Changes in Proline and Polyphenol oxidase enzyme activity in some Banana Cultivars and Hybrids under water stress  

K. Krishna Surendar1 , D. Durga Devi2 , P. Jeyakumar3 , K. Velayudham4 , I. Ravi5
1. Assistant Professor, Crop Physiology, Regional Research Station, TNAU, Paiyur
2. Professor, Department of Crop Physiology, TNAU, Coimbatore. Tamil Nadu, INDIA
3. Professor & Head, Department of Crop Physiology, TNAU, Coimbatore. Tamil Nadu, INDIA
4. Dean, Agriculture College & Research Institute, TNAU, Madurai. Tamil Nadu, INDIA
5. Principal Scientist, National Research Centre for Banana (ICAR), Thiruchirapalli
Author    Correspondence author
Genomics and Applied Biology, 2015, Vol. 6, No. 4   doi: 10.5376/gab.2015.06.0004
Received: 10 Feb., 2015    Accepted: 22 Mar., 2015    Published: 09 Apr., 2015
© 2015 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Surendar et al., 2015, Changes in Proline and Polyphenol oxidase enzyme activity in some Banana Cultivars and Hybrids under water stress, Genomics and Applied Biology, Vol.6, No.4, 1-6 (doi: 10.5376/gab.2015.06.0004)


Plant growth and productivity is adversely affected by abiotic stresses induced changes in proline content and polyphenol oxidase enzyme activity. Field experiments were conducted at the National Research Centre for banana, Thiruchirapalli, during the growing season of 2011-2013 to compare the changes in proline content and PPO enzyme activity in twelve main crop and ratoon crop ofbanana cultivars and hybrids in 5th and 7th MAP (Month after Planting) under two levels of irrigation. The lowest reduction in the bunch yield of tolerant banana cultivars and hybrids viz., Karpuravalli, Karpuravalli x PisangJajee, Saba, and Sannachenkathali with the highest increase in proline and PPO enzyme activity was observed in 5th and 7th MAP stage due to 50 per cent depletion in available soil moisture (ASM) of the stress.

Water deficit; Proline; Polyphenol oxidase; Yield; Banana

In higher plants, proline is a candidate biochemical solute, being well known as a stress indicator, especially of water deficit stress. (Yoshiba et al., 1997). The proline biosynthesis pathway in plant s has been well established via glutamate intermediate, using P5CS (∆1 pyrroline- 5 -carboxylate synthetase) to P5C (∆1 -pyrroline-5-carboxylate), subsequently oxidized to the final product proline by P5CR (∆1-pyrroline- 5-carboxylate reductase). Also, proline degradation has been discovered through ProDH (proline dehydrogenase) from proline to P5C (∆1-pyrroline-5- carboxylate) and then P5CDH (∆1-pyrroline-5- carboxylatedehydrogenase) (Verslues and Sharma, 2010). The function of proline in plant defence responses to water deficit stress has been reported, including signal transduction, osmoregulation and antioxidant systems (Dalauney and Verma, 1993).
Poly Phenol Oxidaseis a copper-containing enzyme and is responsible for the enzymatic browning reaction occurring in many fruits and vegetables. In the presence of molecular oxygen, PPO catalyzes the o-hydroxylation of monophenols to o-diphenols (monophenolase activity) and oxidation of the o-diphenols to o-quinones (diphenolase activity) (Chararra et al., 2001). The synthesis of phenolic compounds is often enhanced in plant tissues under oxidative stresses such as drought and mechanical damage (Reyes and Cisneros-Zevalloss, 2003). Bananas are quite sensitive to drought; however, genotypes with “B” genome are more tolerant to abiotic stresses than those solely based on “A” genome. In particular, bananas with “ABB” genomes are more tolerant to drought and other abiotic stresses than other genotypes (Ravi et al., 2013). Salekdeh et al. (2009) mentioned that the reduction in banana growth and yield may be due to the shortage of water in the root zone.
Drought is one of the important abiotic constraints restricting banana cultivation and its further adoption into non- conventional growing areas. Drought has rarely been addressed in the past, but is gaining importance in the face of depleting natural resources (Ravi et al., 2013). The results of successful cultivation, especially of the water loving Cavendish clones, in drought prone areas with protected irrigation have provided the required momentum to perform research on drought in bananas (Ravi et al., 2013). In subtropical and semi- arid banana cultivation zones have very limited rainy days and also had uneven distribution of rainfall, new crop management practices in terms of varieties selected, soil improvement (in terms of physical properties and nutrient enrichment), water management, etc. are being adopted (Ravi et al., 2013). The aim of this investigation was to screen the twelve banana cultivars and hybrids through the accumulation of proline content and PPO enzyme activity during 50 per cent depletion of ASM at 5th and 7th MAP (Table 1).

