Generation of new rice germplasms with low amylose content by CRISPR/CAS9-targeted mutagenesis of the FLOURY ENDOSPERM 2 gene.

FLOURY ENDOSPERM 2 (FLO2), encoding a tetratricopeptide repeat domain (TPR)-containing protein located in the nucleus, is considered to be a regulatory protein that controls the biosynthesis of seed storage substances. The diversity of flo2 allele is attributable for the variations in grain appearance, amylose content (AC), and physicochemical properties, influencing the eating and cooking quality (ECQ) of rice. In this study, we used CRISPR/Cas9 to introduce loss-of-function mutations into the FLOURY ENDOSPERM 2 gene in Suken118 (SK118), a widely cultivated elite japonica rice variety in Jiangsu, China. Physiochemical analyses of the flo2 mutants were congruent with previous studies, exhibiting lowered AC and viscosity, risen gel consistency (GC) and gelatinization temperature (GT) values, which were all instrumental to the improvement of ECQ. However, the wrinkled opaque appearance and the decrease in grain width, grain thickness and grain weight imply trade-offs in grain yield. Despite the ex-ante estimation for low yielding, the superior ECQ in these novel genotypes generated by using genome editing approach may have the potential for formulating high value specialty food.

Efficient breeding of low glutelin content rice germplasm by simultaneous editing multiple glutelin genes via CRISPR/Cas9.

Chronic kidney disease (CKD) and phenylketonuria (PKU) patients need to eat rice with low glutelin content. Therefore, breeding low glutelin content rice varieties with high yield and delicious taste is one of the major goals of rice breeders due to the high demand for the product. In this study, we designed three sgRNAs targeting nine glutelin genes and generated nine T-DNA-free homozygous editing lines with reduced glutelin content compared with the wild-type due to simultaneous mutation(s) in 5-7 glutelin genes. The glutelin content of two lines is even significantly lower than that of the low glutelin content cultivar, LGC-1. Compared to the wild-type, these low glutelin lines showed similar agronomic traits, including yield components and viscosity properties, and can be used as new varieties or parental materials for further breeding.

Heterodimer formed by ROC8 and ROC5 modulates leaf rolling in rice.

Moderately rolled leaf is one of the target traits of the ideal plant architecture in rice breeding. Many genes, including homeodomain leucine zipper IV transcription factors ROC5 and ROC8, regulating rice leaf rolling have been cloned and functionally analysed. However, the molecular mechanism by which these genes modulate leaf-rolling remains largely elusive. In this study, we demonstrated the transcription activation activity of both ROC8 and ROC5. Overexpressing ROC8 caused adaxially rolled leaves due to decreased number and size of bulliform cells, whereas knockout of ROC8 induced abaxially rolled leaves due to increased number and size of bulliform cells. ROC8 and ROC5 each could form homodimer, but ROC8 interacted preferably with ROC5 to forms a heterodimer. Importantly, we showed that the ROC8-ROC5 heterodimer rather than the homodimer of ROC8 or ROC5 was functional as neither overexpressing ROC8 in the ROC5 mutant nor overexpressing ROC5 in the ROC8-knockout line could rescue the mutant phenotype. This was further partially supported by the identification of a large number of common differentially expressed genes in single and double mutants of roc8 and roc5. ROC8 and ROC5 were functionally additive as the phenotype of abaxially rolled leaves was stronger in the roc5roc8 double mutant than in their single mutants. Our results provide evidence for the role of dimerization of ROC members in regulating leaf rolling of rice.

RGG1, Involved in the Cytokinin Regulatory Pathway, Controls Grain Size in Rice.

Heterotrimeric GTP binding proteins (G proteins) and cytokinin play important roles in regulating plant growth and development. However, little is known about the mechanism by which they coordinate the regulation of grain size in rice. We functionally characterized one gene, RGG1, encoding a type-A Gγ subunit. Strong GUS staining was detected in young panicles and spikelets, suggesting a role for this gene in modulating panicle-related trait development. Overexpression of RGG1 in Nipponbare (NIP) and Wuyunjing 30 (WYJ30) significantly decreased plant height, panicle length and grain length by regulating cell division. However, rgg1 mutants generated by the CRISPR/Cas9 system exhibited no obvious phenotypic differences, which may be due to the extremely low expression level of this gene in vivo. The transcriptomes of young panicles of NIP, the NIP-rgg1-2 mutant and the NIP-OE2 overexpression line were sequenced, and the results showed that many differentially expressed genes (DEGs) were associated with the cytokinin biosynthetic pathway. We confirmed this result by measuring the endogenous cytokinin levels and found that cytokinin content was lower in the overexpression lines. Additionally, increased expression of RGG1 decreased sensitivity to low concentrations of 6-benzylaminopurine (6-BA). Our results reveal a novel G protein-cytokinin module controlling grain size in rice and will be beneficial for understanding the mechanisms by which G proteins regulate grain size and plant development.

