Dicer-like 5 deficiency confers temperature-sensitive male sterility in maize

Three major classes of endogenous small RNAs (sRNAs) exist in plants: microRNAs (miRNAs), heterochromatic small interfering RNAs (hc-siRNAs), and phased, secondary small interfering RNAs (phasiRNAs). PhasiRNAs are abundant in flowers, especially male reproductive organs. Their production begins with miRNA-mediated cleavage of RNA polymerase II transcripts at PHAS loci. DICER-LIKE 4 (DCL4) generates 21-nt phasiRNAs, while DCL5 is proposed to generate 24-nt phasiRNAs. The 21-nt phasiRNAs are crucial for male fertility in rice. The 24-nt phasiRNAs accumulate during meiosis, suggesting a role in its regulation. Five DICER-LIKE (DCL) proteins exist in angiosperms, with DCL1 involved in miRNA biogenesis, DCL2 in processing viral siRNAs, DCL3 producing 24-nt hc-siRNAs, and DCL4 generating 21-nt trans-acting siRNAs and phasiRNAs. DCL5, a monocot-specific gene, is the focus of this study to understand its function in 24-nt phasiRNA biogenesis. Maize *dcl5* mutants were generated using CRISPR-Cas9 and transposon mutagenesis to investigate DCL5's role.

Previous research has extensively documented the presence and diverse roles of small RNAs in plant development. Studies in maize have established the spatiotemporal dynamics of phasiRNAs in anthers, highlighting their cell-type-specific expression and abundance during different developmental stages. In rice, the importance of specific phasiRNAs and the Argonaute family proteins in male fertility has been demonstrated. The roles of various DICER-LIKE (DCL) proteins in different small RNA pathways have been characterized in various plant species. However, the specific functions of DCL5 and its involvement in the biogenesis of 24-nt phasiRNAs, particularly in the context of male fertility, remain largely unexplored.

The study utilized CRISPR-Cas9 gene editing to generate four *dcl5* alleles in maize. A transposon-disrupted allele (*dcl5-mu03*) was also used. The resulting mutants were characterized phenotypically, observing their anther morphology and fertility under different temperature conditions (greenhouse and field). Confocal and transmission electron microscopy were used to examine anther cell structures, focusing on tapetal cell development. Small RNA sequencing (sRNA-seq) was performed to quantify 24-nt phasiRNA levels in the mutants compared to wild-type plants. RNA sequencing (RNA-seq) was used to analyze transcriptomic changes in the mutants. A temperature swap experiment was conducted to determine the specific developmental window in which temperature sensitivity manifested. Statistical analyses were used to compare phenotypic traits and molecular data between mutants and wild-type plants. PlantCV v2 platform was used for quantitative image-based analysis of tassel images to assess male fertility.

The *dcl5* mutants exhibited male sterility, with anthers shorter than those of wild-type plants. The tapetal cells in *dcl5* mutants showed developmental defects, including reduced binucleate status and delayed programmed cell death. 24-nt phasiRNAs were drastically reduced in *dcl5* mutants, confirming DCL5's crucial role in their biogenesis. The *dcl5* mutant's male sterility was temperature-sensitive; under cooler temperatures (23/20 °C), fertility was partially or fully restored. Transcriptomic analysis revealed minimal changes in gene expression in *dcl5* mutants, suggesting that most changes observed were indirect effects of tapetal cell defects. The temperature swap experiment demonstrated that a limited period around meiosis is critical for the requirement of DCL5, and therefore 24-nt phasiRNAs, for male fertility.

The findings demonstrate that DCL5 is essential for robust male fertility in maize. The temperature sensitivity suggests that 24-nt phasiRNAs play a crucial role in tapetal development, which is essential for pollen maturation. The reduced 24-nt phasiRNAs and the tapetal defects likely lead to male sterility at higher temperatures, impacting maize yield potential. The minimal transcriptional changes in *dcl5* mutants suggest a direct role of 24-nt phasiRNAs in tapetal development rather than indirect regulation of gene expression. The temperature-dependent phenotype emphasizes the interaction between genetic factors and environmental conditions in plant reproduction.

This study highlights the importance of DCL5 and its resultant 24-nt phasiRNAs in maize male fertility, particularly at temperatures optimal for crop yield. The temperature sensitivity of the *dcl5* mutants suggests potential strategies for improving crop resilience to climate change. Future research could explore the specific molecular mechanisms by which 24-nt phasiRNAs regulate tapetal development and investigate the potential for manipulating this pathway to enhance fertility and yield in maize and other crops.

The study focused on a single maize inbred line (W23), limiting the generalizability of findings. The transcriptomic analysis identified minimal differences in gene expression, possibly due to the indirect nature of tapetal defects on overall gene expression. Further investigation is needed to fully elucidate the specific molecular targets of 24-nt phasiRNAs and their mode of action.