Arp Schnittger

SUMMARY: The ability to insert, delete, or modify genetic information is crucial for mechanistic studies and biotechnological applications. However, efficient genetic transformation remains a major bottleneck for research in maize and many other crops. Here, we report an optimized Agrobacterium ‐mediated transformation platform based on systematic reconstruction of tissue culture handling in the maize inbred line A188. Refinement of callus induction, selection, and regeneration substantially improved recovery of transgenic plantlets. To distinguish independent T‐DNA insertion events, we developed TAFLP (T‐DNA amplified fragment length polymorphism), a simple and inexpensive assay that amplifies T‐DNA flanking sequences and can be performed using standard laboratory equipment. Our enhanced transformation pipeline was also applicable to the inbred line B104 as well as to hygromycin and G418 selection systems, demonstrating broad utility of our method. We validated the platform for CRISPR/Cas9 mutagenesis and reporter‐line generation. Using this approach, we isolated new loss‐of‐function alleles of the maize‐specific MAC1 ( MULTIPE ARCHESPORIAL CELLS 1 ) and F‐box protein encoding ACOZ1 ( ABNORMAL CHROMOSOME ORGANIZATION IN ZYGOTENE 1 ), a homolog of the Arabidopsis RMF1/2 ( REDUCED MALE FERTILITY 1/2 ) and rice ZYGO1 ( ZYGOTENE 1 ). The concomitant generation of reporter lines for ACOZ1 and the nuclear lamin‐like proteins NCH1 (NMCP/CROWN HOMOLOG1) and NCH2, as well as SUN1 (SUN DOMAIN PROTEIN 1) revealed that the function of this F‐box protein class in releasing the somatic constraints of chromosome organization for meiotic recombination is highly conserved in flowering plants.

Meiotic recombination in reproductive cells is initiated by DNA double-strand breaks (DSBs) and requires the expression of meiosis-specific genes, yet how the transcription of those genes is regulated in either meiotic or somatic cells remains unclear. Here, using a representative meiosis-specific DMC1 gene promoter as a bait, we identified two DREAM factors TCX5 and TCX6 required for direct repression of DMC1 in somatic cells. Transcriptomic analyses show broad ectopic up-regulation of meiotic genes in tcx5/6 leaves . Consistently, tcx5/6 somatic cells form DSBs and exhibit hypersensitivity to DNA damage compared to wild type. Further genetic analyses reveal that SPO11-1 is dispensable for ectopic meiotic gene activation but essential for the DNA damage response in tcx5/6 somatic cells. Last, we demonstrated that TCX5/6 repress meiotic gene expression through H3K4me3 modification. This finding uncovers an unexpected mechanism for suppressing meiotic recombination in somatic cells, thus preventing genetic diversity and genome instability in organisms.

Meiotic chromosome segregation and recombination, essential for sexual reproduction and evolution, require a specialized gene set. Despite its importance, the transcriptional regulators that directly orchestrate meiotic gene expression remain unidentified in plants. We identify the MuvB core components known from the DREAM complex as key transcriptional regulators of meiotic recombination in Arabidopsis . Loss of DREAM components TCX5/6 and ALY1/2 leads to impaired recombination and fertility defects. Meiocyte-specific transcriptome profiling and genome-wide binding analyses reveal that core recombination genes, including the crossover-promoting helicase MER3, are direct targets. Consistently, MER3 overexpression partially rescues recombination defects in DREAM-compromised plants. These findings establish the DREAM complex components as important regulators of the meiotic program in plants. Given that DREAM dysfunction causes reproductive defects in animals, this regulatory role may be conserved across multicellular eukaryotes.

Small RNAs (sRNAs) are widespread across diverse flowering plant species and play important roles regulating reproduction and environmental responses. However, how sRNAs exert their function is largely unknown, likely due to their typical origin in non-genic regions, the lack of distinct gene targets, and the sparse characterization of their associated Argonaute (AGO) effector proteins. Here we show that Arabidopsis male reproductive sRNAs associate with AGO4, 5, 6 and 9, all of which are enriched in meiocytes along with RNA Polymerase V (Pol V). This enables these sRNAs to methylate loci with imperfect sequence homology. AGO4, AGO6 and AGO9 predominantly load 24-nucleotide (nt) Pol IV-derived sRNAs and mediate DNA methylation in meiocytes in a dosage-dependent manner. Despite closer homology to microRNA-associated AGO1 and AGO10, AGO5 primarily binds CLSY3- and Pol IV-derived 21-24 nt sRNAs. Transcriptomic and methylomic analyses of meiocytes from single and compound ago mutants demonstrate distinct yet complementary roles of AGO4/6/9 and AGO5 in regulating protein-coding genes and silencing transposons in the germline. These molecular disruptions are accompanied by significant meiotic defects. Together, our findings reveal how AGO4/6/9-mediated DNA methylation and AGO5, likely acting through post-transcriptional gene silencing, work together to promote male germline development.

