Generation of aroma in three-line hybrid rice through CRISPR/Cas9 editing of BETAINE ALDEHYDE DEHYDROGENASE2 (OsBADH2).

Aroma or fragrance in rice is a genetically controlled trait; Its high appreciation by consumers increases the rice market price. Previous studies have revealed that the rice aroma is controlled by a specific gene called BETAINE ALDEHYDE DEHYDROGENASE (OsBADH2), and mutation of this gene leads to the accumulation of an aromatic substance 2-acetyl-1-pyrroline (2-AP). The use of genetic engineering to produce aroma in commercial and cultivated hybrids is a contemporary need for molecular breeding. The current study reports the generation of aroma in the three-line hybrid restorer line Shu-Hui-313 (SH313). We created knock-out (KO) lines of OsBADH2 through the CRISPR/Cas9. The analysis of KO lines revealed a significantly increased content of 2AP in the grains compared with the control. However, other phenotypic traits (plant height, seed setting rate, and 1000-grain weight) were significantly decreased. These KO lines were crossed with a non-aromatic three-line hybrid rice male sterile line (Rong-7-A) to produce Rong-7-You-626 (R7Y626), R7Y627 and R7Y628. The measurement of 2-AP revealed significantly increased contents in these cross combinations. We compared the content of 2-AP in tissues at the booting stage. Data revealed that young spike stalk base contained the highest content of 2-AP and can be used for identification (by simple chewing) of aromatic lines under field conditions. In conclusion, our dataset offers a genetic source and illustrates the generation of aroma in non-aromatic hybrids, and outlines a straightforward identification under field conditions.

Rapid Visual Detection of Elite Erect Panicle Dense and Erect Panicle 1 Allele for Marker-Assisted Improvement in Rice (Oryza sativa L.) Using the Loop-Mediated Isothermal Amplification Method.

Molecular-assisted breeding is an effective way to improve targeted agronomic traits. dep1 (dense and erect panicle 1) is a pleiotropic gene that regulates yield, quality, disease resistance, and stress tolerance, traits that are of great value in rice (Oryza sativa L.) breeding. In this study, a colorimetric LAMP (loop-mediated isothermal amplification) assay was developed for the detection of the dep1 allele and tested for the screening and selection of the heavy-panicle hybrid rice elite restorer line SHUHUI498, modified with the allele. InDel (Insertion and Deletion) primers (DEP1_F and DEP1_R) and LAMP primers (F3, B3, FIP, and BIP) for genotyping were designed using the Primer3 Plus (version 3.3.0) and PrimerExplore (version 5) software. Our results showed that both InDel and LAMP markers could be used for accurate genotyping. After incubation at a constant temperature of 65 °C for 60 min with hydroxynaphthol blue (HNB) as a color indicator, the color of the LAMP assay containing the dep1 allele changed to sky blue. The SHUHUI498 rice line that was detected in our LAMP assay displayed phenotypes consistent with the dep1 allele such as having a more compact plant architecture, straight stems and leaves, and a significant increase in the number of effective panicles and spikelets, demonstrating the effectiveness of our method in screening for the dep1 allele in rice breeding.

HCC1, a Polygalacturonase, Regulates Chlorophyll Degradation via the Ethylene Synthesis Pathway.

Chlorophyll degradation is an important physiological process and is essential for plant growth and development. However, how chlorophyll degradation is controlled at the cellular and molecular level remains largely elusive. Pectin is a main component of the primary cell wall, and polygalacturonases (PGs) is a group of pectin-hydrolases that cleaves the pectin backbone and release oligogalacturonide. Whether and how PGs affect chlorophyll degradation metabolism and its association with ethylene (ETH) have not been reported before. Here, we report a novel function of PG in a mutant 'high chlorophyll content1' hcc1, which displayed a decrease in growth and yield. Our morphological, biochemical and genetic analyses of hcc1, knockout lines and complementation lines confirm the function of HCC1 in chlorophyll degradation. In hcc1, the PG activity, ETH content and D-galacturonic acid (D-GA) was significantly decreased and showed an increase in the thickness of the cell wall. Exogenous application of ETH and D-GA can increase ETH content and induce the expression of HCC1, which further can successfully induce the chlorophyll degradation in hcc1. Together, our data demonstrated a novel function of HCC1 in chlorophyll degradation via the ETH pathway.

