Creating a zero amylose barley with high soluble sugar content by genome editing.

Amylose biosynthesis is strictly associated with granule-bound starch synthase I (GBSSI) encoded by the Waxy gene. Mutagenesis of single bases in the Waxy gene, which induced by CRISPR/Cas9 genome editing, caused absence of intact GBSSI protein in grain of the edited line. The amylose and amylopectin contents of waxy mutants were zero and 31.73%, while those in the wild type were 33.50% and 39.00%, respectively. The absence of GBSSI protein led to increase in soluble sugar content to 37.30% compared with only 10.0% in the wild type. Sucrose and ß-glucan, were 39.16% and 35.40% higher in waxy mutants than in the wild type, respectively. Transcriptome analysis identified differences between the wild type and waxy mutants that could partly explain the reduction in amylose and amylopectin contents and the increase in soluble sugar, sucrose and ß-glucan contents. This waxy flour, which showed lower final viscosity and setback, and higher breakdown, could provide more option for food processing.

Induce male sterility by CRISPR/Cas9-mediated mitochondrial genome editing in tobacco.

Genome editing has become more and more popular in animal and plant systems following the emergence of CRISPR/Cas9 technology. However, target sequence modification by CRISPR/Cas9 has not been reported in the plant mitochondrial genome, mtDNA. In plants, a type of male sterility known as cytoplasmic male sterility (CMS) has been associated with certain mitochondrial genes, but few genes have been confirmed by direct mitochondrial gene-targeted modifications. Here, the CMS-associated gene (mtatp9) in tobacco was cleaved using mitoCRISPR/Cas9 with a mitochondrial localization signal. The male-sterile mutant, with aborted stamens, exhibited only 70% of the mtDNA copy number of the wild type and exhibited an altered percentage of heteroplasmic mtatp9 alleles; otherwise, the seed setting rate of the mutant flowers was zero. Transcriptomic analyses showed that glycolysis, tricarboxylic acid cycle metabolism and the oxidative phosphorylation pathway, which are all related to aerobic respiration, were inhibited in stamens of the male-sterile gene-edited mutant. In addition, overexpression of the synonymous mutations dsmtatp9 could restore fertility to the male-sterile mutant. Our results strongly suggest that mutation of mtatp9 causes CMS and that mitoCRISPR/Cas9 can be used to modify the mitochondrial genome of plants.

Critical contributions of protein cargos to the functions of macrophage-derived extracellular vesicles.

BACKGROUND: Macrophages are highly plastic innate immune cells that play key roles in host defense, tissue repair, and homeostasis maintenance. In response to divergent stimuli, macrophages rapidly alter their functions and manifest a wide polarization spectrum with two extremes: M1 or classical activation and M2 or alternative activation. Extracellular vesicles (EVs) secreted from differentially activated macrophages have been shown to have diverse functions, which are primarily attributed to their microRNA cargos. The role of protein cargos in these EVs remains largely unexplored. Therefore, in this study, we focused on the protein cargos in macrophage-derived EVs. RESULTS: Naïve murine bone marrow-derived macrophages were treated with lipopolysaccharide or interlukin-4 to induce M1 or M2 macrophages, respectively. The proteins of EVs and their parental macrophages were subjected to quantitative proteomics analyses, followed by bioinformatic analyses. The enriched proteins of M1-EVs were involved in proinflammatory pathways and those of M2-EVs were associated with immunomodulation and tissue remodeling. The signature proteins of EVs shared a limited subset of the proteins of their respective progenitor macrophages, but they covered many of the typical pathways and functions of their parental cells, suggesting their respective M1-like and M2-like phenotypes and functions. Experimental examination validated that protein cargos in M1- or M2-EVs induced M1 or M2 polarization, respectively. More importantly, proteins in M1-EVs promoted viability, proliferation, and activation of T lymphocytes, whereas proteins in M2-EVs potently protected the tight junction structure and barrier integrity of epithelial cells from disruption. Intravenous administration of M2-EVs in colitis mice led to their accumulation in the colon, alleviation of colonic inflammation, promotion of M2 macrophage polarization, and improvement of gut barrier functions. Protein cargos in M2-EVs played a key role in their protective function in colitis. CONCLUSION: This study has yielded a comprehensive unbiased dataset of protein cargos in macrophage-derived EVs, provided a systemic view of their potential functions, and highlighted the important engagement of protein cargos in the pathophysiological functions of these EVs.