The chromosome axis is a proteinaceous scaffold essential for crossover formation in meiosis. While the axis associated HORMA-domain protein ASY1 is known to be necessary for the localization of the recombinase DMC1 to the axis, the molecular mechanism linking ASY1 to DMC1 loading remains unclear. Here, we demonstrate that ASY1 directly recruits the plant-specific cyclin SDS to chromosomes, which ensures efficient DMC1 localization and interhomolog recombination. Live-cell imaging and immunodetection revealed that SDS localizes to chromosomes from leptotene to zygotene and disappears after pachytene resembling DMC1 dynamics. Biochemical assays demonstrated a direct interaction between ASY1 and SDS. Further analyses revealed that ASY1’s closure motif, a conformational regulatory element, is required for SDS recruitment despite being dispensable for ASY1’s own chromosome association. Moreover, unlike in sds mutants, depletion of the DMC1 inhibitor FIGL1-FLIP complex failed to restore DMC1 foci in asy1 mutants, positioning ASY1 upstream of both positive (SDS) and negative (FIGL1-FLIP) regulators of DMC1. These findings establish ASY1 as a key coordinator of interhomolog recombination, bridging chromosome axis architecture with recombinase dynamics to control crossover formation and genome stability.

Female and male meiosis often differ in many aspects, such as their duration and the frequency as well as the positioning of crossovers. However, studying female meiosis is often very challenging and thus, much less is known about female versus male meiosis in many species including plants, where meiosis occurs deep within the ovules. To approach this gap, we developed a live-cell imaging system for female meiocytes in Arabidopsis (Arabidopsis thaliana) in this study. This allowed us to obtain a temporally resolved cytological framework of female meiosis in the wild type that serves as a guiding system for future studies. Subsequently, we have applied this imaging system here to study mutants in cyclin-dependent kinase inhibitors, in which a designated female meiocyte undergoes several mitotic divisions before entering meiosis. This mutant context enabled us to address when a meiocyte is committed to meiosis, a key question during reproductive development and in particular for the analysis of apomictic species in which meiosis is skipped.

Stress granules (SG) are biomolecular condensates that represent an adaptive response of cells to various stresses, including heat. However, the cell type–specific function and relevance of SG formation, especially during reproductive development, are largely not understood. Here, we show that the meiotic A-type cyclin TARDY ASYNCHRONOUS MEIOSIS (TAM) is recruited to SGs in male meiocytes of Arabidopsis after exposure to heat. We find that the amino terminus of TAM is necessary and sufficient for the localization of proteins to meiotic SGs. Swapping the amino terminus of TAM with the one of its sister protein CYCA1;1 resulted in a separation-of-function allele of TAM , which prevents the partitioning of TAM to SGs while restoring a wild-type phenotype in a tam mutant background under nonheat stress conditions. Notably, plants expressing this TAM version prematurely terminate meiosis under heat resulting in unreduced gametes. Thus, the formation of TAM-containing SGs is necessary for genome stability under heat stress.

Meristems are major determinants of plant architecture, diversification, and acclimation to environmental stresses. Moreover, meristems play also a major role during crop domestication and are fundamentally important for the productivity of crop plants as they directly determine biomass and grain yield. While vegetative meristems shape the basic plant body plan and produce all above- and below-ground parts of plants, some vegetative meristems transit to reproductive meristems, forming sexual organs and germ cells. Most knowledge about plant meristems was generated using the model plant Arabidopsis. Compared with Arabidopsis, architecture of grass or cereals, including crops like maize, wheat, barley, rice and sorghum, is more complex: cereals produce additional organs like a coleoptile, seminal roots originating from the scutellar nodes in the embryo and shoot-borne crown roots as well as highly complex inflorescence meristems with meristem types absent in eudicots. Moreover, studies in cereals indicated that paradigms based on studies using Arabidopsis are not universally applicable. This review therefore aims to provide a comprehensive overview about the initiation, establishment, maintenance, and function of the various cereal meristems and their stem cell niches that shape our most important crop plants. Stem cell–like systems involved in leaf pattering and germline formation are also considered. The focus is also on the significant progress that has been made recently using novel tools to elucidate gene regulatory networks underlying the development and function of the various cereal meristems.