The biosynthesis of storage reserves and auxin is coordinated by a hierarchical regulatory network in maize endosperm.

Grain filling in maize (Zea mays) is intricately linked to cell development, involving the regulation of genes responsible for the biosynthesis of storage reserves (starch, proteins, and lipids) and phytohormones. However, the regulatory network coordinating these biological functions remains unclear. In this study, we identified 1744 high-confidence target genes co-regulated by the transcription factors (TFs) ZmNAC128 and ZmNAC130 (ZmNAC128/130) through chromatin immunoprecipitation sequencing coupled with RNA-seq analysis in the zmnac128/130 loss-of-function mutants. We further constructed a hierarchical regulatory network using DNA affinity purification sequencing analysis of downstream TFs regulated by ZmNAC128/130. In addition to target genes involved in the biosynthesis of starch and zeins, we discovered novel target genes of ZmNAC128/130 involved in the biosynthesis of lipids and indole-3-acetic acid (IAA). Consistently, the number of oil bodies, as well as the contents of triacylglycerol, and IAA were significantly reduced in zmnac128/130. The hierarchical regulatory network centered by ZmNAC128/130 revealed a significant overlap between the direct target genes of ZmNAC128/130 and their downstream TFs, particularly in regulating the biosynthesis of storage reserves and IAA. Our results indicated that the biosynthesis of storage reserves and IAA is coordinated by a multi-TFs hierarchical regulatory network in maize endosperm.

Recent advances in exploring transcriptional regulatory landscape of crops.

Crop breeding entails developing and selecting plant varieties with improved agronomic traits. Modern molecular techniques, such as genome editing, enable more efficient manipulation of plant phenotype by altering the expression of particular regulatory or functional genes. Hence, it is essential to thoroughly comprehend the transcriptional regulatory mechanisms that underpin these traits. In the multi-omics era, a large amount of omics data has been generated for diverse crop species, including genomics, epigenomics, transcriptomics, proteomics, and single-cell omics. The abundant data resources and the emergence of advanced computational tools offer unprecedented opportunities for obtaining a holistic view and profound understanding of the regulatory processes linked to desirable traits. This review focuses on integrated network approaches that utilize multi-omics data to investigate gene expression regulation. Various types of regulatory networks and their inference methods are discussed, focusing on recent advancements in crop plants. The integration of multi-omics data has been proven to be crucial for the construction of high-confidence regulatory networks. With the refinement of these methodologies, they will significantly enhance crop breeding efforts and contribute to global food security.

Callus-specific CRISPR/Cas9 system to increase heritable gene mutations in maize.

MAIN CONCLUSION: A callus-specific CRISPR/Cas9 (CSC) system with Cas9 gene driven by the promoters of ZmCTA1 and ZmPLTP reduces somatic mutations and improves the production of heritable mutations in maize. The CRISPR/Cas9 system, due to its editing accuracy, provides an excellent tool for crop genetic breeding. Nevertheless, the traditional design utilizing CRISPR/Cas9 with ubiquitous expression leads to an abundance of somatic mutations, thereby complicating the detection of heritable mutations. We constructed a callus-specific CRISPR/Cas9 (CSC) system using callus-specific promoters of maize Chitinase A1 and Phospholipid transferase protein (pZmCTA1 and pZmPLTP) to drive Cas9 expression, and the target gene chosen for this study was the bZIP transcription factor Opaque2 (O2). The CRISPR/Cas9 system driven by the maize Ubiquitin promoter (pZmUbi) was employed as a comparative control. Editing efficiency analysis based on high-throughput tracking of mutations (Hi-TOM) showed that the CSC systems generated more target gene mutations than the ubiquitously expressed CRISPR/Cas9 (UC) system in calli. Transgenic plants were generated for the CSC and UC systems. We found that the CSC systems generated fewer target gene mutations than the UC system in the T0 seedlings but reduced the influence of somatic mutations. Nearly 100% of mutations in the T1 generation generated by the CSC systems were derived from the T0 plants. Only 6.3-16.7% of T1 mutations generated by the UC system were from the T0 generation. Our results demonstrated that the CSC system consistently produced more stable, heritable mutants in the subsequent generation, suggesting its potential application across various crops to facilitate the genetic breeding of desired mutations.

Expansion and improvement of ChinaMu by MuT-seq and chromosome-level assembly of the Mu-starter genome.

