CRISPR/Cas9 genome editing strategy has been applied to a variety of species and the tRNA-processing system has been used to compact multiple gRNAs into one synthetic gene for manipulating multiple genes in rice. We optimized and introduced the multiplex gene editing strategy based on the tRNA-processing system into maize. Maize glycine-tRNA was selected to design multiple tRNA-gRNA units for the simultaneous production of numerous gRNAs under the control of one maize U6 promoter. We designed three gRNAs for simplex editing and three multiple tRNA-gRNA units for multiplex editing. The results indicate that this system not only increased the number of targeted sites but also enhanced mutagenesis efficiency in maize. Additionally, we propose an advanced sequence selection of gRNA spacers for relatively more efficient and accurate chromosomal fragment deletion, which is important for complete abolishment of gene function especially long non-coding RNAs (lncRNAs). Our results also indicated that up to four tRNA-gRNA units in one expression cassette design can still work in maize. The examples reported here demonstrate the utility of the tRNA-processing system-based strategy as an efficient multiplex genome editing tool to enhance maize genetic research and breeding.

Zeins are the most abundant storage proteins in maize (Zea mays) kernels, thereby affecting the nutritional quality and texture of this crop. 27-kD γ-zein is highly expressed and plays a crucial role in protein body formation. Several transcription factors (TFs) (O2, PBF1, OHP1, and OHP2) regulate the expression of the 27-kD γ-zein gene, but the complexity of its transcriptional regulation is not fully understood. Here, using probe affinity purification and mass spectrometry analysis, we identified ZmbZIP22, a TF that binds to the 27-kD γ-zein promoter. ZmbZIP22 is a bZIP-type TF that is specifically expressed in endosperm. ZmbZIP22 bound directly to the ACAGCTCA box in the 27-kD γ-zein promoter and activated its expression in wild tobacco (Nicotiana benthamiana) cells. 27-kD γ-zein gene expression was significantly reduced in CRISPR/Cas9-generated zmbzip22 mutants. ChIP-seq (chromatin immunoprecipitation coupled to high-throughput sequencing) confirmed that ZmbZIP22 binds to the 27-kD γ-zein promoter in vivo and identified additional direct targets of ZmbZIP22. ZmbZIP22 can interact with PBF1, OHP1, and OHP2, but not O2. Transactivation assays using various combinations of these TFs revealed multiple interaction modes for the transcriptional activity of the 27-kD γ-zein promoter. Therefore, ZmbZIP22 regulates 27-kD γ-zein gene expression together with other known TFs.

Nitrogen assimilation in developing maize seeds is regulated by the SnRK1-ZmRFWD3-O2 signaling axis, which is responsive to diurnal sucrose concentrations.

Cohesin complexes maintain sister chromatid cohesion to ensure proper chromosome segregation during mitosis and meiosis. In plants, the exact components and functions of the cohesin complex remain poorly understood. Here, we positionally cloned the classic maize (Zea mays) mutant defective kernel 15 (dek15), revealing that it encodes a homolog of SISTER CHROMATID COHESION PROTEIN 4 (SCC4), a loader subunit of the cohesin ring. Developing dek15 kernels contained fewer cells than the wild type, but had a highly variable cell size. The dek15 mutation was found to disrupt the mitotic cell cycle and endoreduplication, resulting in a reduced endosperm and embryo lethality. The cells in the dek15 endosperm and embryo exhibited precocious sister chromatid separation and other chromosome segregation errors, including misaligned chromosomes, lagging chromosomes, and micronuclei, resulting in a high percentage of aneuploid cells. The loss of Dek15/Scc4 function upregulated the expression of genes involved in cell cycle progression and stress responses, and downregulated key genes involved in organic synthesis during maize endosperm development. Our yeast two-hybrid screen identified the chromatin remodeling proteins chromatin remodeling factor 4, chromatin remodeling complex subunit B (CHB)102, CHB105, and CHB106 as SCC4-interacting proteins, suggesting a possible mechanism by which the cohesin ring is loaded onto chromatin in plant cells. This study revealed biological functions for DEK15/SCC4 in mitotic chromosome segregation and kernel development in maize.

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.

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.