Transcriptomic analysis of EPFL and Cycloheximide treated clv3 epfl1 epfl2 epfl4 epfl6 mutants
Maintenance and regulation of the shoot apical meristem size and boundaries are essential for plant growth and leaf organogenesis. Canonically, CLAVATA 1, CLAVATA 3 and WUSCHEL control shoot apical meristem (SAM) size and SAM maintenance. Recently it has been shown that ERECTA (ER) family genes also control SAM size at its boundaries. The ERECTA family of receptor like protein kinases are bound by EPIDERMAL PATTERNING FACTOR LIKE (EPFL) protein ligands. Loss of the ER receptor genes or a subset of the EPFL ligands lead to an enlarged meristem. Loss of both CLAVATA genes and ER family genes synergize to produce even larger meristems with a loss of leaf production. In this experiment we perform deep sequencing to measure the transcriptome wide molecular changes in the clv3 epfl1 epfl2 epfl4 epfl6 quintuple mutant under mock, 3h ectopic EPLF6 ligand, 10 minute pretreatment with cycloheximide (CHX) and combined 10 minute CHX pretreatment followed by a 3h EPFL6 treatment. Treatment of the quintuple mutant with EPFL6 leads to a modest but targeted downregulation of MEI2 C-TERMINAL RRM ONLY LIKE 1(MCT1) RNA binding family proteins. CHX treatment was used to inhibit the translation of new proteins. By blocking new protein production, only direct signaling targets of EPFL6 would be seen. The severe toxic effects of CHX leads to a complete rearrangement of the transcriptome. However, EPFL6 still alters some CHX targets, mostly through downregulation. Overall design: Pairwise comparisons on a two factorial design of Mock, EPFL6, cycloheximide and  EPFL6+cycloheximide treatment