The phenylalanine ammonia-lyase gene family in Isatis indigotica Fort.: molecular cloning, characterization, and expression analysis.

Phenolic compounds, metabolites of the phenylpropanoid pathway, play an important role in the growth and environmental adaptation of many plants. Phenylalanine ammonia-lyase (PAL) is the first key enzyme of the phenylpropanoid pathway. The present study was designed to investigate whether there is a multi-gene family in I. Indigotic and, if so, to characterize their properties. We conducted a comprehensive survey on the transcription profiling database by using tBLASTn analysis. Several bioinformatics methods were employed to perform the prediction of composition and physicochemical characters. The expression levels of IiPAL genes in various tissues of I. indigotica with stress treatment were examined by quantitative real-time PCR. Protoplast transient transformation was used to observe the locations of IiPALs. IiPALs were functionally characterized by expression with pET-32a vector in Escherichia colis strain BL21 (DE3). Integration of transcripts and metabolite accumulations was used to reveal the relation between IiPALs and target compounds. An new gene (IiPAL2) was identified and both IiPALs had the conserved enzymatic active site Ala-Ser-Gly and were classified as members of dicotyledon. IiPAL1 and IiPAL2 were expressed in roots, stems, leaves, and flowers, with the highest expression levels of IiPAL1 and IiPAL2 being observed in stems and roots, respectively. The two genes responded to the exogenous elicitor in different manners. Subcellular localization experiment showed that both IiPALs were localized in the cytosol. The recombinant proteins were shown to catalyze the conversion of L-Phe to trans-cinnamic acid. Correlation analysis indicated that IiPAL1 was more close to the biosynthesis of secondary metabolites than IiPAL2. In conclusion, the present study provides a basis for the elucidation of the role of IiPALs genes in the biosynthesis of phenolic compounds, which will help further metabolic engineering to improve the accumulation of bioactive components in I. indigotica.

[Cloning and expression analysis of a hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferasegene(IiHCT) from Isatis indigotica].

Based on the transcriptome data, we cloned the open reading frame of IiHCT gene from Isatis indigotica, and then performed bioinformatic analysis of the sequence. Further, we detected expression pattern in specific organs and hairy roots treated methyl jasmonate( MeJA) by RT-PCR. The IiHCT gene contains a 1 290 bp open reading frame( ORF) encoding a polypeptide of 430 amino acids. The predicted isoelectric point( pI) was 5.7, a calculated molecular weight was about 47.68 kDa. IiHCT was mainly expressed in stem and undetectable in young root, leaf and flower bud. After the treatment of MeJA, the relative expression level of IiHCT increased rapidly. The expression level of IiHCT was the highest at 4 h and maintained two fold to control during 24 h. In this study, cloning of IiHCT laid the foundation for illustrating the biosynthesis mechanism of phenylpropanoids in I. indigotica.

[Effects of different K fertilizer and water level on growth and physiological characteristics of Isatis indigotica].

The experiment included three potassium levels (K0 0 g x kg(-1), K1 0.33 g x kg(-1), K2 0.67 g x kg(-1)) and two water gradients (well watered and drought stress), then measured growth indicators, SOD, POD, CAT activities and concents of osmotic regulation substances. To explore the effects of K fertilizer and water on growth and physiological characteristics of Isatis indigotica, providing reference for improving drought resistance of I. indigotica. The result showed drought stress inhibited the growth and decreased the biomass of I. indigotica but K fertilizer can alleviate the drought stress. Compared with K0 treatment, K1, K2 treatment increased the biomass of overground part of by 89. 13% ,60. 87% under drought stress. The corresponding increase in soluble sugar content was 16.67%, 5.00%, and in proline content was 42.41%, 65.62%, respectively. SOD,POD and CAT activities was significantly improved in K1, K2 treatment in comparison with K0 treatment under drought stress, but soluble protein content significantly reduced. The conclusion is that appropriate amount of K fertilizer can increase the activities of antioxidase and the content of osmoregulation substance under drought stress, and improve drought resistance of I. indigotica.

