All critical developmental and physiological events in a plant's life cycle depend on the proper activation and repression of specific gene sets, and this often involves epigenetic mechanisms. Some mutants with disorders of the epigenetic machinery exhibit pleiotropic defects, including incurved leaves and early flowering, due to the ectopic and heterochronic derepression of developmental regulators. Here, we studied one such mutant class, the () loss-of-function mutants. We have identified as the founding member of a small gene family that we have named (). This family is part of the 2-oxoglutarate/Fe(II)-dependent dioxygenase superfamily. and its closest paralog, have unequally redundant functions: although mutants are phenotypically wild type, double mutants skip vegetative development and flower upon germination. This phenotype is reminiscent of loss-of-function mutants of the Polycomb-group genes () and Double mutants harboring alleles and loss-of-function alleles of genes encoding components of the epigenetic machinery exhibit synergistic, severe phenotypes, and some are similar to those of mutants. Hundreds of genes are misexpressed in plants, including (), and derepression of causes the leaf phenotype of ICU11 and CP2 are nucleoplasmic proteins that act as epigenetic repressors through an unknown mechanism involving histone modification, but not DNA methylation.

The 2-oxoglutarate and Fe (II)-dependent dioxygenase (2OGD) superfamily includes oxidative enzymes with an active site containing two histidines and (in most cases) one aspartic or glutamic acid residue. This conserved motif is termed the 2-His-1carboxylate facial triad, chelates iron, and is housed within a double-stranded b-helix fold, also known as the DSBH, jellyroll, cupin, or Jumonji C fold (Martinez and Hausinger, 2015). The reactions catalyzed by 2OGDs (also called 2ODDs, 2ODOs, and 2OGXs) include but are not limited to demethylation, demethylenation, hydroxylation, halogenation, desaturation, ring cleavage, ring closure, and epimerization (Farrow and Facchini, 2014). The list of plant 2OGDs is long and growing; for example, the Arabidopsis thaliana (hereafter, Arabidopsis) genome contains more than 150 genes encoding proteins containing a 2OGD domain. These Arabidopsis proteins can be classified into the DOXA, DOXB, DOXC (Kawai et al., 2014), and JMJ groups, which include 14, 14, 102, and 21 proteins, respectively. DOXA proteins are homologs of Escherichia coli alpha-ketoglutaratedependent dioxygenase (AlkB), a DNA repair enzyme that reverses the N 1-methyladenine (m 1 A) and N 3-methylcytosine (m 3 C) lesions caused by alkylating agents. Nine AlkB homologs (ALKBHs) have been found in mammals, and their substrates include DNA (m 1 A and m 3 C), RNA (m 6 A, the most abundant RNA methylation mark), and proteins (methylated lysine). Two Arabidopsis DOXAs, the close paralogs ALKBH9B and ALKBH10B, have been recently found to demethylate m 6 A in RNA (Figure 1A). ALKBH9B is the first plant RNA demethylase described that demethylates m 6 A marks of specific foreign RNAs. ALKBH9B physically interacts with the coat protein of Alfalfa mosaic virus (AMV), thereby regulating its capacity for infection. AMV-infected alkbh9b Arabidopsis mutants exhibit reduced systemic infection and lower levels of viral RNA, which was found to be hypermethylated. In addition, ALKBH9B demethylates single-stranded AMV RNA in vitro (Martínez-Pé rez et al., 2017). ALKBH10B demethylates endogenous mRNAs in Arabidopsis. The alkbh10b mutant flowers late due to instability of mRNAs from genes regulating flowering time. ALKBH10B reduces the m 6 A methylation of FLOWERING LOCUS T, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 (SPL3), and SPL9 mRNAs, increasing their stability and capacity to trigger the floral transition (Duan et al., 2017).

The paralogous genesINCURVATA11(ICU11) andCUPULIFORMIS2(CP2) encode components of the epigenetic machinery in Arabidopsis and belong to the 2-oxoglutarate and Fe (II)-dependent dioxygenase superfamily. We previously inferred unequal functional redundancy betweenICU11andCP2from a study of the synergistic phenotypes of the double mutant and sesquimutant combinations oficu11andcp2mutations, although they represented mixed genetic backgrounds. To avoid potential confounding effects arising from different genetic backgrounds, we generated theicu11-5andicu11-6mutants via CRISPR/Cas genome editing in the Col-0 background and crossed them tocp2mutants in Col-0. The resulting mutants exhibited a postembryonic-lethal phenotype reminiscent of strongembryonic flower(emf) mutants. Double mutants involvingicu11-5and mutations affecting epigenetic machinery components displayed synergistic phenotypes, whereascp2-3did not besidesicu11-5. Our results confirmed the unequal functional redundancy betweenICU11andCP2and demonstrated that it is not allele or genetic background specific. An increase in sucrose content in the culture medium partially rescued the post-germinative lethality oficu11 cp2double mutants and sesquimutants, facilitating the study of their morphological phenotypes throughout their life cycle, which include floral organ homeotic transformations. We thus established that theICU11-CP2module is required for proper flower organ identity.