Male sterility (MS) provides a useful breeding tool to harness hybrid vigor for hybrid seed production. It is necessary to generate new male sterile mutant lines for the development of hybrid seed production technology. The CRISPR/Cas9 technology is well suited for targeting genomes to generate male sterile mutants. In this study, we artificially synthesized Streptococcus pyogenes Cas9 gene with biased codons of maize. A CRISPR/Cas9 vector targeting the MS8 gene of maize was constructed and transformed into maize using an Agrobacterium-mediated method, and eight T₀ independent transgenic lines were generated. Sequencing results showed that MS8 genes in these T₀ transgenic lines were not mutated. However, we detected mutations in the MS8 gene in F₁ and F₂ progenies of the transgenic line H17. A potential off-target site sequence which had a single nucleotide that was different from the target was also mutated in the F₂ progeny of the transgenic line H17. Mutation in the MS8 gene and the male sterile phenotype could be stably inherited by the next generation in a Mendelian fashion. Transgene-free ms8 male sterile plants were obtained by screening the F₂ generation of male sterile plants, and the MS phenotype could be introduced into other elite inbred lines for hybrid production.

The “green revolution” gene gibberellin oxidase contributes to the semidwarf phenotype, improving product and lodging resistance. Dissecting the function of GA biosynthetic genes would be helpful for dwarf maize breeding. In this study, we edited the maize GA20ox3 gene and generated semidwarf maize plants using CRISPR/Cas9 technology. Application of exogenous gibberellin can recover the dwarf phenotype, indicating that the mutants are gibberellin deficient. The contents of GA₁₂ and GA₅₃ were elevated in the mutants due to the disruption of GA20 oxidase, whereas the contents of other GA precursors (GA₁₅, GA₂₄, GA₉, GA₄₄, and GA₂₀) were decreased in the mutants, and the accumulation of bioactive GA₁ and GA₄ was also decreased, contributing to the semidwarf phenotype. Transgene-free dwarf maize was selected from T₂-generation plants and might be useful for maize breeding in the future.

Pentatricopeptide repeat (PPR) proteins play crucial roles in intron splicing, which is important for RNA maturation. Identification of novel PPR protein with the function of intron splicing would help to understand the RNA splicing mechanism. In this study, we identified the maize empty pericarp602 (emp602) mutants, the mature kernels of which showed empty pericarp phenotype. We cloned the Emp602 gene from emp602 mutants and revealed that Emp602 encodes a mitochondrial-localized P-type PPR protein. We further revealed that Emp602 is specific for the cis-splicing of mitochondrial Nad4 intron 1 and intron 3, and mutation of Emp602 led to the loss of mature Nad4 transcripts. The loss of function of Emp602 nearly damaged the assembly and accumulation of complex I and arrested mitochondria formation, which arrested the seed development. The failed assembly of complex I triggers significant upregulation of Aox expression in emp602 mutants. Transcriptome analysis showed that the expression of mitochondrial-related genes, e.g. the genes associated with mitochondrial inner membrane presequence translocase complex and electron carrier activity, were extensively upregulated in emp602 mutant. These results demonstrate that EMP602 functions in the splicing of Nad4 intron 1 and intron 3, and the loss of function of Emp602 arrested maize seed development by disrupting the mitochondria complex I assembly.

R2R3-MYB proteins play role in plant development, response to biotic and abiotic stress, and regulation of primary and secondary metabolism. Little is known about the R2R3-MYB proteins in Scutellaria baicalensis which is an important Chinese medical plant. In this paper, nineteen putative SbMYB genes were identified from a S. baicalensis cDNA library, and eleven R2R3-MYBs were clustered into 5 subgroups according to phylogenetic reconstruction. In the S. baicalensis leaves which were sprayed with GA₃, SbMYB2 and SbMYB7 had similar expression pattern with SbPALs, indicating that SbMYB2 and SbMYB7 might be involved in the flavonoid metabolism. Transactivation assay results showed that SbMYB2 and SbMYB7 can function as transcriptional activator. The expression of several flavonoid biosynthesis-related genes were induced or suppressed by overexpression of SbMYB2 or SbMYB7 in transgenic tobacco plants. Consistent with the change of the expression of NtDH29 and NtCHI, the contents of dicaffeoylspermidine and quercetin-3,7-O-diglucoside in SbMYB2-overexpressing or SbMYB7-overexpressing transgenic tobacco plants were decreased. The transcriptional level of NtUFGT in transgenic tobacco overexpressing SbMYB7 and the transcriptional level of NtHCT in SbMYB2-overexpressing tobacco plants were increased; however the application of GA₃ inhibited the transcriptional level of these two genes. These results suggest that SbMYB2 and SbMYB7 might regulate the flavonoid biosynthesis through GA metabolism.

Callus induction is a dedifferentiation process that accompanies a cell fate transition, and epigenetic regulation plays a crucial role in the process. N ⁶ ‐methyladenosine (m6A) methylation is an important mechanism in post‐transcriptional epigenetic regulation and functions in cell reprogramming. However, the function of m6A methylation during callus induction is still unknown. Here, we performed transcriptome‐wide m6A‐seq on immature maize embryos after culturing for 2, 4, or 8 days with or without the auxin analogue 2,4‐D. A total of 26,794 unique m6A peaks were detected from 17,456 maize genes; and 2,338 specific, 2,4‐D‐induced m6A peaks (D‐specific m6A) were detected only in embryos cultured with 2,4‐D. Furthermore, a positive correlation between m6A methylation and mRNA abundance was discovered in the genes with D‐specific m6A deposition, especially at the beginning of callus induction. Key genes involved in callus induction, i.e. BABY BOOM and LBD transcription factors, underwent m6A methylation, increasing their transcript levels, thus improving callus induction. These results revealed the importance of m6A methylation during the early stage of callus induction and provided new insights into the molecular mechanism of callus induction at an epitranscriptomic level.