The miR167-OsARF12 module regulates rice grain filling and grain size downstream of miR159.

Grain weight and quality are always determined by grain filling. Plant microRNAs have drawn attention as key targets for regulation of grain size and yield. However, the mechanisms that underlie grain size regulation remain largely unclear because of the complex networks that control this trait. Our earlier studies demonstrated that suppressed expression of miR167 (STTM/MIM167) substantially increased grain weight. In a field test, the yield increased up to 12.90%-21.94% because of a significantly enhanced grain filling rate. Here, biochemical and genetic analyses revealed the regulatory effects of miR159 on miR167 expression. Further analysis indicated that OsARF12 is the major mediator by which miR167 regulates rice grain filling. Overexpression of OsARF12 produced grain weight and grain filling phenotypes resembling those of STTM/MIM167 plants. Upon in-depth analysis, we found that OsARF12 activates OsCDKF;2 expression by directly binding to the TGTCGG motif in its promoter region. Flow cytometry analysis of young panicles from OsARF12-overexpressing plants and examination of cell number in cdkf;2 mutants verified that OsARF12 positively regulates grain filling and grain size by targeting OsCDKF;2. Moreover, RNA sequencing results suggested that the miR167-OsARF12 module is involved in the cell development process and hormone pathways. OsARF12-overexpressing plants and cdkf;2 mutants exhibited enhanced and reduced sensitivity to exogenous auxin and brassinosteroid (BR) treatment, confirming that targeting of OsCDKF;2 by OsARF12 mediates auxin and BR signaling. Our results reveal that the miR167-OsARF12 module works downstream of miR159 to regulate rice grain filling and grain size via OsCDKF;2 by controlling cell division and mediating auxin and BR signals.

ResNetKhib: a novel cell type-specific tool for predicting lysine 2-hydroxyisobutylation sites via transfer learning.

Lysine 2-hydroxyisobutylation (Khib), which was first reported in 2014, has been shown to play vital roles in a myriad of biological processes including gene transcription, regulation of chromatin functions, purine metabolism, pentose phosphate pathway and glycolysis/gluconeogenesis. Identification of Khib sites in protein substrates represents an initial but crucial step in elucidating the molecular mechanisms underlying protein 2-hydroxyisobutylation. Experimental identification of Khib sites mainly depends on the combination of liquid chromatography and mass spectrometry. However, experimental approaches for identifying Khib sites are often time-consuming and expensive compared with computational approaches. Previous studies have shown that Khib sites may have distinct characteristics for different cell types of the same species. Several tools have been developed to identify Khib sites, which exhibit high diversity in their algorithms, encoding schemes and feature selection techniques. However, to date, there are no tools designed for predicting cell type-specific Khib sites. Therefore, it is highly desirable to develop an effective predictor for cell type-specific Khib site prediction. Inspired by the residual connection of ResNet, we develop a deep learning-based approach, termed ResNetKhib, which leverages both the one-dimensional convolution and transfer learning to enable and improve the prediction of cell type-specific 2-hydroxyisobutylation sites. ResNetKhib is capable of predicting Khib sites for four human cell types, mouse liver cell and three rice cell types. Its performance is benchmarked against the commonly used random forest (RF) predictor on both 10-fold cross-validation and independent tests. The results show that ResNetKhib achieves the area under the receiver operating characteristic curve values ranging from 0.807 to 0.901, depending on the cell type and species, which performs better than RF-based predictors and other currently available Khib site prediction tools. We also implement an online web server of the proposed ResNetKhib algorithm together with all the curated datasets and trained model for the wider research community to use, which is publicly accessible at https://resnetkhib.erc.monash.edu/.

LncRNA FGD5-AS1 reduces cardiomyocyte apoptosis and inflammation by modulating Akt and miR-223-3p expression.

