Amaranth's Growth and Physiological Responses to Salt Stress and the Functional Analysis of AtrTCP1 Gene.

Amaranth species are C4 plants that are rich in betalains, and they are tolerant to salinity stress. A small family of plant-specific TCP transcription factors are involved in the response to salt stress. However, it has not been investigated whether amaranth TCP1 is involved in salt stress. We elucidated that the growth and physiology of amaranth were affected by salt concentrations of 50-200 mmol·L-1 NaCl. The data showed that shoot and root growth was inhibited at 200 mmol·L-1, while it was promoted at 50 mmol·L-1. Meanwhile, the plants also showed physiological responses, which indicated salt-induced injuries and adaptation to the salt stress. Moreover, AtrTCP1 promoted Arabidopsis seed germination. The germination rate of wild-type (WT) and 35S::AtrTCP1-GUS Arabidopsis seeds reached around 92% by the seventh day and 94.5% by the second day under normal conditions, respectively. With 150 mmol·L-1 NaCl treatment, the germination rate of the WT and 35S::AtrTCP1-GUS plant seeds was 27.0% by the seventh day and 93.0% by the fourth day, respectively. Under salt stress, the transformed 35S::AtrTCP1 plants bloomed when they grew 21.8 leaves after 16.2 days of treatment, which was earlier than the WT plants. The transformed Arabidopsis plants flowered early to resist salt stress. These results reveal amaranth's growth and physiological responses to salt stress, and provide valuable information on the AtrTCP1 gene.

Identification of WRKY gene family members in amaranth based on a transcriptome database and functional analysis of AtrWRKY42-2 in betalain metabolism.

Introduction: WRKY TFs (WRKY transcription factors) contribute to the synthesis of secondary metabolites in plants. Betalains are natural pigments that do not coexist with anthocyanins within the same plant. Amaranthus tricolor ('Suxian No.1') is an important leaf vegetable rich in betalains. However, the WRKY family members in amaranth and their roles in betalain synthesis and metabolism are still unclear. Methods: To elucidate the molecular characteristics of the amaranth WRKY gene family and its role in betalain synthesis, WRKY gene family members were screened and identified using amaranth transcriptome data, and their physicochemical properties, conserved domains, phylogenetic relationships, and conserved motifs were analyzed using bioinformatics methods. Results: In total, 72 WRKY family members were identified from the amaranth transcriptome. Three WRKY genes involved in betalain synthesis were screened in the phylogenetic analysis of WRKY TFs. RT-qPCR showed that the expression levels of these three genes in red amaranth 'Suxian No.1' were higher than those in green amaranth 'Suxian No.2' and also showed that the expression level of AtrWRKY42 gene short-spliced transcript AtrWRKY42-2 in Amaranth 'Suxian No.1' was higher than that of the complete sequence AtrWRKY42-1, so the short-spliced transcript AtrWRKY42-2 was mainly expressed in 'Suxian No.2' amaranth. Moreover, the total expression levels of AtrWRKY42-1 and AtrWRKY42-2 were down-regulated after GA3 treatment, so AtrWRKY42-2 was identified as a candidate gene. Therefore, the short splice variant AtrWRKY42-2 cDNA sequence, gDNA sequence, and promoter sequence of AtrWRKY42 were cloned, and the PRI 101-AN-AtrWRKY42-2-EGFP vector was constructed to evaluate subcellular localization, revealing that AtrWRKY42-2 is located in the nucleus. The overexpression vector pRI 101-AN-AtrWRKY42-2-EGFP and VIGS (virus-induced gene silencing) vector pTRV2-AtrWRKY42-2 were transferred into leaves of 'Suxian No.1' by an Agrobacterium-mediated method. The results showed that AtrWRKY42-2 overexpression could promote the expression of AtrCYP76AD1 and increase betalain synthesis. A yeast one-hybrid assay demonstrated that AtrWRKY42-2 could bind to the AtrCYP76AD1 promoter to regulate betalain synthesis. Discussion: This study lays a foundation for further exploring the function of AtrWRKY42-2 in betalain metabolism.

Comparative Transcriptomic Analysis of the Metabolism of Betalains and Flavonoids in Red Amaranth Hypocotyl under Blue Light and Dark Conditions.

