A user-friendly deep learning application for accurate lung cancer diagnosis.

BACKGROUND: Accurate diagnosis and subsequent delineated treatment planning require the experience of clinicians in the handling of their case numbers. However, applying deep learning in image processing is useful in creating tools that promise faster high-quality diagnoses, but the accuracy and precision of 3-D image processing from 2-D data may be limited by factors such as superposition of organs, distortion and magnification, and detection of new pathologies. The purpose of this research is to use radiomics and deep learning to develop a tool for lung cancer diagnosis. METHODS: This study applies radiomics and deep learning in the diagnosis of lung cancer to help clinicians accurately analyze the images and thereby provide the appropriate treatment planning. 86 patients were recruited from Bach Mai Hospital, and 1012 patients were collected from an open-source database. First, deep learning has been applied in the process of segmentation by U-NET and cancer classification via the use of the DenseNet model. Second, the radiomics were applied for measuring and calculating diameter, surface area, and volume. Finally, the hardware also was designed by connecting between Arduino Nano and MFRC522 module for reading data from the tag. In addition, the displayed interface was created on a web platform using Python through Streamlit. RESULTS: The applied segmentation model yielded a validation loss of 0.498, a train loss of 0.27, a cancer classification validation loss of 0.78, and a training accuracy of 0.98. The outcomes of the diagnostic capabilities of lung cancer (recognition and classification of lung cancer from chest CT scans) were quite successful. CONCLUSIONS: The model provided means for storing and updating patients' data directly on the interface which allowed the results to be readily available for the health care providers. The developed system will improve clinical communication and information exchange. Moreover, it can manage efforts by generating correlated and coherent summaries of cancer diagnoses.

Genome-wide association and targeted transcriptomic analyses reveal loci and candidate genes regulating preharvest sprouting in barley.

KEY MESSAGE: Genome-wide association study of diverse barley genotypes identified loci, single nucleotide polymorphisms and candidate genes that control seed dormancy and therefore enhance resistance to preharvest sprouting. Preharvest sprouting (PHS) causes significant yield and quality loss in barley and it is strongly associated with the level of seed dormancy. This study performed genome-wide association study using a collection of 255 diverse barley genotypes grown over four environments to identify loci controlling dormancy/PHS. Our phenotypic analysis revealed substantial variation in germination index/dormancy levels among the barley genotypes. Marker-trait association and linkage disequilibrium (LD) decay analyses identified 16 single nucleotide polymorphisms (SNPs) and two QTLs associated with dormancy/PHS, respectively, on chromosome 3H and 5H explaining 6.9% to 11.1% of the phenotypic variation. QTL.5H consist of 14 SNPs of which 12 SNPs satisfy the FDR threshold of α = 0.05, and it may represent the SD2 locus. The QTL on 3H consists of one SNP that doesn't satisfy FDR (α = 0.05). Genes harbouring the significant SNPs were analyzed for their expression pattern in the seeds of selected dormant and non-dormant genotypes. Of these genes, HvRCD1, HvPSRP1 and HvF3H exhibited differential expression between the dormant and non-dormant seed samples, suggesting their role in controlling seed dormancy/PHS. Three SNPs located within the differentially expressed genes residing in QTL.5H explained considerable phenotypic variation (≥ 8.6%), suggesting their importance in regulating PHS resistance. Analysis of the SNP marker data in QTL.5H identified a haplotype for PHS resistance. Overall, the study identified loci, SNPs and candidate genes that control dormancy and therefore play important roles in enhancing PHS resistance in barley through marker-assisted breeding.

The utility and post-operative evolution of head circumference in nonsyndromic single-suture craniosynostosis: a prospective study in Vietnamese children.