Rapid Visual Detection of High Nitrogen-Use Efficiency Gene OsGRF4 in Rice (Oryza sativa L.) Using Loop-Mediated Isothermal Amplification Method.

The GROWTH-REGULATING FACTOR4 (OsGRF4) allele is an important target for the development of new high nitrogen-use efficiency (NUE) rice lines that would require less fertilizers. Detection of OsGRF4 through PCR (polymerase chain reaction)-based assay is cumbersome and needs advanced laboratory skills and facilities. Hence, a method for conveniently and rapidly detecting OsGRF4 on-field is a key requirement for further research and applications. In this study, we employed cleaved amplified polymorphic sequences (CAPs) and loop-mediated isothermal amplification (LAMP) techniques to develop a convenient visual detection method for high NUE gene OsGRF4NM73 (OsGRF4 from the rice line NM73). The TC→AA mutation at 1187-1188 bp loci was selected as the target sequence for the OsGRF4NM73 allele. We further employed this method of identification in 10 rice varieties that carried the OsGRF4 gene and results revealed that one variety (NM73) carries the target OsGRF4NM73 allele, while other varieties did not possess the osgrf4 genotype. The optimal LAMP reaction using hydroxynaphthol blue (HNB), a chromogenic indicator, was carried out at 65 °C for 60 min, and the presence of OsGRF4NM73 allele was confirmed by color changes from violet to sky blue. The results of this study showed that the LAMP method can be conveniently and accurately used to detect the OsGRF4NM73 gene in rice.

Development of novel palbociclib-based CDK4/6 inhibitors exploring the back pocket behind the gatekeeper.

CDK4/6 inhibitors plus endocrine therapy is a standard therapy for HR+/HER2- breast cancer. Herein, using structure-based drug design strategy, a novel series of palbociclib derivatives were designed and synthesized as CDK4/6 inhibitors, among which compound 17m exhibited more potent CDK4/6 inhibitory activity and in vitro antiproliferative activity against the phosphorylated Rb-positive cell line MDA-MB-453 than the approved drug palbociclib. Moreover, compound 17m possessed remarkable CDK4/6 selectivity over other CDK family members including CDK1, CDK2, CDK3, CDK5, CDK7 and CDK9. The potent and selective CDK4/6 inhibitory activity endowed compound 17m with robust G1 cell cycle arrest ability in MDA-MB-453 cells. The intracellular inhibition of CDK4/6 by 17m was confirmed by western blot analysis of the levels of phosphorylated Rb in MDA-MB-453 cells. With respect to the metabolic stability, compound 17m possessed longer half-life (t1/2) in mouse liver microsome than palbociclib.

A putative SUBTILISIN-LIKE SERINE PROTEASE 1 (SUBSrP1) regulates anther cuticle biosynthesis and panicle development in rice.

INTRODUCTION: Panicle abortion is a severe physiological defect and causes a reduction in grain yield. OBJECTIVES: In this study, we aim to provide the characterization and functional analysis of a mutant apa1331 (apical panicle abortion1331). METHODS: The isolated mutant from an EMS-mutagenized population was subjected to SSR analysis and Mutmap assay for candidate gene mapping. We performed phenotypic analysis, anthers cross-sections morphology, wax and cutin profiling, biochemical assays and phylogenetic analysis for characterization and evaluation of apa1331. We used CRISPR/Cas9 disruption for functional validation of its candidate gene. Furthermore, comparative RNA-seq and relative expression analysis were performed to get further insights into mechanistic role of the candidate gene. RESULTS: The anthers from the apical spikelets of apa1331 were degenerated, pollen-less and showed defects in cuticle formation. Transverse sections of apa1331 anthers showed defects in post-meiotic microspore development at stage 8-9. Gas Chromatography showed a significant reduction of wax and cutin in anthers of apa1331 compared to Wildtype (WT). Quantification of H2O2 and MDA has indicated the excessive ROS (reactive oxygen species) in apa1331. Trypan blue staining and TUNEL assay revealed cell death and excessive DNA fragmentation in apa1331. Map-based cloning and Mutmap analysis revealed that LOC_Os04g40720, encoding a putative SUBTILISIN-LIKE SERINE PROTEASE (OsSUBSrP1), harbored an SNP (A > G) in apa1331. Phenotypic defects were only seen in apical spikelets due to highest expression of OsSUBSrP1 in upper panicle portion. CRISPR-mediated knock-out lines of OsSUBSrP1 displayed spikelet abortion comparable to apa1331. Global gene expression analysis revealed a significant downregulation of wax and cutin biosynthesis genes. CONCLUSIONS: Our study reports the novel role of SUBSrP1 in anther cuticle biosynthesis by ROS-mediated programmed cell death in rice.