Functional R2R3-MYB transcription factor NsMYB1, regulating anthocyanin biosynthesis, was relative to the fruit color differentiation in Nitraria sibirica Pall.

BACKGROUND: Nitraria sibirica Pall. is an economic plant with two kinds of fruit color, widely spreads in the Qinghai Tibet Plateau. The chemical analysis and pharmacological evaluation had been carried out for several tens of years, the mechanism behind the fruit color differentiation is still unclear. RESULTS: In this manuscript, the chemical analysis of the extractions showed that the chemical composition of fruit color was anthocyanin, and two kind of Nitraria sibirica Pall. were caused by the content differentiation with the same anthocyanin kinds. Cyanidin-3-[2"-(6'"-coumaroyl)-glucosyl]-glucoside (C3G) was the major anthocyanin. Transcriptome analysis and the qRT-PCR revealed that the structural genes relative to anthocyanin biosynthesis except CHS, F3'5'H and ANS were up-regulated in the peels of BF (Black fruit) compared with the peels of RF (Red fruit), which indicated that transcript factor should be the reason for the expression difference of the structure genes. In the unigenes of the transcript factor MYB and bHLH, relative to anthocyanin, only NsMYB1 (Cluster 8422.10600), was high-expression and up-expression in the peels of BF. NsMYB1 encoded the same length protein with four amino acid differences in the RF and BF, and both contained the intact DNA, HTH-MYB and SANT domains. NsMYB1 was close to the AtMYB114, AtMYB113 and AtPAP1, regulating anthocyanin biosynthesis, in phylogenetic relationship. Both NsMYB1r and NsMYB1b could promote the transcript of the structural genes, and induced the anthocyanin accumulation in all tissues of transgenic tobacco. The insertion of 'TATA' in the promoter of NsMYB1r gave one more promoter region, and was the reason for higher transcripts in black fruit possibly. CONCLUSIONS: Cyanidin-3-[2''-(6'"-coumaroyl)-glucosyl]-glucoside was the major anthocyanin in black fruit of Nitraria sibirica Pall.. NsMYB1 was a functional R2R3-MYB transcription factor, regulated the anthocyanin biosynthesis, and led to the fruit color differentiation in Nitraria sibirica Pall.

Genotyping-by-sequencing and genome-wide association study reveal genetic diversity and loci controlling agronomic traits in triticale.

KEY MESSAGE: The genetic diversity and loci underlying agronomic traits were analyzed by the reads coverage and genome-wide association study based genotyping-by-sequencing in a diverse population consisting of 199 accessions. Triticale (× Triticosecale Wittmack) is an economically important grain forage and energy crop planted worldwide for its high biomass. Little is known about the genetic diversity and loci underlying agronomic traits in triticale. We performed genotyping-by-sequencing of 199 cultivars and mapped reads to the A, B, D, and R genomes for karyotype analysis. These cultivars could mostly be grouped into five types. Some chromosome abnormalities occurred with high frequency, such as 2D (2R) substitution, deletion of the long arm of chromosome 2D or the short arm of 5R, and translocation of the long arms of 7D/7A, the short arms of 6D/6A, or the long arms of 1D/1A. We chose only widely planted hexaploid triticale cultivars (153) for genome-wide association study. These cultivars could be divided into nine distinct groups, and the linkage disequilibrium decay was 25.4 kb in this population. We identified 253 significant marker-trait associations (MTAs) on 20 chromosomes, except 7R. Twenty-one reliable MTAs were identified repeatedly over two environments. We predicted 16 putative candidate genes involved in plant growth and development using the genome sequences of wheat and rye. These results provide a basis for understanding the genetic mechanisms of agronomic traits and will benefit the breeding of improved hexaploid triticale.

