RNA-Seq-Based Profiling of pl Mutant Reveals Transcriptional Regulation of Anthocyanin Biosynthesis in Rice (Oryza sativa L.).

Purple-colored leaves in plants attain much interest for their important biological functions and could be a potential source of phenotypic marker in selecting individuals in breeding. The transcriptional profiling helps to precisely identify mechanisms of leaf pigmentation in crop plants. In this study, two genetically unlike rice genotypes, the mutant purple leaf (pl) and wild (WT) were selected for RNA-sequencing and identifying the differentially expressed genes (DEGs) that are regulating purple leaf color. In total, 609 DEGs were identified, of which 513 and 96 genes were up- and down-regulated, respectively. The identified DEGs are categorized into metabolic process, carboxylic acid biosynthesis, phenylpropanoids, and phenylpropanoid biosynthesis process enrichment by GO analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) confirmed their association with phenylpropanoid synthesis, flavonoid synthesis, and phenylalanine metabolism. To explore molecular mechanism of purple leaf color, a set of anthocyanin biosynthetic and regulatory gene expression patterns were checked by qPCR. We found that OsPAL (Os02g0626100, Os02g0626400, Os04g0518400, Os05g0427400 and Os02g0627100), OsF3H (Os03g0122300), OsC4HL (Os05g0320700), and Os4CL5 (Os08g0448000) are associated with anthocyanin biosynthesis, and they were up-regulated in pl leaves. Two members of regulatory MYB genes (OsMYB55; Os05g0553400 and Os08g0428200), two bHLH genes (Os01g0196300 and Os04g0300600), and two WD40 genes (Os11g0132700 and Os11g0610700) also showed up-regulation in pl mutant. These genes might have significant and vital roles in pl leaf coloration and could provide reference materials for further experimentation to confirm the molecular mechanisms of anthocyanin biosynthesis in rice.

Identification and Characterization of a Novel Strigolactone-Insensitive Mutant, Dwarfism with High Tillering Ability 34 (dhta-34) in Rice (Oryza sativa L.).

Rice tillering ability and plant height are two of the important traits determining the grain yield. A novel rice (Oryza sativa L.) mutant dhta-34 from an Indica cultivar Zhenong 34 treated by ethyl methy1 sulfonate (EMS) was investigated in this study. The dhta-34 mutant significantly revealed thrifty tillers with reduced plant height, smaller panicles and lighter grains. It also exhibited late-maturing (19.80 days later than the wild type) and withered leaf tip during the mature stage. The length of each internode was reduced compared to the wild type, belonging to the dn type (each internode of the plant stem decreased in the same ratio). The longitudinal section of dhta-34 internodes showed that the length of cells was reduced leading to the dwarfism of the mutant. The F2 population derived from a cross between dhta-34 and an Japonica cultivar Zhenongda 104 were used for gene mapping by using the map-based cloning strategy. The gene DHTA-34 was fine mapped in 183.8kb region flanked by markers 3R-7 and 3R-10. The cloning and sequencing of the target region from the mutant revealed that there was a substitution of G to A in the second exon of LOC_Os03g10620, which resulted in an amino acid substitution arginine to histidine. DHTA-34 encoded a protein of the α/ß-fold hydrolase superfamily, which could suppress the tillering ability of rice. DHTA-34 was a strong loss-of-function allele of the Arabidopsis thaliana D14 gene, which was involved in part of strigolactones (SLs) perception and signaling. Moreover, the relative expression of DHTA-34 gene in leaf was higher than that in bud, internode, root or sheath. This study revealed that DHTA-34 played an important role in inhabiting tiller development in rice and further identifying the function of D14.

Transcriptional Profile Corroborates that bml Mutant Plays likely Role in Premature Leaf Senescence of Rice (Oryza sativa L.).