Investigation of flavonoid components and their associated antioxidant capacity in different pigmented rice varieties.

Black and red rice are flavonoid-rich and nutritious. However, comprehensive information of flavonoid components in different pigmented rice varieties remain unclear. Here, we analyze the differences in flavonoid components in black, red, and white rice by ultra-high-performance liquid chromatography (UPLC) and metabolome analysis. Cyanidin-3-glucoside (Cy-3-G), peonidin-3-glucoside (Pe-3-G), quercetin, dihydromyricetin, naringin, and taxifolin contents were significantly high in black rice. By contrast, catechin and epicatechin contents were substantial in red rice. Cy-3-G was the main anthocyanin and its content was more than four times that of Pe-3-G in black rice varieties. Trifolin hardly showed specificity and exhibited a high content in all rice varieties. The antioxidant capacity of the red and black rice varieties was significantly higher than that of white rice. Moreover, in black and red rice, quercetin and catechin respectively exhibited the strongest antioxidant capacity and a good contribution toward the total flavonoid content, and mean time, white rice possessed antioxidant capacity main derived from quercetin and trifolin. Besides, the study also found that there was slightly inconsistent results between UPLC and metabolome, because certain components with trace by metabolome were not detected by UPLC, but their combination could play a complementary role in the exploration of metabolic components to confirm the ingredients.

Rice ß-Glucosidase 4 (Os1ßGlu4) Regulates the Hull Pigmentation via Accumulation of Salicylic Acid.

Salicylic acid (SA) is a stress hormone synthesized in phenylalanine ammonia-lyase (PAL) and the branching acid pathway. SA has two interconvertible forms in plants: SAG (SA O-ß-glucoside) and SA (free form). The molecular mechanism of conversion of SA to SAG had been reported previously. However, which genes regulate SAG to SA remained unknown. Here, we report a cytoplasmic ß-glucosidase (ß-Glu) which participates in the SA pathway and is involved in the brown hull pigmentation in rice grain. In the current study, an EMS-generated mutant brown hull 1 (bh1) displayed decreased contents of SA in hulls, a lower photosynthesis rate, and high-temperature sensitivity compared to the wild type (WT). A plaque-like phenotype (brown pigmentation) was present on the hulls of bh1, which causes a significant decrease in the seed setting rate. Genetic analysis revealed a mutation in LOC_Os01g67220, which encodes a cytoplasmic Os1ßGlu4. The knock-out lines displayed the phenotype of brown pigmentation on hulls and decreased seed setting rate comparable with bh1. Overexpression and complementation lines of Os1ßGlu4 restored the phenotype of hulls and normal seed setting rate comparable with WT. Subcellular localization revealed that the protein of Os1ßGlu4 was localized in the cytoplasm. In contrast to WT, bh1 could not hydrolyze SAG into SA in vivo. Together, our results revealed the novel role of Os1ßGlu4 in the accumulation of flavonoids in hulls by regulating the level of free SA in the cellular pool.

Phytohormones and Transcriptome Analyses Revealed the Dynamics Involved in Spikelet Abortion and Inflorescence Development in Rice.

