GJA3, GJA8, CRYAA, CRYBB2, CRYGS, CRYGA, GCNT2, CRYGA, and MIP; and three previously reported cataract-causing mutations in GJA8, CRYAA, and CRYBB2. The most commonly mutated genes were those coding for gap junctions and crystallin proteins. Including previous reports of pediatric cataract-associated mutations in our Australian cohort, known genes account for >60% of familial pediatric cataract in Australia, indicating that still more causative genes remain to be identified.Pediatric cataract is a leading cause of childhood blindness. This study aimed to determine the genetic cause of pediatric cataract in Australian families by screening known disease-associated genes using massively parallel sequencing technology. We sequenced 51 previously reported pediatric cataract genes in 33 affected individuals with a family history (cases with previously known or published mutations were excluded) using the Ion Torrent Personal Genome Machine. Variants were prioritized for validation if they were predicted to alter the protein sequence and were absent or rare with minor allele frequency <1% in public databases. Confirmed mutations were assessed for segregation with the phenotype in all available family members. All identified novel or previously reported cataract-causing mutations were screened in 326 unrelated Australian controls. We detected 11 novel mutations in  Pediatric cataract is one of the leading causes of blindness in children. Approximately 200,000 children worldwide are blind from this condition . In AustAround 25–33% of pediatric cataracts are inherited . GeneticMIP, LIM2  sequencing (MPS) technologies are now accessible and are cost-effective tools to screen many candidate genes in parallel. In this study, we screened our repository of South Eastern Australian individuals with familial pediatric cataract for mutations in known causative genes using the Ion Torrent Personal Genome Machine (PGM). We hypothesized that a significant proportion of familial pediatric cataract cases would be accounted for by mutations in known genes, and that screening genes in parallel would be an effective method for genetic testing in this heterogeneous disease.The study adhered to the tenets of the Declaration of Helsinki and was approved by the Southern Adelaide Clinical Human Research Ethics Committee, Adelaide, Australia, and the Royal Victorian Eye and Ear Hospital (RVEEH) Human Research and Ethics Committee, Melbourne, Australia. The probands in each family were recruited from the eye clinic at Flinders Medical Centre (Adelaide), the Women’s and Children’s Hospital (Adelaide), the Royal Children’s Hospital (Melbourne), or the Royal Victorian Eye and Ear Hospital (Melbourne). Written informed consent was obtained from all participants or their guardians if they were under 18 years old. A detailed family history was obtained and additional affected and unaffected family members were invited to participate in the study. An ophthalmologist examined all available family members.Genomic DNA was extracted from either peripheral whole blood using a QiaAmp DNA Blood Maxi Kit  or from saliva using an Oragene DNA saliva collection kit  according to the manufacturers’ protocols. Participants were included in this study if they reported a family history of pediatric cataract and if a causative mutation had not previously been detected in the family by other means.File S1 . A search in PubMed using the terms “paediatric cataract” or “congenital cataracts” in combination with “genetic” or “gene” was used to include additional genes from more recent publications and those genes not covered by the review (n = 12). We focused on those genes known to be associated with nonsyndromic pediatric cataract, as this was the predominant phenotype in our cohort. In addition, we included some syndromic pediatric cataract genes known to cause a predominantly ocular syndrome  or where pediatric cataract is a major diagnostic feature of the syndrome (NHS) as such genes may also contribute to nonsyndromic cataract . The final design consisted of a total of 1216 amplicons ranging from 125 to 225 bp, covering 94.26% of the target sequence. Primers were supplied in two 100 nM pools . Briefly, the concentration of genomic DNA was determined using the dsDNA HS Assay Kit on a Qubit fluorometer (Life Technologies), and libraries were prepared with the Ion AmpliSeq library kit version 2.0 according to manufacturer’s protocols. Libraries were prepared in two pools per individual, and the amplified pools were combined before partially digesting the primers and barcode adaptor ligation. The amplified library was diluted to 10 pM, and 25 μl of the diluted library was used for template preparation using Ion PGM Template OT2 200 Kit (Life Technologies) and the manufacturer’s protocol. The clonally amplified library was then enriched on an Ion OneTouch enrichment system. Samples were barcoded during library preparation using Ion Xpress Barcode Adapters 1–16 kit (Life Technologies) and pooled in groups of 3–5 during template preparation on the Ion OneTouch.PCR primers to amplify coding, 3′-, and 5′-untranslated regions of the 51 genes were designed with the Ion AmpliSeq Designer tool v1.22  using the High Sensitivity DNA Kit (Agilent Technologies), or by qPCR using the Ion library TaqMan quantification kit (Life Technologies). Sequencing was performed on an Ion Torrent PGM using The Ion PGM Sequencing 200 Kit v2 and an Ion 318 chip (Life Technologies).https://ionreporter.lifetechnologies.com/ir/) using the Ion Reporter Uploader plugin for Torrent Suite (v4.1-r79929). Variants were prioritized for further analysis if they were predicted to be protein-changing, and were absent or rare with minor allele frequency (MAF) <1% in dbSNP137 (http://www.ncbi.nlm.nih.gov/projects/SNP/), the Exome Aggregation Consortium (ExAC) (http://exac.broadinstitute.org/), and gnomAD (http://gnomad.broadinstitute.org/). In addition, identified variants were compared with an in-house list of common sequencing errors previously detected with this gene panel.Torrent Suite (version 3.6) was used to align reads to human genome reference sequence 19 (hg19). The Coverage Analysis plugin (v4.0-r77897) was used to calculate the number of mapped reads, the percentage of on-target reads, and the mean depth of reads. Variants were called using the Variant caller plugin (V4.0-r76860) with the germline algorithm . For annotation, variant call format (VCF) files were uploaded to Ion Reporter V4.0 , 0.5 μM of each primer, 0.5 U Hot Star Plus Taq Polymerase (Qiagen), and 40 ng of gDNA. Five times Q Solution (Qiagen) was included at a final concentration of 1× as required, and water volume was adjusted accordingly. PCR was performed on a Palm Cycler  with one cycle at 95° for 5 min, followed by 30 or 35 cycles (Table S2 in File S1) at 95° for 30 sec, 57−65°  for 30 sec, and 72° for 30 sec, and a final extension step at 72° for 5 min. To clean the PCR products for sequencing, 5 μl of PCR product, 2 μl shrimp alkaline phosphatase  and 0.5 μl (20 units/μl) of exonuclease 1 (Exo1)  were mixed. Reactions were incubated at 37° for 1 hr, followed by incubation at 80° for 20 min to inactivate the enzymes.PCR reactions of 20 μl final volume consisted of 1× Coraload PCR buffer (Qiagen), which gave a final concentration of 1.5 mM Mgi.e., 30 ng for 300 bp product) combined with BigDye Terminator v3.1 (Life Technologies) and 5× Sequencing Buffer (Life Technologies), and made up to 20 μl with water. Reactions were taken through a cycle sequencing PCR protocol on a MasterCycler thermal cycler . PCR extension products were purified using Agencourt CleanSeq Magnetic Beads and a SPRI plate, according to the manufacturer’s protocol . Purified extension products were then resolved using POP-7 polymer on the 3130xl Genetic Analyzer (Life Technologies) in the Flinders Sequencing Facility .Sequencing reactions were prepared with the respective forward primer at 5 μM and purified PCR product at 10 ng/100 bp .http://sift.jcvi.org/) (http://genetics.bwh.harvard.edu/pph2/). The conservation of each altered amino acid was calculated using PhyloP as implemented in Mutation Taster (http://www.mutationtaster.org/) and available through the University of California Santa Cruz (UCSC) genome browser. PhyloP values between −14 and +6 indicate conservation at individual nucleotides, ignoring the effects of their neighbors. Amino acid conservation across species was visualized using the Mutation Taster website. Clinical interpretation of genetic variants by the 2015 ACMG/AMP guideline was determined using InterVar (http://wintervar.wglab.org) .Novel variants were screened in 326 unrelated normal Australian controls recruited from Flinders Medical Centre, Adelaide, using the MassArray platform and iPlEX chemistry (Sequenom) at the Australian Genome Research Facility . Variants identified in families CRCH139, CSA133, and CSA95 were screened in controls using custom TaqMan SNP genotyping assays (Life Technologies) on a StepOne Plus real-time PCR instrument (Life Technologies) using standard manufacturer’s protocols. All variants reported in this study have been submitted to the ClinVar database  shows protein sequence alignments demonstrating the conservation of the altered amino acid in families with causative mutations.Table S1 in File S1) while 28/33 (85%) probands had isolated pediatric cataract. Primers were designed for 1216 amplicons in the size range of 125–225 bp to cover the 51 known causative genes. In total, 154.1 kb of target sequence was included in the design process with amplicons designed for 94.3% of the target sequence (8.8 kb not covered). The presence of repetitive sequence, unacceptable GC content, and melting temperatures of the primers outside the optimal range were the main factors limiting primer design for target regions not covered.We sequenced 51 known pediatric cataract genes in 33 unrelated probands using Ion Torrent MPS technology. Syndromic cataract was present in 5/33 (15%) probands (syndromic features described in Table S3 in File S1).The mean number of mapped reads per sample was 1,536,538, with 91% of reads on target. A mean of 1155 reads was achieved per amplicon, with a coverage uniformity of 89%. Of all the amplicons, 96 and 91% were covered at least 20 and 100 fold, respectively. The average coverage per gene is shown in A total of 4726 variants were annotated . In total, 178 variants were absent/rare (MAF <1%) in publically referenced databases, of which 56 were nonsynonymous exonic variants. Twenty-three variants were selected for validation using Sanger sequencing after filtering out the variants in an in-house list of sequencing artifacts (33 variants). Seventeen variants were validated and six were false positives.File S1 and Of the 17 validated variants , 14 appeGJA3, GJA8, CRYAA, CRYBB2, CRYGS, CRYGA, GCNT2, CRYGA, and MIP), three previously reported cataract-causing mutations in three different genes . The phenotype in each of the 14 families is given in In total, as shown in GJA3 and GJA8) in five families. Of the two families (CSA95 and CSA109) with mutations in GJA3, phenotypic information was not available for family CSA95, but variant p.Thr19Met was predicted to be pathogenic and segregated in the two affected individuals  had cataracts described as nuclear, with no phenotype information available for family CRCH162 ] was detected in family CRVEEH111 that segregated with the disease (CRYAA is a novel frameshift deletion [p.