OBJECTIVES: Long non-coding RNAs (lncRNAs) are known to be involved in heart development and function. In this study, we aimed to explore the effect of the lncRNA FGD5 antisense RNA 1 (FGD5-AS1) on acute myocardial infarction (AMI) by targeting miR-223-3p. METHODS: An AMI model was established both in vivo and in vitro. The levels of FGD5-AS1, miR-223-3p and inflammatory factors were detected by real-time quantitative reverse transcription PCR. Cardiomyocyte apoptosis was assessed using TdT-mediated dUTP nick-end labeling assay. The protein levels of cleaved caspase-3, Bcl-2 and Bax were examined using Western blot. Cardiac function was evaluated using hemodynamic analysis and hematoxylin-eosin and Masson's trichrome staining. In addition, an underlying competitive endogenous RNA mechanism was revealed by bioinformatics analysis, dual-luciferase reporter assay and rescue experiments. RESULTS: We found decreased expression of FGD5-AS1 in AMI. Furthermore, FGD5-AS1 expression significantly decreased the infarct size, improved cardiac performance and attenuated cardiac fibrosis by reducing myocardial apoptosis and inflammation. miR-223-3p was a direct target of FGD5-AS1. Moreover, miRNA-223-3p directly downregulated the expression of phosphorylated Akt in primary neonatal rat cardiomyocytes. Further experiments demonstrated that FGD5-AS1 modulated Akt activity to reduce myocardial injury through miR-223-3p. CONCLUSION: The FGD5-AS1/miR-223-3p/Akt pathway is involved in AMI, suggesting that FGD5-AS1 may act as a potential biomarker and therapeutic target for AMI.

miR-92a-3p promotes ox-LDL induced-apoptosis in HUVECs via targeting SIRT6 and activating MAPK signaling pathway.

Atherosclerosis could be induced by multiple factors, including hypertension, hyperlipidemia, and smoking, and its pathogenesis has not been fully elucidated. MicroRNAs have been shown to possess great anti-atherosclerotic potential, but the precise function of miR-92a-3p in atherosclerosis and its potential molecular mechanism have not been well clarified. Flow cytometry assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium bromide (MTT) assay were performed to evaluate effects of oxidized low-density lipoprotein (ox-LDL) on proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs), respectively. Malondialdehyde and superoxide dismutase levels in cell lysate were assessed with biochemical kits. The expression levels of miR-92a-3p and Sirtuin6 (SIRT6) in HUVECs exposed to ox-LDL were estimated by real-time quantitative polymerase chain reaction (RT-qPCR). In addition, the protein levels of SIRT6, c-Jun N-terminal kinase (JNK), phosphorylation JNK (p-JNK), p38 mitogen activated protein kinase (p38 MAPK), and phosphorylation p38 MAPK (p-p38 MAPK) were measured by western blot assays. The relationship between miR-92a-3p and SIRT6 was confirmed by dual-luciferase reporter assay. Ox-LDL induced apoptosis and oxidative stress in HUVECs in concentration- and time-dependent manners. Conversely, miR-92a-3p silencing inhibited apoptosis and SIRT6 expression in HUVECs. The overexpression of miR-92a-3p enhanced apoptosis and phosphorylation levels of JNK and p38 MAPK as well as inhibited proliferation in ox-LDL-induced HUVECs. In addition, SIRT6 was a target of miR-92a-3p. miR-92a-3p negatively regulated SIRT6 expression in ox-LDL-induced HUVECs to activate MAPK signaling pathway in vitro. In summary, miR-92a-3p promoted HUVECs apoptosis and suppressed proliferation in ox-LDL-induced HUVECs by targeting SIRT6 expression and activating MAPK signaling pathway.

miR-92a-3p promotes ox-LDL induced-apoptosis in HUVECs via targeting SIRT6 and activating MAPK signaling pathway

