The EPIDERMAL PATTERNING FACTOR (EPF) and EPF-LIKE (EPFL) family of small secreted peptides act to regulate many aspects of plant growth and development; however, their functions are not widely characterized in rice (Oryza sativa). Here, we used clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) technology to individually knockout each of 11 EPF/EPFL genes in the rice cultivar Kasalath. Loss of function of most OsEPF/EPFL genes generated no obvious phenotype alteration, while disruption of OsEPFL2 in Kasalath caused a short or no awn phenotype and reduced grain size. OsEPFL2 is strongly expressed in the young panicle, consistent with a role in regulating awn and grain development. Haplotype analysis indicated that OsEPFL2 can be classified into six major haplotypes. Nucleotide diversity and genetic differentiation analyses suggested that OsEPFL2 was positively selected during the domestication of rice. Our work to systematically investigate the function of EPF/EPFL peptides demonstrates that different members of the same gene family have been independently selected for their ability to regulate a similar biological function and provides perspective on rice domestication.

Nucleus-encoded circular RNAs (ncircRNAs) have been widely detected in eukaryotes, and most circRNA identification algorithms are designed to identify them. However, using these algorithms, few mitochondrion-encoded circRNAs (mcircRNAs) have been identified in plants, and the role of plant mcircRNAs has not yet been addressed. Here, we developed a circRNA identification algorithm, mitochondrion-encoded circRNA identifier, based on common features of plant mitochondrial genomes. We identified 7,524, 9,819, 1,699, 1,821, 1,809, and 5,133 mcircRNAs in maize (Zea mays), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), tomato (Solanum lycopersicum), cucumber (Cucumis sativus), and grape (Vitis vinifera), respectively. These mcircRNAs were experimentally validated. Plant mcircRNAs had distinct characteristics from ncircRNAs, and they were more likely to be derived from RNA degradation but not intron backsplicing. Alternative circularization was prevalent in plant mitochondria, and most parental genomic regions hosted multiple mcircRNA isoforms, which have homogenous 5′ termini but heterogeneous 3′ ends. By analysis of mitopolysome and mitoribosome profiling data, 1,463 mcircRNAs bound to ribosomes were detected in maize and Arabidopsis. Further analysis of mass spectrometry-based proteomics data identified 358 mcircRNA-derived polypeptides. Overall, we developed a computational pipeline that efficiently identifies plant mcircRNAs, and we demonstrated mcircRNAs are widespread and translated in plants.

Carbohydrates, proteins, lipids, minerals and vitamins are nutrient substances commonly seen in rice grains, but anthocyanidin, with benefit for plant growth and animal health, exists mainly in the common wild rice but hardly in the cultivated rice. To screen the rice germplasm with high intensity of anthocyanidins and identify the variations, we used metabolomics technique and detected significant different accumulation of anthocyanidins in common wild rice (Oryza rufipogon, with purple leaf sheath) and cultivated rice (Oryza sativa, with green leaf sheath). In this study, we identified and characterized a well-known MYB transcription factor, OsC1, through phenotypic (leaf sheath color) and metabolic (metabolite profiling) genome-wide association studies (pGWAS and mGWAS) in 160 common wild rice (O. rufipogon) and 151 cultivated (O. sativa) rice varieties. Transgenic experiments demonstrated that biosynthesis and accumulation of cyanidin-3-Galc, cyanidin 3-O-rutinoside and cyanidin O-syringic acid, as well as purple pigmentation in leaf sheath were regulated by OsC1. A total of 25 sequence variations of OsC1 constructed 16 functional haplotypes (higher accumulation of the three anthocyanidin types within purple leaf sheath) and 9 non-functional haplotypes (less accumulation of anthocyanidins within green leaf sheath). Three haplotypes of OsC1 were newly identified in our germplasm, which have potential values in functional genomics and molecular breeding of rice. Gene-to-metabolite analysis by mGWAS and pGWAS provides a useful and efficient tool for functional gene identification and omics-based crop genetic improvement.

Bacterial leaf blight (BLB), among the most serious diseases in rice production, is caused by Xanthomonas oryzae pv. oryzae (Xoo). Xa23, the broadest resistance gene against BLB in rice, is widely used in rice breeding. In this study, the rice variety CBB23 carrying the Xa23 resistance gene was inoculated with AH28 and PXO99^A to identify differentially expressed genes (DEGs) associated with the resistance. Transcriptome sequencing of the infected leaves showed 7997 DEGs between the two strains at different time points, most of which were up-regulated, including cloned rice anti-blight, peroxidase, pathology-related, protein kinase, glucosidase, and other coding genes, as well as genes related to lignin synthesis, salicylic acid, jasmonic acid, and secondary metabolites. Additionally, the DEGs included 40 cloned, five NBS-LRR, nine SWEET family, and seven phenylalanine aminolyase genes, and 431 transcription factors were differentially expressed, the majority of which belonged to the WRKY, NAC, AP2/ERF, bHLH, and MYB families. Metabolomics analysis showed that a large amount of alkaloid and terpenoid metabolite content decreased significantly after inoculation with AH28 compared with inoculation with PXO99^A, while the content of amino acids and their derivatives significantly increased. This study is helpful in further discovering the pathogenic mechanism of AH28 and PXO99^A in CBB23 rice and provides a theoretical basis for cloning and molecular mechanism research related to BLB resistance in rice.