ChinaMu is the largest sequence-indexed Mutator (Mu) transposon insertional library in maize (Zea mays). In this study, we made significant improvements to the size and quality of the ChinaMu library. We developed a new Mu-tag isolation method Mu-Tn5-seq (MuT-seq). Compared to the previous method used by ChinaMu, MuT-seq recovered 1/3 more germinal insertions, while requiring only about 1/14 of the sequencing volume and 1/5 of the experimental time. Using MuT-seq, we identified 113,879 germinal insertions from 3,168 Mu-active F1 families. We also assembled a high-quality genome for the Mu-active line Mu-starter, which harbors the initial active MuDR element and was used as the pollen donor for the mutation population. Using the Mu-starter genome, we recovered 33,662 (15.6%) additional germinal insertions in 3,244 (7.4%) genes in the Mu-starter line. The Mu-starter genome also improved the assignment of 117,689 (54.5%) germinal insertions. The newly upgraded ChinaMu dataset currently contains 215,889 high-quality germinal insertions. These insertions cover 32,224 pan-genes in the Mu-starter and B73Ref5 genomes, including 23,006 (80.4%) core genes shared by the two genomes. As a test model, we investigated Mu insertions in the pentatricopeptide repeat (PPR) superfamily, discovering insertions for 92% (449/487) of PPR genes in ChinaMu, demonstrating the usefulness of ChinaMu as a functional genomics resource for maize.

Decoding the gene regulatory network of endosperm differentiation in maize.

The persistent cereal endosperm constitutes the majority of the grain volume. Dissecting the gene regulatory network underlying cereal endosperm development will facilitate yield and quality improvement of cereal crops. Here, we use single-cell transcriptomics to analyze the developing maize (Zea mays) endosperm during cell differentiation. After obtaining transcriptomic data from 17,022 single cells, we identify 12 cell clusters corresponding to five endosperm cell types and revealing complex transcriptional heterogeneity. We delineate the temporal gene-expression pattern from 6 to 7 days after pollination. We profile the genomic DNA-binding sites of 161 transcription factors differentially expressed between cell clusters and constructed a gene regulatory network by combining the single-cell transcriptomic data with the direct DNA-binding profiles, identifying 181 regulons containing genes encoding transcription factors along with their high-confidence targets, Furthermore, we map the regulons to endosperm cell clusters, identify cell-cluster-specific essential regulators, and experimentally validated three predicted key regulators. This study provides a framework for understanding cereal endosperm development and function at single-cell resolution.

Ae1/Sbe1 maize-derived high amylose improves gut barrier function and ameliorates type II diabetes in high-fat diet-fed mice by increasing Akkermansia.

Type II diabetes mellitus (T2DM) has its origins in chronic inflammation due to immune dysregulation. Improving chronic inflammation can significantly reduce the probability of T2DM and the rate of disease progression. Resistance to starch 2 (RSII) high-amylose maize starch (HAMS) has been widely implicated in the improvement and regulation of T2DM. However, its exact molecular mechanisms have not been fully discovered. Here, we used CRISPR/Cas9 technology to knock out two starch-branching enzyme genes, Ae1 and Sbe1, in maize to obtain mutants containing higher levels of HAMS. In experiments in which HAMS was fed to mice on a high-fat diet (HFD), we confirmed the function of HAMS in ameliorating hyperglycemia. Mechanistically, we found that HAMS improves the gut barrier function by increasing the Akkermansia abundance in the gut. This increase led to the alleviation of chronic inflammation in mice on a HFD, resulting in improved insulin sensitivity and a decrease in blood glucose.

Endosperm-specific accumulation of human α-lactalbumin increases seed lysine content in maize.

