Elicitation of peroxidase activity in genetically transformed root cultures of Beta vulgaris L.
Sreedhar Reddampalli Venkataramareddy
Ravishankar Gokare Aswathanarayana
support: Senior and Junior Research Fellowship
from the Council of Scientific and Industrial
Keywords: Aspergillus, calcium, culture filtrate, dried cell powder, elicitor, glutathione, methyljasmonate, Rhizophus, thidiazuron.
Genetically transformed roots
of red beet produce copious levels of peroxidase (POD) - a multifunctional
enzyme with a number of commercial applications. In an effort to elicit
the POD activity, the cultures were treated with biotic elicitors
such as dry cell powders of microbial cultures (0.1-0.5% w/v) and
the respective culture filtrates (1-5% v/v). Similarly, abiotic elicitors,
particularly metal ions (2-8 folds of that present in the nutrient
medium), the plant hormone Thidiazuron (at 0.25-1 ppm) and other bio-molecules
such as Glutathione (at 0.5-
Plants are a rich source of various phytochemicals, proteins, enzymes and other secondary products of immense biotechnological applications. The secondary products and the respective enzymes, particularly those of phenylpropanoid pathway are significantly enhanced under the influence of elicitors. Peroxidase (POD) is one such enzyme associated with the plant defense pathway and is elicited when challenged with elicitors (Gómez-Vásquez et al. 2004; Perera and Jones, 2004). Since this enzyme also has various commercial applications, the present communication is focused on developing a strategy for eliciting POD enzyme that is associated with secondary metabolites of defence functions.
Peroxidase (E.C. 188.8.131.52) can oxidise various substrates due to its ability to perform a number of different types of reactions, such as peroxidative oxidation or oxidative and catalytic hydroxylation (Uyama and Kobayashi, 2003; Veitch, 2004). These reactions indicate a wide array of applications for peroxidases as a component of reagents for clinical diagnosis and various laboratory experiments. Although a large number of commercial applications of POD are possible, the high cost of presently available horseradish POD hinders such applications, which indicates the need for alternative sources of POD. We reported high levels of POD activity in cultured hairy roots of red beet (Thimmaraju et al. 2005) and the present study reports screening of a number of biotic and abiotic elicitors and the interaction of two complex elicitors leading to over-expression of POD activity.
A hairy root clone, established by infecting the cotyledonary leaf explants obtained from aseptic seedlings of the red beet variety “Ruby Queen” with A. rhizogenes strain LMG-150, was used throughout the present study. Induction of hairy roots and their maintenance conditions have been explained in an earlier communication (Thimmaraju et al. 2003). For testing growth performance, approximately 50 mg of root tips from hairy root clones were sub-cultured in 50 ml Erlenmeyer’s flasks containing 15 ml of MS basal liquid medium (Murashige and Skoog, 1962) with 3% sucrose and grown on a rotary shaker as described previously. The biomass accumulation and POD level were monitored at 5 day intervals. Fresh weight increase was recorded after removal of the spent medium by suction while the biomass was retained in a Buchner funnel.
elicitors. Based on earlier reports and the availability of cultures,
CF and DCP of various fungi, yeast and bacteria were used. The fungi
used were A. parasiticus, Aspergillus
and yeast. The fungal and yeast cultures were maintained as slants
in potato dextrose agar (PDA). This medium contained
Bacterial culture was maintained on agar slants with solid medium
containing proteose peptone (
cultures maintained on agar slants, were transferred to 100 ml of
the respective liquid medium in 250-ml flasks and incubated at room
temperature. The bacterial and yeast cultures were kept on a shaker
(90 rpm), while the fungal cultures were allowed to stand without
shaking. The cultures were harvested after they reached their stationary
phase (i.e., 3 weeks for fungal cultures, 72 hrs for yeast
culture and 48 hrs for bacterial culture). The flasks were autoclaved
and the fungal mycelial mat which floated at the surface of the medium
was carefully removed and washed multiple times with sterile distilled
water and allowed to dry in a hot air oven at
compounds and their concentrations were selected based on earlier
studies (Suresh et al. 2004). Thus Mej was used at 20, 40, 60, 80,
100 µM whereas GSH was used at different levels such as
Different abiotic elicitors used included metal ions such as calcium and magnesium, and an hormonal elicitor thidiazuron (TDZ). Metal ions were used at various levels such as 2, 4 and 8 folds of their respective concentrations in the normal MS medium. TDZ was used at 0.25 ppm, 0.50 ppm and 1.0 ppm in MS liquid medium.
milligrams of hairy roots had been grown in 15 ml Murashige and Skoog’s
(1962) liquid medium (MS) in an Erlenmeyer flask, incubated in dark
on a rotary shaker with 90 rpm at 25 ±
possible combined effects of the best complex microbial elicitor and
purified biotic elicitor GSH were studied by using the best fungal
elicitors such as 0.25% DCP of R. oligosporus, 5% CF of A.
parasiticus and 0.1% DCP of yeast C. versatilis with different
concentrations of GSH such as 0.5-
was extracted by homogenizing 50 mg fresh tissue of hairy roots, in
1 ml of sodium phosphate buffer (pH 6). After homogenization the homogenate
was centrifuged at 5000 xg twice and the supernatant was assayed for
POD activity by following the method of Wititsuwannakul et al. (1997).