Amaranth plants contain abundant betalains and flavonoids. Anthocyanins are important flavonoids; however, they cannot coexist in the same plant with betalains. Blue light influences metabolite synthesis and hypocotyl elongation; accordingly, analyses of its effects on betalain and flavonoid biosynthesis in Amaranthus tricolor may provide insight into the distribution of these plant pigments. We analyzed the betalain and flavonoid content and transcriptome profiles in amaranth hypocotyls under blue light and dark conditions. Furthermore, we analyzed the expression patterns of key genes related to betalains and flavonoids. Amaranth hypocotyls were shorter and redder and showed higher betalain and flavonoid content under blue light than in dark conditions. Key genes involved in the synthesis of betalains and flavonoids were upregulated under blue light. The gene encoding DELLA was also upregulated. These results suggest that blue light favors the synthesis of both betalains and flavonoids via the suppression of bioactive gibberellin and the promotion of DELLA protein accumulation, which also suppresses hypocotyl elongation. The metabolite profiles differed between plants under blue light and dark conditions. These findings improve our understanding of the environmental cues and molecular mechanisms underlying pigment variation in Amaranthus.

Genome-wide identification, evolution analysis of cytochrome P450 monooxygenase multigene family and their expression patterns during the early somatic embryogenesis in Dimocarpus longan Lour.

Cytochrome P450 (CYP), a multi-gene superfamily, is involved in a broad range of physiological processes, including hormone responses and secondary metabolism throughout the plant life cycle. Longan (Dimocarpus longan), a subtropical and tropical evergreen fruit tree, its embryonic development is closely related to the yield and quality of fruits. And a large number of secondary metabolites, such as flavonoids and carotenoids, are also produced during the longan somatic embryogenesis (SE). It is important, therefore, to study potential functions of CYPs in longan. However, the knowledge of longan CYPs is still very limited. Here, a total of 327 DlCYPs were identified using the genome-search method, which could be classified into nine clans. The expansion of the DlCYP family was mainly caused by tandem duplication (TD) events. Promoter cis-acting elements analysis elucidated that DlCYPs played important roles in hormonal responses. A total of 246 DlCYPs exhibited six different expression patterns during the early SE based on longan transcriptomic data. Eight DlCYPs underwent alternative splicing (AS) events, and they might produce one to six isoforms. And the AS transcript of DlCYP97C1 might act as an alternative to the full-length transcript in ICpEC and GE stages. Finally, protein-protein interaction (PPI) networks and miRNA target prediction elucidated that DlCYPs might be involved in the phenylpropanoid metabolic pathway and primarily regulated and targeted by miR413. In summary, our results provided valuable inventory for understanding the classification and biological functions of DlCYPs and provided insight into further functional verification of DlCYPs during the longan early SE.

Generative Adversarial Construction of Parallel Portfolios.

Since automatic algorithm configuration methods have been very effective, recently there is increasing research interest in utilizing them for automatic solver construction, resulting in several notable approaches. For these approaches, a basic assumption is that the given training set could sufficiently represent the target use cases such that the constructed solvers can generalize well. However, such an assumption does not always hold in practice since in some cases, we might only have scarce and biased training data. This article studies effective construction approaches for the parallel algorithm portfolios that are less affected in these cases. Unlike previous approaches, the proposed approach simultaneously considers instance generation and portfolio construction in an adversarial process, in which the aim of the former is to generate instances that are challenging for the current portfolio, while the aim of the latter is to find a new component solver for the portfolio to better solve the newly generated instances. Applied to two widely studied problem domains, that is, the Boolean satisfiability problems (SAT) and the traveling salesman problems (TSPs), the proposed approach identified parallel portfolios with much better generalization than the ones generated by the existing approaches when the training data were scarce and biased. Moreover, it was further demonstrated that the generated portfolios could even rival the state-of-the-art manually designed parallel solvers.

RNA-sequencing analysis reveals betalains metabolism in the leaf of Amaranthus tricolor L.