PURPOSE: Head circumference (HC) is an important clinical tool for following head growth in children with craniosynostosis (CS). The purpose of this study is to quantify the usefulness of HC along continuum of CS care, from diagnosis to pre- and post-operative (pre-op, post-op) follow-up in Vietnamese children. METHODS: A prospective cohort of 54 nonsyndromic single-suture CS patients undergoing open surgery from January 2015 to January 2020 was collected at Children's Hospital 2, Vietnam. HC z-score on admission was compared with World Health Organization (WHO) standards to evaluate for utility in initial diagnosis. Pre-op and post-op HC were compared to demonstrate the evolution of head growth following reconstruction. RESULTS: Nonsyndromic single-suture CS was more predominant in males (79.6%) than in females (20.4%). The mean HC z-score was - 0.38 [Formula: see text] 1.29 similar to normal WHO standards regardless of which sutural involvement. The HC z-score increased above + 1 standard deviation (SD) significantly at 3 months of follow-up (p < 0.001); however, the trajectory gradually decreased after the first year of surgery. One patient (1.8%, 1/54) demonstrated restenosis and delayed intracranial hypertension (DIH) 4 years after reconstruction. CONCLUSIONS: The HC in nonsyndromic single-suture CS children presents similarly to the values of healthy children. Additionally, HC reliably increased after reconstruction and gradually normalized over subsequent years. This indicator is consistent in Southeast Asian populations and should be used to follow all patients to assess the normal progression of post-op head growth and as a useful indicator of suspected recurrent synostosis.

Jasmonate regulates seed dormancy in wheat via modulating the balance between gibberellin and abscisic acid.

Jasmonate (JA) regulates seed dormancy and germination; however, the underlying mechanisms remain poorly understood. Furthermore, it is unclear if JA is an essential regulator of dormancy and germination. We investigated whether the role of JA in regulating seed dormancy in wheat (Triticum aestivum L.) is mediated by modulation of gibberellin (GA)/abscisic acid (ABA) balance and if the reciprocal modulation of JA level and sensitivity is required for GA-mediated dormancy loss using physiological, pharmacological, and targeted transcriptomic and metabolomic approaches. JA-induced dormancy release in wheat seeds was associated with no change in GA level but up-regulation of GA signaling and ABA catabolism genes, and reduction of the ABA level. Although JA did not affect the expression levels of ABA signaling genes, up-regulation of germination-associated genes indicates a contribution of reduced ABA sensitivity to dormancy release. After-ripening-mediated dormancy loss was also associated with JA-GA synergistic and JA-ABA antagonistic interplays. The prevalence of no effect of GA, which effectively broke dormancy, on the JA-Ile level and expression patterns of JA biosynthesis/signaling and responsive genes reflects that GA-mediated dormancy release occurs independently of JA. Our study concludes that JA induces seed dormancy release in wheat via modulating ABA/GA balance; however, JA is not an essential regulator of dormancy and germination.

Salicylic Acid Enhances Adventitious Root and Aerenchyma Formation in Wheat under Waterlogged Conditions.

The present study investigated the role of salicylic acid (SA) in regulating morpho-anatomical adaptive responses of a wheat plant to waterlogging. Our pharmacological study showed that treatment of waterlogged wheat plants with exogenous SA promotes the formation axile roots and surface adventitious roots that originate from basal stem nodes, but inhibits their elongation, leading to the formation of a shallow root system. The treatment also enhanced axile root formation in non-waterlogged plants but with only slight reductions in their length and branch root formation. Exogenous SA enhanced the formation of root aerenchyma, a key anatomical adaptive response of plants to waterlogging. Consistent with these results, waterlogging enhanced SA content in the root via expression of specific isochorismate synthase (ICS; ICS1 and ICS2) and phenylalanine ammonia lyase (PAL; PAL4, PAL5 and PAL6) genes and in the stem nodes via expression of specific PAL (PAL5 and PAL6) genes. Although not to the same level observed in waterlogged plants, exogenous SA also induced aerenchyma formation in non-waterlogged plants. The findings of this study furthermore indicated that inhibition of ethylene synthesis in SA treated non-waterlogged and waterlogged plants does not have any effect on SA-induced emergence of axile and/or surface adventitious roots but represses SA-mediated induction of aerenchyma formation. These results highlight that the role of SA in promoting the development of axile and surface adventitious roots in waterlogged wheat plants is ethylene independent while the induction of aerenchyma formation by SA requires the presence of ethylene.