(Gln147Argfs*48)] in a consanguineous family (CSA159) displaying autosomal dominant inheritance ]. These individuals also carried a variant in BFSP2 [p.(Ala379Glu)]. However, this variant was reported to be benign by both SIFT and Polyphen-2 and was less conserved. It was therefore considered not pathogenic. A previously reported truncating mutation, p.(Gln155*) (rs74315489), in the CRYBB2 gene was identified in two affected individuals in family CSA133. No information was available regarding the phenotype in this family. This variant has not been reported in normal populations and was not detected in our local controls, thus is likely pathogenic.One novel and one previously reported mutation were detected in CRYGS which was predicted to be damaging ] was detected in family CRCH136 and was inherited from the unaffected mother. No other variant was identified in GCNT2 in this family.Families CRCH89 and CRCH136 displayed an autosomal recessive inheritance pattern of cataract. Affected members of the consanguineous family, CRCH89, were homozygous for a novel variant [p.(Phe364Ser)] in thogenic . The fouthogenic . A singlMIP was detected in family CSA131 ] in y CSA131 . The moty CSA131 . This faMutations meeting the criteria for potential pathogenicity were not identified in the remaining 19 families (57%) or the other 43 genes screened on this gene panel.In this study, we used targeted massively parallel sequencing to identify genetic variants associated with inherited pediatric cataract. We identified likely causative variants in 42% of previously unsolved familial cases. We detected 11 novel mutations contributing to pediatric cataract, confirmed three previously reported mutations, and identified rare coding variants that may be important in the disease. We have previously identified mutations in genes included in this panel in other families in our repository  and CRYGA (CRCH139). This suggests the involvement of modifier genes altering the penetrance of these mutations and the severity of the cataract; however, such genes have not yet been identified  with incomplete penetrance. Other factors, including high level of conservation at this residue and Polyphen-2 prediction of a functional effect, provide support for contribution of this mutation to cataract in this family. The incomplete penetrance is consistent with the presence of this variant at very low levels in public databases and our local controls. If this variant does not always lead to disease, it would be expected to accumulate in such databases. As public databases increase in size, and are generated from unscreened individuals, we expect that, increasingly, disease-causing variants will be found at low frequency in these resources. This scenario of disease-causing variants being represented in public databases of genetic variation due to the large numbers of individuals sequenced is well exemplified by the p.Gln368* mutation in the MYOC gene, which leads to primary open-angle glaucoma with high penetrance and is present in the 1000 Genomes database with a frequency of 0.06% as rs74315329. Such rare variation should not be automatically discounted when evaluating variants as pathogenic in disease cohorts. SIFT and ACMG predictions of benign for the Arg80His variant of CRCH139 make it difficult to comment on the pathogenicity of this variant without any functional studies. Other possibilities, such as the causative variant being intronic, in a novel not-yet-reported gene (or not included in this panel), or a large structural variant should not be ignored.The possible involvement of rare variants present in public databases in pediatric cataract pathogenesis is suggested by this study. Family CRCH139 has a segregating rare variant in In the current study, 2% of amplicons were covered <20 fold, potentially limiting the ability to detect heterozygous mutations in these amplicons. By chance, the majority of the 16 genes containing these low coverage amplicons are involved in syndromic forms of pediatric cataract. In this study, only five probands had syndromic pediatric cataracts and no mutations were identified in these individuals. It may be that mutations were missed due to low coverage, or that these individuals have mutations in genes not targeted by this panel. Furthermore, it is important to comment that this study is not able to detect large insertions or deletions or any structural variants such as copy number variation (CNV) within screened genes. Such variants have been reported in the literature to be associated with the disease (One of the main advantages of the AmpliSeq method on the Ion Torrent PGM is that it only requires 10–40 ng of DNA as starting material. Many of the DNA samples used in this study were >10 years old, and the limited quantity of DNA available was somewhat degraded. The successful sequencing of these samples and identification of likely causative mutations suggests that quality of DNA is not necessarily a crucial factor in obtaining reliable sequencing results using this methodology.Pediatric cataract is a clinically and genetically heterogeneous condition, which makes accurate molecular diagnosis difficult. Genetic linkage analysis and candidate gene screening are the conventional methods for detecting disease-causing genes for familial diseases, but this approach is limited by the size of the families. In small families, as in this current cohort, gene identification has been difficult due to lack of power for linkage and the excessive cost of screening large numbers of candidate genes using traditional Sanger sequencing. MPS platforms are able to target numerous genes in parallel and are cost-effective tools for gene identification in heterogeneous conditions such as pediatric cataract. In this study, we aimed to determine the genetic contribution of the known pediatric cataract genes to inherited pediatric cataract in an Australian cohort. In our experience, both ophthalmologists and their patients are highly motivated to know the genetic basis of their disease. Developing genetic diagnostic panels increases the chance of obtaining a molecular diagnosis, which allows patients and their families to be better educated about the mode of inheritance, and facilitates more accurate genetic counseling about the risk of recurrence for future pregnancies, enabling discussion regarding possible reproductive options. For example, the parents in family CSA159 enrolled in this research study during a pregnancy with a desire to understand the likelihood of the child being affected with the severe disease observed in their son.Although some degree of genotype/phenotype correlation is beginning to emerge for some pediatric cataract genes, the clinical evaluation of a patient is often insufficient to establish which genes are most likely involved in order to initiate specific genetic testing. This is particularly the case in historic samples where surgery was performed prior to enrolment in this study. These genotype-phenotype correlations are worthy of further study, as genetic counseling in the future should take account of the likely severity of affected status, the likelihood of systemic manifestation, and the chance of incomplete penetrance. Gene panel testing, as has been shown in previous studies (www.g3journal.org/lookup/suppl/doi:10.1534/g3.117.300109/-/DC1.Supplemental material is available online at Click here for additional data file.


The present study examined the relations between preschoolers’ television exposure and executive functions (EF). One hundred and nineteen 3- to 6-year-old children and their parents participated. Parents filled in a questionnaire regarding children’s television viewing time, television content and parental mediation behaviors about their child’s television viewing. The children were asked to finish six EF tasks, including the backward digit span task, the spatial span task, the boy–girl Stroop, the Simon task, the flanker task and the Tower of Hanoi task that assessed working memory, inhibition and planning, respectively. Children’s vocabulary was tested using Peabody Picture Vocabulary Test, and included as control variables in addition to socioeconomic status of the participated families. The results showed that television viewing time and child-directed educational programs were positively associated with EF. In addition, television content fully mediated the effect of television viewing time on EF and parental restrictive approach strategies moderated the effect of television viewing time on EF. With the development and widespread use of electronic media, children now are using electronic media more and more, often at a very early age. Electronic media may be especially influential to children during periods of rapid brain development and plasticity  and may While studies of EF development have focused on American or European children, few studies have examined the relation between television exposure and EF in other cultures, including Chinese culture. On the one hand, television programs in China and the amount of time that Chinese children spend watching television may be different from other cultures. In addition, unlike other western nations that provide guidelines about young children’s screen media use, there are no official recommendations regarding children’s digital media use or television viewing in China. On the other hand, Chinese children perform better than their western counterparts on EF tasks . This maVarious theories have been proposed to explain why media exposure including television viewing may influence child development . There aFigure 1) to understand the effect of television exposure on child development outcomes. This model, which can also help analyze and explain the effect of television exposure on EF, suggests that what children watch (content) and how they watch (context) as well as how much they watch are important factors influencing the nature of television exposure effects  by which we examined both the unique effects, namely, time and content, and the indirect effect (mediation effect) between these factors and children’s EF, moderated by parent mediation behaviors impacting their children’s television viewing to obtain a comprehensive understanding of the influence of television exposure. “A mediating variable transmits the effect of an independent variable on a dependent variable”  and viewing of different content , and to explore the moderating role of parental mediation in these relationships. When measuring television content, we differentiated between adult-directed content and child-directed content, and regarding child-directed content, we further differentiated different kinds of programs, such as educational content, recreational content, and violent content. Last and most important, we extended Christakis’ (2009) model by proposing that television content would mediate the effect between television viewing time and EF. Given that viewing time always involves some type of content, the effect of viewing time would then be at least partly mediated by this content. The conceptual model of the present study is illustrated in We formulated and tested the following hypotheses:(1)Time spent television viewing will positively correlate with EF (H1);(2)Television content will correlate with EF. Specifically, child-directed educational programs will positively correlate with EF (H2a) child-directed fast paced or violent programs (H2b) as well as adult-directed programs will negatively correlate with EF (H2c);(3)Television content will mediate the effect of television viewing time on EF (H3);(4)Parental mediation will moderate the direct effect of television viewing time on EF (H4a), and will moderate the indirect effect of television viewing time on EF through television content (H4b).M = 4.64 years old, SD = 0.92) and their parents (73% mothers) participated in this study. Participants are Han Chinese and speak Mandarin. We adopted two widely used markers of SES: maternal education and household income. 