Novel Hina alleles created by genome editing increase grain hardness and reduce grain width in barley.

The hordoindolina genes (Hina and Hinb) are believed to play critical roles in barley (Hordeum vulgare L.) grain texture. In this study, we created novel alleles of the Hina gene using CRISPR/Cas9 (Clustered regularly inter spaced short palindromic repeat-associated protein, CRISPR-Cas) genome editing. Mutagenesis of single bases in these novel alleles led to loss of Hina protein function in edited lines. The grain hardness index of hina mutants was 95.5 on average, while that of the wild type was only 53.7, indicating successful conversion of soft barley into hard barley. Observation of cross-sectional grain structure using scanning electron microscopy revealed different adhesion levels between starch granules and protein matrix. Starch granules were loose and separated from the protein matrix in the wild type, but deeply trapped and tightly integrated with the protein matrix in hina02 mutants. In addition, the grain width and thousand-grain weight of the hina02 mutant were significantly lower than those of the wild type.

The value of color Doppler sonography in diagnosis and hemodynamic analysis of innominate artery occlusion.

The occlusion of the innominate artery caused a significant decrease in the distal end of the right subclavian artery and the right common carotid artery, internal carotid artery, and external carotid artery (ECA). Due to the different pressure and the abundant communicating arteries between the ECA and the bilateral vertebral artery (VA), different paths of blood steal in the anterior and posterior circulation occurred.

De novo genome assembly of a foxtail millet cultivar Huagu11 uncovered the genetic difference to the cultivar Yugu1, and the genetic mechanism of imazethapyr tolerance.

BACKGROUND: Setaria italica is the second-most widely planted species of millets in the world and an important model grain crop for the research of C4 photosynthesis and abiotic stress tolerance. Through three genomes assembly and annotation efforts, all genomes were based on next generation sequencing technology, which limited the genome continuity. RESULTS: Here we report a high-quality whole-genome of new cultivar Huagu11, using single-molecule real-time sequencing and High-throughput chromosome conformation capture (Hi-C) mapping technologies. The total assembly size of the Huagu11 genome was 408.37 Mb with a scaffold N50 size of 45.89 Mb. Compared with the other three reported millet genomes based on the next generation sequencing technology, the Huagu11 genome had the highest genomic continuity. Intraspecies comparison showed about 94.97 and 94.66% of the Yugu1 and Huagu11 genomes, respectively, were able to be aligned as one-to-one blocks with four chromosome inversion. The Huagu11 genome contained approximately 19.43 Mb Presence/absence Variation (PAV) with 627 protein-coding transcripts, while Yugu1 genomes had 20.53 Mb PAV sequences encoding 737 proteins. Overall, 969,596 Single-nucleotide polymorphism (SNPs) and 156,282 insertion-deletion (InDels) were identified between these two genomes. The genome comparison between Huagu11 and Yugu1 should reflect the genetic identity and variation between the cultivars of foxtail millet to a certain extent. The Ser-626-Aln substitution in acetohydroxy acid synthase (AHAS) was found to be relative to the imazethapyr tolerance in Huagu11. CONCLUSIONS: A new improved high-quality reference genome sequence of Setaria italica was assembled, and intraspecies genome comparison determined the genetic identity and variation between the cultivars of foxtail millet. Based on the genome sequence, it was inferred that the Ser-626-Aln substitution in AHAS was responsible for the imazethapyr tolerance in Huagu11. The new improved reference genome of Setaria italica will promote the genic and genomic studies of this species and be beneficial for cultivar improvement.

Functional MYB transcription factor gene HtMYB2 is associated with anthocyanin biosynthesis in Helianthus tuberosus L.