Ethylene regulates post-germination seedling growth in wheat through spatial and temporal modulation of ABA/GA balance.

This study aimed to gain insights into the molecular mechanisms underlying the role of ethylene in regulating germination and seedling growth in wheat by combining pharmacological, molecular, and metabolomics approaches. Our study showed that ethylene does not affect radicle protrusion but controls post-germination endospermic starch degradation through transcriptional regulation of specific α-amylase and α-glucosidase genes, and this effect is mediated by alteration of endospermic bioactive gibberellin (GA) levels, and GA sensitivity via expression of the GA signaling gene, TaGAMYB. Our data implicated ethylene as a positive regulator of embryo axis and coleoptile growth through transcriptional regulation of specific TaEXPA genes. These effects were associated with modulation of GA levels and sensitivity, through expression of GA metabolism (TaGA20ox1, TaGA3ox2, and TaGA2ox6) and signaling (TaGAMYB) genes, respectively, and/or the abscisic acid (ABA) level and sensitivity, via expression of specific ABA metabolism (TaNCED2 or TaCYP707A1) and signaling (TaABI3) genes, respectively. Ethylene appeared to regulate the expression of TaEXPA3 and thereby root growth through its control of coleoptile ABA metabolism, and root ABA signaling via expression of TaABI3 and TaABI5. These results show that spatiotemporal modulation of ABA/GA balance mediates the role of ethylene in regulating post-germination storage starch degradation and seedling growth in wheat.

Spatiotemporal modulation of abscisic acid and gibberellin metabolism and signalling mediates the effects of suboptimal and supraoptimal temperatures on seed germination in wheat (Triticum aestivum L.).

Seed germination is a complex process regulated by intrinsic hormonal cues such as abscisic acid (ABA) and gibberellin (GA), and environmental signals including temperature. Using pharmacological, molecular and metabolomics approaches, we show that supraoptimal temperature delays wheat seed germination through maintaining elevated embryonic ABA level via increased expression of ABA biosynthetic genes (TaNCED1 and TaNCED2), increasing embryo ABA sensitivity through upregulation of genes regulating ABA signalling positively (TaPYL5, TaSnRK2, ABI3 and ABI5) and decreasing embryo GA sensitivity via induction of TaRHT1 that regulates GA signalling negatively. Endospermic ABA and GA appeared to have minimal roles in regulating germination at supraoptimal temperature. Germination inhibition by suboptimal temperature is associated with elevated ABA level in the embryo and endosperm tissues, mediated by induction of TaNCEDs and decreased expression of endospermic ABA catabolic genes (TaCYP707As), and increased ABA sensitivity in both tissues via upregulation of TaPYL5, TaSnRK2, ABI3 and ABI5 in the embryo and TaSnRK2 and ABI5 in the endosperm. Furthermore, suboptimal temperature suppresses GA synthesis in both tissues and GA sensitivity in the embryo via repressing GA biosynthetic genes (TaGA20ox and TaGA3ox2) and inducing TaRHT1, respectively. These results highlight that spatiotemporal modulation of ABA and GA metabolism and signalling in wheat seeds underlies germination response to temperature.

Hormonal regulation in adventitious roots and during their emergence under waterlogged conditions in wheat.