OsPP2C09, a negative regulatory factor in abscisic acid signalling, plays an essential role in balancing plant growth and drought tolerance in rice.

Plants maintain a dynamic balance between plant growth and stress tolerance to optimise their fitness and ensure survival. Here, we investigated the roles of a clade A type 2C protein phosphatase (PP2C)-encoding gene, OsPP2C09, in regulating the trade-off between plant growth and drought tolerance in rice (Oryza sativa L.). The OsPP2C09 protein interacted with the core components of abscisic acid (ABA) signalling and showed PP2C phosphatase activity in vitro. OsPP2C09 positively affected plant growth but acted as a negative regulator of drought tolerance through ABA signalling. Transcript and protein levels of OsPP2C09 were rapidly induced by exogenous ABA treatments, which suppressed excessive ABA signalling and plant growth arrest. OsPP2C09 transcript levels in roots were much higher than those in shoots under normal conditions. After ABA, polyethylene glycol and dehydration treatments, the accumulation rate of OsPP2C09 transcripts in roots was more rapid and greater than that in shoots. This differential expression between the roots and shoots may increase the plant's root-to-shoot ratio under drought-stress conditions. This study sheds new light on the roles of OsPP2C09 in coordinating plant growth and drought tolerance. In particular, we propose that OsPP2C09-mediated ABA desensitisation contributes to root elongation under drought-stress conditions in rice.

OsGASR9 positively regulates grain size and yield in rice (Oryza sativa).

The plant-specific gibberellic acid (GA)-stimulated transcript gene family is critical for plant growth and development. There are 10 family members in rice (Oryza sativa), known as OsGASRs. However, few have been functionally characterized. Here, we investigated the function of OsGASR9 in rice. OsGASR9 transcripts were detected in various tissues, with the lowest and highest levels in leaves and panicles, respectively. Greater mRNA levels accumulated in young, compared with in old, panicles and spikelets. OsGASR9 localized to the plasma membrane, cytoplasm and nucleus. Transgenic RNA interference-derived lines in the Zhonghua 11 background exhibited reduced plant height, grain size and yield compared with the wild-type. The two osgasr9 mutants in the Nipponbare background showed similar phenotypes. Conversely, the overexpression of OsGASR9 in the two backgrounds increased plant height and grain size. A significantly increased grain yield per plant was also observed in the overexpression lines having a Nipponbare background. Furthermore, by measuring the GA-induced lengths of the second leaf sheaths and α-amylase activity levels of seeds, we concluded that OsGASR9 is a positive regulator of responses to GA in rice. Thus, OsGASR9 may regulate plant height, grain size and yield through the GA pathway and could have an application value in breeding.

The Spermine Synthase OsSPMS1 Regulates Seed Germination, Grain Size, and Yield.

Polyamines, including putrescine, spermidine, and spermine, play essential roles in a wide variety of prokaryotic and eukaryotic organisms. Rice (Oryza sativa) contains four putative spermidine/spermine synthase (SPMS)-encoding genes (OsSPMS1, OsSPMS2, OsSPMS3, and OsACAULIS5), but none have been functionally characterized. In this study, we used a reverse genetic strategy to investigate the biological function of OsSPMS1 We generated several homozygous RNA interference (RNAi) and overexpression (OE) lines of OsSPMS1 Phenotypic analysis indicated that OsSPMS1 negatively regulates seed germination, grain size, and grain yield per plant. The ratio of spermine to spermidine was significantly lower in the RNAi lines and considerably higher in the OE lines than in the wild type, suggesting that OsSPMS1 may function as a SPMS. S-Adenosyl-l-methionine is a common precursor of polyamines and ethylene biosynthesis. The 1-aminocyclopropane-1-carboxylic acid (ACC) and ethylene contents in seeds increased significantly in RNAi lines and decreased in OE lines, respectively, compared with the wild type. Additionally, the reduced germination rates and growth defects of OE lines could be rescued with ACC treatment. These data suggest that OsSPMS1 affects ethylene synthesis and may regulate seed germination and plant growth by affecting the ACC and ethylene pathways. Most importantly, an OsSPMS1 knockout mutant showed an increase in grain yield per plant in a high-yield variety, Suken118, suggesting that OsSPMS1 is an important target for yield enhancement in rice.

