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

Abstract

BackgroundThe Arabidopsis microRNA156 (miR156) regulates 11 members of the SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) family by base pairing to complementary target mRNAs. Each SPL gene further regulates a set of other genes; thus, miR156 controls numerous genes through a complex gene regulation network. Increased axillary branching occurs in transgenic Arabidopsis overexpressing miR156b, similar to that observed in loss-of-function max3 and max4 mutants with lesions in carotenoid cleavage dioxygenases. Arabidopsis miR156b was found to enhance carotenoid levels and reproductive shoot branching when expressed in Brassica napus, suggesting a link between miR156b expression and carotenoid metabolism. However, details of the miR156 regulatory network of SPL genes related to carotenoid metabolism are not known.ResultsIn 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.ConclusionsEnhanced 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.

Seasonal Regulation of Petal Number

“…7A), which represent three subgroups of miRNA-regulated SPL genes (Guo et al, 2008). These genes were also chosen because they contribute to the regulation of sepal trichome number in Arabidopsis (Shikata et al, 2009;Yu et al, 2010;Wei et al, 2012). We found that ChSPL3 expression increased stepwise from vegetative to early inflorescence to anthesis stages of development, while ChSPL9 expression increased from early inflorescence to anthesis stages (Fig.…”

Transitioning to the Next Phase: The Role of Sugar Signaling throughout the Plant Life Cycle

“…In addition, miR156 overexpression enhances shoot branching in Arabidopsis (Schwarz et al, 2008;Wei et al, 2012) and Brachypodium distachyon . Whether this regulation is related to sugar signaling is, however, unknown.…”

The MicroRNA156 system: A tool in plant biotechnology

“…Similarly, the transcript level of miR156 is reduced by feedback when the activation-tagged Arabidopsis sk156 mutant overexpresses a miR156-insensitive SPL15 transgene (Wei et al, 2012). In the latter plant, analysis of the miR156 promoter region revealed SPL binding sites with three repeated GTAC core sequences between -200 bp to -220 bp, suggesting that SPL15 is directly (rather than indirectly) controlling miR156 expression (Wei et al, 2012).…”

“…SPL proteins are characterized by the presence of conserved DNA binding domains that allow them to bind and regulate downstream genes containing a consensus SPL Binding Domain (SBD) element in their promoters (Yamaguchi et al, 2009;Wei et al, 2012). So far, miR156 has been shown to target 11 out of the 17 SPL member genes in Arabidopsis, 11 members of the 19 SPLs in rice, three in tomato and alfalfa, and two in L. japonicus and maize (Mica et al, 2006;Xie et al, 2006;Moxon et al, 2008;Nonogaki, 2010;Wang et al, 2014a;Aung et al, 2015).…”

“…In the latter plant, analysis of the miR156 promoter region revealed SPL binding sites with three repeated GTAC core sequences between -200 bp to -220 bp, suggesting that SPL15 is directly (rather than indirectly) controlling miR156 expression (Wei et al, 2012).…”