Isolation and characterization of a gene encoding cinnamoyl-CoA reductase from Isatis indigotica Fort.

A novel cinnamoyl-CoA reductase gene, designated as Iiccr (GenBank Accession No. GQ872418) was cloned from Isatis indigotica Fort. The full-length cDNA of Iiccr was 1368 bp with an ORF of 1026 bp that putatively encoded a polypeptide of 341 amino acids, with a predicted molecular mass of 37.50 kDa. The deduced amino acid sequence of IiCCR shared high homology with other known CCRs. No intron was detected in the genomic sequence of Iiccr. Southern-blot analysis revealed that Iiccr was a high-copy gene and real-time quantitative PCR analysis indicated that Iiccr was constitutively expressed in roots, stems and leaves of I. indigotica, with the highest expression level in roots. The results from treatment experiments using different signaling components for plant defense responses including methyl jasmonate (MeJA), gibberellins (GA(3)), abscisic acid (ABA) and ultraviolet-B revealed that expression of IiCCR had a prominent diversity. The full-length of ORF was sub-cloned into prokaryotic expression vector pET32a(+), which was then transferred into E. coli BL21(DE3). The recombinant protein had high expression level in E. coli BL21(DE3) with IPTG induction. A 2.6 kb long promoter sequence was isolated and its putative regulatory elements and potential specific transcription factor binding sites were analyzed. This study will enable us to further understand the role of IiCCR in the synthesis of phenylpropanoid compounds in I. indigotica Fort. at the molecular level.

IiSDD1, a gene responsive to autopolyploidy and environmental factors in Isatis indigotica.

In plants, stomata play a pivotal role in the regulation of gas exchange and are distributed throughout the aerial epidermis. SDD1, a gene isolated from Arabidopsis thaliana has been demonstrated to specialize in stomatal density and distribution. In our present study, a comprehensive survey of global gene expression performed by using an A. thaliana whole genome Affymetrix gene chip revealed SDD1 tends to be significantly lower in tetraploid Isatis indigotica than in diploid ones. To intensively investigate different SDD1 expression in response to polyploidy, a full-length cDNA clone (IiSDD1) encoding SDD1 was isolated from the traditional Chinese medicinal herb I. indigotica cDNA library. IiSDD1 shared a high level of identity with that from A. thaliana, containing some basic features of subtilases: D, H and S regions, as well as a substrate-binding site. Real-time quantitative PCR analysis indicated that IiSDD1 was constitutively expressed in all tested tissues, including roots, stems and leaves, both in tetraploid and diploid I. indigotica, and with the highest expression in leaves. In addition, IiSDD1 was also found to be down-regulated by signalling molecules for plant defence responses, such as abscisic acid (100 microM) and gibberellin (100 mg/L), as well as by environmental stresses including salt, darkness, coldness and drought. Our study, for the first time, indicates SDD1 participates not only in the defense/stress responsive pathways, but also probably involves in plants polyploidy evolution.

[Effects of NH4(+)-N/NO3(-)-N ratio in applied supplementary fertilizer on nitrogen metabolism, photosynthesis and growth of Isatis indigotica].

OBJECTIVE: To study the effect of NH4(+)-N/NO3(-)-N ratios in the applied supplementary fertilizer on the growth, nitrogen metabolis related enzymes activity and photosynthetic characteristics of Isatis indigotica. METHOD: The sand culture experiment was conducted, and seedling of I. indigotica was fertilized with the mixed nutrition that containing the Hoagland's macro elements and the Aron's micro elements, the additional 63 mmol N was supplementary with the NH4(+)-N/NO3(-)-N ratio of 100:0, 75:25, 50:50, 25:75 and 0:100. RESULT: The biomass of I. indigotica increased at first when the supplementary N of NH4(+)-N/NO3(-)-N ratio changed from 100:0 to 50:50 and decreased afterwards. The maximum value was at 50:50 and the minimum at 100: 0. With increasing the ratio of NO3(-)-N, the activity of nitrate reductase and glutamine synthetase increased and then decreased and the relationship between the activity and the ratio could be described with an approximate parabola curve. The net photosynthetic rate of I. indigotica was the highest at the NH4(+)-N/NO3(-)-N ratio of 75:25 and the lowest at 100:0. CONCLUSION: Increasing the NO3(-)-N ratio properly was beneficial to promote the growth and improve the activity of nitrate reductase and glutamine synthetase and net photosynthetic rate of I. indigotica.

