Oxidising Enzymes, Phenolic Compounds, and Antioxidant Enzymes

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C-II.3 OXIDISING ENZYME AND PHENOLIC COMPOUNDS

C-II.3.1 IAA OXIDASE

Table C-II.3.1a and Figure C-II.3.1 represents the IAA activity in terms of IAA reduced

per gram fresh flower petals in cut Tithonia rotundifolia Blake. IAAOxidase (IAAO) and peroxidase decompose the IAA. It was observed that activity of IAA oxidase reduced till 48 hour of developing stage in shelf life of Tithonia rotundifolia Blake flower. While in senescent stage it increased. Decreased levels of auxins as a result of higher activity of IAA oxidase must have hastened the senescent changes in

the flower petals. The content of IAA-Oxidase was found to be significantly different

among all the stages during statical analysis (Table C-II.3.1b).

Table C-II.3.1a: IAA Oxidase activity (in mg / g fresh petals) in cut flower petals

Stages

Days

IAA Oxidase (mg / g)

1

1

0.71 ± 0.01

2

2

0.59 ± 0.13

3

3

0.47 ± 0.00

4

4

0.37 ± 0.06

Fig C-II.3.1: The IAA Oxidase activity in cut flower petals

Table C-II.3.1b: ANOVA Summary Table for IAA Oxidase activity in cut flower Petals

Source of Variation

Sum of Squares

(SS)

Degree of Freedom

(DF)

Mean Squares

(MS)

F

Ratio

Table value of

F

Between groups

0.201

3

0.067

75.422

4.07*

Within groups

0.007

8

0.001

Total

0.208

11

* at 0.05 level of significance

The activity was found to have decreasing trend till stage 3 that indicating less destruction of IAA at this stage but again at stage 4 the activity was found to increase which suggesting lowered levels of IAA, peroxidase activity was found to have lowered. Consequently this high level enzyme activity were visible only at senescence stage (stage 4) resulting into senescence of petals. Gilbert and Sink (1971) observed that the senescence of Pointsettia flower was delayed by auxin due to production of peroxidase (POD) and inhibition of IAA oxidase and hydrogen peroxide during aging. Mayak and Halevy (1980) reported that the activity of IAA oxidase increased with aging. Similar results were found by Jani (2010) with pink varieties of Cosmos bipinnatus L. according to Barendse (1980) IAA Oxidase activity increased in the aging tuber discs. Wulster et al., (1982b) while working with carnation reported that IAA was found to stimulate ethylene production and consequently senescence and ethylene production was dependent on IAA concentration. IAA Oxidase activity responsible for the biosynthesis of ethylene at last step (Vioque et al., 1983). Due to inhibition of IAA oxidase levels of ABA might have started increasing simultaneously that lead to the process of senescence. LePage-Degivry et al., (1991) reported that ABA levels increase during the senescence of some flowers. The ABA content of both rose and carnation petals were found to increase only late in the senescence process (Mayak and Halevy, 1972). The ABA content of both rose and carnation petals were found to increase only late in the senescence process (Mayak and Halevy, 1972).

C-11.3.2 TOTAL PHENOLS

Table C-II.3.2a and Figure C-II.3.2 represents the amount of total phenols in mg per gram dry flower petals of Tithonia rotundifolia Blake. It was observed that total phenol content decreased till stage 3 followed by the increase in senescent stage i.e. stage 4. However, Table C-11.3.2b shows that the total phenols in different stages were significantly different.

Table C-II.3.2a: Total Phenols (in mg / g dry petals) in cut flower petals

Stages

Days

Total Phenol (mg / g)

1

1

9.43 ± 0.6

2

2

7.46 ± 0.05

3

3

6.82 ± 0.2

4

4

7.25 ± 0.3

Fig C-II.3.2: The Total Phenols in cut flower petals

Table C-II.3.2b: ANOVA Summary Table for Total Phenols in cut flower petals

Source of Variation

Sum of Squares

(SS)

Degree of Freedom

(DF)

Mean Squares

(MS)

F

Ratio

Table value of

F

Between groups

12.069

3

4.023

285.647

4.07*

Within groups

0.011

8

0.001

Total

12.080

11

* at 0.05 level of significance

This decreasing trend possibly because of after detachment from the mother plant, flower survives under stress condition. Vidhya Sankar (2001) and Bhattacharjee (2003) also observed that total phenols in 'Raktagandha' cut flower petals tended to decrease during flower senescence. However, the total phenols at the senescent stage were found to increase. Phenolic compounds serve in plant defense mechanisms to counteract reactive oxygen species (ROS) in order to survive and prevent molecular damage and damage by microorganisms, insects and herbivores (Vaya et al., 1997). Thus, in the present case the increasing phenolic contents indicate that possibly the flower was trying to protect against the free radicals and ROS being generated. The decrease in the POD activity (Table C-II.4.1a) during this phase also supports this report. It also reported by Paull et al., (1985) that the tissue phenols increase during senescence.

Increase in the level of total phenol suggest that possibly at this stage the accumulation of free radicals and ROS was high with much low POD activity favouring the situation. Similar finding was reported by Vidhya Sankar (2001) and Bhattacharjee (2003).

C-11.4 ANTIOXIDANT ENZYMES

C-11.4.1 PEROXIDASE

Table C-II.4.1a and Figure C-II.4.1 shows the peroxidase (POD) activity in terms of ΔO.D./min/gram fresh petals of cut Tithonia rotundifolia Blake flowers. Peroxidase is an antioxidant enzyme. It is known to protect the flower against oxidative stress injury by scavenging the free radicals and ROS. Singlet oxygen, superoxide radical and H2O2 are ROS that are generated when the tissue are exposed to a variety of stresses. Apart from being toxicity various ROS, H2O2 is regarded as a signaling molecule and a regulator of the expression of some genes in cells (Chakrabarty et al., 2009). POD decomposes H2O2 by oxidation of phenolic compounds. Thus, it acts as a protective mechanism to reduce oxidative damage triggered by stress (Scalet et al., 1995).