21.8% mothers held a graduate degree, 67.2% mothers were college educated, and 11% mothers indicated they had completed high school. Parents demonstrated their average monthly household income by choosing one of seven income levels, ranging from “less than RMB3,000” to “RMB30,000 or more.” The largest percentage of participants selected “RMB3,000 to RMB6,000” (33.6%), followed by “RMB6,000 to RMB8,000” (31.9%), “RMB8,000 to RMB10,000” (16.8%), “RMB10,000 to RMB15,000” (11.8%), “RMB15,000 to RMB30,000” (2.5%), and “less than RMB 3,000” (3.4%). The average annual household income of Chinese was 49,318 RMB in 2014  age range from 3.17 to 6.31 years old .Parents estimated the hours their child watched television on an average weekday and on an average weekend day during three time periods, namely, morning, midday, and evening, respectively. Parents were told that viewing estimates should include “programs seen on a television, computer, or via DVD or a portable electronic device.”  The sum Parents reported the age at which their child first began watching television , choosinnever) to 3 , parents reported how often their child watched six different channels when they watched television, including CCTV-14 Children’s Channel, Golden Eagle Cartoon Channel, KAKU Children’s Channel, other cartoon channels, CCTV (other than the children’s channel), and other channels. A principal-components factor analysis with oblimin rotation was used to explore the factors of children’s channel viewing. Two factors were extracted, explaining approximately 58.24% of variance. The first factor consisted of the viewing of CCTV-14 Children’s Channel, KAKU Children’s Channel, Golden Eagle Cartoon Channel, and other cartoons channels, explaining about 35.5% of the variance. The second factor included viewing of CCTV (other than the children’s channel) and other channels, and explained 22.75% of the variance. Two factors were constructed into two scales to represent child-directed programs viewing and adult-directed programs viewing. The Cronbach’s alpha coefficients of the two scales were 0.70 and 0.65.In China, China Central Television (CCTV) and local television stations both have special channels to broadcast children’s programs. The highest ratings and the most popular children’s channels include CCTV-14 Children’s Channel, Golden Eagle Cartoon Channel of Hunan TV, and KAKU Children’s Channel of Beijing TV. On a scale from 0 (never) to 3 , these program categories included “action cartoons ,” “classic cartoons ,” “live educational children’s programs ,” “fast-paced cartoons ,” “situation comedies for children ,” and “educational cartoons .”Based on the categories used in a previous study , parentsnever, seldom, sometimes, and often) to each of 12 questions representing three types of parental mediation approaches. The three types of parental approaches included: (a) Instructive approach ; (b) Coviewing approach ; and (c) Restrictive approach . In this study, the Cronbach’s alpha coefficient of the three sub-scales were 0.80, 0.69, and 0.68.Parents were asked to indicate their mediation behavior regarding to children’s television viewing. They were asked to choose one of four answers   involvedChildren’s age, gender and family SES have been linked to differences in the amount and quality of television exposure  as well Multiple hierarchical linear regression models were computed with SPSS 19.0 to test the direct effect of television viewing time, viewing content and parental mediation on EF (Hypotheses 1 and 2). SPSS macro PROCESS;  was run Table 1.The descriptive statistics for all the measures are displayed in Table 2 presents the bivariate correlations between non-media variables, media exposure variables and EF. Children’s EF was highly positively correlated with their age and vocabulary. Positive correlations were evident among television viewing time, children’s channels and cartoon programs. Moreover, the television viewing time was not significantly correlated with EF. While, child’s channels viewing and certain types of cartoon programs including classic cartoons viewing, fast-paced cartoons viewing, and children’s situation comedies viewing were positively correlated with EF. These zero-order correlations should be interpreted with caution. For instance, older children may watch more television and have better EF development than younger children. The results indeed revealed that child’s age was positively related with child’s channels viewing and certain types of cartoon programs. Thus, further regression analysis with these confounding variables under control was conducted.Table 3). In each model child’s age, gender, vocabulary, maternal education and family income were as control variables entered in first step. Children’s television viewing time and the age start watching television, children’s channel viewing, children’s genre viewing and parental guidance patterns on children’s television use, were entered in second steps of Model 1~Modle 4 separately. These models allowed us to observe the unique contribution of each set of media variables to the variance of EF above and beyond the control variables. VIFs of the four models ranged from 1.03 to 2.60, which were lower than the recommended level (VIF < 10) (p < 0.001), vocabulary  and mother education  as significant predictors. In Model 1, Children’s television viewing time and the age start watching television explained a significant 2% of additional variance with television viewing time related to better EF, Cohen f2 = 0.06. In Model 2, children’s channel viewing did not account for significant variance in EF, Cohen f2 = 0.01. In Model 3, six kinds of children’s cartoons or programs explained a significant 7% of additional variance in EF, Cohen f2 = 0.27. Classic cartoons viewing was significantly positively related with EF. Live educational children’s programs viewing was marginal significantly positively related with EF . While, educational cartoons viewing was marginal significantly negatively related with EF . In Model 4, three kinds of parental guidance patterns account for a significant 3% of additional variance in EF with restrictive approach significantly related with poor EF, Cohen f2 = 0.1. Cohen’s categories about effect size   suggestiect size  indicateab = 0.24, SE = 0.11, 95% bias corrected CI ) and live educational children’s programs viewing  were significant because both 95% bias corrected CI did not contain zero. In each analysis, maternal education and family income, children’s age, gender, and vocabulary were controlled. The mediated model explained 69% variance in EF (p < 0.001). Number of bootstrap samples was 5000.SPSS macro PROCESS (Model 4)  was run B = 0.76, SE = 0.33, 95% CI ). Secondly, restrictive approach strategies moderated the indirect effect of television viewing time on EF through child classic cartoons viewing, with the index of moderated mediation equal to -0.17 . The index was negative, meaning that the indirect effect of television viewing time on EF through child classic cartoons viewing was a decreasing function of restrictive approach strategies. Conditional indirect effect(s) of television viewing time on EF at values of the restrictive approach showed that only at a low to modest level of restrictive approach, there was a positive indirect effect of television viewing time on EF through child classic cartoons viewing . Thirdly, restrictive approach strategies did not moderate the indirect effect of television viewing time on EF through live educational children’s programs viewing, with the index of moderated mediation equal to -0.01 , 95% CI of the indexes contain zero, suggesting that the tests of moderated mediation were not statistically significant. However, the indirect effect of television viewing time on EF through live educational children’s programs viewing was significant only at the modest level of restrictive approach . Results about the moderated mediation model was presented in Table 4 and Figure 3.We further applied the SPSS macro PROCESS (Model 8)  to test The present study is one of few studies conducted to examine the relations between television viewing and cognitive outcomes among Chinese preschoolers. Most importantly, the study extends earlier work by considering multiple variables related to children’s television viewing behavior: how old they were when they started watching, how much time they watch (television viewing time), what they watch (television viewing content), and how they watch , as well as multiple measures of EF. Results of the study yielded significant implications for understanding what, how, and when television viewing affects the development of EF. In addition, the present results also suggest both cultural similarities and differences in the associated patterns reported in the literature.Figure 2) was thus confirmed.The result that television viewing time was positively associated with EF confirms Hypothesis 1. Hypothesis 2 was also supported by the finding that although child-directed live educational television and classical cartoons viewing was positively associated with EF while adult-directed television and other kinds programs were not significantly correlated with EF. In addition, it is evident that child’s live educational program and classical cartoons viewing mediated the effect of television viewing time on EF, and these results confirm Hypothesis 3. Parental restrictive approach strategies were also found to moderate the direct effect of television viewing time on EF, as well as the indirect effect of television viewing time on EF through child classic cartoons. Hypothesis 4 which indicated that parental mediation can moderate the direct effect of television viewing on EF and can moderate the indirect effect of television viewing on EF through television content were confirmed. Our proposed conceptual model   . Other p = 1.99) . Foster  = 1.99)  have shoSecond, what children watch (content) and how they watch (context) are arguably more important television viewing variables than how much children watch. As revealed in the present study, the effect of television viewing time on EF disappeared when television viewing content was included in the regression model, suggesting that television viewing content fully mediated the effect of television viewing time on EF. It is revealed that classical cartoons and live educational programs viewing were positively correlated with EF. There are many live educational programs such as tree of knowledge directed to preschoolers. These high-quality programs may enable children to develop EF skills.Third, it is important to consider children’s media use context as the moderate effect of parental restrictive behaviors was evident in the present study. The design of