Table 1 General observations on germplasm performance under water deficit conditions. (Ravi et al., 2013, Anon, 2007, 2006; Uma and Sathiamoorthy, 2002; Uma et al., 2002)

1 Materials and Methods
Field experiments were conducted at the National Research Centre for banana, Thiruchirapalli, during the growing season of 2011-2013 in a split plot design with three replications. Two levels of irrigation: 80 per cent ASM with soil pressure maintained from -0.69 to -6.00 bar and second level of irrigation at 50 per cent ASM with the soil pressure maintained in -14.00 bar and twelve ratoon banana cultivars and hybrids namely: S1: Karpuravalli (ABB), S2: Karpuravalli x Pisang Jajee, S3: Saba (ABB), S4: Sanna Chenkathali (AA), S5: Poovan (AAB), S6: Ney poovan (AB), S7: Anaikomban (AA),S8: Matti x Cultivar Rose,S9: Matti (AA),S10: Pisang Jajee x Matti,S11: Matti x Anaikomban and S12: Anaikomban x Pisang Jajee were laid out in the main plots and sub plots respectively. The soil pressure was calculated by using the soil moisture release curve (Figure 1) and the soil moisture was measured by using the pressure plate membrane apparatus instrument (Table 2). The water deficit stress was imposed at 5th and 7th MAP and the proline and PPO enzyme activity were recorded during the stress period.

Figure 1 Pressure plate apparatus soil moisture release curve

Table 2 calculated pressure from stress treatment and soil moisture content from regression equation

1.1 Estimation procedure:
1.1.1 Proline
Proline content of the leaf sample was estimated by the method of Bates et al. (1973) and expressed as µg g-1 of fresh weight.
A fresh leaf sample of 0.5g was macerated with 10mL of aqueous sulphosalicylic acid (3%) using a pestle and mortar. The extract was centrifuged at 4000 rpm for 10 minutes. The supernatant solution of 2 ml was taken in a test tube and to this 2 mL of acid ninhydrin and 2 mL of glacial acetic acid was added. The solution was kept in water bath for one hour at 100°C and it was cooled under tap water. After cooling, the solution was transferred into a separating funnel and 4 mL of toluene was added. The funnel was uniformly shacked for 30 seconds. Two different layers were formed. The colorless bottom layer was discarded and the upper pink color layer was collected. The Optical Density was recorded at 520 nm against blank as toluene.
1.1.2 Acid ninhydrin
(2.5g of ninhydrin was taken and mixed with 60 ml of glacial acetic acid and 40 ml of 6 M orthophosphoric acid. The solution was stirred well and slightly warmed in hot water bath until the content dissolved.)
1.2 Polyphenol Oxidase (PPO)
The Poly Phenol Oxidase (PPO) activities of the leaf sample was estimated at all the stages of the crop by the method of Bray and Thrope (1954) and expressed as unit-1 min-1 mg of protein-1.
The leaf sample of 0.5g was macerated with 10 ml of sodium phosphate buffer (0.1M, pH 7.0) using a pestle and mortar. The extract was centrifuged at 10000 rpm at 4°C for 20 minutes. The supernatant solution of 0.5 mL was taken in a test tube and 2 ml of sodium phosphate buffer (125µmoles, pH 6.8) , 0.5 mL of pyrogallol solution (50 µmoles) was added and kept in water bath for 5 minutes at 25 to 30°C or at room temperature and 0.5 ml of H2SO4 was added. The Optical Density was recorded at 420 nm against blank.
2 Result
2.1 Proline content
The proline content showed an increasing trend from 5th MAP to 7th MAP (Table 3). Between the treatments, M1 (control) had higher proline content of than M2 (stress) at 7th MAP. Analyzing the effect of sub-plot treatments, S1 recorded an increased proline accumulation. This treatment was followed by S2 and S3. The treatment, S12 showed a lesser proline accumulation at 7th MAP in main and ratoon crop.
The interaction effects of M at S and S at M revealed significant differences at all the stages of growth. The treatment M2S1 recorded the highest proline content followed by M2S2, M2S3 and M2S4 at 7th MAP. The treatment M2S10, M2S10, M2S11 and M2S12 found to accumulate the proline at significantly lower level than the other treatments at 7th MAP in main and ratoon crop.
2.2 PPO enzyme activity
Polyphenol oxidase activity steadily increased in 7th MAP at main and ratoon crop (Table 3). Main plot treatments differed significantly at 5th and 7th MAP growth stages. Significantly higher enzymatic activity was maintained by M1 than M2 during the growth period. All the sub-plot treatments exhibited their significant differences. Among the subplot treatments, higher polyphenol oxidase activity was registered by S1 followed by S2, S10 and S4 in the given stage. The lowest enzyme activity was, however, showed by S11 and S12 in main and ratoon crop.

