factors. Arabidopsis mARF6 or mARF8 plants with mutated miR167 target sites have defective anther dehiscence and ovule development. Null mir167a mutations recapitulated mARF6 and mARF8 anther and ovule phenotypes, indicating that MIR167a is the main miR167 precursor gene that delimits ARF6 and ARF8 expression in these organs. Anthers of mir167a or mARF6/8 plants overexpressed genes encoding cell wall loosening functions associated with cell expansion, and grew too large starting at flower stage 11. Experimental desiccation enabled dehiscence of miR167-deficient anthers, indicating competence to dehisce. Conversely, high humidity conditions delayed anther dehiscence in wild-type flowers. These results support a model in which miR167-mediated anther growth arrest permits anther dehiscence. Without miR167 regulation, excess anther growth delays dehiscence by prolonging desiccation. Overall design: The dataset includes experiments from two tissues, stamens or gynoecia. Arabidopsis thaliana plants were grown in 14:10 h day:night cycles. Tissue samples for RNA isolation were collected between 1 and 3 hours after dawn, and immediately frozen in liquid nitrogen. Stage 11 flower buds used for stamen isolation (sample groups F, G, H) were 1.5-1.8 mm long, generally corresponding to 3 to 5 flowers before the first open flower. Stamens at this stage still appeared green before turning yellow at flower stage 12. Tissue for the gynoecium experiment (sample groups A, B, C, D, E) was from stage 12 flowers with gynoecia of 0.9 to 1.4 mm long. For RNA-Seq, RNA was isolated using the Rneasy Plant Mini kit (Qiagen) according to manufacturer's instructions. Libraries were constructed with the Truseq mRNA library prep kit (Illumina) according to manufacturer's instructions. Samples were bar-coded and multiplexed, and sequenced using an Illumina HiSeq 2000 machine with single-end 50 base reads. Library construction and sequencing were performed at the UNC-CH high-throughput sequencing facility. Information on genotypes: Two different stable transgenic lines carrying the introduced mARF8 transgene are called mARF8 3B and mARF8 4B. "mARF8 T1s" refers to a pool from several plants carrying the same mARF8 transgene as lines 3B and 4B. These lines carry a genomic mARF8 gene with the target site for the microRNA miR167 mutated. pSTK:axr3-1 lines 15-5 and 25-2 were two different transgenic Arabidopsis lines carrying the same introduced pSTK:axr3-1 transgene. This transgene uses promoter sequences from the SEEDSTICK gene to drive a gain-of-function axr3-1 gene encoding a stabilized AXR3/IAA17 protein. Col X PSTK:axr3-1 refers to F1 plants from a cross between wild type Columbia and pSTK:axr3-1.