BACKGROUND: Tuber color is an important trait for Helianthus tuberosus L. (Jerusalem artichoke). Usually, purple tubers with high anthocyanin content are more nutritious than white tuber. But, the molecular mechanism underlying it is unknown. RESULTS: In the current study, high-throughput RNA-sequencing was used to compare the transcriptomes between plants with tubers with red or white epidermis. Compared with the white-skinned tubers of cultivar QY3, anthocyanin biosynthesis structural genes had greater expression in the red-skinned tubers of cultivar QY1, indicating that the anthocyanin biosynthesis pathway was activated in 'QY1'; quantitative PCR confirmed this difference in expression. HtMYB2 (Unigene44371_All) was the only MYB transcription factor, homologous to the MYB transcription factor regulating anthocyanin biosynthesis, expressed in the red tuber epidermis of 'QY1'. The anthocyanin concentration in the root, stem, leaf, flower, and tuber epidermis of 'QY1' was higher than in 'QY3', especially tuber epidermis. Correspondingly, HtMYB2 had greater expression in these tissues of 'QY1' than in 'QY3'. The expression of HtMYB2 was associated with anthocyanin accumulation in the different tissues. Overexpression of HtMYB2 activated the anthocyanin biosynthesis pathway, accumulating the pigment in leaves of transgenic tobacco, supporting the model that HtMYB2 regulated anthocyanin biosynthesis. Further experiments found that HtMYB2 had the same coding sequence and genomic sequence in 'QY1' and 'QY3', but that there were several single nucleotide polymorphisms and one insertion-deletion (indel) mutation of 21 nucleotides in the promoter region between the two alleles. The deletion of three nucleotides "AAA" made the promoter of 'QY1' predicted to contain one more possible promoter region. A specific primer, based on the indel, could differentiate between cultivars with red or white tuber epidermis. The genetic variation in HtMYB2 was associated with the tuber skin color in a natural population. CONCLUSIONS: RNA-seq can successfully isolate the candidate gene (HTMYB2) controlling anthocyanin biosynthesis in purple epidermis of Jerusalem artichoke tuber. HTMYB2 can regulate anthocyanin biosynthesis in plants and is closely related to the formation of purple phenotype in tubers. This study should be useful in understanding the genetic mechanism underlying different tuber skin colors and in breeding new H. tuberosus cultivars with different tuber skin colors.

A fructan: the fructan 1-fructosyl-transferase gene from Helianthus tuberosus increased the PEG-simulated drought stress tolerance of tobacco.

BACKGROUND: Jerusalem artichoke (Helianthus tuberosus) is a fructan-accumulating plant, and an industrial source of raw material for fructan production, but the crucial enzymes involved in fructan biosynthesis remain poorly understood in this plant. RESULTS: In this study, a fructan: fructan 1-fructosyl-transferase (1-FFT) gene, Ht1-FFT, was isolated from Jerusalem artichoke. The coding sequence of Ht1-FFT was 2025 bp in length, encoding 641 amino acids. Ht1-FFT had the type domain of the 1-FFT protein family, to which it belonged, according to phylogenetic tree analysis, which implied that Ht1-FFT had the function of catalyzing the formation and extension of beta-(2,1)-linked fructans. Overexpression of Ht1-FFT in the leaves of transgenic tobacco increased fructan concentration. Moreover, the soluble sugar and proline concentrations increased, and the malondialdehyde (MDA) concentration was reduced in the transgenic lines. The changes in these parameters were associated with increased stress tolerance exhibited by the transgenic tobacco plants. A PEG-simulated drought stress experiment confirmed that the transgenic lines exhibited increased PEG-simulated drought stress tolerance. CONCLUSIONS: The 1-FFT gene from Helianthus tuberosus was a functional fructan: fructan 1-fructosyl-transferase and played a positive role in PEG-simulated drought stress tolerance. This transgene could be used to increase fructan concentration and PEG-simulated drought stress tolerance in plants by genetic transformation.

Molecular Marker Based Design for Breeding Wheat Lines with Multiple Resistance and Superior Quality.