Leaf senescence is the last period of leaf growth and a dynamic procedure associated with its death. The adaptability of the plants to changing environments occurs thanks to leaf senescence. Hence, transcriptional profiling is important to figure out the exact mechanisms of inducing leaf senescence in a particular crop, such as rice. From this perspective, leaf samples of two different rice genotypes, the brown midrib leaf (bml) mutant and its wild type (WT) were sampled for transcriptional profiling to identify differentially-expressed genes (DEGs). We identified 2670 DEGs, among which 1657 genes were up- and 1013 genes were down-regulated. These DEGs were enriched in binding and catalytic activity, followed by the single organism process and metabolic process through gene ontology (GO), while the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the DEGs were related to the plant hormone signal transduction and photosynthetic pathway enrichment. The expression pattern and the clustering of DEGs revealed that the WRKY and NAC family, as well as zinc finger transcription factors, had greater effects on early-senescence of leaf compared to other transcription factors. These findings will help to elucidate the precise functional role of bml rice mutant in the early-leaf senescence.

CLD1/SRL1 modulates leaf rolling by affecting cell wall formation, epidermis integrity and water homeostasis in rice.

Leaf rolling is considered as one of the most important agronomic traits in rice breeding. It has been previously reported that SEMI-ROLLED LEAF 1 (SRL1) modulates leaf rolling by regulating the formation of bulliform cells in rice (Oryza sativa); however, the regulatory mechanism underlying SRL1 has yet to be further elucidated. Here, we report the functional characterization of a novel leaf-rolling mutant, curled leaf and dwarf 1 (cld1), with multiple morphological defects. Map-based cloning revealed that CLD1 is allelic with SRL1, and loses function in cld1 through DNA methylation. CLD1/SRL1 encodes a glycophosphatidylinositol (GPI)-anchored membrane protein that modulates leaf rolling and other aspects of rice growth and development. The cld1 mutant exhibits significant decreases in cellulose and lignin contents in secondary cell walls of leaves, indicating that the loss of function of CLD1/SRL1 affects cell wall formation. Furthermore, the loss of CLD1/SRL1 function leads to defective leaf epidermis such as bulliform-like epidermal cells. The defects in leaf epidermis decrease the water-retaining capacity and lead to water deficits in cld1 leaves, which contribute to the main cause of leaf rolling. As a result of the more rapid water loss and lower water content in leaves, cld1 exhibits reduced drought tolerance. Accordingly, the loss of CLD1/SRL1 function causes abnormal expression of genes and proteins associated with cell wall formation, cuticle development and water stress. Taken together, these findings suggest that the functional roles of CLD1/SRL1 in leaf-rolling regulation are closely related to the maintenance of cell wall formation, epidermal integrity and water homeostasis.

A guanine insert in OsBBS1 leads to early leaf senescence and salt stress sensitivity in rice (Oryza sativa L.).

KEY MESSAGE: A rice receptor-like kinase gene OSBBS1/OsRLCK109 was identified; this gene played vital roles in leaf senescence and the salt stress response. Early leaf senescence can cause negative effects on rice yield, but the underlying molecular regulation is not fully understood. bilateral blade senescence 1 (bbs1), an early leaf senescence mutant with a premature senescence phenotype that occurs mainly performing at the leaf margins, was isolated from a rice mutant population generated by ethylmethane sulfonate (EMS) treatment. The mutant showed premature leaf senescence beginning at the tillering stage and exhibited severe symptoms at the late grain-filling stage. bbs1 showed accelerated dark-induced leaf senescence. The OsBBS1 gene was cloned by a map-based cloning strategy, and a guanine (G) insertion was found in the first exon of LOC_Os03g24930. This gene encodes a receptor-like cytoplasmic kinase and was named OsRLCK109 in a previous study. Transgenic LOC_Os03g24930 knockout plants generated by a CRISPR/Cas9 strategy exhibited similar early leaf senescence phenotypes as did the bbs1 mutant, which confirmed that LOC_Os03g24930 was the OsBBS1 gene. OsBBS1/OsRLCK109 was expressed in all detected tissues and was predominantly expressed in the main vein region of mature leaves. The expression of OsBBS1 could be greatly induced by salt stress, and the bbs1 mutant exhibited hypersensitivity to salt stress. In conclusion, this is the first identification of OsRLCKs participating in leaf senescence and playing critical roles in the salt stress response in rice (Oryza sativa L.).

The ferredoxin-dependent glutamate synthase (OsFd-GOGAT) participates in leaf senescence and the nitrogen remobilization in rice.

Ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) plays major roles in photorespiration and primary nitrogen assimilation. However, due to no mutant or knockdown lines of OsFd-GOGAT have been reported in rice (Oryza sativa L.), the contribution of OsFd-GOGAT to rice foliar nitrogen metabolism remains little up-to-date. Here, we isolated a rice premature leaf senescence mutant named gogat1, which was reduced in 67% of the total GOGAT enzyme activity in leaves. The gogat1 mutant exhibited chlorosis under natural condition, while showed less extent premature leaf senescence under low light treatment. The gogat1 locus was mapped to a 54.1 kb region on chromosome 7, and the sequencing of OsFd-GOGAT showed one substitution (A to T) at the 3017th nucleotide of the open reading frame, leading to the amino-acid substitution of leucine changed to histidine. The gogat1 mutant showed reduced seed setting rate, while the grain protein content in gogat1 mutant was significantly higher than that in wild type. Meanwhile, during the grain-filling stage, total amino acids in the up three leaves and the upmost internode were increased dramatically. The results in this study suggested that OsFd-GOGAT might participate in nitrogen remobilization during leaf senescence, which provides a potential way to improve nitrogen use efficiency in rice.

QTL mapping with different genetic systems for nine non-essential amino acids of cottonseeds.

Amino acid is an important nutrient resource for both human and animals. Using a set of 188 RILs population derived from an elite hybrid cross of upland cotton cultivars 'HS46' × 'MARCABUCAG8US-1-88' and their immortal F2 (IF2) with reciprocal backcrosses BC1F1 and BC2F1 (BC) populations in two environments, the QTLs located on the embryo genome and maternal plant genome for nine amino acids of cottonseed were studied across environments. The QTL Network-CL-2.0-seed software was used to analyze the QTLs and their genetic effects for nine amino acids. A total of 56 QTLs for nine amino acids were detected in both populations, with many having over 5% of phenotypic variation. Ten of the total QTLs could be simultaneously found in the IF2 and BC populations. For most QTLs, the genetic effects from embryo genome were more important than those from maternal plant genome for the performance of nine amino acids. Significant embryo additive main effects and maternal additive main effect with their environment interaction effects from many QTLs were also found in present experiment. Some QTLs with larger phenotypic variation were important for improving the amino-acid contents in cottonseeds.

QTL mapping based on the embryo and maternal genetic systems for non-essential amino acids in rapeseed (Brassica napus L.) meal.

BACKGROUND: Non-essential amino acids are a good source of nitrogen and also very important contributors to the metabolic process. Analysis of quantitative trait locus (QTL) simultaneously located on the amphidiploid embryo and maternal plant nuclear genomes for non-essential amino acid contents in rapeseed meal across different environments was conducive to further clarify the genetic mechanism of seed quality traits. RESULTS: Twenty-eight QTLs associated with arginine (five QTLs), histidine (four QTLs), glutamic acid (three QTLs), glycine (three QTLs), proline (three QTLs), alanine (four QTLs) and aspartic acid (six QTLs) contents were identified in present study. All of these QTLs had significant additive main effects from embryo and maternal plant nuclear genomes with eight of them showing significant embryo dominance main effects and 12 showing notable QTL × environment interaction effects. Among them, 12 QTLs were major QTLs which could explain 13.27-35.71% of the phenotypic variation. Specially, five QTL clusters associated with several QTLs related to multiple traits were distributed on chromosomes A1, A4, A5, A7 and C2. CONCLUSION: Non-essential amino acids in rapeseed meal could be simultaneously controlled by the genetic effects from the QTLs which were located on the chromosomes both in the embryo and maternal plant genetic systems.

[Exploration for effective teaching methods to promote students' learning interest in genetics experiment].

The students' interest in genetic experiments was promoted effectively by adding more genetic projects with research or autonomous selections, adopting the vivid and interesting case and problem-based teaching methods, using the teaching philosophy of diversity to design experimental teaching tools, and taking other comprehensive teaching methods.

Differential iron-bioavailability with relation to nutrient compositions in polished rice among selected Chinese genotypes using Caco-2 cell culture model.

