Final Report Summary - AMAIZE (Atlas of leaf growth regulatory networks in MAIZE) Genetic and environmental factors control plant organ size, a key component of crop productivity and yield stability. At the onset of the AMAIZE project, limited knowledge was available on how organ size is regulated in monocotyledoneous plants such as maize. The importance of the transition between cell division and cell expansion was underscored by the number of molecular players influencing this transition in Arabidopsis [1] and by the local accumulation of gibberellins (GAs) at this transition in the maize leaf [2]. Therefore, the ambition of the AMAIZE project was to map the dynamics of the cellular and molecular processes specifically at the transition between cell division and cell expansion, using the maize leaf growth zone as a model. The processes of cell division and cell expansion occur physically separated at the base of the maize leaf and the size of the maize leaf allows for high-resolution sampling of tissues enriched for the two processes for molecular techniques. To determine where and when the transition from cell division to cell expansion takes place in the different tissue layers, a 3D reconstruction of the growth zone of maize leaves was made (WP1) using confocal imaging and the MorphoGraphX software [3]. The quantitative information extracted from the obtained 3D model was complemented with ultrastructural information obtained by transmission electron microscopy.A high-resolution transcriptome with five-millimeter intervals for two conditions that, compared to control leaves, oppositely affected the position of the transition zone between cell division and cell expansion was performed to map the transcriptome changes in the growth zone (WP2). These transcriptome data are made accessible to the community through a web interface called Leaf Growth Viewer (LGV; [4]). The correlation between a set of leaf growth traits and transcriptomics of the basal part of the growing maize leaf in two independent recombinant inbred populations, resulted in a set of genes that were consistently (anti-)correlated with growth [5, 6], revealing novel insights in the regulation of growth such as the importance of protein synthesis [5]. We also examined cellularly and molecularly how the introduction of growth-enhancing transgenes affects heterosis in a panel of hybrids [7]. The transient interaction of regulatory proteins with protein complexes changes as cells progress from cell division to cell expansion [8]. Since this initial proof of concept, several growth related proteins were entered in the pipeline as bait proteins to study the dynamics in protein complex composition between dividing and expanding cells (WP3; Bontinck et al., in preparation) and in the target genes of bait proteins with DNA binding characteristics by chromatin immunoprecipitation (ChIP) (WP4; [9]).The growth regulatory network has now evolved toward a roadmap of genome-wide (co-)expressed genes, interacting proteins and target genes, resulting in the uncovering of the complex regulation of the interplay between cell division and cell expansion (WP5; Slabbinck et al., in preparation, De Vos et al., submitted, Band et al., in preparation) and allowing to select interesting candidates for functional analysis (WP6) using the greenhouse infrastructure for automated maize phenotyping (PHENOVISION) equipped with RGB, thermal-IR and hyperspectral imaging as well as field trials (Nelissen et al., submitted, Van Hautegem et al., submitted, Rodrigues et al., in preparation).In summary, the AMAIZE project helped to build a maize growth regulatory network with genome-wide dimensions and to acquire new biological insights. The observation that not only the growth rate, but also the duration of growth, is important for organ size was first made when growing the samples for mild drought conditions and was confirmed in the detailed analysis of 200 RILs for ten leaf size elated traits. Later, our transgenic research identified a molecular player in the process, showing the complementarity of the research spanning the AMAIZE project. The AMAIZE project also resulted in an expanded toolbox (LGV, gene stacking, genome editing, PHENOwell) that will be made available to the maize community. Finally, being awarded the AMAIZE ERC project was an incentive to take the forefront in stimulating an open European community in molecular maize research (e.g. conferences Hamburg (May 2016), Ghent (May 2017), Montpellier (May 2019)).1.Gonzalez et al., 2012 Trends in Plant Science 17(6): p. 332-340; 2.Nelissen H., et al., 2012. Current Biology, 22(13): p. 1183-1187; 3.Barbier de Reuille, P., et al. 2015 eLife, 4: p. 05864-05864; 4.Nelissen H., et al., 2018 Plant biotechnology journal, 16(2): p. 615-627; 5. Baute, J., et al., 2016 Plant physiology, 170(3): p. 1848-1867; 6.Baute J., et al., 2015. Genome biology, 16(1): p. 168-168; 7.Feys K., et al., 2018 Plant, cell & environment, 41(2): p. 374-382; 8. Nelissen, H., et al., 2015. The Plant Cell, 2015. 27(6): p. 1605-1619; 9. Besbrugge, N., et al., 2018. Plant physiology, 177(2): p. 447-464.