There has not been a major wheat stem rust epidemic worldwide since the 1970s, but the emergence of race TTKSK of Puccinia graminis f. sp. tritici in 1998 presented a great threat to the world wheat production. Single disease-resistance genes are usually effective for only several years before the pathogen changes genetically to overcome the resistance. Stripe rust caused by Puccinia striiformis f. sp. tritici (Pst) is one of the most common and persistent wheat diseases worldwide. The development of varieties with multiple resistance is the most economical and effective strategy for preventing stripe rust and stem rust, the two main rust diseases constraining wheat production. Plateau 448 has been widely used in the spring wheat growing region in northwest China, but it has become susceptible to stripe rust and is susceptible to TTKSK. To produce more durable resistance to race TTKSK as well as to stripe rust, four stem rust resistance genes (Sr33, Sr36, Sr-Cad, and Sr43) and three stripe rust resistance genes (Yr5, Yr18, and Yr26) were simultaneously introgressed into Plateau 448 to improve its stem rust (Ug99) and stripe rust resistance using a marker-assisted backcrossing strategy combined with phenotypic selection. We obtained 131 BC1F5 lines that pyramided two to four Ug99 resistance genes and one to two Pst resistance genes simultaneously. Thirteen of these lines were selected for their TTKSK resistance, and all of them exhibited near immunity or high resistance to TTKSK. Among the 131 pyramided lines, 95 showed high resistance to mixed Pst races. Nine lines exhibited not only high resistance to TTKSK and Pst but also better agronomic traits and high-molecular-weight glutenin subunit compositions than Plateau 448.

Mapping Quantitative Trait Loci for 1000-Grain Weight in a Double Haploid Population of Common Wheat.

Thousand-grain weight (TGW) is a very important yield trait of crops. In the present study, we performed quantitative trait locus (QTL) analysis of TGW in a doubled haploid population obtained from a cross between the bread wheat cultivar "Superb" and the breeding line "M321" using the wheat 55-k single-nucleotide polymorphism (SNP) genotyping assay. A genetic map containing 15,001 SNP markers spanning 2209.64 cM was constructed, and 9 QTLs were mapped to chromosomes 1A, 2D, 4B, 4D, 5A, 5D, 6A, and 6D based on analyses conducted in six experimental environments during 2015-2017. The effects of the QTLs qTgw.nwipb-4DS and qTgw.nwipb-6AL were shown to be strong and stable in different environments, explaining 15.31-32.43% and 21.34-29.46% of the observed phenotypic variance, and they were mapped within genetic distances of 2.609 cM and 5.256 cM, respectively. These novel QTLs may be used in marker-assisted selection in wheat high-yield breeding.

Functional MYB transcription factor encoding gene AN2 is associated with anthocyanin biosynthesis in Lycium ruthenicum Murray.

BACKGROUND: Lycium ruthenicum Murray is an important economic plant in China and contains higher levels of anthocyanins in its fruits than other Lyciums. However, the genetic mechanism of anthocyanin production in this plant is unknown. RESULTS: Based on previous transcriptome analysis, LrAN2 and LbAN2, encoding MYB transcription factors, were isolated from L. ruthenicum and L. barbarum, respectively. Both genes contained two introns, encoded 257 amino acids with two-Aa difference, and carried the unabridged HTH-MYB, MYB-like DNA-binding, and SANT domains. In the phylogenetic trees, LrAN2 and LbAN2 were found to be closely related to NtAN2, which regulates anthocyanin biosynthesis in tobacco. Overexpression of LrAN2 and LbAN2 induced anthocyanin biosynthesis in all tissues of tobacco. The anthocyanin content in the leaves of transgenic lines with LbAN2 was lower than LrAN2. It indicated that the function of LbAN2 was weaker than LrAN2. The AN2 transcript could be detected only in the fruits of L. ruthenicum and increased during fruit development, accompanied by anthocyanin accumulation. In natural population, the alleles LrAN2 and LrAN2 were associated strictly with L. ruthenicum and L. barbarum, respectively. Moreover, an AN2 genetic diversity study suggested that Lyciums with yellow, white, purple, and jujube red fruits were derived from L. ruthenicum. CONCLUSIONS: Two AN2 alleles, from L. ruthenicum and L. barbarum, were functional MYB transcriptor regulating anthocyanin biosynthesis. The functional diversity and high expression level of LrAN2 could be the reason for high anthocyanin content in the fruit of L. ruthenicum. Lyciums with yellow, white, purple, and jujube red fruits were derived from L. ruthenicum based on AN2 sequence diversity. The results may be advantageous in identifying new varieties and breeding new cultivars.