Rice RAD51 paralogs play essential roles in somatic homologous recombination for DNA repair.

Synthesis-dependent strand annealing (SDSA) and single-strand annealing (SSA) are the two main homologous recombination (HR) pathways in double-strand break (DSB) repair. The involvement of rice RAD51 paralogs in HR is well known in meiosis, although the molecular mechanism in somatic HR remains obscure. Loss-of-function mutants of rad51 paralogs show increased sensitivity to the DSB-inducer bleomycin, which results in greatly compromised somatic recombination efficiencies (xrcc3 in SDSA, rad51b and xrcc2 in SSA, rad51c and rad51d in both). Using immunostaining, we found that mutations in RAD51 paralogs (XRCC3, RAD51C, or RAD51D) lead to tremendous impairment in RAD51 focus formation at DSBs. Intriguingly, the RAD51C mutation has a strong effect on the protein loading of its partners (XRCC3 and RAD51B) at DSBs, which is similar to the phenomenon observed in the case of blocking PI3K-like kinases in wild-type plant. We conclude that the rice CDX3 complex acts in SDSA recombination while the BCDX2 complex acts in SSA recombination in somatic DSB repair. Importantly, RAD51C serves as a fulcrum for the local recruitment of its partners (XRCC3 for SDSA and RAD51B for SSA) and is positively modulated by PI3K-like kinases to facilitate both the SDSA and SSA pathways in RAD51 paralog-dependent somatic HR.

Characterisation of a novel quantitative trait locus, GN4-1, for grain number and yield in rice (Oryza sativa L.).

KEY MESSAGE: A novel QTL for grain number, GN4-1, was identified and fine-mapped to an ~ 190-kb region on the long arm of rice chromosome 4. Rice grain yield is primarily determined by three components: number of panicles per plant, grain number per panicle and grain weight. Among these traits, grain number per panicle is the major contributor to grain yield formation and is a crucial trait for yield improvement. In this study, we identified a major quantitative trait locus (QTL) responsible for rice grain number on chromosome 4, designated GN4-1 (a QTL for Grain Number on chromosome 4), using advanced segregating populations derived from the crosses between an elite indica cultivar 'Zhonghui 8006' (ZH8006) and a japonica rice 'Wuyunjing 8' (WYJ8). GN4-1 was delimited to an ~ 190-kb region on chromosome 4. The genetic effect of GN4-1 was estimated using a pair of near-isogenic lines. The GN4-1 gene from WYJ8 promoted accumulation of cytokinins in the inflorescence and increased grain number per panicle by ~ 17%. More importantly, introduction of the WYJ8 GN4-1 gene into ZH8006 increased grain yield by ~ 14.3 and ~ 11.5% in the experimental plots in 2014 and 2015, respectively. In addition, GN4-1 promoted thickening of the culm and may enhance resistance to lodging. These results demonstrate that GN4-1 is a potentially valuable gene for improvement of yield and lodging resistance in rice breeding.

Identification of QTL Associated with Nitrogen Uptake and Nitrogen Use Efficiency Using High Throughput Genotyped CSSLs in Rice (Oryza sativa L.).

Nitrogen (N) availability is a major factor limiting crop growth and development. Identification of quantitative trait loci (QTL) for N uptake (NUP) and N use efficiency (NUE) can provide useful information regarding the genetic basis of these traits and their associated effects on yield production. In this study, a set of high throughput genotyped chromosome segment substitution lines (CSSLs) derived from a cross between recipient 9311 and donor Nipponbare were used to identify QTL for rice NUP and NUE. Using high throughput sequencing, each CSSL were genotyped and an ultra-high-quality physical map was constructed. A total of 13 QTL, seven for NUP and six for NUE, were identified in plants under hydroponic culture with all nutrients supplied in sufficient quantities. The proportion of phenotypic variation explained by these QTL for NUP and NUE ranged from 3.16-13.99% and 3.76-12.34%, respectively. We also identified several QTL for biomass yield (BY) and grain yield (GY), which were responsible for 3.21-45.54% and 6.28-7.31%, respectively, of observed phenotypic variation. GY were significantly positively correlated with NUP and NUE, with NUP more closely correlated than NUE. Our results contribute information to NUP and NUE improvement in rice.