our study enabled us to examine the relation between television viewing and EF while taking context variables into consideration, and our findings suggest a more complex picture: the relation patterns between television viewing and EF also depend on parental restrictive behaviors. Restrictive mediation includes parents’ rules regarding the amount of viewing, permissible and forbidden types of content, and the use of viewing as a behavioral reward or punishment. A significant positive relation between television viewing time and EF was evident only when restrictive mediation was at a low level. In addition, only when restrictive approaches were at low or moderate levels, there was a positive indirect effect of television viewing time on EF through child classical cartoon and live educational programs viewing. Combined with the results of bivariate correlation analyses, it is evident that a restrictive approach was negatively correlated with television viewing time and most kinds of child programs. Although the correlations were not significant, they suggest that parental restrictive behaviors tend to be associated with less television viewing.A particularly interesting finding of this study was the main negative effect of parental restrictive approach strategies on EF. There are several possible explanations. A parental restrictive approach is associated with an arbitrary parenting style, which involves depriving children’s opportunities to regulate their own behavior and requiring them to adapt continually to another’s perspective . Such anAlthough not part of our major concern of the study, an unexpected result involving the relation between mother education and child’s EF. Contrary to prior study , we founIn sum, the present study was conducted to examine the relations between children’s television viewing and the development of EF with various features and strengths. The quality of the measures is one advantage. Our EF tasks were measured directly by children’s responses instead of using parent report. Moreover, in the present study, we controlled other factors that could influence both children’s EF and television viewing. Finally, our research was guided by a theoretical framework and the results largely confirmed the proposed conceptual model. The findings of this study suggest that EF is associated with differences in both television viewing time and content. These findings form an important first step toward the conceptualization and investigation of more nuanced models on the relationship between television viewing and EF. Future research should not only distinguish between the different types of content that children are exposed to, but must also more systematically conceptualize and model mediating and moderating factors in the investigation of relations between EF and television. Building such indirect and conditional effects models will enable us to gain a deeper understanding of the relationship between media use and EF.Although our findings contribute to our understanding of the relation between young children’s television viewing and the development of EF, there are still limitations in our study. First, as with most previous studies, the cross-sectional nature of this study prevents us from drawing conclusions about the direction and causality of the current results. Although television viewing might well affect the development of EF, we cannot exclude the possibility that children with higher EF ability are more capable of choosing child directed educational television programs and spend more time watching them . ResearcThis study reveals that television exposure is related to children’s EF. Furthermore, the relation between television viewing and EF development depend on programming content and parents’ media monitoring behaviors. Given the important role of EF in child’s social function development and school achievement, as well as the widely use of electronic media by young children, increasing our understanding of the links between children’s television viewing as well as the new interactive media and EF is urgently needed.XY designed the study, collected and analyzed data, and drafted the paper. LZ co-designed and revised the paper critically. ZC and ZW revised the paper critically.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Soda lignin does not have thermal flowing characteristics and it is impossible for it to be further thermally molded. To achieve the fusibility of soda lignin for fiber preparation by melt-spinning, an effective method for soda lignin modification was conducted by cooking it with polyethylene glycol (PEG) 400 at various ratios. The higher the ratio of PEG that was used, the more PEG molecular chains were grafted at the alpha carbon of the soda lignin through ether bonds, resulting in lower thermal transition temperatures and more excellent fusibility. The modified soda lignin with a weight ratio of lignin to PEG of 1:4 exhibited a relative thermal stability of molten viscosity at selected temperatures. Thereafter, the resultant fusible soda lignin was successfully melt-spun into filaments with an average diameter of 33 ± 5 μm, which is smaller than that of some industrial lignins. Accordingly, it is possible to utilize soda lignin to produce fibrous carbonaceous materials. As the second most abundant renewable bio-resource, next to cellulose, on the earth, lignin was biosynthesized from the precursor of three monolignols and acted as one of the major cell wall components in higher plants. Although small amounts of lignin in nature were isolated as by-products from pulp and paper manufacturing processes, the amounts of obtained lignin were extremely huge. Generally, around 50 million tons of technical lignins, so-called lignosulfonate, kraft lignin, soda lignin, organosolv lignin, steam explosion lignin, and acid hydrolysis lignin, were produced yearly with various kinds of chemical reagents . HoweverLignin is complexly constructed by three monolignols via the main carbon-carbon bonds and ether bonds. It possesses a hexagonal carbon structure and shows a high carbon content of around 60%–65%, implying it is an appropriate precursor for carbonaceous material preparation. Besides, carbon fibers (CFs), as one of the fibrous carbon materials, have excellent mechanical properties and are usually made into composites with thermal setting resin in the applications of aerospace, the military, construction as well as medical and sporting goods . The preLignin-based CFs have been successfully prepared by spinning and thermostabilization, followed by carbonization. In the case of spinning, several methods were proposed to produce fibers ,9,10,11.However, there were few investigations on the fiber preparation from hardwood and softwood soda lignin by melt-spinning because of a more condensed structure and poorer thermal fusibility. Accordingly, in this article we describe the efforts directed at the modification and characterization of soda lignin with polyethylene glycol (PEG) 400, and attempt to melt-spin the resultant soda lignin for fiber production. Soda lignin powder was isolated from black liquor and subjected to modification with PEG-400 at various weight ratios. The resultant soda lignin exhibited differences in thermal stability by TG measurements as shown in Td), which was defined as a temperature that gives a 5% weight loss [Td of modified soda lignin were 267 °C, 262 °C and 253 °C, respectively, which are higher than that of soda lignin at 209 °C. This result was similar to the changes in the glass transition temperature (Tg) of modified soda lignin, which were determined by DSC. As shown in Tg of soda lignin gradually declined with increasing the weight ratios of PEG. Besides, the PEG contents in each modified soda lignin prepared with the ratios of 1:2, 1:3 and 1:4 were 19.4%, 24.5% and 31.1%, respectively. Accordingly, the high weight ratio of PEG gave rise to the low Td and Tg, which was probably attributed to a greater PEG moiety existing in modified soda lignin prepared with more PEG-400. This was also confirmed by FTIR measurements.Moreover, the high weight loss process was mainly induced by the thermal degradation starting from the decomposition temperature  [FTIR provides an extremely useful method to investigate functional groups. l groups . After mructures ,24,25. Ires [−1) . −1 and 1119 cm−1, assigned to the C–H stretching vibration of the methylene groups and the stretching vibration of the C–O groups, respectively, was likely to become stronger than that of the soda lignin without modification with PEG, indicating the greater abundance of methylene groups and ether bonds existing in the modified soda lignin’s molecular structure. Furthermore, as shown in −1, 2873 cm−1 and 3421 cm−1 to that at 1600 cm−1 (C=C ring stretching vibration), respectively, quantitatively showed the increase with the weight ratio of PEG increasing. Accordingly, the PEG molecular chains were successfully introduced in the lignin structure, and the PEG content increased with the weight ratio of the PEG increasing.Further, the relative peak intensity of all modified soda lignin B–D at 2813C-NMR was performed to elucidate the bonded position of the PEG molecule with respect to the lignin. 13C-NMR spectrum of modified soda lignin with a weight ratio of 1:3. The signals at 39.9 ppm . The content of lignin in black liquor powder is about 30%. Firstly, the powder was homogeneously dissolved in purified water to prepare black liquor. Prior to use, the black liquor was filtrated through filter paper to separate filtrate and residue. After three times filtration, 6 M HCl aqueous solution was slowly added into resultant filtrate with continuous stirring until the pH was around two. The precipitate was then collected by centrifugation at 2270 G for 15 min. Finally, the soda lignin was obtained by freeze drying. Polyethylene glycol (PEG) 400 and sulfuric acid were purchased from SINOPHARM group Chemical Reagent Co., Ltd. Twenty grams of dried soda lignin was blended with PEG 400 at various weight ratios  and 95% aqueous sulfuric acid (1.5 wt % on PEG 400) in a round-bottom flask. The flask was immersed in an oil bath that was preheated at 175 °C with continuous mechanical stirring for 2 h. After the reaction, the flask was cooled with cold water and then the cooking product was poured into distilled water with continuous stirring for 30 min Finally, the modified soda lignin was obtained from resultant mixture by centrifugation at 2270 G for 15 min and then freeze drying.Fusible soda lignin was subjected to melt-spinning using a single-hole nozzle (diameter: 0.8 mm) at the nozzle temperature of 225 °C and winding rates of 56 m/min under a nitrogen pressure to yield thin filaments.Td) of soda lignin were measured using TG thermal analyzer  from room temperature to 300 °C with the heating rate of 10 °C /min in high pure nitrogen stream (30 mL/min). Differential scanning calorimetry (DSC) was performed to measure the glass transition temperature (Tg) of soda lignin using the same machine as TG thermal analyzer with a scan rate of 10 °C/min under nitrogen (30 mL/min). Dried soda lignin samples (4 ± 0.5 mg) were placed in a standard aluminum pan with lid and firstly heated from room temperature to 100 °C to expel any remaining moisture in the sample. After cooling, a second DSC trace was then obtained on heating the sample to 300 °C. The glass transition temperature