Comparative transcriptome analysis of two selenium-accumulating genotypes of Aegilops tauschii Coss. in response to selenium.

BACKGROUND: Selenium (Se), an essential micronutrient in both animals and humans, has various biological functions, and its deficiency can lead to various diseases. The most common method for increasing Se uptake is the consumption of Se-rich plants, which transform inorganic Se into organic forms. Wheat is eaten daily by many people. The Se content of Aegilops tauschii (Ae. tauschii), one of the ancestors of hexaploid common wheat, is generally higher than that of wheat. In this study, two genotypes of Ae. tauschii with contrasting Se-accumulating abilities were subjected to different Se treatments followed by high-throughput transcriptome sequencing. RESULTS: Sequencing of 12 transcriptome libraries of Ae. tauschii grown under different Se treatments produced about a total of 47.72 GB of clean reads. After filtering out rRNA sequences, approximately 19.3 million high-quality clean reads were mapped to the reference genome (ta IWGSC_MIPSv2.1 genome DA). The total number of reference genome gene is 32,920 and about 26,407 known genes were detected in four groups. Functional annotation of these mapped genes revealed a large number of genes and some pivotal pathways that may participate in Se metabolism. The expressions of several genes potentially involved in Se metabolism were confirmed by quantitative real-time PCR. CONCLUSIONS: Our study, the first to examine Se metabolism in Ae. tauschii, has provided a theoretical foundation for future elucidation of the mechanism of Se metabolism in this species.

Expression of the high molecular weight glutenin 1Ay gene from Triticum urartu in barley.

In this study, we successfully expressed the active 1Ay subunit of Triticum urartu in barley by Agrobacterium-mediated transformation with a transformation efficiency of 19.9%. The results of SDS-PAGE revealed that the expressed proteins of 1Ay subunit were present at some grains of each of 46 original T0 plants, showing identical mobility to those of positive standards of T. urartu grain protein and bacteria expressional proteins. In the T2 generation, three homozygous lines, 2-28, 3-11, and 5-6, were identified. The results of scanning electron microscopy showed an increased amount of protein bodies in these transgenic lines. The main effects in the expression of the 1Ay subunits was a considerable increase in the glutenin content, but a decrease in the contents of gliadins while there were no effects in the contents of albumin, globulin and the total protein. We found that the gluten could not be washed out from the flour obtained from transgenic barley lines when using a Gluten index analyzer and a Farinograph indicating that the transgenic barley lines could not form dough. The lack of x-type HMW-GS and the reduction in number of subunit were inferred as the possible reasons for the inability to form gluten polymer.

A bHLH transcription factor TsMYC2 is associated with the blue grain character in triticale (Triticum × Secale).

KEY MESSAGE: RNA-Seq was employed to compare the transcriptome differences between the triticale lines and to identify the key gene responsible for the blue aleurone trait. The accumulation of anthocyanins in the aleurone of triticale results in the formation of the blue-grained trait, but the identity of the genes associated with anthocyanin biosynthesis in the aleurone has not yet been reported. In this manuscript, RNA-Seq was employed to compare the transcriptome differences between the triticale lines HM13 (blue aleurone) and HM5 (white aleurone), and to identify the key genes responsible for the blue aleurone trait. There were 32,406 differentially expressed genes between HM13 and HM5. Seventy-three unigenes were homologous to the structural genes related to anthocyanin biosynthesis, and the average transcript level of the structural genes was higher in HM13 than in HM5, so that quantitative differences between the two lines in transcription rates could be the cause of the blue aleurone. The MYB and bHLH transcription factors had two homologous unigenes, but contained only one differentially expressed unigene each. The relative transcript level of bHLH Unigene5672_All (TsMYC2) in HM13 was 42.71 times that in HM5, while the relative transcript level of the MYB transcription factor Unigene12228_All in HM13 was 2.20 times that in HM5. qPCR experiments determined the relative transcript level of TsMYC2 in developing grain, with the expression of TsMYC2 in grain being the highest compared with that in root, stem or leaf tissue. TsMYC2 was homologous to the bHLH transcription factor regulating anthocyanin biosynthesis and contained three entire functional domains: bHLH-MYC_N, HLH and ACT-like, which were important for exercising regulation of anthocyanin biosynthesis as a bHLH transcription factor. Transient expression of ZmC1 and TsMYC2 could induce anthocyanin biosynthesis in white wheat coleoptile cells, demonstrating that TsMYC2 was a functional bHLH transcription factor. These results indicated that TsMYC2 was associated with the blue aleurone trait and could prove to be a valuable gene with which to breed new triticale cultivars with the blue aleurone trait.

