STAMENLESS1 activates SUPERWOMAN 1 and FLORAL ORGAN NUMBER 1 to control floral organ identities and meristem fate in rice.

Floral patterns are unique to rice and contribute significantly to its reproductive success. SL1 encodes a C2H2 transcription factor that plays a critical role in flower development in rice, but the molecular mechanism regulated by it remains poorly understood. Here, we describe interactions of the SL1 with floral homeotic genes, SPW1, and DL in specifying floral organ identities and floral meristem fate. First, the sl1 spw1 double mutant exhibited a stamen-to-pistil transition similar to that of sl1, spw1, suggesting that SL1 and SPW1 may located in the same pathway regulating stamen development. Expression analysis revealed that SL1 is located upstream of SPW1 to maintain its high level of expression and that SPW1, in turn, activates the B-class genes OsMADS2 and OsMADS4 to suppress DL expression indirectly. Secondly, sl1 dl displayed a severe loss of floral meristem determinacy and produced amorphous tissues in the third/fourth whorl. Expression analysis revealed that the meristem identity gene OSH1 was ectopically expressed in sl1 dl in the fourth whorl, suggesting that SL1 and DL synergistically terminate the floral meristem fate. Another meristem identity gene, FON1, was significantly decreased in expression in sl1 background mutants, suggesting that SL1 may directly activate its expression to regulate floral meristem fate. Finally, molecular evidence supported the direct genomic binding of SL1 to SPW1 and FON1 and the subsequent activation of their expression. In conclusion, we present a model to illustrate the roles of SL1, SPW1, and DL in floral organ specification and regulation of floral meristem fate in rice.

NONSTOP GLUMES1 Encodes a C2H2 Zinc Finger Protein That Regulates Spikelet Development in Rice.

The spikelet is an inflorescence structure unique to grasses. The molecular mechanisms underlying spikelet development and evolution are unclear. In this study, we characterized three allelic recessive mutants in rice (Oryza sativa): nonstop glumes 1-1 (nsg1-1), nsg1-2, and nsg1-3 In these mutants, organs such as the rudimentary glume, sterile lemma, palea, lodicule, and filament were elongated and/or widened, or transformed into lemma- and/or marginal region of the palea-like organs. NSG1 encoded a member of the C2H2 zinc finger protein family and was expressed mainly in the organ primordia of the spikelet. In the nsg1-1 mutant spikelet, LHS1 DL, and MFO1 were ectopically expressed in two or more organs, including the rudimentary glume, sterile lemma, palea, lodicule, and stamen, whereas G1 was downregulated in the rudimentary glume and sterile lemma. Furthermore, the NSG1 protein was able to bind to regulatory regions of LHS1 and then recruit the corepressor TOPLESS-RELATED PROTEIN to repress expression by downregulating histone acetylation levels of the chromatin. The results suggest that NSG1 plays a pivotal role in maintaining organ identities in the spikelet by repressing the expression of LHS1, DL, and MFO1.

MULTI-FLORET SPIKELET 2, a MYB Transcription Factor, Determines Spikelet Meristem Fate and Floral Organ Identity in Rice.

An understanding of flower and panicle development is crucial for improving yield and quality in majority of grass crops. In this study, we used mapping-based cloning to identify MULTI-FLORET SPIKELET2 (MFS2), which encodes a MYB transcription factor and regulates flower and spikelet development in rice (Oryza sativa). In the mfs2 mutant, specification of palea identity was severely disturbed and showed degradation or transformation into a lemma-like organ, and the number of all floral organs was increased to varying degrees. Due to the increase in the number of floral organs and development of extra transformed palea/marginal region of the palea-like organs, some mfs2 spikelets had a tendency to produce two florets. These defects implied that the mfs2 mutation caused abnormal specification of palea identity and partial loss of spikelet determination. We confirm that MFS2 is a transcriptional repressor that shows strong repression activity by means of two typical ethylene-responsive element binding factor-associated amphiphilic motifs, one of which locates at the C terminus and is capable of interaction with three rice TOPLESS and TOPLESS-related proteins. The results indicate that MFS2 acts as a repressor that regulates floral organ identities and spikelet meristem determinacy in rice by forming a repression complex with rice TOPLESS and TOPLESS-related proteins.

[Effect of nitrogen, phosphorus and potassium fertilizers on Flos Sophorae Immaturus yield and quality in hilly area of Chongqing].

With Sophora japonica at the flowering stage as the object, the effect of nitrogen, phosphorus and potassium fertilizers on the yield composition factors, yield and quality of Flos Sophorae Immaturus (FSI) was studied. The results indicated that in early spring, nitrogen, phosphorus and potassium fertilizer on the amplification rate of S. japonica, FSI yield composition, yield and quality were different significantly, middle to high nitrogen (1.5-2.0 kg/plant) significantly increased the level of panicled clusters, raceme and flower bud number and yield. Phosphorus (1.5-2.0 kg/plant) could significantly increase the total buds of flower number and yield, potassium showed no significant increase in yield and yield components. Comprehensively considering yield and quality of FSI, nitrogen 1.5-2.0 kg/plant, phosphorus 1.5-2.0 kg/plant and potassium 0.6-0.9 kg/plant are appropriate.

