Low temperatures occurring at the booting stage in rice (Oryza sativa L.) often result in yield loss by impeding male reproductive development. However, the underlying mechanisms by which rice responds to cold at this stage remain largely unknown. Here, we identified MITOCHONDRIAL ACYL CARRIER PROTEIN 2 (OsMTACP2), the encoded protein of which mediates lipid metabolism involved in the cold response at the booting stage. Loss of OsMTACP2 function compromised cold tolerance, hindering anther cuticle and pollen wall development, resulting in abnormal anther morphology, lower pollen fertility and seed setting. OsMTACP2 was highly expressed in tapetal cells and microspores during anther development, with the encoded protein localizing to both mitochondria and the cytoplasm. Comparative transcriptomic analysis revealed differential expression of genes related to lipid metabolism between the wild type and the Osmtacp2-1 mutant in response to cold. Through a lipidomic analysis, we demonstrated that wax esters, which are the primary lipid components of the anther cuticle and pollen walls, function as cold-responsive lipids. Their levels increased dramatically in the wild type but not in Osmtacp2-1 when exposed to cold. Additionally, mutants of two cold induced genes of wax ester biosynthesis, ECERIFERUM1 and WAX CRYSTAL-SPARSE LEAF2, showed decreased cold tolerance. These results suggest that OsMTACP2-mediated wax ester biosynthesis is essential for cold tolerance in rice at the booting stage.

The WUSCHEL-related homeobox (WOX) belongs to a plant-specific transcription factor gene family that plays crucial roles in plant growth and development. Barley ranks as the fourth global cereal crop and is recognized as a model crop for the study of cereal genetics. However, genome-wide characterization, functional validation, and stress-related studies of the WOX gene family in barley remain limited, hindering efforts to leverage their potential for improving salt tolerance and regeneration efficiency in breeding programs. In this study, we identified 12 HvWOX genes assigned from chromosome 1 to chromosome 5. Phylogenetic analysis revealed that these HvWOX genes can be classified into three clades (WUS, ancient, and intermediate). Gene structure analysis revealed that the exon numbers of HvWOX genes varied in the WUS and intermediate clades but were highly conserved in the ancient clade. Tissue-specific analysis revealed that the most common HvWOX genes were highly expressed in reproductive tissues such as anthers or ovaries. Cis-element analysis suggested that there were multiple stress- and hormone-responsive elements in the HvWOX gene promoters. In addition, overexpression of HvWOX8 in Arabidopsis significantly enhanced root elongation under salt stress (50–100 mM NaCl), suggesting its direct role in salt tolerance. Transcriptomic analysis further revealed that HvWOX8 modulates hormone signaling and electron transfer pathways during ATP synthesis under stress conditions. In conclusion, our results provided a comprehensive understanding of the gene characteristics, expression patterns, and potential roles of barley WOX genes.

Abiotic stress can reprogram the gametophytic pathway; the mechanisms by which floral bud pre-treatment influences microspore embryogenesis initiation remain unclear. In this study, we use bisulfite sequencing, sRNA-seq, and RNA-seq to analyze the dynamic changes in rice microspores under different cold treatment durations. Our results showed that a 10-day cold treatment is essential for CXJ microspore embryogenesis initiation. DNA methylation levels showed a slight change at CG, CHG, and CHH sites under cold treatment. The number of both hyper- and hypomethylated DMRs increased over cold treatment, with more hypermethylated DMRs at 5 and 10 dpt. Hypermethylated DMRs were more frequently in the TSS region compared to hypomethylated DMRs. The proportion of 24 nt sRNAs increased upon cold stress, with more downregulated than upregulated sRNAs at 10 dpt. The number of DMR target DEGs increased from 5 to 10 dpt. Promoter hypomethylation at the CHH site was more frequently associated with DEGs. These outcomes suggested that the RdDM pathway participates in the initiation of rice ME. GO analysis indicated that DMR target DEGs at 10 dpt were enriched in responses to chemical stimuli, biological processes, and stress responses. An auxin-related gene, OsHOX28, was further identified. Its upregulation, potentially mediated by the RdDM pathway, may play a crucial role in the initiation of rice ME. This study provides more information on epigenetic mechanisms during rice ME.

This study elucidates the key molecular features underlying the embryogenic initiation divergence between japonica rice Chongxiangjing (CXJ) and indica rice 9311 during isolated microspore culture. Comparative transcriptome analysis across critical timepoints (0, 5, and 10 days post-culture initiation) revealed that while both varieties initially exhibit comparable microspore viability, CXJ maintains transcriptional stability and activates developmental programs (e.g., hormone signaling, DNA replication, cell morphogenesis), enabling sustained callus formation. In contrast, 9311 undergoes drastic transcriptome reorganization by 5 days, characterized by maladaptive activation of stress-response pathways (glutathione metabolism, MAPK signaling, ER stress) and futile metabolic reactivation (photosynthesis, starch degradation), culminating in near-total cell death and failed callus induction. Transcription factor dynamics further explain this divergence: CXJ specifically upregulates regulators coordinating development and stress resilience (NAC, ERF, HSF, GRAS, bZIP), while 9311 exhibits detrimental upregulation of FAR1 and B3, leading to catastrophic energy misallocation. These findings identify master transcriptional networks and stress-response pathways as pivotal indicators of embryogenic initiation efficiency, providing strategic targets for enhancing indica rice microspore culture technology.