Panicle degeneration, sometimes known as abortion, causes heavy losses in grain yield. However, the mechanism of naturally occurring panicle abortion is still elusive. In a previous study, we characterized a mutant, apical panicle abortion1331 (apa1331), exhibiting abortion in apical spikelets starting from the 6 cm stage of panicle development. In this study, we have quantified the five phytohormones, gibberellins (GA), auxins (IAA), abscisic acid (ABA), cytokinins (CTK), and brassinosteroids (BR), in the lower, middle, and upper parts of apa1331 and compared these with those exhibited in its wild type (WT). In apa331, the lower and middle parts of the panicle showed contrasting concentrations of all studied phytohormones, but highly significant changes in IAA and ABA, compared to the upper part of the panicle. A comparative transcriptome of apa1331 and WT apical spikelets was performed to explore genes causing the physiological basis of spikelet abortion. The differential expression analysis revealed a significant downregulation and upregulation of 1587 and 978 genes, respectively. Hierarchical clustering of differentially expressed genes (DEGs) revealed the correlation of gene ontology (GO) terms associated with antioxidant activity, peroxidase activity, and oxidoreductase activity. KEGG pathway analysis using parametric gene set enrichment analysis (PGSEA) revealed the downregulation of the biological processes, including cell wall polysaccharides and fatty acids derivatives, in apa1331 compared to its WT. Based on fold change (FC) value and high variation in expression during late inflorescence, early inflorescence, and antherdevelopment, we predicted a list of novel genes, which presumably can be the potential targets of inflorescence development. Our study not only provides novel insights into the role of the physiological dynamics involved in panicle abortion, but also highlights the potential targets involved in reproductive development.

PHDMF: A Flexible and Scalable Personal Health Data Management Framework Based on Blockchain Technology.

Currently, most of the personal health data (PHD) are managed and stored separately by individual medical institutions. When these data need to be shared, they must be transferred to a trusted management center and approved by data owners through the third-party endorsement technology. Therefore, it is difficult for personal health data to be shared and circulated over multiple medical institutions. On the other hand, the use of directly exchanging and sharing the original data has become inconsistent with the data rapid growth of medical institutions because of the need of massive data transferring across agencies. In order to secure sharing and managing the mass personal health data generated by various medical institutions, a federal personal health data management framework (PHDMF, https://hvic.biosino.org/PHDMF) has been developed, which had the following advantages: 1) the blockchain technology was used to establish a data consortium over multiple medical institutions, which could provide a flexible and scalable technical solution for member extension and solve the problem of third-party endorsement during data sharing; 2) using data distributed storage technology, personal health data could be majorly stored in their original medical institutions, and the massive data transferring process was of no further use, which could match up with the data rapid growth of these institutions; 3) the distributed ledger technology was utilized to record the hash value of data, given the anti-tampering feature of the technology, malicious modification of data could be identified by comparing the hash value; 4) the smart contract technology was introduced to manage users' access and operation of data, which made the data transaction process traceable and solved the problem of data provenance; and 5) a trusted computing environment was provided for meta-analysis with statistic information instead of original data, the trusted computing environment could be further applied to more health data, such as genome sequencing data, protein expression data, and metabolic profile data through combining the federated learning and blockchain technology. In summary, the framework provides a convenient, secure, and trusted environment for health data supervision and circulation, which facilitate the consortium establish over medical institutions and help achieve the value of data sharing and mining.

Disruption of LLM9428/OsCATC Represses Starch Metabolism and Confers Enhanced Blast Resistance in Rice.

Catalases (CATs) are important self-originating enzymes and are involved in many of the biological functions of plants. Multiple forms of CATs suggest their versatile role in lesion mimic mutants (LMMs), H2O2 homeostasis and abiotic and biotic stress tolerance. In the current study, we identified a large lesion mimic mutant9428 (llm9428) from Ethyl-methane-sulfonate (EMS) mutagenized population. The llm9428 showed a typical phenotype of LMMs including decreased agronomic yield traits. The histochemical assays showed decreased cell viability and increased reactive oxygen species (ROS) in the leaves of llm9428 compared to its wild type (WT). The llm9428 showed enhanced blast disease resistance and increased relative expression of pathogenesis-related (PR) genes. Studies of the sub-cellular structure of the leaf and quantification of starch contents revealed a significant decrease in starch granule formation in llm9428. Genetic analysis revealed a single nucleotide change (C > T) that altered an amino acid (Ala > Val) in the candidate gene (Os03g0131200) encoding a CATALASE C in llm9428. CRISPR-Cas9 targetted knockout lines of LLM9428/OsCATC showed the phenotype of LMMs and reduced starch metabolism. Taken together, the current study results revealed a novel role of OsCATC in starch metabolism in addition to validating previously studied functions of CATs.

Design, synthesis and biological evaluation of dual HDAC and VEGFR inhibitors as multitargeted anticancer agents.