Optimization of Microwave-Assisted Extraction Conditions for Five Major Bioactive Compounds from Flos Sophorae Immaturus (Cultivars of Sophora japonica L.) Using Response Surface Methodology.

Microwave-assisted extraction was applied to extract rutin; quercetin; genistein; kaempferol; and isorhamnetin from Flos Sophorae Immaturus. Six independent variables; namely; solvent type; particle size; extraction frequency; liquid-to-solid ratio; microwave power; and extraction time were examined. Response surface methodology using a central composite design was employed to optimize experimental conditions (liquid-to-solid ratio; microwave power; and extraction time) based on the results of single factor tests to extract the five major components in Flos Sophorae Immaturus. Experimental data were fitted to a second-order polynomial equation using multiple regression analysis. Data were also analyzed using appropriate statistical methods. Optimal extraction conditions were as follows: extraction solvent; 100% methanol; particle size; 100 mesh; extraction frequency; 1; liquid-to-solid ratio; 50:1; microwave power; 287 W; and extraction time; 80 s. A rapid and sensitive ultra-high performance liquid chromatography method coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (EIS-Q-TOF MS/MS) was developed and validated for the simultaneous determination of rutin; quercetin; genistein; kaempferol; and isorhamnetin in Flos Sophorae Immaturus. Chromatographic separation was accomplished on a Kinetex C18 column (100 mm × 2.1 mm; 2.6 µm) at 40 °C within 5 min. The mobile phase consisted of 0.1% aqueous formic acid and acetonitrile (71:29; v/v). Isocratic elution was carried out at a flow rate of 0.35 mL/min. The constituents of Flos Sophorae Immaturus were simultaneously identified by EIS-Q-TOF MS/MS in multiple reaction monitoring mode. During quantitative analysis; all of the calibration curves showed good linear relationships (R² > 0.999) within the tested ranges; and mean recoveries ranged from 96.0216% to 101.0601%. The precision determined through intra- and inter-day studies showed an RSD% of <2.833%. These results demonstrate that the developed method is accurate and effective and could be readily utilized for the comprehensive quality control of Flos Sophorae Immaturus.

Identification and gene mapping of a novel mutant supernumerary lodicules (snl) in rice.

In order to gain a better understanding of rice flower development, a rice flower mutant supernumerary lodicules (snl), which was identified from ethyl methane sulfonate (EMS)-treated Jinhui10 (Oryza sativa L. ssp. indica) was used in the present study. In the snl mutant, the palea obtained lemma identity, additional glume-like organs formed, lodicules increased and elongated, stamens decreased, and a few aberrant carpels formed. These phenotypes suggest that SNL is involved in the entire rice flower development. SNL was mapped between two simple sequence repeat markers RM3512 and RM1342 on chromosome 2, an approximate 800 kb region, and it co-segregated with SSR215. We conclude that SNL is a novel gene involved in flower development in rice. The present study will be useful for further cloning of the SNL gene, which will contribute to the elucidation of rice flower development.

[Influence of Carbonization Temperature on Bacterial Community of the Biological Carbon Electrode Based on High-throughput Sequencing Technology].

Microbial diversity of anodic biofilm in bioelectrochemical systems with hemp rod carbonized at 1000 and 1800℃ as anode was investigated using Solexa high-throughput sequencing technology. The results showed that a total of 4231 and 5263 optimized 16S rRNA gene sequences were gained from the electrode biofilm on the hemp rod carbonized at 1000 and 1800℃, respectively. At the level of 97% similarity, 1187 and 1338 OTUs were obtained for electrode biofilm carbonized at 1000 and 1800℃, respectively. The result of α diversity analysis showed that microbial diversity increased with decreasing carbonization temperature. Dominant phylum for both biofilms were Proteobacteria, Firmicutes and Bacteroidetes, which accounted for 66%, 10% and 9%, respectively for 1000℃, while 71%, 7% and 9%, respectively for 1800℃. Beside the coexisting phylum, some unique species were also discovered, demonstrating that carbonization temperature did not only influence the electrode structure, but also affected the microbial community structure.

[Mapping of two fertility-restoring gene for WA cytoplasmic male sterility in minghui63 using SSR markers].