To gain insights into the molecular mechanisms underlying hormonal regulation in adventitious roots and during their emergence under waterlogged conditions in wheat, the present study investigated transcriptional regulation of genes related to hormone metabolism and transport in the root and stem node tissues. Waterlogging-induced inhibition of axile root elongation and lateral root formation, and promotion of surface adventitious and axile root emergence and aerenchyma formation are associated with enhanced expression levels of ethylene biosynthesis genes, ACS7 and ACO2, in both tissues. Inhibition of axile root elongation is also related to increased root indole acetic acid (IAA) and jasmonate (JA) levels that are associated with up-regulation of specific IAA biosynthesis/transport (TDC, YUC1, and PIN9) and JA metabolism (LOX8, AOS1, AOC1, and JAR1) genes, and transcriptional alteration of gibberellin (GA) metabolism genes (GA3ox2 and GA2ox8). Adventitious root emergence from waterlogged stem nodes is associated with increased levels of IAA and GA but decreased levels of cytokinin and abscisic acid (ABA), which are regulated through the expression of specific IAA biosynthesis/transport (TDC, YUC1, and PIN9), cytokinin metabolism (IPT5-2, LOG1, CKX5, and ZOG2), ABA biosynthesis (NCED1 and NCED2), and GA metabolism (GA3ox2 and GA2ox8) genes. These results enhance our understanding of the molecular mechanisms underlying the adaptive response of wheat to waterlogging.

Dormancy-Associated MADS-Box (DAM) and the Abscisic Acid Pathway Regulate Pear Endodormancy Through a Feedback Mechanism.

In the pear 'Kosui' (Pyrus pyrifolia Nakai), the dormancy-associated MADS-box (PpDAM1 = PpMADS13-1) gene has been reported to play an essential role in bud endodormancy. Here, we found that PpDAM1 up-regulated expression of 9-cis-epoxycarotenoid dioxygenase (PpNCED3), which is a rate-limiting gene for ABA biosynthesis. Transient assays with a dual luciferase reporter system (LUC assay) and electrophoretic mobility shift assay (EMSA) showed that PpDAM1 activated PpNCED3 expression by binding to the CArG motif in the PpNCED3 promoter. PpNCED3 expression was increased toward endodormancy release in lateral flower buds of 'Kosui', which is consistent with the induced levels of ABA, its catabolism (ABA 8'-hydroxylase) and signaling genes (type 2C protein phosphatase genes and SNF1-related protein kinase 2 genes). In addition, we found that an ABA response element (ABRE)-binding transcription factor, PpAREB1, exhibiting high expression concomitant with endodormancy release, bound to three ABRE motifs in the promoter region of PpDAM1 and negatively regulated its activity. Taken together, our results suggested a feedback regulation between PpDAM1 and the ABA metabolism and signaling pathway during endodormancy of pear. This first evidence of an interaction between a DAM and ABA biosynthesis in vitro will provide further insights into bud endodormancy regulatory mechanisms of deciduous trees including pear.

Repression of TERMINAL FLOWER1 primarily mediates floral induction in pear (Pyrus pyrifolia Nakai) concomitant with change in gene expression of plant hormone-related genes and transcription factors.

Floral induction is an important event in the annual growth cycle of perennial fruit trees. For pear, this event directly affects fruit production in the following year. The flower buds in many species are induced by FLOWERING LOCUS T (FT), whose effect is repressed by the meristem-expressed gene TERMINAL FLOWER1 (TFL1). In this study, we investigated the functions of pear FT and TFL1 genes during floral development. Expression of pear FTs (PpFT1a and PpFT2a) in reproductive meristems was not obviously induced prior to floral initiation, while expression of TFL1s (PpTFL1-1a and PpTFL1-2a) rapidly decreased. The induction of the productive meristem identity MADS-box gene AP1 after repression of PpTFL1s suggested a primary role for PpTFL1 in floral induction. RNA-seq analysis suggested that plant hormone-related genes and several transcription factors that were coexpressed with PpTFL1 were potentially involved in the PpTFL1-mediated floral induction. Our data indicate the essential function of TFL1 in pear floral induction and add another species in the family Rosaceae in addition to strawberry and rose that shows a role for TFL1 in floral induction.

Small RNA and PARE sequencing in flower bud reveal the involvement of sRNAs in endodormancy release of Japanese pear (Pyrus pyrifolia 'Kosui').