Herein, a novel series of dual histone deacetylase (HDAC) and vascular endothelial growth factor receptor (VEGFR) inhibitors were designed, synthesized and biologically evaluated based on previously reported pazopanib-based HDAC and VEGFR dual inhibitors. Most target compounds showed significant HDAC1, HDAC6 and VEGFR2 inhibition, which contributed to their potent antiproliferative activities against multiple cancer cell lines and significant antiangiogenic potencies in both human umbilical vein endothelial cell (HUVEC) tube formation assays and rat thoracic aorta ring assays. Further HDAC selectivity evaluations indicated that hydroxamic acids 5 and 9e possessed HDAC isoform selectivity profiles similar to that of the approved HDAC inhibitor suberoylanilide hydroxamic acid(SAHA), while hydrazide12 presented an HDAC isoform selectivity profilesimilar to that of the clinical HDAC inhibitor MS-275. The VEGFR inhibition profiles of 5, 9e and 12 were similar to that of the approved VEGFR inhibitor pazopanib. The intracellular target engagements of Compounds 5 and 12 were confirmed by western blot analysis. The metabolic stabilities of 5, 9e and 12 in mouse liver microsomes were inferior to that of pazopanib. These dual HDAC and VEGFR inhibitors provide lead compounds for further structural optimization to obtainpolypharmacological anticancer agents.

Rice hybrid mimics have stable yields equivalent to those of the F1 hybrid and suggest a basis for hybrid vigour.

MAIN CONCLUSION: We have developed long term stable high yielding rice lines, Hybrid Mimics, from commercial hybrids. The vigour of the Mimic and the hybrid are developmental changes. These Mimics could substitute for hybrid seed for planting. We have used two pre-existing high-yielding hybrid systems (FLY1 and DY527) to develop Hybrid Mimics. In the FLY1 hybrid system we selected, under field conditions, F6 lines which have high grain yields and biomass equivalent to the F1 hybrids, stable over subsequent F7, F8 and later generations. We have termed these lines Hybrid Mimics. The mimics are mostly homozygous as a consequence of selfing in each generation. We have repeated this selection procedure in the second independent hybrid system DY527, producing Mimics with similar characteristics to the F1 hybrid. In both hybrid systems the selection criterion, based on the phenotype of the F1 hybrid, results in the Mimics having grain yield and biomass similar to that of the F1 hybrid. In each generation of the breeding program the plant population has increased phenotypic homogeneity. The genomes of the Mimic plants do not contain any common heterozygous segments negating claims that the vigour of hybrids depends upon heterozygosity of particular loci. Both hybrids and Mimics have early germination and commence photosynthesis before the parents, providing enhanced growth which is maintained throughout the life cycle. The biochemical parameters of photosynthesis in the hybrids and Mimics do not differ from those of the parents. Grain quality and resistance to the two major diseases, bacterial blight and rice blast are similar in the Mimics and hybrids. The Mimics overcome the major disadvantage of hybrids where F2 phenotypic segregation prevents their use as a crop beyond the F1 generation.

A homologous gene of OsREL2/ASP1, ASP-LSL regulates pleiotropic phenotype including long sterile lemma in rice.

BACKGROUND: Panicle is a harvesting organ of rice, and its morphology and development are closely associated with grain yield. The current study was carried on a mutant screened through an EMS (ethyl-methane sulphonate) mutagenized population of a Japonica cultivar Kitaake (WT). RESULTS: A mutant, named as asp-lsl (aberrant spikelet-long sterile lemma), showed a significant decrease in plant height, number of tillers, thousand-grains weight, seed setting rate, spikelet length, kernel length and effective number of grains per panicle as compared to WT. Asp-lsl showed a pleiotropic phenotype coupled with the obvious presence of a long sterile lemma. Cross-sections of lemma showed an increase in the cell volume rather than the number of cells. Genetic segregation analysis revealed its phenotypic trait is controlled by a single recessive nuclear gene. Primary and fine mapping indicated that candidate gene controlling the phenotype of asp-lsl was located in an interval of 212 kb on the short arm of chromosome 8 between RM22445 and RM22453. Further sequencing and indels markers analysis revealed LOC_Os08g06480 harbors a single base substitution (G→A), resulting in a change of 521st amino acid(Gly→Glu. The homology comparison and phylogenetic tree analysis revealed mutation was occurred in a highly conserved domain and had a high degree of similarity in Arabidopsis, corn, and sorghum. The CRISPR/Cas9 mutant line of ASP-LSL produced a similar phenotype as that of asp-lsl. Subcellular localization of ASP-LSL revealed that its protein is localized in the nucleus. Relative expression analysis revealed ASP-LSL was preferentially expressed in panicle, stem, and leaves. The endogenous contents of GA, CTK, and IAA were found significantly decreased in asp-lsl as compared to WT. CONCLUSIONS: Current study presents the novel phenotype of asp-lsl and also validate the previously reported function of OsREL2 (ROMOSA ENHANCER LOCI2), / ASP1(ABERRANT SPIKELET AND PANICLE 1).

