The full-length Auxin Response Factor 8 isoform ARF8.1 controls pollen cell wall formation and directly regulates TDF1, AMS and MS188 expression.

Auxin Response Factor 8 plays a key role in late stamen development: its splice variants ARF8.4 and ARF8.2 control stamen elongation and anther dehiscence. Here, we characterized the role of ARF8 isoforms in pollen fertility. By phenotypic and ultrastructural analysis of arf8-7 mutant stamens, we found defects in pollen germination and viability caused by alterations in exine structure and pollen coat deposition. Furthermore, tapetum degeneration, a prerequisite for proper pollen wall formation, is delayed in arf8-7 anthers. In agreement, the genes encoding the transcription factors TDF1, AMS, MS188 and MS1, required for exine and pollen coat formation, and tapetum development, are downregulated in arf8-7 stamens. Consistently, the sporopollenin content is decreased, and the expression of sporopollenin synthesis/transport and pollen coat protein biosynthetic genes, regulated by AMS and MS188, is reduced. Inducible expression of the full-length isoform ARF8.1 in arf8-7 inflorescences complements the pollen (and tapetum) phenotype and restores the expression of the above transcription factors. Chromatin immunoprecipitation-quantitative polymerase chain reaction assay revealed that ARF8.1 directly targets the promoters of TDF1, AMS and MS188. In conclusion, the ARF8.1 isoform controls pollen and tapetum development acting directly on the expression of TDF1, AMS and MS188, which belong to the pollen/tapetum genetic pathway.

Arabidopsis TWISTED DWARF1 regulates stamen elongation by differential activation of ABCB1,19-mediated auxin transport.

Despite clear evidence that a local accumulation of auxin is likewise critical for male fertility, much less is known about the components that regulate auxin-controlled stamen development. In this study, we analyzed physiological and morphological parameters in mutants of key players of ABCB-mediated auxin transport, and spatially and temporally dissected their expression on the protein level as well as auxin fluxes in the Arabidopsis stamens. Our analyses revealed that the FKBP42, TWISTED DWARF1 (TWD1), promotes stamen elongation and, to a lesser extent, anther dehiscence, as well as pollen maturation, and thus is required for seed development. Most of the described developmental defects in twd1 are shared with the abcb1 abcb19 mutant, which can be attributed to the fact that TWD1-as a described ABCB chaperone-is a positive regulator of ABCB1- and ABCB19-mediated auxin transport. However, reduced stamen number was dependent on TWD1 but not on investigated ABCBs, suggesting additional players downstream of TWD1. We predict an overall housekeeping function for ABCB1 during earlier stages, while ABCB19 seems to be responsible for the key event of rapid elongation at later stages of stamen development. Our data indicate that TWD1 controls stamen development by differential activation of ABCB1,19-mediated auxin transport in the stamen.

A Newly Identified Flower-Specific Splice Variant of AUXIN RESPONSE FACTOR8 Regulates Stamen Elongation and Endothecium Lignification in Arabidopsis.

In addition to the full-length transcript ARF8.1, a splice variant (ARF8.2) of the auxin response factor gene ARF8 has been reported. Here, we identified an intron-retaining variant of ARF8.2, ARF8.4, whose translated product is imported into the nucleus and has tissue-specific localization in Arabidopsis thaliana By inducibly expressing each variant in arf8-7 flowers, we show that ARF8.4 fully complements the short-stamen phenotype of the mutant and restores the expression of AUX/IAA19, encoding a key regulator of stamen elongation. By contrast, the expression of ARF8.2 and ARF8.1 had minor or no effects on arf8-7 stamen elongation and AUX/IAA19 expression. Coexpression of ARF8.2 and ARF8.4 in both the wild type and arf8-7 caused premature anther dehiscence: We show that ARF8.2 is responsible for increased expression of the jasmonic acid biosynthetic gene DAD1 and that ARF8.4 is responsible for premature endothecium lignification due to precocious expression of transcription factor gene MYB26 Finally, we show that ARF8.4 binds to specific auxin-related sequences in both the AUX/IAA19 and MYB26 promoters and activates their transcription more efficiently than ARF8.2. Our data suggest that ARF8.4 is a tissue-specific functional splice variant that controls filament elongation and endothecium lignification by directly regulating key genes involved in these processes.

An auxin maximum in the middle layer controls stamen development and pollen maturation in Arabidopsis.

Here, we investigated the role of auxin distribution in controlling Arabidopsis thaliana late stamen development. We analysed auxin distribution in anthers by monitoring DR5 activity: at different flower developmental stages; inhibiting auxin transport; in the rpk2-3 and ems1 mutants devoid of middle layer (ML) or tapetum, respectively; and in the auxin biosynthesis yuc6 and perception afb1-3 mutants. We ran a phenotypic, DR5::GUS and gene expression analysis of yuc6rpk2 and afb1rpk2 double mutants, and of 1-N-naphthylphthalamic acid (NPA)-treated flower buds. We show that an auxin maximum, caused by transport from the tapetum, is established in the ML at the inception of late stamen development. rpk2-3 mutant stamens lacking the ML have an altered auxin distribution with excessive accumulation in adjacent tissues, causing non-functional pollen grains, indehiscent anthers and reduced filament length; the expression of genes controlling stamen development is also altered in rpk2-3 as well as in NPA-treated flower buds. By decreasing auxin biosynthesis or perception in the rpk2-3 background, we eliminated these developmental and gene expression anomalies. We propose that the auxin maximum in the ML plays a key role in late stamen development, as it ensures correct and coordinated pollen maturation, anther dehiscence and filament elongation.