Genotypic variation of iron bioavailability and the relationship between iron bioavailability and nutrient composition in polished rice among 11 rice genotypes were assessed using an in vitro digestion/Caco-2 cell model. The results indicated that significant differences in iron bioavailability were detected among tested rice genotypes, with a 3-fold range, suggesting a possibility of selecting high bioavailable iron by plant breeding. Although iron bioavailability was not significantly correlated with Fe concentration in polished rice among tested rice genotypes, the results also indicated that most of the iron dense genotypes showed relatively high ferritin formation in Caco-2 cell and transported iron. Additionally, iron bioavailability in polished rice was enhanced by addition of ascorbic acid, with a much wider range of Fe bioavailability variation in polished rice with addition of ascorbic acid than that without addition of ascorbic acid. The positive relationship between iron bioavailability in polished rice and cysteine concentration (R = 0.669) or sulfur (S) concentration (R = 0.744) among tested rice genotypes, suggests that cysteine and sulfur concentration in polished rice could be used as an indicator for high iron bioavailability.

[Promotion of tuition quality in Genetics by developing an excellent instructional system].

This article, which is based on the requirements for developing the course of Genetics as an outstanding course, summarizes the experience of engendering an excellent instructional system, expatiates on the effects of its application to the teaching process of "Genetics" and next step work for continuing this instructional system. The course quality of "Genetics" has been improved under the excellent instructional system by including different teaching methods, renovating the teaching contents, innovating the teaching means, developing a practical courseware, writing extractive textbooks, reforming experimental teaching, and constructing an instructional network, together with teaching methods related to scientific research.

[Morphological characteristics and gene mapping of a novel bent pedicel branch (bpb1) mutant in rice].

Rice pedicels are tightly associated with the yield of grain. In the present study, a novel and stable pedicel mutant bpb1 (bent pedicel branch 1) was obtained from the wild type "Zhenong 7" after 60Co γ-ray treatment. The mutant had the typical phenotype of bent pedicel branches with multiple abnormal phenotypes, such as longer pedicels, short panicles, and dwarfism. Detail examination using scanning electron microscopy revealed that the pedicel epidermal hairs and stomas in the mutant were smaller than those in the wild type. The epidermal and sclerenchymatous cells were arranged irregularly, and the cells in the bend region of pedicels became smaller and arranged closely. The transverse observation of the mutant pedicel branches showed that the small vascular bundles arranged differently from those of the wild type. Genetic analysis indicated that the abnormal phenotypes were controlled by a single recessive gene. Using the F2 mapping population from the bpb1 mutant crossed with the japonica rice variety "Zhenongda 104", the bpb1 gene was mapped in a 343 kb region between two SSR markers, RM21537 and RM21552, at the long arm of chromosome 7. Because no homologous gene was found in this region until now, bpb1 might be a novel gene related to the pedicel development and growth. This study could be beneficial to future cloning and functional analysis of the bpb1 gene.

Enhanced inactivation of Escherichia coli by ultrasound combined with peracetic acid during water disinfection.

Peracetic acid (PAA) is a desirable disinfectant for municipal wastewater because of its potent disinfection performance and limited toxic by-products. This study explored the efficiency and mechanism of Escherichia coli inactivation by PAA combined with ultrasound simultaneously (ultrasound + PAA) or (ultrasound → PAA) sequentially. The result showed that 60 kHz ultrasound combined with PAA sequentially (60 kHz → PAA) had excellent inactivation performance on E. coli, up to 4.69-log10. The result also showed that the increase of pH and humic acid concentration in solution significantly reduced the inactivation efficiency of 60 kHz → PAA treatment. We also observed that the increase of temperature was beneficial to the disinfection, while anions (Cl-; HCO3-) had little effect. With 60 kHz → PAA, the PAA and the synergism between PAA and ultrasound played major contribution to the inactivation, which we assumed might be due to both the diffusion of PAA into the cells and the damage to the cytomembrane by ultrasound, as evidenced through the laser confocal microscopy (LSCM), scanning electron microscope (SEM) and transmission electron microscope (TEM). The inactivation mechanism involved the destruction of cell membrane and loss of intracellular material. Empirically, 60 kHz → PAA was found to be effective for the inactivation of E. coli in actual wastewater, and the regrowth potential of E. coli treated by 60 kHz → PAA was significantly lower than that treated only by PAA.

Genotypic differences in arsenic, mercury, lead and cadmium in milled rice (Oryza sativa L.).