OsINV3 and Its Homolog, OsINV2, Control Grain Size in Rice.

Vacuolar invertase is involved in sugar metabolism and plays a crucial role in plant growth and development, thus regulating seed size. However, information linking vacuolar invertase and seed size in rice is limited. Here we characterized a small grain mutant sg2 (grain size on chromosome 2) that showed a reduced in grain size and 1000-grain weight compared to the wild type. Map-based cloning and genetic complementation showed that OsINV3 is responsible for the observed phenotype. Loss-of-function of OsINV3 resulted in grains of smaller size when compared to the wild type, while overexpression showed increased grain size. We also obtained a T-DNA insertion mutant of OsINV2, which is a homolog of OsINV3 and generated double knockout (KO) mutants of OsINV2 and OsINV3 using CRISPR/Cas9. Genetic data showed that OsINV2, that has no effect on grain size by itself, reduces grain length and width in the absence of OsINV3. Altered sugar content with increased sucrose and decreased hexose levels, as well as changes vacuolar invertase activities and starch constitution in INV3KO, INV2KO, INV3KOINV2KO mutants indicate that OsINV2 and OsINV3 affect sucrose metabolism in sink organs. In summary, we identified OsINV3 as a positive regulator of grain size in rice, and while OsINV2 has no function on grain size by itself. In the absence of OsINV3, it is possible to detect a role of OsINV2 in the regulation of grain size. Both OsINV3 and OsINV2 are involved in sucrose metabolism, and thus regulate grain size. Our findings increase our understanding of the role of OsINV3 and its homolog, OsINV2, in grain size development and also suggest a potential strategy to improve grain yield in rice.

Transcriptome and Proteome Profiling of Different Colored Rice Reveals Physiological Dynamics Involved in the Flavonoid Pathway.

Black and red rice are rich in both anthocyanin and proanthocyanin content, which belong to a large class of flavonoids derived from a group of phenolic secondary metabolites. However, the molecular pathways and mechanisms underlying the flavonoid biosynthetic pathway are far from clear. Therefore, this study was undertaken to gain insight into physiological factors that are involved in the flavonoid biosynthetic pathway in rice cultivars with red, black, and white colors. RNA sequencing of caryopsis and isobaric tags for relative and absolute quantification (iTRAQ) analyses have generated a nearly complete catalog of mRNA and expressed proteins in different colored rice cultivars. A total of 31,700 genes were identified, of which 3417, 329, and 227 genes were found specific for red, white, and black rice, respectively. A total of 13,996 unique peptides corresponding to 3916 proteins were detected in the proteomes of black, white, and red rice. Coexpression network analyses of differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) among the different rice cultivars showed significant differences in photosynthesis and flavonoid biosynthesis pathways. Based on a differential enrichment analysis, 32 genes involved in the flavonoid biosynthesis pathway were detected, out of which only CHI, F3H, ANS, and FLS were detected by iTRAQ. Taken together, the results point to differences in flavonoid biosynthesis pathways among different colored rice cultivars, which may reflect differences in physiological functions. The differences in contents and types of flavonoids among the different colored rice cultivars are related to changes in base sequences of Os06G0162500, Os09G0455500, Os09G0455500, and Os10G0536400. Current findings expand and deepen our understanding of flavonoid biosynthesis and concurrently provides potential candidate genes for improving the nutritional qualities of rice.

Rice Calcineurin B-Like Protein-Interacting Protein Kinase 31 (OsCIPK31) Is Involved in the Development of Panicle Apical Spikelets.