Atherosclerosis could be induced by multiple factors, including hypertension, hyperlipidemia, and smoking, and its pathogenesis has not been fully elucidated. MicroRNAs have been shown to possess great anti-atherosclerotic potential, but the precise function of miR-92a-3p in atherosclerosis and its potential molecular mechanism have not been well clarified. Flow cytometry assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium bromide (MTT) assay were performed to evaluate effects of oxidized low-density lipoprotein (ox-LDL) on proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs), respectively. Malondialdehyde and superoxide dismutase levels in cell lysate were assessed with biochemical kits. The expression levels of miR-92a-3p and Sirtuin6 (SIRT6) in HUVECs exposed to ox-LDL were estimated by real-time quantitative polymerase chain reaction (RT-qPCR). In addition, the protein levels of SIRT6, c-Jun N-terminal kinase (JNK), phosphorylation JNK (p-JNK), p38 mitogen activated protein kinase (p38 MAPK), and phosphorylation p38 MAPK (p-p38 MAPK) were measured by western blot assays. The relationship between miR-92a-3p and SIRT6 was confirmed by dual-luciferase reporter assay. Ox-LDL induced apoptosis and oxidative stress in HUVECs in concentration- and time-dependent manners. Conversely, miR-92a-3p silencing inhibited apoptosis and SIRT6 expression in HUVECs. The overexpression of miR-92a-3p enhanced apoptosis and phosphorylation levels of JNK and p38 MAPK as well as inhibited proliferation in ox-LDL-induced HUVECs. In addition, SIRT6 was a target of miR-92a-3p. miR-92a-3p negatively regulated SIRT6 expression in ox-LDL-induced HUVECs to activate MAPK signaling pathway in vitro. In summary, miR-92a-3p promoted HUVECs apoptosis and suppressed proliferation in ox-LDL-induced HUVECs by targeting SIRT6 expression and activating MAPK signaling pathway.

Seed-Specific Overexpression of SPL12 and IPA1 Improves Seed Dormancy and Grain Size in Rice.

Pre-harvest sprouting (PHS) often results in reduced grain yield and quality and is a major problem in cereal production. Improved seed dormancy would inhibit PHS. Here we show that seed-specific overexpression of two SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) genes SPL12 and IPA1 enhances seed dormancy and inhibits PHS without noticeable effects on shoot architecture in rice. In addition, seed-specific overexpression of IPA1 also increases grain size and thus improves grain productivity. Furthermore, our results suggest that SPL12 enhances the seed dormancy through directly regulating many genes in the gibberellin (GA) pathway. This research provides an efficient method to suppress PHS and will facilitate breeding elite crop varieties.

OsRAD51D promotes homologous pairing and recombination by preventing nonhomologous interactions in rice meiosis.

Homologous recombination is carefully orchestrated to maintain genome integrity. RAD51D has been previously shown to be essential for double-strand break repair in mammalian somatic cells. However, the function of RAD51D during meiosis is largely unknown. Here, through detailed analyses of Osrad51d single and double mutants, we pinpoint the specific function of OsRAD51D in coordinating homologous pairing and recombination by preventing nonhomologous interactions during meiosis. OsRAD51D is associated with telomeres in both meiocytes and somatic cells. Loss of OsRAD51D leads to significant induction of nonhomologous pairing and chromosome entanglements, suggesting its role in suppressing nonhomologous interactions. The failed localization of OsRAD51 and OsDMC1 in Osrad51d, together with the genetic analysis of Osrad51d Osdmc1a Osdmc1b, indicates that OsRAD51D acts at a very early stage of homologous recombination. Observations from the Osrad51d pair1 and Osrad51d ku70 double mutants further demonstrate that nonhomologous interactions require double-strand break formation but do not depend on the KU70-mediated repair pathway. Moreover, the interplay between OsRAD51D and OsRAD51C indicates both conservation and divergence of their functions in meiosis. Altogether, this work reveals that OsRAD51D plays an essential role in the inhibition of nonhomologous connections, thus guaranteeing faithful pairing and recombination during meiosis.

Mutations in MIR396e and MIR396f increase grain size and modulate shoot architecture in rice.

Grain size and plant architecture are critical factors determining crop productivity. Here, we performed gene editing of the MIR396 gene family in rice and found that MIR396e and MIR396f are two important regulators of grain size and plant architecture. mir396ef mutations can increase grain yield by increasing grain size. In addition, mir396ef mutations resulted in an altered plant architecture, with lengthened leaves but shortened internodes, especially the uppermost internode. Our research suggests that mir396ef mutations promote leaf elongation by increasing the level of a gibberellin (GA) precursor, mevalonic acid, which subsequently promotes GA biosynthesis. However, internode elongation in mir396ef mutants appears to be suppressed via reduced CYP96B4 expression but not via the GA pathway. This research provides candidate gene-editing targets to breed elite rice varieties.