The contents of arsenic, mercury, lead and cadmium in milled rice were determined. Among 216 genotypes, the As, Hg, Pb and Cd contents were ranged from 5.06 to 296.45, 2.46 to 65.85, 4.16 to 744.95 and 5.91 to 553.40 ng/g, respectively. Six genotypes with lower contents of toxic metal elements were selected. The averages of As and Pb contents for indica rice were higher than those of japonica rice, while the averages of Hg and Cd contents were in contrast. Compared with white brown rice, the milled rice from black and red brown rice contained lower contents of four elements. Significant negative correlation was found between As content and alkaline spread value. Significant correlations were observed between As and aspartic acid (Asp) content, Hg and Asp or leucine contents, Pb and cysteine or methionine contents. Cd content was significantly negatively correlated with protein and 14 amino acid contents.

Fe/N codoped porous graphitic carbon derived from macadamia shells as an efficient cathode oxygen reduction catalyst in microbial fuel cells.

In this study, Fe/N codoped porous graphitic carbon derived from macadamia shells was prepared at different temperatures as cathodic catalysts for microbial fuel cells (MFCs), with K2FeO4 as a bifunctional catalyst for porosity and graphitization. The catalyst prepared at 750 °C (referred to as MSAC-750) showed a large specific surface area (1670.3 m2 g-1), graphite structure, and high pyridine-N and Fe-N X contents. Through the electrochemical workstation test, MSAC-750 shows excellent oxygen reduction reaction (ORR) activity, with an onset potential of 0.172 V and a half-wave potential of -0.028 V (vs. Ag/AgCl) in a neutral medium, and the ORR electron transfer number is 3.89. When applied to the MFCs as cathodic catalysts, a higher maximum power density and voltage of 378.68 mW m-2 and 0.425 V were achieved with the MSAC-750 catalyst and is superior to that of the Pt/C catalyst (300.85 mW m-2 and 0.402 V). In this case, a promising method is hereby established for the preparation of an excellent electrochemical catalyst for microbial fuel cells using inexpensive and easily available macadamia shells.

[Improving experimental teaching facilities and opening up of laboratories in order to raise experimental teaching quality of genetics].

Advanced teaching facilities and the policy of opening laboratories to students play an important role in raising the quality in the experimental teaching of Genetics. This article introduces the superiority of some advanced instruments and equipment (such as digital microscope mutual laboratory system, flow cytometry, and NIRSystems) in the experimental teaching of genetics, and illustrates with examples the significance of exposing students to experiments in developing their creative consciousness and creative ability. This article also offers some new concepts on the further improvement upon teaching in the laboratory.

[Study on the global and local calibration methods of tryptophan content in rice by near infrared reflectance spectroscopy].

Tryptophan is one of the essential amino acids in human, and also considered as an important limiting amino acid in rice. Alkali hydrolysis method was used to determine the tryptophan content of 272 representative samples selected from the 1 256 samples in four years. Based on the milled rice flour spectroscopy, by comparing the prediction abilities of the equations with global calibrations and local calibration, the global calibration method with MPLS and the local calibration method were better than others. Their calibration models could be introduced to application because of low standard errors of prediction for both methods (0.007%) and high coefficients of determination (87.1% and 87.4%, respectively). Furthermore, the prediction ability of the equation based on the brown rice spectroscopy could be still used for determination application though their effects were worse than that of the milled rice flour spectroscopy. It was concluded that the equation could be directly used to estimate the content of tryptophan in milled rice, and the technology of near infrared reflectance spectroscopy developed in this study could be applied in selecting breeding materials for higher tryptophan content and in quality control during rice processing.

Identification and characterization of dwarf 62, a loss-of-function mutation in DLT/OsGRAS-32 affecting gibberellin metabolism in rice.