Panicle apical abortion (PAA) causes severe yield losses in rice production, but details about its development and molecular basis remain elusive. Herein, a PAA mutant, paa1019, was identified among the progeny of an elite indica maintainer rice line Yixiang 1B (YXB) mutagenized population obtained using ethyl methyl sulfonate. The abortion rate of spikelets in paa1019 was observed up to 60%. Genetic mapping combined with Mutmap analysis revealed that LOC_Os03g20380 harbored a single-bp substitution (C to T) that altered its transcript length. This gene encodes calcineurin B-like protein-interacting protein kinase 31 (OsCIPK31) localized into the cytoplasm, and is preferentially expressed in transport tissues of rice. Complementation of paa1019 by transferring the open reading frame of LOC_Os03g20380 from YXB reversed the mutant phenotype, and conversely, gene editing by knocking out of OsCIPK31 in YXB results in PAA phenotype. Our results support that OsCIPK31 plays an important role in panicle development. We found that dysregulation is caused by the disruption of OsCIPK31 function due to excessive accumulation of ROS, which ultimately leads to cell death in rice panicle. OsCIPK31 and MAPK pathway might have a synergistic effect to lead ROS accumulation in response to stresses. Meanwhile the PAA distribution is related to IAA hormone accumulation in the panicle. Our study provides an understanding of the role of OsCIPK31 in panicle development by responding to various stresses and phytohormones.

Mutation in Rice Abscisic Acid2 Results in Cell Death, Enhanced Disease-Resistance, Altered Seed Dormancy and Development.

Lesion mimic mutants display spontaneous cell death, and thus are valuable for understanding the molecular mechanism of cell death and disease resistance. Although a lot of such mutants have been characterized in rice, the relationship between lesion formation and abscisic acid (ABA) synthesis pathway is not reported. In the present study, we identified a rice mutant, lesion mimic mutant 9150 (lmm9150), exhibiting spontaneous cell death, pre-harvest sprouting, enhanced growth, and resistance to rice bacterial and blast diseases. Cell death in the mutant was accompanied with excessive accumulation of H2O2. Enhanced disease resistance was associated with cell death and upregulation of defense-related genes. Map-based cloning identified a G-to-A point mutation resulting in a D-to-N substitution at the amino acid position 110 of OsABA2 (LOC_Os03g59610) in lmm9150. Knock-out of OsABA2 through CRISPR/Cas9 led to phenotypes similar to those of lmm9150. Consistent with the function of OsABA2 in ABA biosynthesis, ABA level in the lmm9150 mutant was significantly reduced. Moreover, exogenous application of ABA could rescue all the mutant phenotypes of lmm9150. Taken together, our data linked ABA deficiency to cell death and provided insight into the role of ABA in rice disease resistance.

Reversal effect of a macrocyclic bisbibenzyl plagiochin E on multidrug resistance in adriamycin-resistant K562/A02 cells.

Plagiochin E is a new macrocyclic bisbibenzyl compound isolated from Marchantia polymorpha. In the previous studies, we reported that when combined with fluconazole, plagiochin E had synergetic effects against the resistant strain of Candida albicans. Herein, we examined the reversal effect of plagiochin E on multidrug resistance in adriamycin-induced resistant K562/A02 cells and the parental K562 cells. Its cytotoxicity and reversal effects on multidrug resistance were assessed by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide) assay. Apoptosis percentage of cells was obtained from Annexin V/fluorescein isothiocyanate (FITC) and propridium iodide (PI) double-staining. The effects of plagiochin E on P-glycoprotein activity were evaluated by measuring rhodamine 123 (Rh123)-associated mean fluorescence intensity and P-glycoprotein expression on the basis of the flow cytometric technology, respectively. The results showed that plagiochin E ranging from 2 to 12 mug/ml had little cytotoxicity against K562/A02 cells. When combined with adriamycin, it significantly promoted the sensitivity of K562/A02 cells toward adriamycin through increasing intracellular accumulation of adriamycin in a dose-dependent manner. Further study demonstrated that the inhibitory effect of plagiochin E on P-glycoprotein activity was the major cause of increased stagnation of adriamycin inside K562/A02 cells, indicating that plagiochin E, as a new class of mutidrug resistance inhibitor, may effectively reverse the multidrug resistance in K562/A02 cells via inhibiting expression and drug-transport function of P-glycoprotein.