Lokhandwala and Bora

so terminating the chain reaction of lipid peroxidation)

β- carotene and ten times as higher than α-tocopherol.

or by any of the combination from the above (Niwa et

As, Lycopene is associated with red colour of tomato, its

al., 2001). Antioxidants can be classified into three main

studies have been focused in fruits only. (Karakaya and

types: first line defense antioxidants, second line defense

Yilmaz, 2007)

antioxidants and third line defense antioxidants. Oxidases

Considering the above facts; present study was conducted

and Peroxidases come under first line of defense and

to explicate the presence of Lycopene at various vegetative

carotenoids come under second line of defense.

stages along with two antioxidant enzymes to know

Polyphenol Oxidases (catechol oxidase; E.C. 1.10.3.2)

which equivalent (enzymatic or non-enzymatic) can be

are widely distributed and well-studied oxidative

the share of anti-oxidant network at different stages of

enzymes and their effects on discoloration in damaged

growth studied. Present study also aimed in optimizing

and diseased plant tissues have been known for many

the physical conditions at seedling stage for partially

years. Polyphenol Oxidases (PPOs) are copper containing

purified PPOs and CAT.

ubiquitous enzymes which utilize molecular oxygen to

oxidize common ortho-diphenolic compounds such as

Materials and Methods

catechol to their respective quinones. Quinones generated

by PPOs are highly reactive and may cross-link or

Plant material

alkylate proteins which lead to brown pigment formation

Seeds of Lycopersicon esculentum Mill were bought from

in damaged plant tissues. Based on these browning

Seedco Company, Jalna, India. Seeds were stored at 4 o C

reactions, PPOs have been suggested to play a defensive

until used.

role in plants (Constabel and Barbehenn, 2008).

CAT (EC 1.11.1.6) is an iron containing enzyme. CATs

Seed germination

are most notably distinguished from the other enzymes in

Seeds of Lycopersicon esculentum Mill were germinated

not requiring a reductant as they catalyze a dismutation

in autoclaved sand containing 0.5X MS salts (Murashige

reaction and readily degrade hydrogen peroxide.

and Skoog, 1962). Seeds were sown in medium sized

Oxidative stress is cumulative biological effect of oxygen

plastic cups; 0.5 inch deep inside autoclaved sand (10

radicals on plants. To minimize its effect plants have

seeds per pot). These pots were kept in dark until they

evolve various enzymatic defenses, CAT activity is one

started germinating; afterwards pots were transferred to

of them. It has also been reported that CAT mutant plants

natural conditions (16hr/8hr light and dark).

show a decrease in Pathogenesis Related (PR) proteins;

which indirectly correlate role of CAT in plant defense

Preparation of Homogenate for Enzyme Assay

mechanism. (Sanchez-Casas and Klessig, 1994; Chen et

Homogenate for PPO and CAT enzyme extraction was

al., 1995)

prepared according to Christopher et al., (2010). For this,

Tomato is one of the most important horticulture crops

1 gm of root sample was crushed with 2 ml of 0.1 M

in India. According to Indian Agricultural Research

sodium citrate buffer (pH 5.0) at 4°C. The homogenate

Databook (2012); Area coverage of tomato has been

was centrifuged for 2.0 minutes at 10000 rpm. The

increased from 5, 99,000 HA to 8, 65,000 HA in four

supernatant obtained was used for enzyme assay. Enzyme

years with 32.53% increase in production and 2.6%

homogenates were stored in deep freezer (-70°C) until

increase in productivity (Joshi et al., 2012). Studies on

utilized for biochemical analysis.

tomato have been extensively done in regard to Lycopene.

Lycopene is a pigment principally responsible for deep

Ammonium sulphate fractionation and dialysis

red colour of tomato. It has attracted attention because of

PPO’s and CAT’s crude homogenate were subjected to

its biochemical and physiological significance as natural

0-80% (w/v) saturation with ammonium sulphate at cold

antioxidant. Although it has no provitamin A activity, it

conditions. The saturated solutions were left overnight

has a physical quenching rate constant double to that of

at 4 o C and the precipitated proteins were separated

410