KEY MESSAGE: This study demonstrated high expression and accumulation of human α-lactalbumin in transgenic maize, and significant improvement of lysine content in maize endosperm. As a high-yield crop, lack of lysine in endosperm storage protein is a major defect of maize (Zea mays L.). Specifically expression of foreign proteins is a potential way to improve lysine content in maize endosperm. Human α-lactalbumin is such a protein with high lysine content and high nutritional value. In this study, the codon-optimized human lactalbumin alpha (LALBA) gene was driven by maize endosperm-specific 27 kD γ-zein promoter, and transformed into maize. Five independent transgenic lines were obtained, and LALBA was highly expressed in endosperm in all these lines. Protein assay indicated that human α-lactalbumin was highly accumulated in maize endosperm. Immuno-localization assay indicated that human α-lactalbumin was mainly deposited into the protein body (PB). Protein interaction assay showed that human α-lactalbumin interacted with 16 kD γ-zein, which might lead to its deposition to the PBs. Amino acid analysis of two independent transgenic lines showed significant increase of lysine contents in transgenic endosperm, with 47.26% and 45.15% increase to their non-transgenic seeds, respectively. We obtained transgenic maize with endosperm-specific accumulation of human α-lactalbumin at high level and increased the lysine content in maize endosperm. This study demonstrated an effective way to improve the nutritional value of maize seeds.

Accumulation of 22 kDa α-zein-mediated nonzein protein in protein body of maize endosperm.

Protein bodies (PBs), the major protein storage organelle in maize (Zea mays) endosperm, comprise zeins and numerous nonzein proteins (NZPs). Unlike zeins, how NZPs accumulate in PBs remains unclear. We characterized a maize miniature kernel mutant, mn*, that produces small kernels and is embryo-lethal. After cloning the Mn* locus, we determined that it encodes the mitochondrial 50S ribosomal protein L10 (mRPL10). MN* localized to mitochondria and PBs as an NZP; therefore, we renamed MN* Non-zein Protein 1 (NZP1). Like other mutations affecting mitochondrial proteins, mn* impaired mitochondrial function and morphology. To investigate its accumulation mechanism to PBs, we performed protein interaction assays between major zein proteins and NZP1, and found that NZP1 interacts with 22 kDa α-zein. Levels of NZP1 and 22 kDa α-zein in various opaque mutants were correlated. Furthermore, NZP1 accumulation in induced PBs depended on its interaction with 22 kDa α-zein. Comparative proteomic analysis of PBs between wild-type and opaque2 revealed additional NZPs. A new NZP with plastidial localization was also found to accumulate in induced PBs via interaction with 22 kDa α-zein. This study thus reveals a mechanism for accumulation of NZPs in PBs and suggests a potential application for the accumulation of foreign proteins in maize PBs.

ENB1 encodes a cellulose synthase 5 that directs synthesis of cell wall ingrowths in maize basal endosperm transfer cells.

Development of the endosperm is strikingly different in monocots and dicots: it often manifests as a persistent tissue in the former and transient tissue in the latter. Little is known about the controlling mechanisms responsible for these different outcomes. Here we characterized a maize (Zea mays) mutant, endosperm breakdown1 (enb1), in which the typically persistent endosperm (PE) was drastically degraded during kernel development. ENB1 encodes a cellulose synthase 5 that is predominantly expressed in the basal endosperm transfer layer (BETL) of endosperm cells. Loss of ENB1 function caused a drastic reduction in formation of flange cell wall ingrowths (ingrowths) in BETL cells. Defective ingrowths impair nutrient uptake, leading to premature utilization of endosperm starch to nourish the embryo. Similarly, developing wild-type kernels cultured in vitro with a low level of sucrose manifested early endosperm breakdown. ENB1 expression is induced by sucrose via the BETL-specific Myb-Related Protein1 transcription factor. Overexpression of ENB1 enhanced development of flange ingrowths, facilitating sucrose transport into BETL cells and increasing kernel weight. The results demonstrated that ENB1 enhances sucrose supply to the endosperm and contributes to a PE in the kernel.

shrunken4 is a mutant allele of ZmYSL2 that affects aleurone development and starch synthesis in maize.

Minerals are stored in the aleurone layer and embryo during maize seed development, but how they affect endosperm development and activity is unclear. Here, we cloned the gene underlying the classic maize kernel mutant shrunken4 (sh4) and found that it encodes the YELLOW STRIPE-LIKE oligopeptide metal transporter ZmYSL2. sh4 kernels had a shrunken phenotype with developmental defects in the aleurone layer and starchy endosperm cells. ZmYSL2 showed iron and zinc transporter activity in Xenopus laevis oocytes. Analysis using a specific antibody indicated that ZmYSL2 predominately accumulated in the aleurone and sub-aleurone layers in endosperm and the scutellum in embryos. Specific iron deposition was observed in the aleurone layer in wild-type kernels. In sh4, however, the outermost monolayer of endosperm cells failed to accumulate iron and lost aleurone cell characteristics, indicating that proper functioning of ZmYSL2 and iron accumulation are essential for aleurone cell development. Transcriptome analysis of sh4 endosperm revealed that loss of ZmYSL2 function affects the expression of genes involved in starch synthesis and degradation processes, which is consistent with the delayed development and premature degradation of starch grains in sh4 kernels. Therefore, ZmYSL2 is critical for aleurone cell development and starchy endosperm cell activity during maize seed development.

