NAKED ENDOSPERM1, NAKED ENDOSPERM2, and OPAQUE2 interact to regulate gene networks in maize endosperm development

Abstract

NAKED ENDOSPERM1 (NKD1), NKD2 and OPAQUE2 are transcription factors important for cell patterning and nutrient storage in maize (Zea mays) endosperm. To study the complex regulatory interrelationships among these three factors in co-regulating gene networks, we developed a set of nkd1, nkd2 and o2 homozygous lines, including all combinations of mutant and wild-type genes. Among the eight genotypes tested, we observed diverse phenotypes and gene interactions affecting cell patterning, starch content, and storage proteins. From ∼8 to ∼16 days after pollination, maize endosperm undergoes a transition from cellular development to nutrient accumulation for grain filling. Gene network analysis showed that NKD1, NKD2 and O2 dynamically regulate a hierarchical gene network during this period, directing cellular development early, then transitioning to constrain cellular development while promoting the biosynthesis and storage of starch, proteins and lipids. Genetic interactions regulating this network are also dynamic. The assay for transposase-accessible chromatin using sequencing (ATAC-seq) showed that O2 influences the global regulatory landscape, decreasing NKD1 and NKD2 target site accessibility, while NKD1 and NKD2 increase O2 target site accessibility. In summary, interactions of NKD1, NKD2 and O2 dynamically affect the hierarchical gene network and regulatory landscape during the transition from cellular development to grain filling in maize endosperm.

Transcription factor binding site divergence across maize inbred lines drives transcriptional and phenotypic variation

“…Specifically, we tested 300 unique TFs and obtained high quality data for 166 maize TFs. Combined with data from previous studies (Galli et al 2018) (Ricci et al 2019) (Dong et al 2020) (Dai et al 2022) (Wu et al 2023) (Bang et al, 2024), this provided DAP-seq data for 30 unique DNA-binding TF families out of 36 total families tested (Supplemental Figure 1a). The number of peaks identified for each TF ranged from 1,153 to 122,695 giving an average of ~33,000 peaks (Supplemental Figure 1b, Supplemental Table 1).…”

“…This panel consisted of 66 TFs and TF combinations (i.e. trimers of NFY-A, NFY-B, and NFY-C, and dimers of BHLH67/BA1 and ZmSPTs discussed above) from this study and several previously published maize DAP-seq datasets ( Galli et al 2018 ) ( Ricci et al 2019 ) ( Dong et al 2020 ) ( Dai et al 2022 ) ( Wu et al 2023 ) (Bang et al, 2024). Only datasets that showed unique binding motifs, belonged to a distinct subfamily, and/or had a Pearson correlation less than 0.5 were included.…”

Establishing an optimized ATAC-seq protocol for the maize

“…Unlike animal samples, where cells can be directly used as input for the transposition reaction immediately after lysis with a gentle detergent, plant tissues require physical disruption to release cell contents from the rigid cell walls. Thus, various methods were attempted to break down plant cell walls for plant ATAC-seq, such as grinding samples in liquid nitrogen ( Galli et al., 2018 ; Deschamps et al., 2021 ; Wu et al., 2023 ), digesting cell walls for protoplast preparation ( Dong et al., 2017 ; Dai et al., 2022 ), or disrupting plant tissues using homogenizer or razor blade ( Lu et al., 2017 ; Ricci et al., 2019 ). In addition to cell walls, another obstacle in plants is the higher quantity of organelles (i.e.…”

“…As Tn5p targets not only nuclear DNA but also organellar DNA, the presence of a significant amount of organellar DNA can lead to up to 50% of sequencing reads being unusable ( Montefiori et al., 2017 ). To purify plant nuclei from organelles, flow cytometry is commonly used, where nuclei stained with DAPI or expressing fluorescent proteins are sorted ( Lu et al., 2017 ; Galli et al., 2018 ; Noshay et al., 2019 ; Ricci et al., 2019 ; Wu et al., 2023 ). Alternatively, Deal and Henikoff developed a method, named the isolation of nuclei tagged in specific cell types (INTACT), which involves applying streptavidin-coated magnetic beads to isolate biotin-labeled nuclei extracted from plant samples ( Deal and Henikoff, 2011 ).…”

“…ATAC-seq has been employed in maize genome research, where most ATAC-seq results derive from tens of thousands of nuclei isolated by flow cytometry or prepared from mesophyll protoplasts ( Dong et al., 2017 ; Galli et al., 2018 ; Noshay et al., 2019 ; Ricci et al., 2019 ; Crisp et al., 2020 ; Wu et al., 2023 ). To make the ATAC-seq method more applicable easily, we optimize critical steps in this study and develop a robust and efficient methodology for ATAC-seq analysis in maize.…”