The grain yield modulator miR156 regulates seed dormancy through the gibberellin pathway in rice.

The widespread agricultural problem of pre-harvest sprouting (PHS) could potentially be overcome by improving seed dormancy. Here, we report that miR156, an important grain yield regulator, also controls seed dormancy in rice. We found that mutations in one MIR156 subfamily enhance seed dormancy and suppress PHS with negligible effects on shoot architecture and grain size, whereas mutations in another MIR156 subfamily modify shoot architecture and increase grain size but have minimal effects on seed dormancy. Mechanistically, mir156 mutations enhance seed dormancy by suppressing the gibberellin (GA) pathway through de-represssion of the miR156 target gene Ideal Plant Architecture 1 (IPA1), which directly regulates multiple genes in the GA pathway. These results provide an effective method to suppress PHS without compromising productivity, and will facilitate breeding elite crop varieties with ideal plant architectures.

Characterization of a new semi-dominant dwarf allele of SLR1 and its potential application in hybrid rice breeding.

The widespread introduction of semi-dwarf1 (sd1), also known as the 'Green Revolution' gene, has dramatically increased rice yield. However, the extensive use of limited sources of dwarf genes may cause 'bottleneck' effects in breeding new rice varieties. Alternative dwarf germplasms are quite urgent for rice breeding. Here, we characterized a new allele of the rice Slr1-d mutant, Slr1-d6, which reduced plant height by 37%, a much milder allele for dwarfism. Slr-d6 was still responsive to gibberellin (GA) to a reduced extent. The mutation site in Slr1-d6 was less conserved in the TVHYNP domain, leading to the specific semi-dominant dwarf phenotype. Expression of SLR1 and five key GA biosynthetic genes was disturbed in Slr1-d6, and the interaction between Slr1-d6 and GID1 was decreased. In the genetic background of cultivar 9311 with sd1 eliminated, Slr1-d6 homozygous plants were ~70 cm tall. Moreover, Slr1-d6 heterozygous plants were equivalent in height to the standard sd1 semi-dwarf 9311, but with a 25% yield increase, showing its potential application in hybrid rice breeding.

Mutations in a subfamily of abscisic acid receptor genes promote rice growth and productivity.

Abscisic acid (ABA) is a key phytohormone that controls plant growth and stress responses. It is sensed by the pyrabactin resistance 1 (PYR1)/PYR1-like (PYL)/regulatory components of the ABA receptor (RCAR) family of proteins. Here, we utilized CRISPR/Cas9 technology to edit group I (PYL1-PYL6 and PYL12) and group II (PYL7-PYL11 and PYL13) PYL genes in rice. Characterization of the combinatorial mutants suggested that genes in group I have more important roles in stomatal movement, seed dormancy, and growth regulation than those in group II. Among all of the single pyl mutants, only pyl1 and pyl12 exhibited significant defects in seed dormancy. Interestingly, high-order group I mutants, but not any group II mutants, displayed enhanced growth. Among group I mutants, pyl1/4/6 exhibited the best growth and improved grain productivity in natural paddy field conditions, while maintaining nearly normal seed dormancy. Our results suggest that a subfamily of rice PYLs has evolved to have particularly important roles in regulating plant growth and reveal a genetic strategy to improve rice productivity.

OsRAD51C is essential for double-strand break repair in rice meiosis.

RAD51C is one of the RAD51 paralogs that plays an important role in DNA double-strand break repair by homologous recombination. Here, we identified and characterized OsRAD51C, the rice homolog of human RAD51C. The Osrad51c mutant plant is normal in vegetative growth but exhibits complete male and female sterility. Cytological investigation revealed that homologous pairing and synapsis were severely disrupted. Massive chromosome fragmentation occurred during metaphase I in Osrad51c meiocytes, and was fully suppressed by the CRC1 mutation. Immunofluorescence analysis showed that OsRAD51C localized onto the chromosomes from leptotene to early pachytene during prophase I, and that normal loading of OsRAD51C was dependent on OsREC8, PAIR2, and PAIR3. Additionally, ZEP1 did not localize properly in Osrad51c, indicating that OsRAD51C is required for synaptonemal complex assembly. Our study also provided evidence in support of a functional divergence in RAD51C among organisms.