Table C-II.4.1a: Peroxidase activity (in ΔO.D./min/g fresh petals) in cut flower Petals

Stages

Days

Peroxidase (mg / g)

1

1

0.094 ± 0.000

2

2

0.075 ± 0.002

3

3

0.059 ± 0.001

4

4

0.041 ± 0.002

Fig C-II.4.1: The Peroxidase activity in cut flower petals

Table C-II.4.1b: ANOVA Summary Table for Peroxidase activity in cut flower petals

Source of Variation

Sum of Squares

(SS)

Degree of Freedom

(DF)

Mean Squares

(MS)

F

Ratio

Table value of

F

Between groups

0.005

3

0.002

313.960

4.07*

Within groups

0.000

8

0.000

Total

0.005

11

* at 0.05 level of significance

The POD activity was found to have decreasing trend till the senescence of Tithonia rotundifolia Blake and analyzed to prove that at different stages the peroxidase activity is significantly different as shown in table C-II.4.1b. As said above, the production of ROS and low activity of POD facilitated accumulation is facilitate due to stress condition and these free radicals possibly caused damage to the enzymes. This view is also supported by decrease in the values of enzyme proteins during this phase (Table C-II.2.2a). Reddy and Srivastava (2003) have also reported that may be due to disintegration of enzyme proteins, the activities of POD enzymes decreased with the progress of ripening in Mango. Peroxidase activity of high in stage 1 was observed which goes with the findings of Fridovich (1975) that the increased activity of peroxidase apparently related to an increase in peroxidase and free radicals, which react with cellular constituents and are involved in promotion of senescence (Brennan and Frenkel, 1977; Nichols, 1973) and possibly also in ethylene production (Borochov et al., 1976). Peroxidase has been implicated in plant senescence mainly due to its ability to oxidize IAA.

C-11.4.2 POL YPHENOL OXIDASE (PPO)

Table C-II.4.2a and Figure C-II.4.2 shows the PPO activity in terms of ΔO.D./min/g fresh flower petals of Tithonia rotundifolia Blake. PPO is known to be involved in oxidation of phenolic and also known be involved in scavenging ROS compounds.

Table C-II.4.2a: Polyphenol Oxidase activity (in Δ O.D./min/g fresh petals) in cut flower petals

Stages

Days

Polyphenol Oxidase (mg / g)

1

1

0.042 ± 0.005

2

2

0.037 ± 0.004

3

3

0.019 ± 0.001

4

4

0.011 ± 0.001

Fig C-II.4.2: The Polyphenol Oxidase activity in cut flower petals

Table C-II.4.2b: ANOVA Summary Table for Polyphenol Oxidase activity in cut flower petals

Source of Variation

Sum of Squares

(SS)

Degree of Freedom

(DF)

Mean Squares

(MS)

F

Ratio

Table value of

F

Between groups

0.002

3

0.001

40.583

4.07*

Within groups

0.000

8

0.000

Total

0.002

11

* at 0.05 level of significance

PPO had a decreasing trend till the senescence stage. Table C-II.4.2b reveals that the PPO activity was significantly different at different stages of Tithonia rotundifolia Blake. This indicates that possibly the enzyme was getting degraded or become inactive due to some reason. A lower level of enzyme protein during this period also supports this view (Table C-II.2.2a). According to Rivero et al. (2001) activity of the enzyme was increased in response to different types of stresses. However, in the present case though the flower was under stress, the activity of the enzyme kept decreasing. Therefore, more in depth experimentation is required to understand the physiology. The general decrease in activity suggests that either the substrate was inadequate in amount for the enzyme to act upon it or the enzyme was getting degraded gradually as the protein was getting broken down.

C-11.4.3 CATALASE

Catalase is an important free-radical scavenging enzyme known to increase during senescence. Table C-II.4.3a and Figure C-II.4.3 represents the catalase (CAT) activity in cut flower petals of Tithonia rotundifolia Blake. It was observed that catalase activity followed the decreasing trend towards senescence. It was also found to be significantly different at various stages (Table C-II.4.3b).

Table C-II.4.3a: Catalase activity (in ml H202 reduced /g fresh petals) in cut flower Petals

Stages

Days

Catalase (mg / g)

1

1

0.723 ± 0.001

2

2

0.592 ± 0.002

3

3

0.432 ± 0.001

4

4

0.327 ± 0.004

Fig C-II.4.3: The catalase activity in cut flower petal

Table C-II.4.3b: ANOVA Summary Table for Catalase activity in cut flower petals

Source of Variation

Sum of Squares

(SS)

Degree of Freedom

(DF)

Mean Squares

(MS)

F

Ratio

Table value of

F

Between groups

0.278

3

0.093

171.188

4.07*

Within groups

0.000

8

0.000

Total

0.278

11

* at 0.05 level of significance

It was reported that CAT reacts with H2O2 (Monk et al., 1989) and breaks it to produce water and oxygen (Scandalias, 1993; Agarwal and Pandey, 2003). So it keeps checking the accumulation of H202 to toxic levels. Peroxidase activity was observed remarkably less at senescent stage (0.327 mg /g) as compare with the first stage (0.723 mg/ g) of flower development this suggest that more and more breakdown of H2O2 towards senescence. Kraus et al., (1995) reported that increase in CAT activity is related with increase in stress tolerance which seems to be true even for the present investigation.

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