of the samples were recorded as the onset temperatures of the step change in heat capacity in the DSC trace on the second heating process.The weight loss behaviors and decomposition temperature  in the scanning range of 4000 to 400 cmd6) as the solvent. The concentration of soda lignin solution was 20 to 30 wt %. The 13C-NMR spectrum of the soda lignin solution was recorded using JNM-ECZ400R NMR spectrometer  operating at 150.91 MHz. The spectrum was registered at 25 °C.The chemical structure of soda lignin was estimated by NMR analysis. The soda lignin (200 mg) was dissolved in 600 μL of deuterated dimethyl sulfoxide  at magnifications of 150 to 2000 X and at an accelerating voltage of 5 kV. Before observation, the samples were coated with a thin layer by spraying gold metal using Ion Sputter  before searching morphology. The average diameter of fiber was calculated from the individual measurement value of 30 filaments.Td and Tg. Modified soda lignin prepared with the weight ratio of 1:4 exhibited a relative thermally stable dynamic viscosity, and it was melt-spun into the soda lignin-based fibers with an average diameter of around 33 μm, which is smaller than those of lignin fibers derived from several types of industrial lignins. Therefore, the modified soda lignin could be considered as a promising raw material for preparing fibrous carbon materials such as carbon fibers and activated carbon fibers.Soda lignin was successfully modified with PEG-400 at various weight ratios. The amount of PEG moieties grafted at the alpha carbon position of the modified soda lignin structure increased as the weight ratio of the PEG used increased, resulting in the low thermal transition temperatures of


Chloroflexi (54%), while Firmicutes was dominant in McoDi (60%). For the mesophilic reactors, the dominant archaea was Methanosaeta in MDi, while Methanobacterium and Methanosaeta had similar abundance in McoDi. In the thermophilic systems, the dominant bacteria were Thermotogae, Firmicutes and Synergistetes in both digesters, however, the relative abundance of these phyla were different. For archaea, the genus Methanothermobacter were entirely dominant in both TDi and TcoDi.In this work, performance and microbial structure of a digestion (food waste-only) and a co-digestion process (mixture of cow manure and food waste) were studied at mesophilic (37 °C) and thermophilic (55 °C) temperatures. The highest methane yield (480 mL/g VS) was observed in the mesophilic digester (MDi) fed with food waste alone. The mesophilic co-digestion of food waste and manure (McoDi) yielded 26% more methane than the sum of individual digestions of manure and food waste. The main volatile fatty acid (VFA) in the mesophilic systems was acetate, averaging 93 and 172 mg/L for McoDi and MDi, respectively. Acetate (2150 mg/L) and propionate (833 mg/L) were the main VFAs in the thermophilic digester (TDi), while propionate (163 mg/L) was the major VFA in the thermophilic co-digester (TcoDi). The dominant bacteria in MDi was  Anaerobic digestion process has widely been employed for treatment of various organic wastes because the process can be used for production of value-added products such as an energy-rich gas and bio-fertilizer. This process is carried out by a complex microbial community which degrade various organic compounds into final products such as methane and carbon dioxide, collectively called biogas.3, while digestion processes with food waste as the sole substrate were often found to be unstable5. Several researchers have reported the benefits of using mixed feedstocks, including increased biogas production, enhanced degradation rates and higher digester capacity7. The beneficial effects of co-digestion are mostly related to a balanced availability of macro- and micronutrient required by the microbial community, optimal moisture content, buffer capacity and dilution of inhibitory or toxic compounds. Additionally, co-digestion may improve the process kinetics rather than the bioavailability of the feedstock. Ebner et al.8 measured hydrolysis rates using bio-methane potential assays, and found that co-digestion increased hydrolysis rates when food waste and manure was co-digested compared to mono-digestion in BMP assays. The synergistic effect was attributed to dilution of inhibitory compounds and improved nutrient balance due to co-digestion9. The enzymes involved in hydrogenotrophic methanogenesis and syntrophic acetate oxidation requires trace elements such as selenium (Se), molybdenum (Mo) tungsten (W), cobolt (Co), nickel (Ni) and iron (Fe)4. Lack of these trace elements can limit the syntrophic acetate oxidation as well as formate oxidation5. The resulting accumulation of formate may again inhibit propionic acid oxidation. This will result in an overall acid accumulation, which eventually can cause the pH in the digester to drop, severely affecting or completely stopping the methanogenesis. Notably, the toxicity of intermediate compounds also increases with increasing temperature10 and thermophilic digesters are commonly considered to be more prone to process inhibition than mesophilic digesters7. Moreover, anaerobic digestion processes operating at high-temperature often selects for a less diverse microbial community, which is more vulnerable to stress and operational changes12. Most studies in the literature have focused on enhancing functionality and operation of anaerobic co-digesters using food waste and other feedstocks. Several studies have compared performance of mesophilic and thermophilic digesters3, but comparison of community structures and diversity in anaerobic digesters and anaerobic co-digesters at these different temperatures are rare in the literature.There are presently many research efforts worldwide on anaerobic digestion of food waste to improve process efficiency, stability and economic competitiveness. Studies of co-digestion of food waste generally found that inclusion of food waste was beneficial for methane yieldAccordingly, the aim of this study was to investigate the microbial structure of co-digestion of food waste and cow manure under mesophilic (37 °C) and thermophilic (55 °C) conditions. Additionally, we compared the microbial structure of the co-digestion process to that of food waste digestion alone to determine how the co-digestion process influences the microbial communities. Also, performance parameters were studied under the various conditions and it was attempted to explain performance efficiencies using microbial data.8 that are released as ammonia during the fermentation process and acts as a buffering system.The average pH values for MDi, McoDi, TDi and TcoDi were 7.7 ± 0.1, 7.9 ± 0.1, 7.8 ± 0.2 and 8.2 ± 0.1, respectively. As illustrated in Fig. 13. MDi had the highest methane yield of all four digesters with 479.5 ± 33.9 ml CH4/g VSfeed, which was 11.5%, 7.0% and 31.6% higher than the McoDi, TDi and TcoDi, respectively  digester. The methane yields of manure-fed digester and McoDi were 133 ± 18 and 430 ± 28 mL CH4/g VSfeed, respectively. Based on the measured specific methane yield from MDi and the manure-only reactor, the expected methane yield for McoDi without any synergistic effects would be 341 mL CH4/g VSfeed. However, our results showed that the observed methane yield of McoDi was 430 mL CH4/g VSfeed, meaning that the co-digestion of food waste and manure (McoDi) resulted in 26% higher methane production than the sum of digestions of individual substrates.It has been observed that co-digestion of food waste and manure may enhance biogas production, and lead to more stable digestion processesvalue = 0.05).The solubilization values estimated for MDi, McoDi, TDi and TcoDi were 56, 55, 63 and 48%, respectively . Analysis of the VFAs measurements Fig.  revealedles Fig.  revealedvalue < 0.005) higher microbial richness compared to the digesters fed with food waste alone  was the dominant phylum in McoDi, while the relative abundance of Chloroflexi (22%) and Bacteroidetes (8%) was noticeably lower in McoDi than MDi. Additionally, the candidate phylum WWE1 was identified in McoDi and accounted for 5% of the relative abundance. Limam et al.21 investigated the metabolic function of WWE1 members and suggested that the members of this division were involved in hydrolysis of cellulosic materials. WWE1 was also found in mesophilic co-digestion studies of manure with various agricultural residues23. Thus, the addition of cow manure to the co-digestion system seems to spur the growth of WWE1 members, probably involved in decomposition of cellulose content of the manure. It should be noted that WWE1 was not detected in the cow manure in the current study. The dominance of Chloroflexi in MDi . Anaerolinacea are mostly saccharolytic anaerobes and use a number of carbohydrates for growth25. Use of manure in the feedstock of the co-digestion systems resulted in a different relative abundance of bacterial communities in McoDi and prompted the prevalence of Firmicutes, which include members with very versatile metabolic characteristics and more potential to degrade the recalcitrant manure27. Firmicutes has been reported as one of the major microbial contributors in several studies carried out on anaerobic digesters, indicating that the phylum is common in both mesophilic and thermophilic processes29. Additionally, Firmicutes dominance has also been linked to better reactor performance20. The higher relative abundance of Bacteroidetes in MDi, which was fed with the preprocessed food waste, probably indicates involvement of their members in degradation of intermediate degradation products of carbohydrates and proteins.Statistical analysis demonstrated that the anaerobic co-digestion process resulted in a significantly  where the main genus was Clostridium (42% of phylum). In compliance, three OTUs assigned to Clostridium were significantly more abundant in McoDi compared to MDi  was used to investigate possible links between microbiome and performance. Based on this analysis an association of Clostridium to the concentration of n-Butyrate was observed, although only low levels of butyrate were measured in both mesophilic digesters  of the archaeal sequences. Methanosaeta was also prominent in the McoDi , yet significantly lower compared to MDi . Methanobrevibacter most likely originated from the manure used in this study as the analysis of manure samples showed Methanobrevibacter as the only dominant archaea  and partly due to the continual addition of Methanobrevibacter through manure. This agrees with previous studies indicating the dominance of hydrogen utilizing methanogens in manure-fed digesters33.Archaea belonging to the phylum oDi Fig. . The genaea Fig. . While MThermotogae, Firmicutes, Synergistetes and Bacteroidetes  and a decreased relative abundance of Thermotogae . In comparison, the relative abundance of Firmicutes and Thermotogae was 35% and 40% in the TDi, respectively. Notably, the distribution of Synergistetes, mainly represented by genus Anaerobaculum, differed significantly in the thermophilic digesters  tes Fig. . HoweverCoprothermobacter accounted for more than 50% of the sequences assigned to Firmicutes in the initial samples of both TDi and TcoDi, this portion was largely reduced in the final phase . Instead, a more even distribution of several genera was observed in the final phase , Thermactogenium (9% and 10% in TDi and TcoDi respectively) and