Comparative analysis of the miRNA171-mediated gene regulation in land plants
Final Report Abstract
MicroRNAs (miRNAs) are small non-coding RNAs which bind to complementary sequence motifs within their target RNAs to direct RNA cleavage or translational inhibition. To analyze whether a sequence-based conservation provokes similar functions we studied conserved miR171-regulated networks in moss and seed plants. In tomato we identified eleven miR171 loci coding for two groups of sly-miR171 that were silenced by overexpression of a short tandem target mimic (STTM171). Resulting transgenic lines showed significantly reduced sly-miR171 levels and increased target SlHAMs expression provoking abnormal leaf development, increased axillary branch formation and sterility caused by abnormal tapetum development. Tomato encodes three HAM homologs (SlHAM, SlHAM2 and SlHAM4) out of which SlHAM and SlHAM2 (SlHAMs) are guided for cleavage by both sly-miR171 subgroups. SlHAMs, but not SlHAM4, are abundant in the shoot and floral meristems as well as in the compound leaf primordia and silencing them in these tissues by sly-miR171 overexpression led to the formation of ectopic meristematic cell clusters (EMCC). Similarly, leaf-specific silencing of SlHAMs provoked EMCC formation in the organogenic compound leaf rachis. Further, we demonstrated that EMCC formation in both meristems and leaves was due to the misexpression of the stem cell regulator WUSCHEL (SlWUS), previously shown to be induced by cytokinin. Strikingly, reduction of cytokinin levels in SlHAMs-silenced leaves completely suppressed the formation of EMCC, suggesting a regulatory link between SlHAMs and cytokinin. To decipher the genetic network underlying thee formation of EMCC we performed RNAseq of 35S>>MIR171b revealing the upregulation of SlWUS and orthologs of CLV3 and KNOX consistent with the increase in stem cell numbers and their meristematic descendants in the 35S>>MIR171b SAM. More importantly, Plant MetaGenMAP analysis indicated a significant downregulation of the cytokinin degradation pathway supporting our hypothesis that SlHAMs may promote cellular differentiation in the SAM by negative regulation of SAM cytokinin levels. In the moss Physcomitrella patens we identified two miR171 targets that encode GRAS domain transcription factors (PpGRAS1 and PpGRAS2). Surprisingly, we observed only mild phenotypic changes in targeted single (ΔPpGRAS1, ΔPpGRAS2) as well as double (ΔPpGRAS1/2) knockout mutants which strongly contrasts the phenotypes observed in tomato. However, inducible overexpression of miR171-resistant versions of PpGRAS1 and PpGRAS2 caused severe, but distinct phenotypes where only PpGRAS1 overexpression lines are able to recover after release to non-inducing conditions. Elevated levels of PpGRAS1 cause the formation of multiple, but arrested apical meristems which develop into miniature gametophores upon release to non-inducing conditions. Increased levels of PpGRAS2 lead to irreversible defects and the degradation of chloroplasts. Based on these results we postulate key regulatory functions of PpGRAS1 in the control of meristem identity and the requirement of PpGRAS2 in plastid maintenance and homeostasis. We further generated P. patens lines harboring inducible expression constructs of the two closest A. thaliana PpGRAS homologs. Strikingly, these homologs are not miR171-regulated, but their inducible overexpression led to similar developmental phenotypes as observed in PpGRAS1 and 2 overexpression lines. Thus, we propose a complex evolution and diversification of miR171-dependent regulatory networks in land plants.
Publications
- (2016) The tomato HAIRY MERISTEM genes are involved in meristem maintenance and compound leaf morphogenesis. Journal of Experimental Botany, 67, 6187-6200
Hendelman, A., Kravchik, M., Stav, R., Frank, W., Arazi, T.
(See online at https://doi.org/10.1093/jxb/erw388)