Alpha-tryptophan synthase of Isatis tinctoria: gene cloning and expression.

Indole producing reaction is a crux in the regulation of metabolite flow through the pathways and the coordination of primary and secondary product biosynthesis in plants. Indole is yielded transiently from indole-3-glycerol phosphate and immediately condensed with serine to give tryptophan, by the enzyme tryptophan synthase (TS). There is evidence that plant TS, like the bacterial complex, functions as an alpha beta heteromer. In few species, e.g. maize, are known enzymes, related with the TS alpha-subunit (TSA), able to catalyse reaction producing indole, which is free to enter the secondary metabolite pathways. In this contest, we searched for TSA and TSA related genes in Isatis tinctoria, a species producing the natural blue dye indigo. The It-TSA cDNA and the full-length exons/introns genomic region were isolated. The phylogenetic analysis indicates that It-TSA is more closely related to Arabidopsis thaliana At-T14E10.210 TSA (95.7% identity at the amino acid level) with respect to A. thaliana At-T10P11.11 TSA1-like (63%), Zea mays indole-3-glycerol phosphate lyase (54%), Z. mays TSA (53%), and Z. mays indole synthase (50%). The It-TSA cDNA was also able to complement an Escherichia coli trpA mutant. To examine the involvement of It-TSA in the biosynthesis of secondary metabolism compounds, It-TSA expression was tested in seedling grown under different light conditions. Semi-quantitative RT-PCR showed an increase in the steady-state level of It-TSA mRNA, paralleled by an increase of indigo and its precursor isatan B. Our results appear to indicate an involvement for It-TSA in indigo precursor synthesis and/or tryptophan biosynthesis.

[Primary effects on Isatis indigotica after spaceflight].

Isatis indigotica carried by the Chinese first spaceship "Shenzhou" was studied in order to find the mutation after spaceflight. TLC differentiation experiments showed no distinct discrepancy among the samples of spaceflight and non-space-flight, and the same color spot appeared corresponding to the location of the arginine. Isoenzymes of esterase and peroxidase were studied with PAGE. Isoenzymes of esterase were difference among the samples. To peroxidases, little difference was found with them. The ratio of dry weight and extract contents showed out the mutation has emerged after spaceflight, but some characters were unstable. It is necessary for further study.

Molecular cloning and characterization of a novel calcium-dependent protein kinase gene IiCPK2 Responsive to polyploidy from tetraploid Isatis indigotica.

A novel calcium-dependent protein kinase gene (designated as IiCPK2) was cloned from tetraploid Isatis indigotica. The full-length cDNA of IiCPK2 was 2585 bp long with an open reading frame (ORF) of 1878 bp encoding a polypeptide of 625 amino acid residues. The predicted IiCPK2 polypeptide included three domains: a kinase domain, a junction domain (or autoinhibitory region), and a C-terminal calmodulin-like domain (or calcium-binding domain), which presented a typical structure of plant CDPKs. Further analysis of IiCPK2 genomic DNA revealed that it contained 7 exons, 6 introns and the length of most exons was highly conserved. Semi-quantitative RTPCR revealed that the expression of IiCPK2 in root, stem and leaf were much higher in tetraploid sample than that in diploid progenitor. Further expression analysis revealed that gibberellin (GA3), NaCl and cold treatments could upregulate the IiCPK2 transcription. All our findings suggest that IiCPK2 might participate in the cold, high salinity and GA3 responsive pathways.