BACKGROUND: In woody perennial plants, including deciduous fruit trees, such as pear, endodormancy is a strategy for surviving the cold winter. A better understanding of the mechanism underlying the endodormancy phase transition is necessary for developing countermeasures against the effects of global warming. In this study, we analyzed the sRNAome of Japanese pear flower buds in endodormant and ecodormant stages over two seasons by implementing of RNA-seq and degradome-sequencing. RESULTS: We identified 137 conserved or less conserved miRNAs and 50 pear-specific miRNAs. However, none of the conserved microRNAs or pear-specific miRNAs was differentially expressed between endodormancy and ecodormancy stages. On the contrast, 1540 of 218,050 loci that produced sRNAs were differentially expressed between endodormancy and ecodormancy, suggesting their potential roles on the phase transition from endodormancy to ecodomancy. We also characterized a multifunctional miRNA precursor MIR168, which produces two functional miR168 transcripts, namely miR168.1 and miR168.2; cleavage events were predominantly mediated by the non-conserved variant miR168.2 rather than the conserved variant miR168.1. Finally, we showed that a TAS3 trans-acting siRNA triggered phased siRNA within the ORF of one of its target genes, AUXIN RESPONSE FACTOR 4, via the analysis of phased siRNA loci, indicating that siRNAs are able to trigger phased siRNAs in pear. CONCLUSION: We analyzed the sRNAome of pear flower bud during dormant phase transition. Our work described the sRNA profiles of pear winter buds during dormant phase transition, showing that dormancy release is a highly coordinated physiological process involving the regulation of sRNAs.

Epigenetic regulation of MdMYB1 is associated with paper bagging-induced red pigmentation of apples.

MAIN CONCLUSION: Paper-bagging treatment can transform non-transcribed MdMYB1 - 2 and MdMYB1 - 3 alleles into transcribed alleles through epigenetic regulations, resulting in the red pigmentation of a normally non-red apple cultivar 'Mutsu.' Anthocyanin biosynthesis in apples is regulated by MdMYB1/A/10, an R2R3-Type MYB gene. 'Mutsu,' a triploid apple cultivar harboring non-transcribed MdMYB1-2 and MdMYB1-3 alleles, retains green skin color under field conditions. However, it can show red/pink pigmentation under natural or artificial ultraviolet-B (UV-B) light exposure after paper-bagging and bag removal treatment. In the present study, we found that in 'Mutsu,' paper bagging-induced red pigmentation was due to the activation of non-transcribed MdMYB1-2/-3 alleles, which triggered the expression of downstream anthocyanin biosynthesis genes in a UV-B-dependent manner. By monitoring the epigenetic changes during UV-B-induced pigmentation, no significant differences in DNA methylation and histone modifications in the 5' upstream region of MdMYB1-2/-3 were recorded between the UV-B-treated fruit skin (red) and the fruit skin treated only by white light (green). In contrast, bag treatment lowered the DNA methylation in this region of MdMYB1-2/-3 alleles. Similarly, higher levels of histone H3 acetylation and trimethylation of H3 tail at lysine 4, and lower level of trimethylation of H3 tail at lysine 27 were observed in the 5' upstream region of MdMYB1-2/-3 in the skin of the fruit immediately after bag removal. These results suggest that bagging treatment can induce epigenetic changes, facilitating the binding of trans factor(s) to MdMYB1-2/-3 alleles, resulting in the activation of these MYBs after bag removal.

The crucial role of PpMYB10.1 in anthocyanin accumulation in peach and relationships between its allelic type and skin color phenotype.

