Arabidopsis mutant sk156 reveals complex regulation of SPL15 in a miR156-controlled gene network

In this study, an Arabidopsis T-DNA enhancer mutant, sk156, was identified due to its altered branching and trichome morphology and increased seed carotenoid levels compared to wild type (WT) ecovar Columbia. Enhanced miR156b expression due to the 35S enhancers present on the T-DNA insert was responsible for these phenotypes. Constitutive and leaf primodium-specific expression of a miR156-insensitive (mutated) SPL15 (SPL15m) largely restored WT seed carotenoid levels and plant morphology when expressed in sk156. The Arabidopsis native miR156-sensitive SPL15 (SPL15n) and SPL15m driven by a native SPL15 promoter did not restore the WT phenotype in sk156. Our findings suggest that SPL15 function is somewhat redundant with other SPL family members, which collectively affect plant phenotypes. Moreover, substantially decreased miR156b transcript levels in sk156 expressing SPL15m, together with the presence of multiple repeats of SPL-binding GTAC core sequence close to the miR156b transcription start site, suggested feedback regulation of miR156b expression by SPL15. This was supported by the demonstration of specific in vitro interaction between DNA-binding SBP domain of SPL15 and the proximal promoter sequence of miR156b.Enhanced miR156b expression in sk156 leads to the mutant phenotype including carotenoid levels in the seed through suppression of SPL15 and other SPL target genes. Moreover, SPL15 has a regulatory role not only for downstream components, but also for its own upstream regulator miR156b.The Arabidopsis miR156 family has eight members and is highly conserved in the plant kingdom. It has been identified in 45 plant species [1]. MiR156 is known to repress SPL (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE) genes [2-4], which are plant-specific transcription factors containing the SBP (SQUAMOSA promoter binding protein) box [5]. In Arabidopsis, 11 SPL genes are targeted by miR156 [2,4,6,7], and in silico full genome analysis showed that no other genes in Arabidopsis