Amaranth plants contain large amounts of betalains, including betaxanthins and betacyanins. Amaranthin is a betacyanin, and its molecular structure and associated metabolic pathway differ from those of betanin in beet plants. The chlorophyll, carotenoid, betalain, and flavonoid contents in amaranth leaves were analyzed. The abundance of betalain, betacyanin, and betaxanthin was 2-5-fold higher in the red leaf sectors than in the green leaf sectors. Moreover, a transcriptome database was constructed for the red and green sectors of amaranth leaves harvested from 30-day-old seedlings. 22 unigenes were selected to analyze the expression profiles in the two leaf sectors. The RNA-sequencing data indicated that many unigenes are involved in betalain metabolic pathways. The potential relationships between diverse metabolic pathways and betalain metabolism were analyzed. The validation of the expression of 22 selected unigenes in a qRT-PCR assay revealed the genes that were differentially expressed in the two leaf sectors. Betalains were biosynthesized in specific tissues of the red sectors of amaranth leaves. Almost all of the genes related to betalain metabolism were identified in the transcriptome database, and the expression profiles were different between the red sectors and green sectors in the leaf. Amaranth plants consist of diverse metabolic pathways, and the betalain metabolic pathway is linked to a group of other metabolic pathways.

Genome-wide sequencing of longan (Dimocarpus longan Lour.) provides insights into molecular basis of its polyphenol-rich characteristics.

Longan (Dimocarpus longan Lour.), an important subtropical fruit in the family Sapindaceae, is grown in more than 10 countries. Longan is an edible drupe fruit and a source of traditional medicine with polyphenol-rich traits. Tree size, alternate bearing, and witches' broom disease still pose serious problems. To gain insights into the genomic basis of longan traits, a draft genome sequence was assembled. The draft genome (about 471.88 Mb) of a Chinese longan cultivar, "Honghezi," was estimated to contain 31 007 genes and 261.88 Mb of repetitive sequences. No recent whole-genome-wide duplication event was detected in the genome. Whole-genome resequencing and analysis of 13 cultivated D. longan accessions revealed the extent of genetic diversity. Comparative transcriptome studies combined with genome-wide analysis revealed polyphenol-rich and pathogen resistance characteristics. Genes involved in secondary metabolism, especially those from significantly expanded (DHS, SDH, F3΄H, ANR, and UFGT) and contracted (PAL, CHS, and F3΄5΄H) gene families with tissue-specific expression, may be important contributors to the high accumulation levels of polyphenolic compounds observed in longan fruit. The high number of genes encoding nucleotide-binding site leucine-rich repeat (NBS-LRR) and leucine-rich repeat receptor-like kinase proteins, as well as the recent expansion and contraction of the NBS-LRR family, suggested a genomic basis for resistance to insects, fungus, and bacteria in this fruit tree. These data provide insights into the evolution and diversity of the longan genome. The comparative genomic and transcriptome analyses provided information about longan-specific traits, particularly genes involved in its polyphenol-rich and pathogen resistance characteristics.

Baicalein suppresses the proliferation of acute T-lymphoblastic leukemia Jurkat cells by inhibiting the Wnt/ß-catenin signaling.

Although the response rates of chemotherapy in patients with acute T-lymphoblastic leukemia (T-ALL) have improved significantly, the outcome of these patients is still poor. Previous studies suggested that baicalein could inhibit the growth of several cancers, while its effect on T-ALL cells remains unclear. We used Jurkat cells as an in vitro model of T-ALL. Cell counting kit-8 assay and cytometric analysis with Annexin V-FITC/PI double staining were used to investigate the proliferation and apoptosis of Jurkat cells treated with increasing concentration of baicalein for indicated time. RT-PCR and western blotting was used to test the expression of Wnt/ß-catenin associated genes and proteins. In cell viability assay, baicalein could inhibit the proliferation of Jurkat cells both in dose- and time-dependent manners. In cell apoptosis assay, baicalein could stimulate apoptosis of Jurkat cells both in dose- and time-dependent manners. Moreover, we demonstrated that baicalein could down-regulated the mRNA and protein levels of ß-catenin and its widely accepted downstream targets (c-Myc, cyclin D1, and Axin2) in dose-dependent manners. These results proved that baicalein might be a potential choice for the treatment of T-ALL.

Cloning and expression analysis of betalain biosynthesis genes in Amaranthus tricolor.