A dwarf mutant, dwarf 62 (d62), was isolated from rice cultivar 93-11 by mutagenesis with γ-rays. Under normal growth conditions, the mutant had multiple abnormal phenotypes, such as dwarfism, wide and dark-green leaf blades, reduced tiller numbers, late and asynchronous heading, short roots, partial male sterility, etc. Genetic analysis indicated that the abnormal phenotypes were controlled by the recessive mutation of a single nuclear gene. Using molecular markers, the D62 gene was fine mapped in 131-kb region at the short arm of chromosome 6. Positional cloning of D62 gene revealed that it was the same locus as DLT/OsGRAS-32, which encodes a member of the GRAS family. In previous studies, the DLT/OsGRAS-32 is confirmed to play positive roles in brassinosteroid (BR) signaling. Sequence analysis showed that the d62 carried a 2-bp deletion in ORF region of D62 gene which led to a loss-of-function mutation. The function of D62 gene was confirmed by complementation experiment. RT-PCR analysis and promoter activity analysis showed that the D62 gene expressed in all tested tissues including roots, stems, leaves and panicles of rice plant. The d62 mutant exhibited decreased activity of α-amylase in endosperm and reduced content of endogenous GA(1). The expression levels of gibberellin (GA) biosynthetic genes including OsCPS1, OsKS1, OsKO1, OsKAO, OsGA20ox2/SD1 and OsGA2ox3 were significantly increased in d62 mutant. Briefly, these results demonstrated that the D62 (DLT/OsGRAS-32) not only participated in the regulation of BR signaling, but also influenced GA metabolism in rice.

Characterization and fine mapping of the glabrous leaf and hull mutants (gl1) in rice (Oryza sativa L.).

The glabrous leaf and hull (gl1) mutants were isolated from M(2) generation of indica cultivar 93-11. These mutants produced smooth leaves and hairless glumes under normal growth conditions. By analyzing through scanning electron microscope, it was revealed that the leaf trichomes, including macro and micro hairs, were deficient in these mutants. Genetic analysis indicated that the mutation was controlled by a single recessive gene. Using nine SSR markers and one InDel marker, the gl1 gene was mapped between RM1200 and RM2010 at the short arm of chromosome 5, which was consistent with the mapping of gl1 in previous studies. To facilitate the map-based cloning of the gl1 gene, 12 new InDel markers were developed. A high-resolution genetic and physical map was constructed by using 1,396 mutant individuals of F(2) mapping population. Finally, the gl1 was fine mapped in 54-kb region containing 10 annotated genes. Cloning and sequencing of the target region from four gl1 mutants (gl1-1, gl1-2, gl1-3 and gl1-4) and four glabrous rice varieties (Jackson, Jefferson, Katy and Lemont) all showed that the same single point mutation (A-->T) occurred in the 5'-untranslated region (UTR) of the locus Os05g0118900 (corresponding to the 3'-UTR of STAR2). RT-PCR analysis of the locus Os05g0118900 revealed that its mRNA expression level was normal in gl1 mutant. RNA secondary structure prediction showed that the single point mutation resulted in a striking RNA conformational change. These results suggest that the single point mutation is most likely responsible for the glabrous leaf and hull phenotypes in rice.

The Purple Leaf (pl6) Mutation Regulates Leaf Color by Altering the Anthocyanin and Chlorophyll Contents in Rice.

The anthocyanin biosynthesis attracts strong interest due to the potential antioxidant value and as an important morphological marker. However, the underlying mechanism of anthocyanin accumulation in plant tissues is not clearly understood. Here, a rice mutant with a purple color in the leaf blade, named pl6, was developed from wild type (WT), Zhenong 41, with gamma ray treatment. By map-based cloning, the OsPL6 gene was located on the short arm of chromosome 6. The multiple mutations, such as single nucleotide polymorphism (SNP) at -702, -598, -450, an insertion at -119 in the promoter, three SNPs and one 6-bp deletion in the 5'-UTR region, were identified, which could upregulate the expression of OsPL6 to accumulate anthocyanin. Subsequently, the transcript level of structural genes in the anthocyanin biosynthesis pathway, including OsCHS, OsPAL, OsF3H and OsF3'H, was elevated significantly. Histological analysis revealed that the light attenuation feature of anthocyanin has degraded the grana and stroma thylakoids, which resulted in poor photosynthetic efficiency of purple leaves. Despite this, the photoabatement and antioxidative activity of anthocyanin have better equipped the pl6 mutant to minimize the oxidative damage. Moreover, the contents of abscisic acid (ABA) and cytokanin (CK) were elevated along with anthocyanin accumulation in the pl6 mutant. In conclusion, our results demonstrate that activation of OsPL6 could be responsible for the purple coloration in leaves by accumulating excessive anthocyanin and further reveal that anthocyanin acts as a strong antioxidant to scavenge reactive oxygen species (ROS) and thus play an important role in tissue maintenance.