Table 3 Effect of water stress on at proline and polyphenol oxidase enzyme activity and yield at different growth stages of banana cultivar and hybrids in main crop and ratoon crop

The significant variations among the interaction treatments revealed the influence of main plots on sub plot for regulating the enzyme activity. The treatment M1S1 showed a higher value of 1.36, followed by M1S2, M1S3 and M1S4. However a considerable reduction in PPO activity could also be observed due to interaction with M2 and subplot treatments. M2S1, M2S2, M2S3, and M2S4 recorded about 6.3 to 9.8 per cent reduction. M2S5, M2S6, M2S7, and M2S8 showed about 12.4 to 15.0 per cent reduction, whereas, M2S9, M2S10, M2S11 and M2S12 registered about 20.8 to 22.6 per cent reduction over the M1 and subplot treatments in main and ratoon crop.
3 Discussion
In the present study, proline content increased relative to the degree of water deficit stress. Proline acts as an osmolyte and helps the plants to maintain tissue water potential under all kinds of stresses. Proline, as an osmoprotectant, is largely confined to the cytoplasm and is mostly absent from the vacuole (Mc Neil et al., 1999). It plays a key role in the cytoplasm as a scavenger of free radicals as well as a mediator in osmotic adjustment and also increases the solubility of sparingly soluble proteins (Saradhi et al., 1995). Shen et al. (1990) advocated that water stress enhanced the accumulation of proline in many plant species and it might function as a source of solute for intercellular osmotic adjustment under water stress. Stewart (1978) suggested that proline might severe as a storage compounds for reduced carbon and nitrogen during stress. Proline might regulate the osmotic balance of the cell thus relieving the negative effect of stress (Reddy et al., 2004). In the present study also, cultivars like Karpuravalli, Karpuravalli x Pisang jajee, Saba and Sannachenkathali had higher amount of proline accumulation particularly at 7th MAP followed by Poovan, Ney Poovan, Anaikomban and Anaikomban x Pisang jajee than cultivars of Matti, Matti x Anaikomban, Matti x cultivar rose and Pisang jajee x Matti. These findings are further supported by the results of Mohd Razi Ismail (2004) in banana, which explained that the enhancement in free proline content could occur either due to ‘de novo’ synthesis of proline or breakdown of proline-rich protein or shift in metabolism.
3.1 Polyphenol Oxidase
(PPO) is a copper-containing enzyme, responsible for the enzymatic browning reaction occurring in many fruits and vegetables damaged by improper handling etc. (Meyer and Boyer, 1976). Accumulation of polyphenols in the plants is controlled by PPO, also known as phenolase, catalyzing the oxidation of o-diphenols to o-diquinons, as well as hydroxylation of monophenols. Activities of these enzymes increase in response to different types of stresses, both biotic and abiotic (Farooq, 2009). Chararra et al., (2001) reported that PPO activity in banana converted certain phenol compounds to highly reactive quinones in the presence of molecular oxygen. Quinones readily bound to proteins to form complexes, which were more resistant to breakdown by plant and microbial enzymes. Fukumoto et al. (2002) reported that decreased activity under oxidative stress period led to forming symptoms such as brown pitting, necrosis, deterioration of mitochondrial activity and cell damage associated with increased deposition of phenolic compounds. In the present study, a significantly higher rate of PPO activity was observed under water deficit conditions. The enzyme activity was however increased when the twelve cultivars were influenced with water stress. The cultivars of Matti, Matti x Anaikomban, Matti x cultivar rose and Pisang jajee x Matti had increased PPO activity of about 56 per cent over control, whereas cultivars of Karpuravalli, Karpuravalli x Pisang jajee, Saba and Sannachenkathali resulted in 9 to 10 per cent increase in enzyme activity, indicating higher increase in enzyme activity of susceptible cultivars to the water deficit treatment. Similar results were made by Keshavkant (2000); Ose et al., (1999) who found that the considerable reduction in PPO activity during oxidative stress period, particularly in leaves can be explained by the location of this enzyme in the leaf tissue, the membrane of which is the primary targets during oxidative stress induced photo oxidation.
4 Conclusion
Proline synthesis and accumulation was increased due to water deficit stress especially in 50 per cent depletion of ASM in all the twelve banana cultivars and hybrids. But higher proline acculmulation were present in tolerant cultivars and hybrids of Karpuravalli, Karpuravalli x Pisang Jajee, Saba, and Sannachenkathali with lesser reduction in PPO enzyme activity due to water deficit of banana cultivars and hybrids.
The research have been supported and facilitated by National Research Centre for Banana (ICAR), Trichy. Tamil Nadu. India. I extend my sincere thanks to Dr. M. M. Mustaffa (Director) NRC for banana, Dr. D. Durga Devi (Professor) TNAU and Dr. I. Ravi (Sr. Scientist) NRC for banana for given proper guidance during research.
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