Baphicacanthus cusia (Nees) Bremek is an herb widely used for the clinical treatment of colds, fever, and influenza in Traditional Chinese Medicine. The roots, stems and leaves can be used as natural medicine, in which indigo and indirubin are two main active ingredients. In this study, quantification of indigo, indirubin, indican and adenosine among various tissues of B. cusia was conducted using HPLC-DAD. Leaves have significantly higher contents than stems and roots (380.66, 315.15, 20,978.26, 4323.15 μg/g in leaves, 306.36, 71.71, 3,056.78, 139.45 μg/g in stems, and 9.31, 7.82, 170.45, 197.48 μg/g in roots, respectively). De novo transcriptome sequencing of B. cusia was performed for the first time. The sequencing yielded 137,216,248, 122,837,394 and 140,240,688 clean reads from leaves, stems and roots respectively, which were assembled into 51,381 unique sequences. A total of 33,317 unigenes could be annotated using the databases of Nr, Swiss-Prot, KEGG and KOG. These analyses provided a detailed view of the enzymes involved in indican backbone biosynthesis, such as cytochrome P450, UDP-glycosyltransferase, glucosidase and tryptophan synthase. Analysis results showed that tryptophan synthase was the candidate gene involved in the tissue-specific biosynthesis of indican. We also detected sixteen types of simple sequence repeats in RNA-Seq data for use in future molecular mark assisted breeding studies. The results will be helpful in further analysis of B. cusia functional genomics, especially in increasing biosynthesis of indican through biotechnological approaches and metabolic regulation.
Baphicacanthus cusia (Nees) Bremek (B. cusia) is an effective herb for the treatment of acute promyelocytic leukemia and psoriasis in traditional Chinese medicine. Methyl jasmonate (MeJA) is a well-known signaling phytohormone that triggers gene expression in secondary metabolism. Currently, MeJA-mediated biosynthesis of indigo and indirubin in B. cusia is not well understood. In this study, we analyzed the content of indigo and indirubin in leaf and root tissues of B. cusia with high-performance liquid chromatography and measured photosynthetic characteristics of leaves treated by MeJA using FluorCam6 Fluorometer and chlorophyll fluorescence using the portable photosynthesis system CIRAS-2. We performed de novo RNA-seq of B. cusia leaf and root transcriptional profiles to investigate differentially expressed genes (DEGs) in response to exogenous MeJA application. The amount of indigo in MeJA-treated leaves were higher than that in controled leaves (p = 0.004), and the amounts of indigo in treated roots was higher than that in controlled roots (p = 0.048); Chlorophyll fluorescence of leaves treated with MeJA were significantly decreased. Leaves treated with MeJA showed lower photosynthetic rate compared to the control in the absence of MeJA. Functional annotation of DEGs showed the DEGs related to growth and development processes were down-regulated in the treated leaves, while most of the unigenes involved in the defense response were up-regulated in treated roots. This coincided with the effects of MeJA on photosynthetic characteristics and chlorophyll fluorescence. The qRT-PCR results showed that MeJA appears to down-regulate the gene expression of tryptophan synthase β-subunits (trpA-β) in leaves but increased the gene expression of anthranilate synthase (trp 3) in roots responsible for increased indigo content. The results showed that MeJA suppressed leaf photosynthesis for B. cusia and this growth-defense trade-off may contribute to the improved adaptability of B. cusia in changing environments.
BackgroundIndigo alkaloids, such as indigo, indirubin and its derivatives, have been identified as effective antiviral compounds in Baphicacanthus cusia. Evidence suggests that the biosynthesis of indigo alkaloids in plants occurs via the shikimate pathway. The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is involved in plant metabolism; however, its underlying putative mechanism of regulating the production of indigo alkaloids is currently unknown.ResultsOne gene encoding EPSPS was isolated from B. cusia. Quantitative real-time PCR analysis revealed that BcEPSPS was expressed at the highest level in the stem and upregulated by methyl jasmonate (MeJA), salicylic acid (SA) and abscisic acid (ABA) treatment. The results of subcellular localization indicated that BcEPSPS is mainly expressed in both the plastids and cytosol, which has not been previously reported. An enzyme assay revealed that the heterogeneously expressed BcEPSPS protein catalysed the generation of 5-enolpyruvyl shikimate-3-phosphate. The overexpression of BcEPSPS in Isatis indigotica hairy roots resulted in the high accumulation of indigo alkaloids, such as indigo, secologanin, indole and isorhamnetin.ConclusionsThe function of BcEPSPS in catalysing the production of EPSP and regulating indigo alkaloid biosynthesis was revealed, which provided a distinct view of plant metabolic engineering. Our findings have practical implications for understanding the effect of BcEPSPS on active compound biosynthesis in B. cusia.
Strobilanthes cusia (Nees) Kuntze is an important plant used to process the traditional Chinese herbal medicines “Qingdai” and “Nanbanlangen”. The key active ingredients are indole alkaloids (IAs) that exert antibacterial, antiviral, and antitumor pharmacological activities and serve as natural dyes. We assembled the S. cusia genome at the chromosome level through combined PacBio circular consensus sequencing (CCS) and Hi-C sequencing data. Hi-C data revealed a draft genome size of 913.74 Mb, with 904.18 Mb contigs anchored into 16 pseudo-chromosomes. Contig N50 and scaffold N50 were 35.59 and 68.44 Mb, respectively. Of the 32,974 predicted protein-coding genes, 96.52% were functionally annotated in public databases. We predicted 675.66 Mb repetitive sequences, 47.08% of sequences were long terminal repeat (LTR) retrotransposons. Moreover, 983 Strobilanthes-specific genes (SSGs) were identified for the first time, accounting for ~2.98% of all protein-coding genes. Further, 245 putative centromeric and 29 putative telomeric fragments were identified. The transcriptome analysis identified 2,975 differentially expressed genes (DEGs) enriched in phenylpropanoid, flavonoid, and triterpenoid biosynthesis. This systematic characterization of key enzyme-coding genes associated with the IA pathway and basic helix-loop-helix (bHLH) transcription factor family formed a network from the shikimate pathway to the indole alkaloid synthesis pathway in S. cusia. The high-quality S. cusia genome presented herein is an essential resource for the traditional Chinese medicine genomics studies and understanding the genetic underpinning of IA biosynthesis.
An efficient protein extraction method for twodimensional gel electrophoresis (2-DE) from plant samples is usually challenging due to the low protein content and high level of interfering compounds. Proteomic analyses of rice (Oryza sativa L.) roots are limited by the lack of an efficient protein extraction method. To establish an effective protocol of protein extraction suitable for 2-DE analysis in rice roots, we evaluated three protein extraction methods (trichloroacetic acid [TCA]/acetone, Mg/NP-40/ TCA, and tris-base/acetone). Our results showed that the Mg/NP-40/TCA extraction method had the highest protein yield and is the best resolution of protein separation among the three methods. The TCA/acetone method exhibited clear protein profiles and detected more protein spots with the highest intensity in the region of high Mr (above 45 kDa) than the other methods. However, this method was unable to detect proteins with low-Mr (less than 24.0 kDa). The Tris-base/acetone method showed the poorest resolution of protein separation. Our results suggest that the Mg/ NP-40/TCA method was the most effective among the three methods and may provide enhanced proteomic information for rice and other crop roots.
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