Overexpression of ThMYC4E Enhances Anthocyanin Biosynthesis in Common Wheat.

The basic helix-loop helix (bHLH) transcription factor has been inferred to play an important role in blue and purple grain traits in common wheat, but to date, its overexpression has not been reported. In this study, the bHLH transcription factor ThMYC4E, the candidate gene controlling the blue grain trait from Th. Ponticum, was transferred to the common wheat JW1. The positive transgenic lines displayed higher levels of purple anthocyanin pigments in their grains, leaves and glumes. Stripping the glumes (light treatment) caused white grains to become purple in transgenic lines. RNA-Seq and qRT-PCR analysis demonstrated that the transcript levels of structural genes associated with anthocyanin biosynthesis were higher in transgenic wheat than the wild-type (WT), which indicated that ThMYC4E activated anthocyanin biosynthesis in the transgenic lines. Correspondingly, the anthocyanin contents in grains, roots, stems, leaves and glumes of transgenic lines were higher than those in the WT. Metabolome analysis demonstrated that the anthocyanins were composed of cyanidin and delphinidin in the grains of the transgenic lines. Moreover, the transgenic lines showed higher antioxidant activity, in terms of scavenging DPPH radicals, in the ethanol extracts of their grains. The overexpression of ThMYC4E sheds light on the traits related to anthocyanin biosynthesis in common wheat and provide a new way to improve anthocyanin content.

Comprehensive Influences of Overexpression of a MYB Transcriptor Regulating Anthocyanin Biosynthesis on Transcriptome and Metabolome of Tobacco Leaves.

Overexpression of R2R3-MYB transcriptor can induce up-expression of anthocyanin biosynthesis structural genes, and improve the anthocyanin content in plant tissues, but it is not clear whether the MYB transcription factor overexpression does effect on other genes transcript and chemical compounds accumulation. In this manuscript, RNA-sequencing and the stepwise multiple ion monitoring-enhanced product ions (stepwise MIM-EPI) strategy were employed to evaluate the comprehensive effect of the MYB transcription factor LrAN2 in tobacco. Overexpression of LrAN2 could promote anthocyanin accumulation in a lot of tissues of tobacco cultivar Samsun. Only 185 unigenes express differently in a total of 160,965 unigenes in leaves, and 224 chemical compounds were differently accumulated. Three anthocyanins, apigeninidin chloride, pelargonidin 3-O-beta-D-glucoside and cyanidin 3,5-O-diglucoside, were detected only in transgenic lines, which could explain the phenotype of purple leaves. Except for anthocyanins, the phenylpropanoid, polyphenol (catechin), flavonoid, flavone and flavonol, belong to the same subgroups of flavonoids biosynthesis pathway with anthocyanin and were also up-accumulated. Overexpression of LrAN2 activated the bHLH (basic helix-loop-helix protein) transcription factor AN1b, relative to anthocyanin biosynthesis and the MYB transcription factor MYB3, relative to proanthocyanin biosynthesis. Then, the structural genes, relative to the phenylpropanoid pathway, were activated, which led to the up-accumulation of phenylpropanoid, polyphenol (catechin), flavonoid, flavone, flavonol and anthocyanin. The MYB transcription factor CPC, negative to anthocyanin biosynthesis, also induced up-expression in transgenic lines, which implied that a negative regulation mechanism existed in the anthocyanin biosynthesis pathway. The relative contents of all 19 differently accumulated amino and derivers were decreased in transgenic lines, which meant the phenylalanine biosynthesis pathway completed the same substrates with other amino acids. Interestingly, the acetylalkylglycerol acetylhydrolase was down-expressed in transgenic lines, which caused 19 lyso-phosphatidylcholine and derivatives of lipids to be up-accumulated, and 8 octodecane and derivatives were down-accumulated. This research will give more information about the function of MYB transcription factors on the anthocyanin biosynthesis and other chemical compounds and be of benefit to obtaining new plant cultivars with high anthocyanin content by biotechnology.