BACKGROUND: Red coloration of fruit skin is one of the most important traits in peach (Prunus persica), and it is mainly due to the accumulation of anthocyanins. Three MYB10 genes, PpMYB10.1, PpMYB10.2, and PpMYB10.3, have been reported as important regulators of red coloration and anthocyanin biosynthesis in peach fruit. In this study, contribution of PpMYB10.1/2/3 to anthocyanin accumulation in the fruit skin was investigated in the Japanese peach cultivars, white-skinned 'Mochizuki' and red-skinned 'Akatsuki'. We then investigated the relationships between allelic type of PpMYB10.1 and skin color phenotype in 23 Japanese peach cultivars for future establishment of DNA-marker. RESULTS: During the fruit development of 'Mochizuki' and 'Akatsuki', anthocyanin accumulation was observed only in the skin of red 'Akatsuki' fruit in the late ripening stages concomitant with high mRNA levels of the last step gene leading to anthocyanin accumulation, UDP-glucose:flavonoid-3-O-glucosyltransferase (UFGT). This was also correlated with the expression level of PpMYB10.1. Unlike PpMYB10.1, expression levels of PpMYB10.2/3 were low in the skin of both 'Mochizuki' and 'Akatsuki' throughout fruit development. Moreover, only PpMYB10.1 revealed expression levels associated with total anthocyanin accumulation in the leaves and flowers of 'Mochizuki' and 'Akatsuki'. Introduction of PpMYB10.1 into tobacco increased the expression of tobacco UFGT, resulting in higher anthocyanin accumulation and deeper red transgenic tobacco flowers; however, overexpression of PpMYB10.2/3 did not alter anthocyanin content and color of transgenic tobacco flowers when compared with wild-type flowers. Dual-luciferase assay showed that the co-infiltration of PpMYB10.1 with PpbHLH3 significantly increased the activity of PpUFGT promoter. We also found close relationships of two PpMYB10.1 allelic types, MYB10.1-1/MYB10.1-2, with the intensity of red skin coloration. CONCLUSION: We showed that PpMYB10.1 is a major regulator of anthocyanin accumulation in red-skinned peach and that it activates PpUFGT transcription. PpMYB10.2/3 may be involved in functions other than anthocyanin accumulation in peach. The peach cultivars having two MYB10.1-2 types resulted in the white skin color. By contrast, those with two MYB10.1-1 or MYB10.1-1/MYB10.1-2 types showed respective red or pale red skin color. These findings contribute to clarifying the molecular mechanisms of anthocyanin accumulation and generating gene-based markers linked to skin color phenotypes.

A model immunization programme to control Japanese encephalitis in Viet Nam.

In Viet Nam, an inactivated, mouse brain-derived vaccine for Japanese encephalitis (JE) has been given exclusively to ≤ 5 years old children in 3 paediatric doses since 1997. However, JE incidence remained high, especially among children aged 5-9 years. We conducted a model JE immunization programme to assess the feasibility and impact of JE vaccine administered to 1-9 year(s) children in 3 standard-dose regimen: paediatric doses for children aged <3 years and adult doses for those aged ≥ 3 years. Of the targeted children, 96.2% were immunized with ≥ 2 doses of the vaccine. Compared to the national immunization programme, JE incidence rate declined sharply in districts with the model programme (11.32 to 0.87 per 100,000 in pre-versus post-vaccination period). The rate of reduction was most significant in the 5-9 years age-group. We recommend a policy change to include 5-9 years old children in the catch-up immunization campaign and administer a 4th dose to those aged 5-9 years, who had received 3 doses of the vaccine during the first 2-3 years of life.

Molecular cloning and characterization of genes involved in rosmarinic acid biosynthesis from Prunella vulgaris.