unclassified phylotypes assigned to the candidate divisions SHA-98 (17% and 9% in TDi and TcoDi respectively) and MBA08 (8% and 28% in TDi and TcoDi respectively). Coprothermobacter is a proteolytic bacterium involved in the syntrophic fermentation of polypeptides, and the high dominance in the initial phase was most likely reflected by a strong dominance of the Coprothermobacter population in the seed culture from the FREVAR biogas plant, as reported in a previous study35. The OTUs assigned to genus Thermacetogenium were in all probability affiliated to Thermacetogenium phaeum35, a bacterium able to oxidize acetate syntrophically and grow acetogenically on organic acids and alcohols37.While phylotypes assigned to genus ase Fig. , with pr38 also reported the candidate order MBA08 as one of the major bacterial groups in the thermophilic reactor of a staged system used for the co-digestion of whey and manure. However, none of the Firmicutes-associated sequences obtained from cow manure were related to MBA08 in the current study. On the contrary, the relative abundance of the candidate order SHA-98 was greater in the food waste-fed digester . There is almost no knowledge regarding the function of the members of the unclassified order SHA-98 and no genera could be assigned in this group.The phylotype affiliated to candidate order MBA08 was noticeably higher in the TcoDi compared to TDi. This probably suggests the role of the members of the candidate group MBA08 in the co-digestion of food waste and manure. LiClostridiales were probably involved in the degradation of polysaccharides, fermentable carbohydrates and syntrophic oxidation of saturated fatty acids26. Members of Syntrophomonas are believed to oxidize anaerobically C4-C18 saturated fatty acids39. Clostridium consists of bacteria that display metabolic versatility40. Ruminococccaceae, Caldicoprobacteriaceae and Lachnospiraceae were less dominant families. Caldicoprobacter, which exclusively was represented by Caldicoprobacteriaceae in both digesters, ferments xylan and simple sugars to lactate, acetate, H2 and CO241. Interestingly, the family Lachnospiracea, although less abundant, differed in the genus and was mainly composed of Butyrivibrio in TcoDi and of Coprococcus in TDi. Members of the Butyrivibrio and Coprococcus both use fermentable carbohydrates, however, the Butyrivibrio members are also involved in degradation of plant materials and are a major component of rumen microbiota41. The Butyrivibrio members probably came from cow manure and were able to retain their activities and growth in TcoDi.Most of the phylotypes that could be assigned to a known genus in the order Firmicutes, the second most dominant phylum, Thermotogae  and TcoDi . As described earlier42, the members of Thermotoga are capable to grow on the various simple  and complex  polysaccharides. The lower abundance of Thermotoga species may explain the lower methane yield in the TcoDi (23% less) than the TDi, especially considering the higher amount of particulate COD (44% higher) that left the TcoDi as compared to that of the TDi. Presence of the higher particulate COD might be due to an inefficient conversion of the complex carbohydrates in the feed, in particular in the recalcitrant manure. Additionally, principle component analysis  than TDi (198 mg/L), suggesting that ammonium may possibly influence the abundance of the Thermotoga species43.Unlike the large diversity observed within gae Fig. , demonstsis Fig.  showed aAnaerobaculum, Coprothermobacter, Thermotoga and Syntrophomonas in TDi might indirectly imply an enhanced hydrolysis and acidogenesis of the food waste as compared to the co-digestion of food waste and manure. This might further be supported by the presence of significantly greater amount of VFAs  in TDi than TcoDi , from where the inoculum was taken for the start-up of the thermophilic digesters used in this study35. The lack of Methanosaetaceae species reflected an unfavorable environment  for their activities in TDi and TcoDi, suggesting the prevalence of the hydrogenotrophic methanogensis pathway in both digesters. In addition to an unfavorable environment, the prevalence of Methanothermobacter members might be due to the improved hydrolysis and fermentation at the elevated temperature that required syntrophic reactions to efficiently convert intermediates such as H2 and carboxylic organic acids.Analysis of the archaeal sequences Fig.  showed tion Fig. . Such coClostridium in both McoDi and TcoDi indicates that the establishment of this population is a direct result of microbiome transmission from the cow manure. Carry-over of methanogens from the cow manure, represented by Methanobrevibacter was also suggested for the mesophilic co-digestion system (McoDi), while only to a minor extent in the thermophilic co-digestion system (TcoDi). As higher microbial diversity often is associated with a microbiome that is more resilient to environmental changes and stress, co-digestion could potentially enhance the robustness of the anaerobic digestion process. Additionally, co-digestion at mesophilic temperature clearly showed a synergistic effect, yielded more methane than the digestion of manure-alone.The anaerobic digesters fed solely food waste performed better than the co-digesters (food waste and cow manure), most probably due to the addition of a more recalcitrant material in the form of cow manure in the co-digesters. Nevertheless, co-digestion resulted in a higher microbial diversity at both temperatures, compared to anaerobic digestion of food waste as sole substrate. This could be a reflection of the increased complexity of feedstocks in co-digestion, selecting for a richer microbial community. Although similar in the initial phase, the microbial community compositions diverged when cow manure was added at both temperatures. Based on our observations, we speculate that this variation is mostly explained by cow manure providing trace minerals and a balanced C/N ratio, rather than carry-over of microorganisms from the cow manure. However, the increased population Food waste (FW) was shipped from Norsk Matretur AS , which is a central food waste pre-treatment plant which reduces particle size to <7 mm and sanitizes the waste at 70 °C for 1 hour, as required by Norwegian health regulations. Dairy cow manure was collected at the farm of the Norwegian University of Life Sciences . Both manure and FW were stored at 4 °C and diluted using tap water to achieve the targeted organic loading rate. The waste batch were characterized on a weekly basis to ensure a constant organic loading. The average characteristics of the substrates are shown in Table et al.45. Seed sludge for the mesophilic reactors came from the Oslo EGE biogas plant ; a full scale mesophilic anaerobic digester with food waste as its sole substrate. Seed sludge for the thermophilic reactors came from the FREVAR biogas plant ; a full scale thermophilic reactor with sludge and food waste as its substrates. The feeding of the experimental reactors was done manually once per day, 6 days a week. Hydraulic retention time (HRT) and organic loading rate (OLR) for all the digesters were 20 days and 3 g VS/L/d, respectively. Temperature, pH, biogas volume and stirrer speed (set at 100 rpm) of the digesters were monitored and recorded online using BIOPHANTOM software . Additionally, samples of the effluent were regularly taken to monitor the performance of the digesters and co-digesters.Four completely mixed reactors  with a working volume of 6 L were used in this study. Two of the digesters were only fed with food waste, where one of them was kept at mesophilic temperature , while the other one was kept at thermophilic temperature . The Co-digestion systems consisted of one mesophilic co-digester (McoDi) and one thermophilic co-digester (TcoDi), which both were fed with a mixture of food waste and cow manure in a ratio of 60:40 on volatile solid (VS) basis. The start-up of the reactors was performed as previously described by Estevez 46. Chemical oxygen demand was measured using Merck Spectroquant® COD Cell test with measuring range 0.5–10 g/L. The extent of solubilization was calculated using Equation , volatile solids (VS), total chemical oxygen demand (TCOD) and soluble chemical oxygen demand (SCOD) were measured following Standard Methods4+) was measured with an ammonium ion selective electrode according to the company’s manual . Biogas composition was analyzed online for methane and carbon dioxide as previously described Zamanzadeh et al.15, with the use of an SRI gas chromatograph (Model 8610 C) equipped with a thermal conductivity detector  and a 2 m Haysep-D column. Volatile fatty acids (VFAs) were determined by high-pressure liquid chromatography  with a Zorbax Eclipse Plus C18 column  as previously described by Zamanzadeh et al.15. The samples were centrifuged at 14000 rpm for 5 min, adjusted to pH < 2.5 using 95–98% H2SO4 and then filtered through a 0.45 µm cellulose acetate syringe filter.Ammonium  and in the final phase (after 152 days) from reactors MDi, McoDi, TDi and TcoDi, in addition to the cow manure used for co-digestion. Food waste was also sampled for the same purpose, but genomic DNA was not successfully recovered, likely due to the sanitization treatment (70 °C for 1 hour). All samples were frozen immediately after sampling, and stored at −20 °C until DNA extraction. For DNA extraction, thawed samples were centrifuged at 18 800 x g for 7 min. to remove the liquid. The pellet was then re-suspended in S.T.A.R buffer  to stabilize nucleic acid and prevent bacterial growth. The suspension was vortexed followed by a subsequently slow spin to dissociate cells from large particles. The cell-containing suspension was transferred to FastPrep24 tubes with acidic washed glass beads for mechanical lysis. DNA was extracted using a commercial DNA extraction kit , and DNA concentration measured with Qubit™ fluorometer and Quant-iT™ dsDNA BR Assay Kit . The DNA samples were stored at −20 °C until sequencing preparation.et al.15. In brief, 16S rRNA gene PCR amplification was carried out using the Pro341F/Pro805R primer set selected from Takahsahi et al.47 modified with an Illumina adapter overhang in, and iProof HF DNA polymerase . A second PCR was carried out to attach unique 8-bp indices (Nextera XT Index Kit) to the Illumina sequencing adaptors to allow multiplexing of samples. A clean-up step  was preformed after each PCR. The amplicons were quantified , normalized and pooled to equimolar concentration, and then spiked with 30% PhiX control. A final concentration of 8 pm denaturated DNA was sequenced on an Illumina MiSeq instrument using the MiSeq reagent kit V3.The 16S rRNA gene amplicons were prepared for the Illumina MiSeq system (Illumina Inc.) as described in Zamanzadeh 48. Single-ends were trimmed to 200 bp and quality filtered as follows: only three sequential low-quality  bases were allowed per sequence before truncating, and sequences with <75%  consecutive high-quality base calls were discarded. No N characters or barcodes were allowed in the sequence. Chimeric sequences were removed from the dataset using UCHIME incorporated in USEARH49 and a threshold of 3% dissimilarity between 16S rRNA gene sequences was used to cluster sequences into de novo operational taxonomic units (OTUs)49. Taxonomy (up to rank ‘genus’) was assigned to each OTU using the uclust-based consensus taxonomy assigner implemented in QIIME with default parameters. Alpha rarefaction plot of the phylogenetic diversity was generated using the script alpha_rarefaction.py with default parameters in QIIME. Low abundant OTUs  and singletons were filtered out. Statistical analysis and visualization was carried out using Calypso version 8.2051. The alpha diversity of the microbial communities was measured by OTU richness in addition to the Shannon index. The diversity were further compared with ANOVA to evaluate the significance between the different subgroups . ANOVA was also used to compare taxa abundance across the different digesters. Finally, associations between reactor performance and the community composition were assessed. For this, a principle component analysis (PCA) was applied to examine how much of the variance in the 16S rRNA gene sequencing dataset could explain the process variables .All 16S rRNA gene sequences were processed using the QIIME version 1.8.0 software packageSequence data are available at NCBI Sequence Read Archive under accession number SRP123045.Figures S1 and S2Table S1


