CONFERENCE PRESENTATIONS:
Chapter 4: Evaluation of mycorrhiza as an elicitor for rosmarinic acid in a co-culture system with hairy roots of
4.2.5 Study on Rhizophagus irregularis isolate 1 as an elicitor .1 Experimental set up .1 Experimental set up
Growth kinetics studies were performed to study the effect of mycorrhiza on the growth and metabolite content of the selected hairy root lines and its role as an elicitor. Rhizophagus irregularis isolate 1 colonized roots of HR 2, HR 4 and HR 5 were used for this study. Method described herein was for HR 2 - Rhizophagus
158 irregularis isolate 1 and similar steps were repeated for the other two root lines. M media plates (150 mm, Corning incorporate, Corning, U.S.A.) having 135 mL media in each were used for this study. To each Petri plate, a 3 cm2 square shaped segment having mycorrhizal structures and six hairy root tips (each approximately 3 cm in length) were inoculated to the centre of each Petri plate. Control (non-mycorrhized) hairy root lines) were also prepared as above. Petri plates were then incubated in the dark at 26 °C in an incubator (ET-650-8, Lovibond, Dortmund, Germany) for three time periods (30 d, 60 d and 90 d). The plates were monitored weekly for growth of symbionts and any contamination. For each time period five replicates were prepared.
4.2.5.2 Effect of mycorrhization on root growth and biomass
To study the effect of Rhizophagus irregularis isolate 1 on root growth and its biomass, the roots were harvested after 30 d, 60 d and 90 d in citrate buffer as described in the section 4.2.3. After each time period the freshly harvested control and colonized root lines were scanned for their root diameter, length and number of root tips using image analysis software (WinRHIZO® version Pro2007; Regent Instruments Inc, Quebec, Canada) and a scanner (EPSON Perfection V 700, Delhi, India). The characterized roots were then lyophilized (Labconco lyophilizer, Kansas City, U.S.A.) and their dry weight were recorded.
4.2.5.3 Extraction of roots for total phenolics, caffeic and rosmarinic acids
Total phenolics and the concentration of caffeic and rosmarinic acids were determined in extracts prepared by homogenizing 50 mg of the lyophilized root in
159 500 μL of 60 % (v/v) ethanol in water (AR grade, Merck, Mumbai, India). To the homogenized mixture, 14.5 mL of 60 % (v/v) ethanol in water was added and the mixture was sonicated (B3510E-DTH, Branson Ultrasonics, Danbury, U.S.A.) at 25
°C for 10 min. The sonicated extract was then centrifuged (HeraeusTM BiofugeTM StratosTM Centrifuge, Buckinghamshire, England) at 10,000 rpm for 5 min and the supernatant was collected in a 25 mL volumetric flask (Borosil, New Delhi, India) and the left over residue was re-extracted in 10 mL of 60 % (v/v) ethanol in water for five min and then centrifuged as previously described. The supernatant was then pooled in volumetric flasks that contained the first fraction and the final volume was made up to 25 mL with 60 % (v/v) ethanol in water. The extracts were then filtered and used directly for total phenolics, caffeic and rosmarinic acids quantification.
4.2.5.4 Effect of mycorrhization on total phenolics
The Folin-Ciocalteau colorimetric assay (Singleton and Rossi 1965) was used for the quantification of total phenolics and to study the effect of mycorrhization on the total phenolic content in comparison to control hairy root lines. Briefly, to 1 mL of the methanolic extract, 4 mL of distilled water, 2.5 mL of Folin-Ciocalteau reagent (SRL, Ranbaxy, Delhi, India), 1.25 mL of 2.1 % aqueous sodium carbonate (Qualigens, Mumbai, India) was added and incubated in the dark for 30 min and absorbance of the resulting mixture was taken at 735 nm using UV-Vis Spectrophotometer (UV-Vis 2450, Shimadzu, Kyoto, Japan) against the same mixture without sample. The total phenolic concentration was quantified from the standard curve prepared for gallic acid within the range of 20 - 100 mg/ L and the
160 final concentration of total phenolics in the sample was reported as gallic acid equivalents (GAE mg/ g DW).
4.2.5.5 Effect of mycorrhization on individual polyphenolics: HPLC analysis HPLC analysis was performed on root extracts of the control and mycorrhized hairy root lines for the quantification of RA and CA. HPLC (CBM-20A, Shimadzu, Kyoto, Japan), equipped with a quaternary pump (LC - 20AT), solvent degasser system (DGU - 20 A5), autosampler (SIL – 20A) and diode array detector (SPDM – 20A) was used. Inbuilt software (Shimadzu, LC solution) was used to control the HPLC pump and acquire data from the diode array detector. Separations were performed on a C18 Phenomenex column (Gemini-NX 250 mm × 4.6 mm × 5 μm).