Prunella vulgaris L., commonly known as "self-heal" or "heal-all," is a perennial herb with a long history of medicinal use. Phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate:coenzyme-A (CoA) ligase (4CL) are important enzymes in the phenylpropanoid pathway and in the accumulation of rosmarinic acid (RA), which is a major secondary metabolite in P. vulgaris. In this study, we isolated cDNAs encoding PvPAL, PvC4H, and Pv4CL from P. vulgaris using rapid amplification of cDNA ends polymerase chain reaction (PCR). The amino acid sequence alignments of PvPAL, PvC4H, and Pv4CL showed high sequence identity to those of other plants. Quantitative real-time PCR analysis was used to determine the transcript levels of genes involved in RA biosynthesis in the flowers, leaves, stems, and roots of P. vulgaris. The transcript levels of PvPAL, PvC4H, and Pv4CL1 were the highest in flowers, whereas Pv4CL2 was the highest in roots. High-performance liquid chromatography analysis also showed the highest RA content in the flowers (3.71 mg/g dry weight). We suggest that the expression of the PvPAL, PvC4H, and Pv4CL1 genes is correlated with the accumulation of RA. Our results revealed that P. vulgaris flowers are appropriate for medicinal usage, and our findings provide support for increasing RA production in this plant.

Cloning and characterization of a flavonol synthase gene from Scutellaria baicalensis.

Flavonols are the most abundant of all the flavonoids and play pivotal roles in a variety of plants. We isolated a cDNA clone encoding flavonol synthase from Scutellaria baicalensis (SbFLS). The SbFLS cDNA is 1011 bp long, encodes 336 amino acid residues, and belongs to a family of 2-oxoglutarate-dependent dioxygenases. The overall structure of SbFLS is very similar to that of Arabidopsis thaliana anthocyanidin synthase (AtANS), with a ß jelly-roll fold surrounded by tens of short and long α-helices. SbFLS was constitutively expressed in the roots, stems, leaves, and flowers, with particularly high expression in the roots and flowers. SbFLS transcript levels in the roots were 376-, 70-, and 2.5-fold higher than in the leaves, stems, and flowers. The myricetin content was significantly higher than that of kaempferol and quercetin. Therefore, we suggest that SbFLS mediates flavonol formation in the different organs of S. baicalensis. Our study may contribute to the knowledge of the role of FLS in S. baicalensis.

Enhancement of chlorogenic acid production in hairy roots of Platycodon grandiflorum by over-expression of an Arabidopsis thaliana transcription factor AtPAP1.

To improve the production of chlorogenic acid (CGA) in hairy roots of Platycodon grandiflorum, we induced over-expression of Arabidopsis thaliana transcription factor production of anthocyanin pigment (AtPAP1) using an Agrobacterium rhizogenes-mediated transformation system. Twelve hairy root lines showing over-expression of AtPAP1 were generated. In order to investigate the regulation of AtPAP1 on the activities of CGA biosynthetic genes, the expression levels of seven P. grandiflorum CGA biosynthetic genes were analyzed in the hairy root line that had the greatest accumulation of AtPAP1 transcript, OxPAP1-1. The introduction of AtPAP1 increased the mRNA levels of all examined CGA biosynthetic genes and resulted in a 900% up-regulation of CGA accumulation in OxPAP1-1 hairy roots relative to controls. This suggests that P. grandiflorum hairy roots that over-express the AtPAP1 gene are a potential alternative source of roots for the production of CGA.

Molecular characterization of carotenoid biosynthetic genes and carotenoid accumulation in Lycium chinense.

Lycium chinense is a shrub that has health benefits and is used as a source of medicines in Asia. In this study, a full-length cDNA clone encoding ß-ring carotene hydroxylase (LcCHXB) and partial-length cDNA clones encoding phytoene synthase (LcPSY), phytoene desaturase (LcPDS), ξ-carotene desaturase (LcZDS), lycopene ß-cyclase (LcLCYB), lycopene ε-cyclase (LcLCYE), ε-ring carotene hydroxylase (LcCHXE), zeaxanthin epoxidase (LcZEP), carotenoid cleavage dioxygenase (LcCCD1), and 9-cis epoxycarotenoid dioxygenase (LcNCED) were identified in L. chinense. The transcripts were constitutively expressed at high levels in leaves, flowers and red fruits, where the carotenoids are mostly distributed. In contrast, most of the carotenoid biosynthetic genes were weakly expressed in the roots and stems, which contained only small amounts of carotenoids. The level of LcLCYE transcripts was very high in leaves and correlated with the abundance of lutein in this plant tissue. During maturation, the levels of lutein and zeaxanthin in L. chinense fruits dramatically increased, concomitant with a rise in the level of ß-cryptoxanthin. LcPSY, LcPDS, LcZDS, LcLCYB, and LcCHXE were highly expressed in red fruits, leading to their substantially higher total carotenoid content compared to that in green fruits. Total carotenoid content was high in both the leaves and red fruits of L. chinense. Our findings on the biosynthesis of carotenoids in L. chinense provide insights into the molecular mechanisms involved in carotenoid biosynthesis and may facilitate the optimization of carotenoid production in L. chinense.