GNS4, a novel allele of DWARF11, regulates grain number and grain size in a high-yield rice variety.

BACKGROUND: Rice plays an extremely important role in food safety because it feeds more than half of the world's population. Rice grain yield depends on biomass and the harvest index. An important strategy to break through the rice grain yield ceiling is to increase the biological yield. Therefore, genes associated with organ size are important targets for rice breeding. RESULTS: We characterized a rice mutant gns4 (grain number and size on chromosome 4) with reduced organ size, fewer grains per panicle, and smaller grains compared with those of WT. Map-based cloning indicated that the GNS4 gene, encoding a cytochrome P450 protein, is a novel allele of DWARF11 (D11). A single nucleotide polymorphism (deletion) in the promoter region of GNS4 reduced its expression level in the mutant, leading to reduced grain number and smaller grains. Morphological and cellular analyses suggested that GNS4 positively regulates grain size by promoting cell elongation. Overexpression of GNS4 significantly increased organ size, 1000-grain weight, and panicle size, and subsequently enhanced grain yields in both the Nipponbare and Wuyunjing7 (a high-yielding cultivar) backgrounds. These results suggest that GNS4 is key target gene with possible applications in rice yield breeding. CONCLUSION: GNS4 was identified as a positive regulator of grain number and grain size in rice. Increasing the expression level of this gene in a high-yielding rice variety enhanced grain yield. GNS4 can be targeted in breeding programs to increase yields.

Exploitation of heterosis loci for yield and yield components in rice using chromosome segment substitution lines.

We constructed 128 chromosome segment substitution lines (CSSLs), derived from a cross between indica rice (Oryza sativa L.) 9311 and japonica rice Nipponbare, to investigate the genetic mechanism of heterosis. Three photo-thermo-sensitive-genic male sterile lines (Guangzhan63-4s, 036s, and Lian99s) were selected to cross with each CSSL to produce testcross populations (TCs). Field experiments were carried out in 2009, 2011, and 2015 to evaluate yield and yield-related traits in the CSSLs and TCs. Four traits (plant height, spikelet per panicle, thousand-grain weight, and grain yield per plant) were significantly related between CSSLs and TCs. In the TCs, plant height, panicle length, seed setting rate, thousand-grain weight, and grain yield per plant showed partial dominance, indicating that dominance largely contributes to heterosis of these five traits. While overdominance may be more important for heterosis of panicles per plant and spikelet per panicle. Based on the bin-maps of CSSLs and TCs, we detected 62 quantitative trait loci (QTLs) and 97 heterotic loci (HLs) using multiple linear regression analyses. Some of these loci were clustered together. The identification of QTLs and HLs for yield and yield-related traits provide useful information for hybrid rice breeding, and help to uncover the genetic basis of rice heterosis.

Correction: Mapping Quantitative Trait Loci Associated with Toot Traits Using Sequencing-Based Genotyping Chromosome Segment Substitution Lines Derived from 9311 and Nipponbare in Rice (Oryza sativa L.).

[This corrects the article DOI: 10.1371/journal.pone.0151796.].

Mapping Quantitative Trait Loci Associated with Toot Traits Using Sequencing-Based Genotyping Chromosome Segment Substitution Lines Derived from 9311 and Nipponbare in Rice (Oryza sativa L.).

Identification of quantitative trait loci (QTLs) associated with rice root morphology provides useful information for avoiding drought stress and maintaining yield production under the irrigation condition. In this study, a set of chromosome segment substitution lines derived from 9311 as the recipient and Nipponbare as donor, were used to analysis root morphology. By combining the resequencing-based bin-map with a multiple linear regression analysis, QTL identification was conducted on root number (RN), total root length (TRL), root dry weight (RDW), maximum root length (MRL), root thickness (RTH), total absorption area (TAA) and root vitality (RV), using the CSSL population grown under hydroponic conditions. A total of thirty-eight QTLs were identified: six for TRL, six for RDW, eight for the MRL, four for RTH, seven for RN, two for TAA, and five for RV. Phenotypic effect variance explained by these QTLs ranged from 2.23% to 37.08%, and four single QTLs had more than 10% phenotypic explanations on three root traits. We also detected the correlations between grain yield (GY) and root traits, and found that TRL, RTH and MRL had significantly positive correlations with GY. However, TRL, RDW and MRL had significantly positive correlations with biomass yield (BY). Several QTLs identified in our population were co-localized with some loci for grain yield or biomass. This information may be immediately exploited for improving rice water and fertilizer use efficiency for molecular breeding of root system architectures.

