Most molecular processes during plant development occur with a particular spatio-temporal specificity. Thus far, it has remained technically challenging to capture dynamic protein-protein interactions within a growing organ, where the interplay between cell division and cell expansion is instrumental. Here, we combined high-resolution sampling of the growing maize (Zea mays) leaf with tandem affinity purification followed by mass spectrometry. Our results indicate that the growthregulating SWI/SNF chromatin remodeling complex associated with ANGUSTIFOLIA3 (AN3) was conserved within growing organs and between dicots and monocots. Moreover, we were able to demonstrate the dynamics of the AN3-interacting proteins within the growing leaf, since copurified GROWTH-REGULATING FACTORs (GRFs) varied throughout the growing leaf. Indeed, GRF1, GRF6, GRF7, GRF12, GRF15, and GRF17 were significantly enriched in the division zone of the growing leaf, while GRF4 and GRF10 levels were comparable between division zone and expansion zone in the growing leaf. These dynamics were also reflected at the mRNA and protein levels, indicating tight developmental regulation of the AN3-associated chromatin remodeling complex. In addition, the phenotypes of maize plants overexpressing miRNA396a-resistant GRF1 support a model proposing that distinct associations of the chromatin remodeling complex with specific GRFs tightly regulate the transition between cell division and cell expansion. Together, our data demonstrate that advancing from static to dynamic protein-protein interaction analysis in a growing organ adds insights in how developmental switches are regulated.

DNA methylation is an important and widespread epigenetic modification in plant genomes, mediated by DNA methyltransferases (DMTs). DNA methylation is known to play a role in genome protection, regulation of gene expression, and splicing and was previously associated with major developmental reprogramming in plants, such as vernalization and transition to flowering. Here, we show that DNA methylation also controls the growth processes of cell division and cell expansion within a growing organ. The maize (Zea mays) leaf offers a great tool to study growth processes, as the cells progressively move through the spatial gradient encompassing the division zone, transition zone, elongation zone, and mature zone. Opposite to de novo DMTs, the maintenance DMTs were transcriptionally regulated throughout the growth zone of the maize leaf, concomitant with differential CCGG methylation levels in the four zones. Surprisingly, the majority of differentially methylated sequences mapped on or close to gene bodies and not to repeat-rich loci. Moreover, especially the 59 and 39 regions of genes, which show overall low methylation levels, underwent differential methylation in a developmental context. Genes involved in processes such as chromatin remodeling, cell cycle progression, and growth regulation, were differentially methylated. The presence of differential methylation located upstream of the gene anticorrelated with transcript expression, while gene body differential methylation was unrelated to the expression level. These data indicate that DNA methylation is correlated with the decision to exit mitotic cell division and to enter cell expansion, which adds a new epigenetic level to the regulation of growth processes.DNA methylation is the covalent modification of nucleotides in DNA by the addition of a methyl group. In the nuclear genome of higher eukaryotes, 5-methylcytosine is the most important DNA modification (Goll and Bestor, 2005). It is a phenomenon of ancient origin predating plant-animal diversification. However, some differences exist between plant and animal methylome patterning and function, and DNA methylation has been found to be evolutionarily lost in a few species (Feng et al., 2010;Zemach et al., 2010). Eukaryotic DNA methylation is established by DNA methyltransferase (DMT) enzymes that transfer a methyl group from S-adenosyl Met to the fifth carbon of cytosine. These enzymes can largely be subdivided in maintenance and de novo DMTs, depending on whether the recognition site is already methylated or not. Maintenance DMTs conserve the methylation status of symmetrical (palindromic) sites after DNA replication, by recognizing the hemimethylated locus and methylating the newly synthesized strand. In plants, there are two types of maintenance DMTs: DNA METHYLTRANSFERASE (MET) and CHROMOMETHYLASE (CMT). The former methylates CG sites during DNA replication, whereas the latter methylates CHG (H = A, C, or T) sites located in chromatin in which histone 3 is dimethylated on Lys-9 (Goll and Bestor, 2005). De no...