Oryza sativa) is steadily being replaced by direct seeding due to the progressive scarcity of these resources. However, the alternate dry direct seeding leads to competition with weeds and poor establishment when soils are flooded. Direct seeded rice capable of anaerobic germination  is ideal, which under rainfed ecosystems would also overcome waterlogging during germination. AG tolerance is associated with faster germination and faster elongation of coleoptiles, with the activities of alcoholic fermentation enzymes replacing aerobic respiration as a source of energy. To better understand the variability in the morpho-physiological responses and in the nature of the alcoholic fermentation enzymes during AG, 21 rice genotypes were studied. The genotypes Khao Hlan On (KHO) and IR42 were used as the tolerant and susceptible checks, respectively. KHO exhibited faster germination, with 82.5% of the coleoptiles emerging out of 10 cm of water within 8 days, whereas IR42 exhibited 20% germination and limited coleoptile growth. Among the test genotypes, four performed well, including two that are drought tolerant. Increased content and activity of the alcoholic fermentation enzymes, alcohol dehydrogenase (ADH1) and acetaldehyde dehydrogenase (ALDH2a and ALDH2b), was noted in KHO under anaerobic than under aerobic conditions and also in comparison with IR42 under AG. Gene transcripts for these enzymes were also more in KHO undergoing AG. However, no major differences were observed between KHO and IR42 in the critical cis-acting regulatory elements, such as the auxin, light, and sugar response elements, in the promoters of ADH1, ALDH2a, and ALDH2b genes. Post-transcriptional and post-translational regulatory mechanisms were implicated for the increased transcript and protein content/activity of the enzymes in KHO by observing four different transcripts of ALDH2a and a unique non-glycosylated form of ADH1 under AG. IR42 lacked the non-glycosylated ADH1 and contained only a truncated form of ALDH2a, which lacked the active site. Additionally, KHO exhibited increased activity and more isoforms for reactive oxygen species detoxifying enzymes under AG compared to IR42. These results highlight the need for a deeper functional understanding of the critical enzymes involved in AG.The water-, energy-, and labor-intensive system of transplanted puddled rice ( Flooding during germination results in poor crop establishment and yield in rice and other cereal crops, as seeds of most crops are sensitive to low oxygen stress during germination and early seedling growth. Rice is the only cereal that exhibits a degree of tolerance to anaerobic conditions during germination (AG), which is limited to coleoptile emergence and partial growth, but not sufficient to overcome the stress. Flooded conditions during seed germination can be a consequence of uneven leveling of the field or early rain, or sometimes, when rice fields are purposely flooded after sowing to combat weeds.AG1) was characterized and the underpinning gene, threalose-6-phosphate phosphatase (OsTTP7) proven to be functionally responsible for tolerance to anaerobic stress , alcohol dehydrogenase , and aldehyde dehydrogenase . These have been previously studied in relation to rice seed germination in flooded soils . HoweverThe enzyme at the center of the alcoholic fermentation process is ADH1, which is crucial for tolerance of low oxygen stress during germination in rice . ADH1 raThe aim of this study was to develop a better understanding of the differences in expression and functionality of ADH and ALDH during alcoholic fermentation under AG, by comparing a tolerant (KHO) and sensitive (IR42) rice genotypes. The upregulation of ADH and ALDH under AG was confirmed. Notably, however, ALDH2b isoform did not react differentially during AG in the contrasting genotypes. The upregulation of ADH1 and ALDH2a during AG in KHO compared to IR42 does not seem to depend on the respective genes’ promoters. Instead, our observations of the differential splice-variants of ALDH2a and post-translational modifications (PTMs) of ADH1 may underlie important differences in the content and activities of these enzymes. In parallel, differential activation of the ROS detoxifying enzymes in the two genotypes might contribute to the contrasting phenotype during AG. Taken together our results pave the way for exploring the avenues of splice-variants and protein modification as regulatory mechanisms underpinning differential content/activity of particular isoforms of the enzymes critical for rice seed germination under water.This paper has two sets of experiments, a physiological characterization with 21 genotypes and a molecular characterization with three genotypes (included within the 21 genotypes). Growth conditions of plant material for both sets followed the same protocol as detailed below. Any differences were detailed in the pertinent section.Seeds of all rice genotypes studied (Supplementary Table n = 20). Germinated seeds were those with emerging radicle and coleoptile visible to the naked eye. Length measurements in cm were recorded using a ruler and in mm using a caliper  and sent for sequencing.Around 500 mg of each sample was pulverized in liquid nitrogen and DNA was extracted using the CTAB method. Kapa HiFi PCR kit was used for cloning (Kapa Biosystems). The reaction volume was 20 μL and the cycling conditions were as recommended by the manufacturer. Primer sets were: TM Reverse Transcription System, Promega, United States).The method used for RNA extraction was a modification of ALDH2a 5′TCTTCTTCAACCAGGGGCAA3′, 5′TGGCCTTCTCCACGAACTC3′, and ALDH2b 5′TTGAACAGGGCCCTCAGATT3′, 5′TAATCTGTCGCCACCAGTCA3′.A 10 μl reaction volume consisted of 1.0 μl normalized cDNA, 5 μl 2X SYBR green PCR master mix  and 0.4 μl of 10 mM primer for each primer pair. Reactions were run in triplicate in a 7500 Fast Real-Time PCR System . Amplification conditions were 50°C for 2′, 95°C for 2′, 40 cycles of denaturing at 95°C for 10″ and a combined annealing and extension step at 55°C for 30″, followed by a disassociation stage from 55 to 95°C . The comparative threshold cycle (ΔΔCt) method was used to quantify the relative expression levels. Primers used were (forward and reverse): g at 4°C for 20 min. The supernatant was used as crude extract. Protein concentration was determined using the Bradford reagent (Sigma B 6916) with bovine serum albumin as standard  prior to in-gel detection. All water used for buffer and solution preparations was autoclaved prior to use.Aldehyde dehydrogenase: The gel was immersed in 200 mL staining solution  at 30°C for 30 min in the dark or until dark blue bands appeared.Ascorbate peroxidase: Protein extracts were added with 5 mM L-ascorbate immediately after extraction and gels were run with 5 mM L-ascorbate in the running buffer. After electrophoresis, the gel was submerged in 50 mM phosphate buffer pH 7 containing 2 mM L-ascorbate for 30 min, with buffer changes every 10 min. The buffer was finally discarded and the gel was incubated in solution A  for 20 min. The gel was rinsed in 50 mM phosphate buffer pH 7 for 1 min and finally incubated in solution B . After 5 min, light yellow bands appear against a dark green–blue background.Superoxide dismutase: After electrophoresis the gel was incubated in the darkness for 10 min in a solution , and then exposed to light until clear bands appeared against a dark purple background.g for 20 min at 10°C. The supernatant was used as crude extract. The protein was isolated first with ammonium sulfate fractionation .For protein purification, 500 g of fresh seedlings grown for 5 days under flooded conditions were ground to a powder in liquid nitrogen, added to cold buffer composed of 50 mM TRIS pH 8.0 and 2 mM DTT and centrifuged at 9000 × TM IEF Cell  and immobilized pH gradient (IPG) strips of 7 cm with a linear pH range from 3 to 10. Before the second dimension, the IPG strips were equilibrated in buffer. Finally, proteins were separated on 15% SDS polyacrylamide gels, followed by staining with silver stain.The lower activity band excised from the gel (∼200 μg protein) was prepared by using a ReadyPrep 2-D Cleanup Kit . The isoelectric focusing was done using PROTEANpost hoc pairwise t-test with the Bonferroni correction. Since the samples per group were small , a non-parametric Kruskal–Wallis test was also performed, with equal results as with the ANOVA. Therefore, only ANOVA results are discussed in this paper. When indicated, a t-test was also performed, and treatment means were compared using LSD (P = 0.05). All statistical analyses were performed using R software , two-way factorial ANOVA, and software .1. Protein identification was performed using MASCOT software. Glycosylation sites were analyzed with NetOGlyc 4.0  evaluated for AG are listed in Supplementary Table The 21 diverse rice genotypes representing different subpopulations . In this analysis also KHO and IR42 were found on opposite sides of the first PC dimension. Again ‘Bomba’ was positively correlated to KHO while ‘FR13A’ and ‘Dular’ to IR42 . These analyses indicated that the first component in the PCA classified the genotypes according to their degree of tolerance; sensitive on the left and tolerant on the right . Most traits, especially in roots, were positively correlated . Only the second component separated the studied traits.The most differential traits under stress for aerial parts were the SL, IL, PAW, and ADW. For roots, the most differential traits under stress were LENGTH, LENVOL, PROJAREA, and SURFAREA (japonica), Senia (japonica), and CO39 (indica) may have breeding value toward discovering new QTL/genes/mechanisms for AG. These genotypes could be used in generating populations for identifying positively contributing factors for AG.These studies suggested that along with the established methods of screening for tolerance to flooding through assessing coleoptile length, additional shoot, and root features correlated with tolerance could be included to identify genotypes that might exhibit additive effects. Taken together the aerial and root trait analysis suggested that genotypes such as Bomba (Table 1). Prophyllum length and root dry weight values were similar between KHO and IR42. Number of internodes and root length values were closer between