Maize endosperm development.

Recent breakthroughs in transcriptome analysis and gene characterization have provided valuable resources and information about the maize endosperm developmental program. The high temporal-resolution transcriptome analysis has yielded unprecedented access to information about the genetic control of seed development. Detailed spatial transcriptome analysis using laser-capture microdissection has revealed the expression patterns of specific populations of genes in the four major endosperm compartments: the basal endosperm transfer layer (BETL), aleurone layer (AL), starchy endosperm (SE), and embryo-surrounding region (ESR). Although the overall picture of the transcriptional regulatory network of endosperm development remains fragmentary, there have been some exciting advances, such as the identification of OPAQUE11 (O11) as a central hub of the maize endosperm regulatory network connecting endosperm development, nutrient metabolism, and stress responses, and the discovery that the endosperm adjacent to scutellum (EAS) serves as a dynamic interface for endosperm-embryo crosstalk. In addition, several genes that function in BETL development, AL differentiation, and the endosperm cell cycle have been identified, such as ZmSWEET4c, Thk1, and Dek15, respectively. Here, we focus on current advances in understanding the molecular factors involved in BETL, AL, SE, ESR, and EAS development, including the specific transcriptional regulatory networks that function in each compartment during endosperm development.

Maize kernel development.

Maize (Zea mays) is a leading cereal crop in the world. The maize kernel is the storage organ and the harvest portion of this crop and is closely related to its yield and quality. The development of maize kernel is initiated by the double fertilization event, leading to the formation of a diploid embryo and a triploid endosperm. The embryo and endosperm are then undergone independent developmental programs, resulting in a mature maize kernel which is comprised of a persistent endosperm, a large embryo, and a maternal pericarp. Due to the well-characterized morphogenesis and powerful genetics, maize kernel has long been an excellent model for the study of cereal kernel development. In recent years, with the release of the maize reference genome and the development of new genomic technologies, there has been an explosive expansion of new knowledge for maize kernel development. In this review, we overviewed recent progress in the study of maize kernel development, with an emphasis on genetic mapping of kernel traits, transcriptome analysis during kernel development, functional gene cloning of kernel mutants, and genetic engineering of kernel traits.

Maize pentatricopeptide repeat protein DEK53 is required for mitochondrial RNA editing at multiple sites and seed development.

Pentatricopeptide repeat (PPR) proteins were identified as site-specific recognition factors for RNA editing in plant mitochondria and plastids. In this study, we characterized maize (Zea mays) kernel mutant defective kernel 53 (dek53), which has an embryo lethal and collapsed endosperm phenotype. Dek53 encodes an E-subgroup PPR protein, which possesses a short PLS repeat region of only seven repeats. Subcellular localization analysis indicated that DEK53 is localized in the mitochondrion. Strand- and transcript-specific RNA-seq analysis showed that the dek53 mutation affected C-to-U RNA editing at more than 60 mitochondrial C targets. Biochemical analysis of mitochondrial protein complexes revealed a significant reduction in the assembly of mitochondrial complex III in dek53. Transmission electron microscopic examination showed severe morphological defects of mitochondria in dek53 endosperm cells. In addition, yeast two-hybrid and luciferase complementation imaging assays indicated that DEK53 can interact with the mitochondrion-targeted non-PPR RNA editing factor ZmMORF1, suggesting that DEK53 might be a functional component of the organellar RNA editosome.

A SnRK1-ZmRFWD3-Opaque2 Signaling Axis Regulates Diurnal Nitrogen Accumulation in Maize Seeds.

