Genetic tango: unveiling maize’s endosperm filling regulation orchestrated by NAKED ENDOSPERM1, NAKED ENDOSPERM2, and OPAQUE2

The cereal endosperm is a vital energy store for germination and growth and serves as a key resource for human and animal consumption and industry. After fertilization, the trip-loid endosperm experiences coenocytic growth ( ∼ 1 to 3 days after pollination [DAP]) and cellularization ( ∼ 3 to 4 DAP), followed by differentiation ( ∼ 4 DAP). This phase gives rise to diverse cell types with unique features and functions. Roughly spanning 8 to 16 DAP, a pivotal shift occurs, transitioning from cellular growth to grain filling. In orchestrating this shift, transcription factors (TFs) NAKED ENDOSPERM1 (NKD1


In Brief
The cereal endosperm is a vital energy store for germination and growth and serves as a key resource for human and animal consumption and industry.After fertilization, the triploid endosperm experiences coenocytic growth (∼1 to 3 days after pollination [DAP]) and cellularization (∼3 to 4 DAP), followed by differentiation (∼4 DAP).This phase gives rise to diverse cell types with unique features and functions.Roughly spanning 8 to 16 DAP, a pivotal shift occurs, transitioning from cellular growth to grain filling.In orchestrating this shift, transcription factors (TFs) NAKED ENDOSPERM1 (NKD1), NAKED ENDOSPERM2 (NKD2), and OPAQUE2 (O2) are key players (Schmidt et al. 1990).In this issue of The Plant Cell, Hao Wu and colleagues (Wu et al. 2023) found the intricate regulatory connections among these 3 TFs, as these factors collaboratively control gene networks during the shift from cellular development to grain-filling in maize endosperm (Fig.).
Previous studies have shown that NKD1, NKD2, and O2 are involved in grain-filling and exhibit interconnected relationships.nkd1 and nkd2 double mutants (nkd1;2) impact endosperm development and cause multiple compromised peripheral cell layers.This suggests that NKD1 and NKD2 restrict cell count while promoting differentiation.Also, in grain-filling, NKD1 and NKD2 positively influence storage protein genes.The nkd1;2 mutant displays reduced starch, changes in starch branching, and irregular granules (Gontarek et al. 2016).Mutations in o2 lead to an opaque kernel appearance by reducing storage protein levels.Target genes of O2 are primarily related to nutrient reservoir functions like zein storage proteins, establishing its significance during grain filling.Single nkd1 or nkd2 mutants show increased expression of NKD2 or NKD1, respectively (Yi et al. 2015).NKD2 directly represses NKD1 expression, forming a feedback loop that could regulate NKD TF levels.NKD1 and NKD2 activate O2 while nkd1;2 mutants downregulate O2 (Gontarek et al. 2016).O2 activates NKD2 and, together with NKD2, co-activates genes important for storage protein biosynthesis.Wu et al. (2023) generated homozygous mutant combinations for nkd1, nkd2, and o2 in a W22 inbred background to investigate their combined impact.The o2 mutants displayed kernels of normal size but reduced translucency.The nkd1;2 double mutant exhibited small, wrinkled kernels with reduced translucency.Remarkably, the nkd1;nkd2;o2 triple mutant showed a highly shrunken, wrinkled phenotype.Furthermore, the nkd1;o2 double-mutant kernels displayed a unique indentation below the crown region, which was absent in other genotypes.These observations imply an interaction between the NKD1, NKD2, and O2 genes during grain development.
RNAseq was performed on developing endosperm tissues from each genotype from 8 DAP to 16 DAP to compare gene regulatory networks among the 3 TFs.The analysis showed that the 8-DAP transcriptomes were distinctly separated from later samples, indicating a significant shift between 8 and 12 DAP as the endosperm progresses from initial cell processes to grain-filling.Genotype impact was more pronounced at 12 and 16 DAP than at 8 DAP.Notably, genotypes with o2 gene mutations were clearly separated from O2+ genotypes (homozygous wild-type O2 allele) at 12 and 16 DAP, highlighting the vital role of O2+ in grain-filling.As expected, doublemutant nkd1;2 genotypes consistently differed from single mutants and Nkd1+;Nkd2+ genotypes due to redundancy.Triple-mutant nkd1;nkd2;o2 transcriptomes clustered with earlier time points, possibly indicating delayed development in the triple-mutant endosperm.The authors then deployed Weighted Gene Co-expression Network Analysis to explore the relationships between the 3 TFs.From this analysis, the differentially expressed genes were sorted into co-expression modules, linked to nkd1, nkd2, and/or o2 expression.At 8 DAP, nkd1 exhibited a positive correlation with module that linked to starch metabolism.Conversely, at 16 DAP, nkd2 and o2 collaboratively influenced the module that is associated with storage protein production.
DNA Affinity Purification Sequencing (DAP-seq) was conducted on in vitro-produced NKD1 and NKD2 proteins to identify the DNA regulatory elements and potential target genes of these 2 TFs.This analysis showed that there was a significant overlap between DAP-seq targets of NKD2 and differentially expressed genes associated with nkd1, o2, nkd1;o2, and nkd1;nkd2;o2.Additionally, hub genes were identified, indicating strong connectivity within O2, NKD1, and NKD2.O2's targeted hub genes encompassed TFs like bZip17 and GBF1, underlining O2's hierarchical role.Among possible NKD2 targets were ARF1 and ARF29.
Together, Wu et al. (2023) demonstrate NKD1, NKD2, and O2 function dynamically, regulating functions primarily associated with cellular development at early stages and transitioning to functions associated with nutrient accumulation and storage during grain-filling in maize.These functions have been demonstrated at both the phenotypic and transcriptomic levels.They identified gene co-expression modules that are associated with biological processes, along with hub genes including potential direct targets of NKD1, NKD2, and O2.