Dynamic Changes in ANGUSTIFOLIA3 Complex Composition Reveal a Growth Regulatory Mechanism in the Maize Leaf

Abstract

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 growth-regulating 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.

Figure 1.

Model of the Protein Complex Associated with AN3 as Identified in the TAP Experiments. (A) Schematic representation of the core complex, based on data from Arabidopsis (Vercruyssen et al., 2014) and maize (leaf division zone [DZ], leaf elongation zone [EZ], and ear TAPs), and the maize tissue- or organ-specific GRFs as determined by quantitative analysis (leaf) and identification (ear). (B) Schematic representation of the AN3/GIF1-associated chromatin remodeling core complex at the different positions in the leaf. The core complex is present at dividing and expanding tissue but is recruited to the target DNA by growth process-specific GRFs (blue is division zone specific; green is expansion zone specific). The components of the core complex, as well as the GRFs, are depleted from the mature leaf samples. For several components, differential phosphorylation throughout the leaf developmental gradient has been shown (Facette et al., 2013). (C) Model of how the specific interaction of the GRFs with the AN3-associated chromatin remodeling complex regulates the transition from cell division to cell expansion, based on the phenotypes of the GRF10 (Wu et al., 2014) and the GRF1^R overexpression lines.

Figure 2.

Relative Intensity-Based Label-Free Quantification in Maize Leaf: AN3/GIF1 TAP Copurified Proteins from Division Zone (1st cm) Compared with Expansion Zone (4th cm). Significantly changing interactors of AN3/GIF1 are identified by a permutation-based FDR-corrected t test (threshold FDR = 0.01 and S0 = 0.9; Supplemental Table 4). The difference in average log₂ LFQ intensities between division zone (group 1) and expansion zone (group 2) is plotted versus the significance (–log₁₀ [P value]). The curve indicates the permutation-based FDR threshold. Positions of AN3/GIF1 bait, tag, GRFs, and core subunits (in green) are marked on the plot.

Figure 3.

Phenotypes of Nontransgenic and Transgenic GRF1^R Segregating Plants of the Three Independent GRF1^R Overexpression Lines. (A) Expression levels of GRF1 in the GRF1^R-overexpressing transgenic plants and the nontransgenic siblings over the maize leaf growth zone for three independent transgenic events. Quantitative RT-PCR was performed with primers specific for GRF1 and normalized relative to 18S rRNA. NTG, nontransgenic siblings; TG, transgenic siblings. Error bars represent se. (B) Seedling phenotype of line 2. The arrows indicate leaf 3. (C) Final length of leaf 4 of two independent transgenic events. Error bars represent se. (D) Size of the division zone of two independent transgenic events. Three asterisks indicate P value < 0.01, and one asterisk indicates P value < 0.05. Error bars represent se. (E) Overall plant height phenotype of line 1. (F) Tassel phenotype of line 2. (G) Ears of crosses between B104 and nontransgenic or transgenic plants and crosses between the nontransgenic and transgenic plants with B104. The inset panel shows an enlargement of the macrohairs formed on and between files of spikelets.