Zeins are the predominant storage proteins in maize (Zea mays) seeds, while Opaque2 (O2) is a master transcription factor for zein-encoding genes. How the activity of O2 is regulated and responds to external signals is yet largely unknown. Here, we show that the E3 ubiquitin ligase ZmRFWD3 interacts with O2 and positively regulates its activity by enhancing its nuclear localization. Ubiquitination of O2 enhances its interaction with maize importin1, the α-subunit of Importin-1 in maize, thus enhancing its nuclear localization ability. We further show that ZmRFWD3 can be phosphorylated by a Suc-responsive protein kinase, ZmSnRK1, which leads to its degradation. We demonstrated that the activity of O2 responds to Suc levels through the ZmSnRK1-ZmRFWD3-O2 signaling axis. Intriguingly, we found that Suc levels, as well as ZmRFWD3 levels and the cytonuclear distribution of O2, exhibit diurnal patterns in developing endosperm, leading to the diurnal transcription of O2-regulated zein genes. Loss of function in ZmRFWD3 disrupts the diurnal patterns of O2 cytonuclear distribution and zein biosynthesis, and consequently changes the C/N ratio in mature seeds. We therefore identify a SnRK1-ZmRFWD3-O2 signaling axis that transduces source-to-sink signals and coordinates C and N assimilation in developing maize seeds.

The regulation of zein biosynthesis in maize endosperm.

KEY MESSAGE: We review the current knowledge regarding the regulation of zein storage proteins biosynthesis and protein body formation, which are crucial processes for the successful accumulation of nutrients in maize kernels. Storage proteins in the seeds of crops in the grass family (Poaceae) are a major source of dietary protein for humans. In maize (Zea mays), proteins are the second largest nutrient component in the kernels, accounting for ~ 10% of the kernel weight. Over half of the storage proteins in maize kernels are zeins, which lack two essential amino acids, lysine and tryptophan. This deficiency limits the use of maize proteins in the food and feed industries. Zeins are encoded by a large super-gene family. During endosperm development, zeins accumulate in protein bodies, which are derived from the rough endoplasmic reticulum. In recent years, our knowledge of the pathways of zein biosynthesis and their deposition within the endosperm has been greatly expanded. In this review, we summarize the current understanding of zeins, including the genes encoding these proteins, their expression patterns and transcriptional regulation, the process of protein body formation, and other biological processes affecting zein accumulation.

A Sequence-Indexed Mutator Insertional Library for Maize Functional Genomics Study.

Sequence-indexed insertional libraries are important resources for functional gene study in model plants. However, the maize (Zea mays) UniformMu library covers only 36% of the annotated maize genes. Here, we generated a new sequence-indexed maize Mutator insertional library named ChinaMu through high-throughput sequencing of enriched Mu-tagged sequences. A total of 2,581 Mu F2 lines were analyzed, and 311,924 nonredundant Mu insertion sites were obtained. Based on experimental validation, ChinaMu contains about 97,000 germinal Mu insertions, about twice as many as UniformMu. About two-thirds (66,565) of the insertions are high-quality germinal insertions (positive rate > 90%), 89.6% of which are located in genic regions. Furthermore, 45.7% (20,244) of the 44,300 annotated maize genes are effectively tagged and about two-thirds (13,425) of these genes harbor multiple insertions. We tested the utility of ChinaMu using pentatricopeptide repeat (PPR) genes. For published PPR genes with defective kernel phenotypes, 17 out of 20 were tagged, 11 of which had the previously reported mutant phenotype. For 16 unstudied PPR genes with both Mu insertions and defective kernel phenotypes, 6 contained insertions that cosegregated with the mutant phenotype. Our sequence-indexed Mu insertional library provides an important resource for functional genomics study in maize.

Uncovering kappa-opioid receptor agonist-induced PAK1/2 phosphorylation by quantitative phosphoproteomics.

Kappa-opioid receptor (KOR) is a member of G-protein coupled receptors (GPCRs) expressed in serotonergic neurons and neuronal terminals. The involvement of KOR ligands in nociception, diuresis, emotion, cognition, and immune system has been extensively studied. Omics-based methods are preferable to understand the signaling cascade after KOR activation in a systematic manner. In this study, an in-depth quantitative phosphoproteomic analysis resulted in 305 phosphosites, which were significantly changed in three KOR-overexpressed cells upon treatment with two KOR agonists. The subsequent substrate-kinase prediction analysis revealed that 18 potential kinases might be activated under stimulation of the agonists. We found that phosphorylation of PAK1/2 (p21-activated kinase 1/2) was induced by KOR agonists, resulting in reduced actin stress fibers and cytoskeletal reorganization. In summary, this quantitative phosphoproteomics-based research studied the downstream phosphorylation events upon KOR activation, which may shed light on the investigations of KOR signaling pathway and targeted therapy for KOR-related diseases.