Transcriptome Analysis Identifies Key Genes Responsible for Red Coleoptiles in Triticum Monococcum.

Red coleoptiles can help crops to cope with adversity and the key genes that are responsible for this trait have previously been isolated from Triticum aestivum, Triticum urartu, and Aegilops tauschii. This report describes the use of transcriptome analysis to determine the candidate gene that controls the trait for white coleoptiles in T. monococcum by screening three cultivars with white coleoptiles and two with red coleoptiles. Fifteen structural genes and two transcription factors that are involved in anthocyanin biosynthesis were identified from the assembled UniGene database through BLAST analysis and their transcript levels were then compared in white and red coleoptiles. The majority of the structural genes reflected lower transcript levels in the white than in the red coleoptiles, which implied that transcription factors related to anthocyanin biosynthesis could be candidate genes. The transcript levels of MYC transcription factor TmMYC-A1 were not significantly different between the white and red coleoptiles and all of the TmMYC-A1s contained complete functional domains. The deduced amino acid sequence of the MYB transcription factor TmMYB-A1 in red coleoptiles was homologous to TuMYB-A1, TaMYB-A1, TaMYB-B1, and TaMYB-D1, which control coleoptile color in corresponding species and contained the complete R2R3 MYB domain and the transactivation domain. TmMYB-a1 lost its two functional domains in white coleoptiles due to a single nucleotide deletion that caused premature termination at 13 bp after the initiation codon. Therefore, TmMYB-A1 is likely to be the candidate gene for the control of the red coleoptile trait, and its loss-of-function mutation leads to the white phenotype in T. monococcum.

Transcriptome Analysis Clarified Genes Involved in Betalain Biosynthesis in the Fruit of Red Pitayas (Hylocereus costaricensis).

The red flesh trait gives red pitayas more healthful components and a higher price, while the genetic mechanism behind this trait is unknown. In this manuscript, transcriptome analysis was employed to discover the genetic differences between white and red flesh in pitayas. A total of 27.99 Gb clean data were obtained for four samples. Unigenes, 79,049 in number, were generated with an average length of 1333 bp, and 52,618 Unigenes were annotated. Compared with white flesh, the expression of 10,215 Unigenes was up-regulated, and 4853 Unigenes were down-regulated in red flesh. The metabolic pathways accounted for 64.6% of all differentially expressed Unigenes in KEGG pathways. The group with high betalain content in red flesh and all structural genes, related to betalain biosynthesis, had a higher expression in red flesh than white flesh. The expression of the key gene, tyrosine hydroxylase CYP76AD1, was up-regulated 245.08 times, while 4,5-DOPA dioxygenase DODA was up-regulated 6.46 times. Moreover, the special isomers CYP76AD1α and DODAα were only expressed in red flesh. The competitive anthocyanin biosynthesis pathway had a lower expression in red flesh. Two MYB transcription factors were of the same branch as BvMYB1, regulating betalain biosynthesis in beet, and those transcription factors had expression differences in two kinds of pitayas, which indicated that they should be candidate genes controlling betalain accumulation in red pitayas. This research would benefit from identifying the major gene controlling red flesh trait and breed new cultivars with the red flesh trait. Future research should aim to prove the role of each candidate gene in betalain biosynthesis in red pitayas.