Accumulation of astragalosides and related gene expression in different organs of Astragalus membranaceus Bge. var mongholicus (Bge.).

Astragalus membranaceus is one of the most important traditional Korean and Chinese medicinal herbs because it contains triterpenoid saponins (astragaloside I, II, III, and IV), which have beneficial and pharmacological effects on health. In this study, we analyzed 10 mevalonate pathway genes that are involved in astragaloside biosynthesis using the Illumina/Solexa HiSeq2000 platform. We determined the expression levels of the 10 genes using quantitative real-time PCR, and analyzed the accumulation of astragalosides in different organs using high-performance liquid chromatography. Genes related to the mevalonate pathway were expressed in different levels in different organs. Almost all genes showed high transcript levels in the stem and leaf, with the lowest transcript levels being recorded in the root. In contrast, most astragalosides accumulated in the root. In particular, the astragaloside IV content was distributed in the following order: root (0.58 mg/g DW) > flower (0.27 mg/g DW) > stem (0.23 mg/g DW) > leaf (0.04 mg/g DW). In the root, astragaloside II exhibited the highest content (2.09 mg/g DW) compared to astragaloside I, III, and IV. Notably, gene expression did not follow the same pattern as astragaloside accumulation. We suggest carefully that astragalosides are synthesized in the leaves and stem and then translocated to the root. This study contributes towards improving our understanding of astragaloside biosynthesis in A. membranaceus.

Riboflavin accumulation and molecular characterization of cDNAs encoding bifunctional GTP cyclohydrolase II/3,4-dihydroxy-2-butanone 4-phosphate synthase, lumazine synthase, and riboflavin synthase in different organs of Lycium chinense plant.

Riboflavin (vitamin B2) is the precursor of flavin mononucleotide and flavin adenine dinucleotide-essential cofactors for a wide variety of enzymes involving in numerous metabolic processes. In this study, a partial-length cDNA encoding bifunctional GTP cyclohydrolase II/3,4-dihydroxy-2-butanone-4-phosphate synthase (LcRIBA), 2 full-length cDNAs encoding lumazine synthase (LcLS1 and LcLS2), and a full-length cDNA encoding riboflavin synthase (LcRS) were isolated from Lycium chinense, an important traditional medicinal plant. Sequence analyses showed that these genes exhibited high identities with their orthologous genes as well as having the same common features related to plant riboflavin biosynthetic genes. LcRIBA, like other plant RIBAs, contained a DHBPS region in its N terminus and a GCHII region in its C-terminal part. LcLSs and LcRS carried an N-terminal extension found in plant riboflavin biosynthetic genes unlike the orthologous microbial genes. Quantitative real-time polymerase chain reaction analysis showed that 4 riboflavin biosynthetic genes were constitutively expressed in all organs examined of L. chinense plants with the highest expression levels found in the leaves or red fruits. LcRIBA, which catalyzes 2 initial reactions in riboflavin biosynthetic pathway, was the highest transcript in the leaves, and hence, the richest content of riboflavin was detected in this organ. Our study might provide the basis for investigating the contribution of riboflavin in diverse biological activities of L. chinense and may facilitate the metabolic engineering of vitamin B2 in crop plants.