Effects of 2-aminoindan-2-phosphonic acid treatment on the accumulation of salidroside and four phenylethanoid glycosides in suspension cell culture of Cistanche deserticola.

2-Aminoindan-2-phosphonic acid (AIP), a specific competitive phenylalanine ammonia lyase (PAL) inhibitor was applied to a suspension cell culture of Cistanche deserticola. The effects of AIP treatment on cell growth, PAL activity, contents and yields of total phenolic compound, salidroside and four phenylethanoid glycosides (PheGs) are investigated. The results demonstrated that, 0.5 and 2.0 µM AIP treatments had similar effects on the measurements investigated in this study. AIP treatment resulted in significant decreases in PAL activity, total phenolic compounds content, and PheGs content. Linear regression analysis showed that PAL activity had a high correlation coefficient with the total phenolic compound content and the four PheGs contents. Total PAL activity-time area under curve (AUC) had a high correlation coefficient with the total phenolic compound yield and the yields of five tested compounds in untreated cell samples. In AIP-treated cells, total PAL activity-time AUC retained a high correlation with the total phenolic compound yield and the yields of three tested compounds, echinacoside, acteoside, and tubuloside A, but not salidroside and cistanoside A. The difference could be caused by the different biosynthetic origins of each of the tested compounds. These results demonstrate the important role of PAL in the biosynthesis of PheGs in the suspension cell culture of C. deserticola.

Molecular characterization of phenylalanine ammonia lyase gene from Cistanche deserticola.

We cloned the gene, CdPAL1, from Cistanche deserticola callus using RACE PCR with degenerate primers that were designed based on a multiple sequence alignment of known PAL genes from other plant species. The gene shows high homology to other known PAL genes registered in GenBank. The recombinant protein exhibited Michaelis-Menten kinetics with a Km of 0.1013 mM, Vmax of 4.858 µmol min(-1), Kcat of 3.36 S(-1), and Kcat/Km is 33,168 M(-1) S(-1). The enzyme had an optimal pH of 8.5 and an activation energy of 38.92 kJ mol(-1) when L-Phenylalanine was used as a substrate; L-tyrosine cannot be used as substrate for this protein. The optimal temperature was 55°C, and the thermal stability results showed that, after a treatment at 70°C for 20 min, the protein retained 87% activity, while a treatment at 75°C for 20 min resulted in a loss of over 85% of the enzyme activity. Treatment with heavy metal ions (Hg2+, Pb2+, and Zn2+) showed remarkable inhibitory effects. Among the intermediates from the lignin (cinnamyl alcohol, cinnamyl aldehyde, coniferyl aldehyde, coniferyl alcohol), phenylpropanoid (cinnamic acid, coumaric acid, caffeic acid, and chlorogenic acid) and phenylethanoid (tyrosol and salidroside) biosynthetic pathways, only cinnamic acid showed strong inhibitory effects against CdPAL1 activity with a Ki of 8 µM. Competitive inhibitor AIP exhibited potent inhibition with Ki=0.056 µM.

[Effects of methyl jasmonate and salicylic acid on phenylethanoid glycosides synthesis in suspension cultures of Cistanche deserticola].

The present study investigated the influence of the methyl jasmonate and salicylic acid elicitors on the formation of phenylethanoid glycosides (PeG) in the suspension cultures of Cistanche deserticola. The results showed that methyl jasmonate and salicylic acid enhanced greatly the accumulation of PeG and echinacoside (Echin), but their optimum elicitation dosage and addition time were different. The yields of PeG and Echin were significantly increased in the presence of 5 micromol/L methyl jasmonate on day 14 (up to 2.59-fold and 3.82-fold, respectively), whereas treated with 50 micromol/L salicylic acid on day 28, the maximum content of them were, respectively, 2.71 and 3.16-fold higher than the untreated cell cultures.

Formation of phenylethanoid glycosides by Cistanche deserticola callus grown on solid media.

The optimal growth of Cistanche deserticola callus and formation of phenylethanoid glycosides (PeG) was at 25 degrees C with light irradiation intensity of 24 micromol m(-2) s(-1) on solidified B5 media supplemented with 0.5 mg 6-benzylaminopurine l(-1), 10 mg gibberellin l(-1), 800 mg casein hydrolysate l(-1) and 20 g sucrose l(-1). After 30 d culture, the biomass reached 15.5 g dry wt callus l(-1) medium and its PEG content was 10.7% (w/w). The PeG content was 42%-127% higher than those in explants.

Effects of rare earth elements on the growth of Cistanche deserticola cells and the production of phenylethanoid glycosides.

The rare earth elements Nd, La, Ce at proper concentrations had positive effects on the cell growth of Cistanche deserticola and production of phenylethanoid glycosides (PeG). A mixture of rare earth elements (MRE, La(2)O(3):CeO(2):Pr(6)O(11):Sm(2)O(3)=255:175:3:1, mol/mol) showed the most remarkable effects. After 30 day's culture, 0.02 mmoll(-1) MRE gave the highest content (20.8%) and production (1.6 gl(-1)) of PeG, which were 104 and 167% higher than those obtained in control (without rare earth elements).