NB and IR42. Seedling base diameter was the lowest in KHO, but not significantly different from the other two genotypes. For the rest of the features, values in KHO were similar to those in NB. Thus, under control conditions, values for morphological features relevant to seed germination and growth did not convincingly discriminate the tolerant from the sensitive genotype.The tolerant KHO and the sensitive IR42, known to be contrasting for seed germination underwater, were further studied along with the moderately tolerant genotype Nipponbare (NB). Under control conditions only 10 of the 12 traits were measured since two traits namely, number of leaves above water and leaf portion above water, were specific to flooded conditions. One of the 10 features, i.e., the ML was zero in all three genotypes. For each of the remaining nine features, at least two of the three genotypes exhibited similar values (Table 2). The number of internodes in IR42, i.e., one, was identical to KHO and NB. Only the IR42 mesocotyl was significantly longer than that of KHO. Most importantly, the germination percentage for KHO remained similar under the two conditions while it dropped significantly for IR42, with a germination rate drop from 90% to 20%, of which only 10% emerged above the water level. NB germination rates also dropped, but not as drastically as in IR42. These results clearly separated the tolerant KHO and the sensitive IR42 and reiterated their contrasting responses to AG . For ALDH2b although there was a trend for increase in its transcript under AG in both genotypes, the increase was insignificant. These results were in accordance with earlier reports in the same two genotypes, where ALDH2a quickly increases under AG in KHO  and could be a target of the ‘nonsense-mediated decay’ (NMD) process of transcript degradation, which is a regulatory mechanism for transcripts that need not be translated . Most prominent differences were, firstly, that under AG conditions, KHO exhibited a doublet of the ca. 60 kDa band , which was visible only as a single band in IR42. Secondly, KHO exhibited a ca. 95 kDa band under control and AG conditions (white arrowhead with black outline), which was somewhat pronounced under AG. However, IR42 completely lacked this band under AG conditions whereas it appeared faintly under control conditions only 4 days after germination. Thirdly, the AG-specific doublet in KHO clearly exhibited a sequential increase till 5 DAS followed by its decrease. Along with the dark bands, there were white bands as well . Once again, these were more pronounced in KHO under AG. In IR42, these became pronounced again only 5 days after germination, in control conditions.In order to assess the correspondence between the Immuno-blot assay with monoclonal anti-ALDH2 antibody was used to assess which bands were really the ALDH2. Although the expected ca. 95 kDa band was readily identified, the high molecular weight white band of more than ca. 250 kDa also hybridized, while the AG-specific ca. 60 kDa doublet yielded no signal in KHO or IR42 . The LOC_Os11g10510 and LOC_Os11g10520 codes for ADH2 and ADH3 protein, respectively, with the predicted splice variants of two for the first gene (LOC_Os11g10510.1 and LOC_Os11g10510.2) and three for the second gene . The protein products of two splice variants in each of the two  are close to the predicted molecular weight , which may form the protein doublet on the gel. Concordance in the original predicted molecular weight of 40.9 kDa to the LOC_Os11g10480.1 protein suggested that the bands may belong to the two splice forms of LOC_Os11g10480. However, qRT-PCR-based attempts at observing the variant CDS in KHO under AG were not successful when tested with different sets of primers. Additionally, qRT-PCR for ADH2 (LOC_Os11g10510) and ADH3 (LOC_Os11g10520) were also performed to check the possibility that the smaller band would belong to a splice form from one of these genes, but there was no amplification. All splice forms were also tested through PCR with 5′ and 3′ UTR primers. The lack of an amplification of any shorter form suggested that the doublet might not be the result of a splice variant.Protein doublets mostly occur due to one of the following reasons; translation products of alternative splice forms, signal peptide processing or protein post-translation modifications. The putative ADH identified by mass spectrometry  maps to The predicted protein of neither LOC_Os11g10480, LOC_Os11g10510 nor LOC_Os11g10520 contain a signal peptide, which can generally be expected due to the cytoplasmic location of ADH. Hence, signal peptide processing also cannot be the reason behind the doublet.in silico prediction of the possible PTMs of the ADH. Common PTMs that readily differentiate the same protein as multiple bands on the gel are protein acetylation, methylation, myristoylation, phosphorylation, glycosylation, and ubiquitination. The in silico analysis predicted a single O-glycosylation at position T120 and multiple myristoylation and phosphorylation sites of the ADH protein sequence of NB, the reference genome.The option of protein PTMs causing the doublet was explored by LOC_Os11g10480 CDS was cloned and sequenced from the two genotypes. There were four SNPs between IR42 and NB in the CDS region. Predicted protein showed that only one of these SNPs translated into a non-synonymous change of V291I. There were more non-synonymous SNPs between KHO and NB and these translated into amino acid changes shown in Figure 5. However, none of the changes were within the dehydrogenase domain, or the NAD+ or zinc binding sites. Protein modifications predicted in IR42 and NB ADH were identical. However, changes in the KHO protein sequence led to differences in the predicted PTMs such that in KHO the T120 O-glycosylation could not happen; there were four additional possible phosphorylation sites and one additional myristoylation site. These additional PTMs could not account for the doublet in KHO because the PTMs lead to slower movement of the protein and hence would result in the upper band of the doublet, which was anyway common to the three genotypes . This common upper band could not be due to O-glycosylation since KHO ADH is predicted not to be O-glycosylated. It could not be due only to myristoylation because it would need to be demyristoylated to lead to the lower band, and myristoylation is an irreversible process. Although AG-specific dephosphorylation could lead to the lower band in KHO, given that the ADH1 in KHO putatively has four additional phosphorylation sites, its phosphorylation status and pattern would need to be uniquely regulated in order to result in a band of molecular weight similar to IR42 and NB under control conditions. To confirm protein PTMs the ADH1 lower activity band was eluted from the gel and run on a 2D denaturing gel. The isoelectric focusing during the first dimension separated the modified proteins based on the altered charge profile. For example, a protein population differentially phosphorylated at its multiple putative phosphorylation sites would sequentially become more negatively charged. In the second dimension these modified proteins were separated according to the molecular weights. Phosphorylated proteins would appear as a series of spots along the horizontal axis around the similar molecular weight region on the vertical axis since the SDS gel will not show the small change in molecular weight , it was easy to identify the putatively modified ADH1 spots (red circle). These were missing in IR42 . The minor discrepancy in pI and Mw between the predicted and experimental spots could be due to the non-ideal experimental conditions. This data only proves the existence of PTMs but not the nature of these PTMs, which, however, is rather likely to be phosphorylation because protein modification by phosphorylation, if possible, is very common under submergence. However, they could also be acetylation  mutant . The panel for indica-aus  did not yield any peak around the ADH region on chromosome 11. Another study on AG also detected below threshold peaks along chromosome 11, however, they were not further studied  mutant , which c) mutant  have notro panel . This pa studied . A simil3. The hypothesis that follows is that allelic variation in enzymes that are involved in PTMs of ADH could underlie differential response. Although this hypothesis also needs to be tested in future research, it is encouraging to note that kinases and phosphatases are known to be associated with AG  of the gene promoters. Thus the 1 kb upstream sequences of the genes for ALDH2a and ADH1 were cloned and sequenced from KHO to be compared with such sequences available for NB from the reference genome sequence.Differential expression of the ADH1 in NB and KHO, respectively. Qualitatively most CAREs were shared between the two genotypes but quantitatively some motifs abounded in one or the other variety . The second most abundant group of motifs was of CAAT and CACT elements . Both genotypes had elements related to response to O2, low CO2, and sugar levels. Curiously, binding sites for transcription factors (TFs) related to dehydration response were more (ca. 5%) compared to those for TFs related to other stresses (ca. 2–3%). Comparative excess of the ethylene response element (ERE) in KHO was noteworthy, especially together with the uniquely present auxin response elements. These two hormones have been identified to increase coleoptile elongation related to cell extension in rice  which regulates signaling through the T6P/sucrose ratios (O-linked glycosylation whereby the protein fluctuates between the O-glycosylated to non-glycosylated (the predominant) form, which may underlie modulation of the enzyme activity. ALDH2a, on the other hand, seemed more regulated by splice forms, which were stress specific. In addition to that, both enzymes had ethylene and auxin response elements; the hormones intrinsically linked to coleoptile elongation. By examining the genes of interest in a pathway, we provide new avenues for queries on the mechanistic aspects of the pathway, suggesting a common activation under stress. However, these avenues should be further explored and validated. In addition, ROS scavenging enzymes were more active in KHO than in IR42. These activities relate to increased metabolism under flooded conditions and have a strong link to tolerance. Moreover, they are directly related to detoxification when the coleoptile reaches the more aerated zone at the point of emergence from the water. These findings provide mechanistic and biological evidence of the linkage between the tolerant phenotype with its genotype.Fast germination and coleoptile elongation are essential for rice seedling to survive flooding during germination and early seedling establishment. One of the major bottlenecks for coleoptile elongation is the metabolic reactions of alcoholic fermentation. Recent research showed a relationship between AG tolerance and a threalose-6-phosphate phosphatase (Oe ratios . HoweverBM undertook the physiology, molecular biology, enzyme activity, and computational analyses. TL undertook the protein purification and part in the molecular biology. FE took part in the physiology and molecular biology analyses. AK, AI, BM, and TL planned the research, design of experiments and handled the interpretation and preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.