Maize Dek44 Encodes Mitochondrial Ribosomal Protein L9 and Is Required for Seed Development.

Mitochondrial respiration depends on proteins encoded by the nuclear and mitochondrial genomes. Many respiratory chain-related proteins are encoded by the mitochondrial genome and undergo translation by mitochondrial ribosomes. The newly identified maize (Zea mays) defective kernel44 (dek44) mutant produces small kernels showing embryo-lethal phenotypes. We cloned Dek44 by isolating the Mutator tag that produced the mutation and identified it as encoding a putative 50S ribosomal protein L9. Subcellular fractionation by ultracentrifugation confirmed that DEK44 is a mitochondrial ribosomal protein. DEK44 is highly conserved in monocots and only accumulates in kernels. Transcriptome and reverse transcription quantitative PCR analyses revealed that loss of DEK44 function affects the expression of genes encoding respiratory chain-related proteins from the mitochondrial and nuclear genomes. Blue native-PAGE revealed significantly reduced assembly of respiratory chain complexes in dek44 mutant kernels. Transmission electron microscopy indicated that the biogenesis and morphology of mitochondria were strongly affected in dek44 mutant kernels. Furthermore, DEK44 might regulate cell growth and kernel development via cyclin/cyclin-dependent kinase-mediated activities. This study provides insight into the regulation of kernel development based on mitochondrial ribosomal protein function.

Dek40 Encodes a PBAC4 Protein Required for 20S Proteasome Biogenesis and Seed Development.

The 26S proteasome, an essential protease complex of the ubiquitin-26S proteasome system (UPS), controls many cellular events by degrading short-lived regulatory proteins marked with polyubiquitin chains. The 20S proteolytic core protease (CP), the catalytic core of the 26S proteasome, is a central enzyme in the UPS. Its biogenesis proceeds in a multistep and orderly fashion assisted by a series of proteasome assembly chaperones. In this study, we identified a novel maize (Zea mays) kernel mutant named defective kernel40 (dek40), which produces small, collapsed kernels and exhibits delayed embryo and endosperm development. Dek40 was identified by map-based cloning and confirmed by transgenic functional complementation. Dek40 encodes a putative cytosol-localized proteasome biogenesis-associated chaperone4 (PBAC4) protein. DEK40 participates in the biogenesis of the 20S CP by interacting with PBAC3. Loss-of-function of DEK40 substantially affected 20S CP biogenesis, resulting in decreased activity of the 26S proteasome. Ubiquitylome analysis indicated that DEK40 influences the degradation of ubiquitinated proteins and plays an essential role in the maintenance of cellular protein homoeostasis. These results demonstrate that Dek40 encodes a PBAC4 chaperone that affects 20S CP biogenesis and is required for 26S proteasome function and seed development in maize.

Maize Dek33 encodes a pyrimidine reductase in riboflavin biosynthesis that is essential for oil-body formation and ABA biosynthesis during seed development.

The maize (Zea mays) defective kernel 33 (dek33) mutant produces defective and occasionally viviparous kernel phenotypes. In this study, we cloned Dek33 by positional cloning and found that it encodes a pyrimidine reductase in riboflavin biosynthesis. In dek33, a single-base mutation (G to A) in the C-terminal COG3236 domain caused a premature stop codon (TGA), producing a weak mutant allele with only a truncated form of the DEK33 protein that occurred at much lower levels that the completed WT form, and with a reduced riboflavin content. The dek33 mutation significantly affected oil-body formation and suppressed endoreduplication. It also disrupted ABA biosynthesis, resulting in lower ABA content that might be responsible for the viviparous embryo. In addition, our results indicated that the COG3236 domain is important for the protein stability of DEK33. Yeast two-hybrid experiments identified several proteins that interacted with DEK33, including RGLG2 and SnRK1, suggesting possible post-translational regulation of DEK33 stability. The interaction between DEK33 and these proteins was further confirmed by luciferase complementation image assays. This study provides a weak mutant allele that can be utilized to explore cellular responses to impaired riboflavin biosynthesis during seed development. Our findings indicate that the COG3236 domain might be an essential regulatory structure for DEK33 stability in maize.