OBJECTIVES: To clone genes involved in the betalain biosynthesis pathway and to assess the effects of phytohormones on betalain biosynthesis in Amaranthus tricolor. RESULTS: Five betalain biosynthesis genes were cloned by reverse transcription PCR and rapid amplification of cDNA ends. Betacyanin analyses revealed that pigments accumulated differently in various tissues and under different phytohormone treatments. Quantitative RT-PCR analysis indicated that gene expression levels did not correlate with pigment accumulation. Notably, gene expression and pigment accumulation were negatively regulated by 2,4-dichlorophenoxyacetic acid. The expression of AmaDOPA5-GT, AmaDODA, and AmaB5-GT was induced by pigmentation-promoting 6-benzyl aminopurine (6-BA). and pigmentation-inhibiting gibberellin A3 while AmaTyDC expression was suppressed. AmaTyDC expression was also suppressed by pigmentation-promoting kinetin. Additionally, the expression of AmaB6-GT was suppressed by 6-BA. CONCLUSIONS: The changes in betacyanin levels among various tissues and following phytohormone treatments were related to the differences in betalain biosynthesis gene expression levels.

Transcriptome analysis by Illumina high-throughout paired-end sequencing reveals the complexity of differential gene expression during in vitro plantlet growth and flowering in Amaranthus tricolor L.

Amaranthus tricolor L. is a C4 plant, which is consumed as a major leafy vegetable in some tropical countries. Under conditions of high temperature and short daylight, Am. tricolor readily bolts and blooms, degrading leaf quality. A preliminary in vitro flowering study demonstrated that the flowering control pathway in Am. tricolor may differ from that of Arabidopsis. Nevertheless, no transcriptome analysis of the flowering process in Amaranthus has been conducted. To study Am. tricolor floral regulatory mechanisms, we conducted a large-scale transcriptome analysis--based on Illumina HiSeq sequencing of cDNA libraries generated from Am. tricolor at young seedling (YSS), adult seedling (ASS), flower bud (FBS), and flowering (FS) stages. A total of 99,312 unigenes were obtained. Using BLASTX, 43,088 unigenes (43.39%) were found to have significant similarity with accessions in Nr, Nt, and Swiss-Prot databases. Of these unigenes, 11,291 were mapped to 266 KEGG pathways. Further analysis of the four digital transcriptomes revealed that 735, 17,184, 274, and 206 unigenes were specifically expressed during YSS, ASS, FBS, and FS, respectively, with 59,517 unigenes expressed throughout the four stages. These unigenes were involved in many metabolic pathways related to in vitro flowering. Among these pathways, 259 unigenes were associated with ubiquitin-mediated proteolysis, indicating its importance for in vitro flowering in Am. tricolor. Other pathways, such as circadian rhythm and cell cycle, also had important roles. Finally, 26 unigenes were validated by qRT-PCR in samples from Am. tricolor at YSS, ASS, FBS, and FS; their differential expressions at the various stages indicate their possible roles in Am. tricolor growth and development, but the results were somewhat similar to Arabidopsis. Because unigenes involved in many metabolic pathways or of unknown function were revealed to regulate in vitro plantlet growth and flowering in Am. tricolor, the process appears to be highly complex in this species.

Bayesian composite model space approach for mapping quantitative trait Loci in variance component model.

In this article, we successfully apply the novel model selection method, Bayesian composite model space approach which has been used to map quantitative trait loci (QTL) for allelic substitution model, to map QTL for variance component model. The novel model selection approach has two advantages compared to the reversible jump Markov chain Monte Carlo method. Firstly, it mixes well due to the fixedness of the model dimension; secondly, it can map multiple QTL with higher power especially in genome-wide QTL mapping; finally, in the new method, it is also easy to incorporate our prior information about the variance components, which may bring precise estimate for variance components. A series of simulation experiments were conducted to demonstrate the general characters of the proposed method. The computer program is written in FORTRAN language, which is also built into a software "BayesMapQTL", and they also can be used for real data analysis and are available for request.

Bayesian analysis of genetic architecture of quantitative trait using data of crosses of multiple inbred lines.

Using the data of crosses of multiple of inbred lines for mapping QTL can increase QTL detecting power compared with only cross of two inbred lines. Although many fixed-effect model methods have been proposed to analyze such data, they are largely based on one-QTL model or main effect model, and the interaction effects between QTL are always neglected. However, effectively separating the interaction effects from the residual error can increase the statistical power. In this article, we both extended the novel Bayesian model selection method and Bayesian shrinkage estimation approaches to multiple inbred line crosses. With two extensions, interacting QTL are effectively detected with high solution; in addition, the posterior variances for both main effects and interaction effects are also subjected to full Bayesian estimate, which is more optimal than two step approach involved in maximum-likelihood. A series of simulation experiments have been conducted to demonstrate the performance of the methods. The computer program written in FORTRAN language is freely available on request.