Briefly, 1 ml assay mixture was prepared which consisted of 100 µl
The data presented is an average of at least six replicates from two individual experiments. The data was further analyzed for variance using Microsoft Excel 2000 version, followed by a Student t test (Sokal and Rolf, 1995).
Elicitors are compounds of biological or non-biological origin, which upon contact with plant cells, trigger defense-related compounds through over-expression of relevant enzymes (Singh, 1999;Gómez-Vásquez et al. 2004). It is well established that these defence-related responses are activated through a signal transduction pathway that includes the recognition of elicitors by receptors located in the plasma-membrane, activation of ion fluxes across the membrane, induction of down-stream functions such as oxidative bursts (free radical formation) and formation of secondary messengers (Radman et al. 2003). Due to such high specificities of actions, failure of one elicitor does not necessarily mean that the metabolic pathway cannot be triggered indicating that a large number of elicitors have to be screened for accomplishing effective elicitation process. Conversely, a combination of inappropriate medium and elicitor, as well as unsuitable concentration of the latter can result in ineffective elicitation. For example, the phenylpropanoid (PP) pathway was not induced in any cultures of Vanilla planifolia by yeast extract, whereas the same elicitor was used by the same group of workers to induce phytoalexin in cultures of Glycine max. The PP pathway could however be triggered in V. planifolia by using chitosan as an elicitor (Gómez-Vázquez et al. 2004). Thus, successful application of elicitation requires extensive screening. Most of the earlier studies on elicitation of peroxidase enzyme involved in vivo plants in response to pathogenic organisms (Gomez-Vazquez et al. 2004; Perera and Jones, 2004). However, a few studies focused on the elicitation of POD in in vitro cultures using mostly one elicitor (Agostini et al. 1997; Flocco et al. 1998; Flocco and Giulietti, 2003; Xu et al. 2004) but so far no report exists on elicitation of POD from red beet hairy roots.
The results of the present study show (Table 1) that among the fungal elicitors, the majority of the treatments resulted in elicited levels of enzyme activities. The highest were those with DCPs of both R. oligosporus (added on 15th day) and P. notatum (added on 20th day) causing enhancements to the tune of 3-fold higher activity than the control. High levels (5% v/v) of culture filtrates of A. parasiticus and P. notatum (2.5-5% v/v) enhanced POD activity by nearly 3-fold. However, a low level of (1%) culture filtrate of R oligosporus was needed to cause similar levels of elicitation in a short period of 5 days.
The dry cell powders of yeast C. versatilis elicited the activity of POD up to 3.5-fold at very low concentration whereas the culture filtrates suppressed the turn over of enzyme activity (Table 2). Dry cell powder of L. helveticus elicited nearly 3-fold when added on 15th day, only at a low concentration of 0.10%. Higher levels of yeast DCP were however inhibitory in a dose-dependent manner (Table 2). Similar to most of the earlier studies where fungal elicitors have been found feasible for elicitation, the results of present work also indicated that the biotic elicitors caused elicitation of POD. An important observation made from the present screening study was that the concentration and time of elicitor contact were very critical factors for efficient elicitation of POD as observed in the elicitation of secondary metabolites in other species (Singh, 1999). CF of C. versatilis (Table 2) and L. helviticus did not cause elicitation of POD at any of the levels used. Instead there was a concentration-dependent suppression of POD, therefore, the results of the present study could indicate that the receptors for elicitation in this case are responsive for high molecular weight cell wall polysaccharides that are present in DCP rather than in CF.
horseradish hairy roots, significant enhancement of POD (100%) occurred
upon treatment of cultures with certain metal ions such as AgNO3,
CuSO4 and fungal extracts such as Verticillium sp.,
Monodyctis cataneae and Aspergillus
the pure compounds (Table 3), GSH used at 5 concentrations
at three different treatment time caused highest elicitation at lowest
TDZ caused the highest elicitation of POD activity at 1 ppm whereas a low level of 0.25 ppm also caused a similar elicitation when added at a later stage i.e., on the 20th day. Conclusively, the present study has established that among the abiotic elicitors only GSH has significantly enhanced POD activity and more so in the presence of DCP of R. oligosporus (as explained below).
various combinations of the three elicitors such as GSH, CF of A.
parasiticus, DCPs of R. oligosporus, and C. versatilis,
only the combination of
Although many researchers have studied elicitation in various systems and recorded the suppression of biomass in elicitor treatment, no attempt had been made to check the possibility of effectively using an elicitor at the late exponential growth phase. For pigment we observed that the addition of elicitor at late exponential phase could enhance the overall productivity (Savitha et al. 2006). The activity of POD was generally high at the early exponential phase (Thimmaraju et al. 2005) and therefore, maximum elicitation also occurred at treatments on the 15th and 18th days rather than on 20th day. Therefore, by judiciously selecting and timing the addition of an elicitor, there is a possibility of enhancing the production of both POD and betalain in the same process, in which case the process of online recovery of pigment developed earlier by us (Thimmaraju et al. 2004) could further be extended for the online recovery of POD, as proposed by Agostini et al. (1997) for turnip hairy roots.
Encouragement by Dr. V. Prakash, Director, CFTRI for research activities is gratefully acknowledged.
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