Natural Variations in SLG7 Regulate Grain Shape in Rice.

Rice (Oryza sativa) grain shape, which is controlled by quantitative trait loci (QTL), has a strong effect on yield production and quality. However, the molecular basis for grain development remains largely unknown. In this study, we identified a novel QTL, Slender grain on chromosome 7 (SLG7), that is responsible for grain shape, using backcross introgression lines derived from 9311 and Azucena. The SLG7 allele from Azucena produces longer and thinner grains, although it has no influence on grain weight and yield production. SLG7 encodes a protein homologous to LONGIFOLIA 1 and LONGIFOLIA 2, both of which increase organ length in Arabidopsis. SLG7 is constitutively expressed in various tissues in rice, and the SLG7 protein is located in plasma membrane. Morphological and cellular analyses suggested that SLG7 produces slender grains by longitudinally increasing cell length, while transversely decreasing cell width, which is independent from cell division. Our findings show that the functions of SLG7 family members are conserved across monocots and dicots and that the SLG7 allele could be applied in breeding to modify rice grain appearance.

MicroRNA-34a suppresses the breast cancer stem cell-like characteristics by downregulating Notch1 pathway.

MicroRNAs play pivotal roles in cancer stem cell regulation. Previous studies have shown that microRNA-34a (miR-34a) is downregulated in human breast cancer. However, it is unknown whether and how miR-34a regulates breast cancer stem cells. Notch signaling is one of the most important pathways in stem cell maintenance and function. In this study, we verified that miR-34a directly and functionally targeted Notch1 in MCF-7 cells. We reported that miR-34a negatively regulated cell proliferation, migration, and invasion and breast cancer stem cell propagation by downregulating Notch1. The expression of miR-34a was negatively correlated with tumor stages, metastasis, and Notch1 expression in breast cancer tissues. Furthermore, overexpression of miR-34a increased chemosensitivity of breast cancer cells to paclitaxel (PTX) by downregulating the Notch1 pathway. Mammosphere formation and expression of the stemness factor ALDH1 were also reduced in the cells treated with miR-34a and PTX compared to those treated with PTX alone. Taken together, our results indicate that miR-34a inhibited breast cancer stemness and increased the chemosensitivity to PTX partially by downregulating the Notch1 pathway, suggesting that miR-34a/Notch1 play an important role in regulating breast cancer stem cells. Thus miR-34a is a potential target for prevention and therapy of breast cancer.

Smoothened activates breast cancer stem-like cell and promotes tumorigenesis and metastasis of breast cancer.

Smoothened (Smo) is a G protein-coupled receptor protein encoded by the Smo gene of the hedgehog signalling pathway, which is thought to play an important role in maintaining organ patterning, cell differentiation and self-renewal. The possible role of Smo in the process of tumorigenesis and metastasis of breast cancer still remains unclear. The present experiments were to investigate the effect of Smo on activating breast cancer stem-like CD44(+)CD24(-) cells and the tumorigenesis and metastasis of breast cancer. By injected CD44(+)CD24(-) cells (1×10(4)) into the cleared fat pad of NOD/SCID mice, it was observed that CD44(+)CD24(-) cells possess higher tumor-initiating capacity and metastasis properties than equal numbers of non-CD44(+)CD24(-) cells. The mRNA and protein expressions of Smo in CD44(+)CD24(-) cells were higher than those in non-CD44(+)CD24(-) cells, indicating that Smo may play a role in maintaining breast cancer stem cell features. qRT-PCR results revealed that expressions of STAT3, Bcl-2 and cyclinD1 mRNA in MCF-7 cells were decreased after transfected by Smo siRNA. In addition, the expressions of MMP-2 and MMP-9 were downregulated in MCF-7 cells after Smo expression was inhibited. Smo inhibition may be a possible therapeutic target that potentially suppresses breast tumor formation and development.