Central region component1, a novel synaptonemal complex component, is essential for meiotic recombination initiation in rice.

In meiosis, homologous recombination entails programmed DNA double-strand break (DSB) formation and synaptonemal complex (SC) assembly coupled with the DSB repair. Although SCs display extensive structural conservation among species, their components identified are poorly conserved at the sequence level. Here, we identified a novel SC component, designated central region component1 (CRC1), in rice (Oryza sativa). CRC1 colocalizes with ZEP1, the rice SC transverse filament protein, to the central region of SCs in a mutually dependent fashion. Consistent with this colocalization, CRC1 interacts with ZEP1 in yeast two-hybrid assays. CRC1 is orthologous to Saccharomyces cerevisiae pachytene checkpoint2 (Pch2) and Mus musculus THYROID receptor-interacting protein13 (TRIP13) and may be a conserved SC component. Additionally, we provide evidence that CRC1 is essential for meiotic DSB formation. CRC1 interacts with homologous pairing aberration in rice meiosis1 (PAIR1) in vitro, suggesting that these proteins act as a complex to promote DSB formation. PAIR2, the rice ortholog of budding yeast homolog pairing1, is required for homologous chromosome pairing. We found that CRC1 is also essential for the recruitment of PAIR2 onto meiotic chromosomes. The roles of CRC1 identified here have not been reported for Pch2 or TRIP13.

The role of rice HEI10 in the formation of meiotic crossovers.

HEI10 was first described in human as a RING domain-containing protein that regulates cell cycle and cell invasion. Mice HEI10(mei4) mutant displays no obvious defect other than meiotic failure from an absence of chiasmata. In this study, we characterize rice HEI10 by map-based cloning and explore its function during meiotic recombination. In the rice hei10 mutant, chiasma frequency is markedly reduced, and those remaining chiasmata exhibit a random distribution among cells, suggesting possible involvement of HEI10 in the formation of interference-sensitive crossovers (COs). However, mutation of HEI10 does not affect early recombination events and synaptonemal complex (SC) formation. HEI10 protein displays a highly dynamic localization on the meiotic chromosomes. It initially appears as distinct foci and co-localizes with MER3. Thereafter, HEI10 signals elongate along the chromosomes and finally restrict to prominent foci that specially localize to chiasma sites. The linear HEI10 signals always localize on ZEP1 signals, indicating that HEI10 extends along the chromosome in the wake of synapsis. Together our results suggest that HEI10 is the homolog of budding yeast Zip3 and Caenorhabditis elegans ZHP-3, and may specifically promote class I CO formation through modification of various meiotic components.

OsSGO1 maintains synaptonemal complex stabilization in addition to protecting centromeric cohesion during rice meiosis.

Shugoshin is a conserved protein in eukaryotes that protects the centromeric cohesin of sister chromatids from cleavage by separase during meiosis. In this study, we identify the rice (Oryza sativa, 2n=2x=24) homolog of ZmSGO1 in maize (Zea mays), named OsSGO1. During both mitosis and meiosis, OsSGO1 is recruited from nucleoli onto centromeres at the onset of prophase. In the Tos17-insertional Ossgo1-1 mutant, centromeres of sister chromatids separate precociously from each other from metaphase I, which causes unequal chromosome segregation during meiosis II. Moreover, the release of OsSGO1 from nucleoli is completely blocked in Ossgo1-1, which leads to the absence of OsSGO1 in centromeric regions after the onset of mitosis and meiosis. Furthermore, the timely assembly and maintenance of synaptonemal complexes during early prophase I are affected in Ossgo1 mutants. Finally, we found that the centromeric localization of OsSGO1 depends on OsAM1, not other meiotic proteins such as OsREC8, PAIR2, OsMER3, or ZEP1.