Separation of the individual polyphenolics was performed using HPLC grade water + 0.1 % (v/v) ortho Phosphoric Acid (OPA) in water (Mobile phase A) and Methanol (HPLC grade, Merck, Mumbai, India) + 0.1 % (v/v) OPA in methanol (Mobile phase B) as mobile phase. A gradient program was developed for CA and RA quantification: 0 - 2 min isocratic 0 % B, 2 - 5 min linear gradient to 40 % B, 5 - 10 min a linear gradient to 50 % B, from 10 - 18 min isocratically maintained at 50 % B and 18 - 23 min a decreasing gradient from 50 % to 40 % and finally from 23 - 25 minute 0 % B for column washing. The flow rate of the mobile phase was 1.0 mL/
min and the wavelength used for detection of all three acids was 280 nm with an injection volume of 20 μL. Unknown samples were identified by comparison of the retention times with those of the commercial standard of RA and CA (Sigma, Bangalore, India). Quantification of the samples was determined by comparison of
161 integrated peak area for each sample with a standard calibration curve prepared for RA and CA over a concentration range from 20 -100 mg/ L.
4.2.5.6 Effect of mycorrhization on activity of Phenylalanine ammonia-lyase (PAL) activity
4.2.5.6.1 Extraction of Phenylalanine ammonia-lyase (PAL) and estimation of protein content
Enzyme extraction was performed from the control and mycorrhized root samples of HR 2, 4 and 5 after 30 d, 60 d and 90 d. Fresh roots were extracted in 100 mM sodium borate buffer (pH 8.8) containing 2 mM EDTA (Sigma, Bangalore, India), 4 mM dithiothreitol (Sigma, Bangalore, India) and 2 % (w/w) polyvinylpyrrolidone (Sigma, Bangalore, India). For enzyme extraction, 200 mg of fresh root sample was ground in the extraction buffer for 5 min with a mortar and pestle on ice, followed by centrifugation at 13,000 rpm (HeraeusTM BiofugeTM StratosTM Centrifuge, Buckinghamshire, England), 4 °C for 25 min to obtain a solid-free extract (Yan et al. 2006). The extract was used for protein quantification using Bradford reagent (Sigma, Bangalore, India). Protein content was quantified using bovine albumin (Serva Electrophoresis GmbH, Hiedelberg, Germany) as a standard in the concentration of 20 μg/ mL to 100 μg/ mL.
4.2.5.6.2 Estimation of PAL activity
The reaction mixture (3 mL) for PAL activity assessment contained 500 μL of enzyme extract, 1.9 mL of Milli Q water, 300 μL of 300 μM sodium borate buffer and 300 μL of 30 μM L-phenylalanine. The reaction mixture was then incubated for
162 2 h at 40 °C. The enzymatic activity was measured by recording absorbance at 290 nm using a spectrophotometer (UV-Vis 2450, Shimadzu, Kyoto, Japan) against the same volume of the reaction mixture without enzyme extract. The enzymatic activity was expressed in terms of μmol of trans-cinnamic acid produced min−1 mg -1 of protein from L-phenylalanine due to enzymatic activity. Standards of cinnamic acid (Sigma, Bangalore, India) were prepared at a range of molarities from 1 × 10-5 M to 5 × 10-5 M.
4.2.6 Statistical analysis
All data presented in this study is expressed in terms of mean ± SEM. Raw data was analyzed using a commercial statistical package (GraphPad Prism 6). One way analysis of variance with a Tukey’s HSD test of significance at p ≤ 0.05 was used to determine the difference between the different hairy root lines for colonization percentage for the three different mycorrhiza used in the study. One way analysis was also performed to determine the difference between the three ages of co-cultures set for the elicitation study for parameters such as root morphology, biomass, total phenol and individual polyphenolics. Two way analysis of variance (ANOVA) was performed to determine difference between the control and mycorrhized hairy root lines for root morphology parameters, biomass, total phenol, individual polyphenolic and enzymatic activity. Correlation between rosmarinic acid and enzymatic activity was also tested using the Pearson test at a significance level of 0.05.
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4.3 Results
4.3.1. Development of hairy root-mycorrhiza co-culture system and colonization