F2 population derived from Shanyou63, F1 hybrid developed from the cross Zhenshan97 A/Zhenshan97B, was used in this study. Fertile bulk was constructed by polling equal amount of 15 highly fertile lines. Sterile bulk was obtained by pooling equal amount of 15 highly sterile lines. Minghui63 and Zhenshan97A, parents of Shanyou63, were analyzed with 302 pairs of SSR primers. 244 pairs of primers gave amplification products, of which 58 pairs of primers on 12 different chromosomes showed polymorphism between the two parents with polymorphic frequency up to 23.77%. Gene bulks were further assayed with the 5 pairs of primers. RM1 on chromosome 1 and RM258, RM304 on chromosome 10 was found to be polymorphic between the two gene bulks. In theory, there should be no difference detected between the two gene bulks except for the target traits governed by fertility-restoring genes. RM1, RM258 and RM304 were probably related to the restorer genes. Ten highly fertile and ten highly sterile lines were selected from F2 population of Shanyou63 to screen the gene bulks. The results showed that specific bands of Minghui63 were detected in all ten highly fertile lines while not observed in all the sterile lines. It indicated that the three SSR markers might be linked to fertility-restoring genes. Dominant lines were not selected due to their inalbility to distinguish recombinant lines from non-recombinant lines. Pure recessive lines were chosen to conduct mapping analysis. A total of 53 highly sterile lines were selected from 900 lines of Shanyou63 F2 population to estimate the genetic distance between three SSR markers and fertility-restoring genes respectively. The results demonstrated that recombination occurred in 2, 3, lines with RM1 and RM258 while no one with RM304. Using MAPMAKER/EXP 3.0, the genetic distance between RM1, RM258, RM304 and the related restorer genes were calculated as 1.9, 2.9 and 0.0 cM, respectively. It is possible that the fertility restoring gene(s) on chromosome 10 for three different types of cytoplasmic male sterility(WA, BT and HL) are of the same, or belong to a gene family.

[Analysis on the inheritance of inhibition and anti-inhibition of coleoptile purple line in rice and the SSR location of Ai(t) gene].

The ratio of purple line: no-purple line(13:3) was observed in six different F2 populations produced by crossing between parents with purple line and no-purple line in coleoptile. The backcross of XNA//XNA/ 21A150 (XNA, no-purple line and CMS, as the recurrent parent) resulted in a ratio of 1:1 (purple line: no-purple line). Genetic analysis showed that the expression of rice coleoptile purple line was influenced by two genes, inhibiting gene I and anti-inhibiting gene Ai(t). I gene inhibits P gene of C_A_P_ system and Ai(t) inhibits / gene, respectively. The gene pools of Ai(t) ai(t) and ai(t) ai(t) were constructed with BF1 of XNA//XNA/21A150. SSR analysis indicated that Ai(t) gene was linked with the markers of RM335, RM295, RM287 and RM21 and the genetic distance from Ai(t) to these four markers were 2.8 cM, 10.2 cM, 13.9 cM, 26.1 cM, respectively.

[Prediction of yield and yield components in hybrid rice by using molecular markers].

Yield and yield components in hybrid rice were investigated using AFLP, RAPD and SSR markers. Ten restorer and five male-sterile lines were crossed in all possible pairs resulting in 50 crosses. Positive loci, effect-increasing loci, effect-decreasing loci and non-environmental loci were selected from the 931 marker loci surveyed in the 15 parental lines and their correlation with yield and yield components were analyzed. The results indicated as follows (1) The correlation between genetic difference and yield and yield components calculated on all three molecular loci failed to reach significant level for most of the traits investigated and can not be used to predict yield and yield components directly. (2) Positive loci were of limited usefulness in the prediction of yield and yield components for their variation with different traits investigated despite that they can improve the correlation coefficient in some degree. (3) Effect-increasing and effect-decreasing loci can greatly improve correlation coefficient and may be used to predict the yield and yield components for their consistence with the environment. (4) The coefficient based on non-environmental loci was high though it was a bit lower than that based on effect-increasing and effect-decreasing loci. It indicated that environment had great effect on yield and yield components in rice.

[Polymorphisms in semilate indica hybrid rice as revealed by AFLPs].

The polymorphisms were analyzed in 15 semilate indica parental materials with AFLPs. The genetic distances between parental materials were small with an average of 0.2033, ranged from 0.0589 to 0.3913. Fifteen parental materials were classified into two groups,cytoplasmic male-sterile lineIand restorer line II. The latter were classified into two subgroups, II-1 and II-2. SubgroupII-2 was consanguinity with Minghui63. The genetic distances between male-sterile line and two restorer line subgroups(II-1 and II-2) did not show significant difference, indicating that the genetic bases of restorer lines were similar,which maybe one of major reasons for the yield still not surpassing Shanyou63 in indica hybrid rice presently. To increase the heterosis of hybrid rice, we must enrich the genetic diversity and expand the genetic differences between parental lines.

[The simple gain of templates of rice genomes DNA for PCR].

The solution of alkali-treated fresh rice leaves was used directly as the templates of PCR. The amplified results were stable, reliable, and had no difference compared with that amplified with rice total DNA extracted by common method. The stable results can still be obtained based on the templates kept at 25 degrees for tow weeks, at 4 degrees for three weeks, at -20 degrees for over four months. With this technique, less material and only common reagent are required, which is especially adapted to the screening of the precious transgenic rice in advance and large-scale PCR tests.